S4 Rev Iberoam Micol 2008; 25: S4-S12 Review Diversity of the neoformans- species complex Marjan Bovers1, Ferry Hagen1 and Teun Boekhout1,2

1Yeast Research Group, CBS Fungal Diversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The ; 2Department of Internal Medicine and Infectious Diseases, University Medical Centre Utrecht, The Netherlands

Summary More than 110 years of study of the and Cryptococcus gattii species complex has resulted in an enormous accumulation of fundamental, applied biological and clinical knowledge. Recent developments in our understanding of the diversity within the species complex are presented, emphasizing the intraspecific complexity, which includes species, microspecies, hybrids, serotypes and genotypes. Each of these may have different roles in disease. An overview of obsolete and current names is presented.

Key words Cryptococcus neoformans, Cryptococcus gattii, , , Pathogen. Diversidad del complejo de especies Cryptococcus neoformans-Cryptococcus gattii Resumen Más de 110 años de estudio sobre el complejo de especies Cryptococcus neoformans y Cryptococcus gattii han dado lugar a un gran acúmulo de conocimiento básico, aplicado y clínico. En este artículo se describen los avances recientes en la comprensión de su diversidad, haciendo énfasis en la complejidad intraespecífica que presenta y que llega a englobar especies, microespecies, híbridos, serotipos y genotipos. Cada uno de estos grupos puede jugar un papel diferente en la enfermedad. Se presenta también una visión global de los nombres obsoletos y actuales de estos taxones.

Palabras clave Cryptococcus neoformans, Cryptococcus gattii, Levaduras, Patógeno, Taxonomía.

Introduction racteristics of the Saccharomyces were not present, he placed these species in the genus Cryptococcus [166]. Studies on Cryptococcus neoformans and Crypto- Cryptococcus hominis was later described as a synonym to coccus gattii started in 1894, when Sanfelice first isolated C. neoformans [102]. In 1970, an atypical strain of C. neo- Saccharomyces neoformans from peach juice in Italy [139]. formans was isolated from a leukemic patient and des- In the same year Saccharomyces hominis was observed in cribed as C. neoformans var. gattii [162]. The teleomorph an infection of a German patient [10,11] and two years of C. neoformans was discovered when two serotype D later an encapsulated bacilliform yeast, which was named strains were crossed and was named neofor- Saccharomyces subcutaneous tumefaciens, was isolated mans [74]. However, when serotype B and C strains were from an otherwise healthy man in France [35]. Vuillemin crossed, a teleomorph that clearly differed from F. neofor- examined several of these cultures, but as the ascospore cha- mans was observed. This resulted in the description of Filobasidiella bacillispora [76]. In addition, several physiological assays showed that C. neoformans serotype B and C strains differed from C. neoformans serotype A and D isolates, and the name Cryptococcus bacillisporus was proposed for serotype B and C isolates [81]. Although previous mating experiments with C. neoformans var. gattii had not been successful [81], viable basidiospores were pro- Corresponding author duced when strains of C. neoformans var. gattii were cros- Dr. Teun Boekhout sed with isolates of F. bacillispora and F. neoformans [141]. Yeast Research Group CBS Fungal Diversity Centre Furthermore, mating of isolates of F. bacillispora with Uppsalalaan 8 F. neoformans resulted in the formation of viable basidios- 3584 CT Utrecht pores [80,141]. Kwon-Chung et al. [80] therefore proposed The Netherlands Tel.: +31 30 2122671 to treat the two species as varieties of C. neoformans, Fax:+31 30 2512097 namely var. and E-mail: [email protected] C. neoformans neoformans C. neoformans var. gattii. In 1999, based on phenotypic and genotypic ©2008 Revista Iberoamericana de Micología data Franzot et al. [51] proposed to install a third variety, Apdo. 699, E-48080 Bilbao (Spain) 1130-1406/01/10.00  namely C. neoformans var. grubii, corresponding to se- Diversity of Cryptococcus neoformans Bovers M, et al. S5 rotype A. Despite the results of earlier mating experiments, C. gattii has recently been described as a separate species, because of the increasing amount of data suggesting the distinctiveness of C. neoformans and C. neoformans var. gattii [82]. Table 1 presents an overview of names in old and current use within the species complex. Currently, two species are recognized within the C. neoformans - C. gattii species complex. These species are the asexual yeast C. neoformans (Sanfelice) Vuillemin [166], with teleomorph F. neoformans Kwon-Chung [74] and the asexual yeast C. gattii (Vanbreuseghem and Ta- kashio) Kwon-Chung et al. [82] with F. bacillispora Kwon-Chung as teleomorph [76]. Both C. neoformans and C. gattii belong to the Filobasidiella-clade of the (, Agaricomycotina, ) [47,63,143]. Crypto- coccus amylolentus, Tsuchiyaea wingfieldii and Filobasi- diella depauperata, species which are closely related to C. neoformans and C. gattii [47,48,60,83,113,143], have been isolated either from insect frass or from dead insects (www.cbs.knaw.nl/yeast/BioloMICS.aspx).

Infection and virulence A pulmonary infection with or C. neoformans Figure 1. India ink stained capsule around budding yeast cell of C. gattii most likely starts with inhalation of infectious Cryptococcus gattii CBS 883 after two days at room temperature on particles, which have not been identified with certainty, Littmann agar. although basidiospores seem to be the most likely candi- date [20]. Yeast cells are too large to penetrate into the alveoli [62] where infection starts and desiccated yeast cells display poor viability [136, 169]. Inhaled basidiospo- res may cause infection in mice and are more effective than yeast cells in causing [152]. Interes- tingly, humans without a history of cryptococcal disease have antibodies against C. neoformans [30]. Furthermore, the majority of children have been exposed to C. neofor- mans before they reach the age of five [56]. These results show that humans frequently come into contact with C. neo- formans. In immunocompetent individuals C. neoformans infection is either cleared or remains dormant [55]. In immunocompromised individuals, however, C. neofor- mans can disseminate to other organs. Infection of the cen- tral nervous system may result in meningoencephalitis, a Figure 2. Melanin (JEC 21, right) and non-melanin (JEC 20, left) forming condition that is fatal if left untreated. Contrary to C. neo- colonies of C. neoformans serotype D on L-DOPA medium after 5 days at formans, C. gattii primarily infects otherwise healthy indi- 25 ºC. viduals [31,111,112,135,147], but serotype C isolates of C. gattii were found to be implicated in HIV-associated infections in California, Botswana and Malawi [27,97]. grow optimally at temperatures above 30 °C [86,129] and temperature sensitive mutants of are The reported incubation period for C. gattii serotype B, C. neoformans genotype AFLP/6/VGII of up to six or seven months [104] attenuated in virulence [128]. Another important virulen- or as short as two months [96], is consistent with infection ce factor is the production of a large polysaccharide cap- by a primary pathogen. Although both species infect the sule (Fig. 1). Acapsular mutants are avirulent in mouse models [22-25]. Capsule size increases during infec- central nervous system, C. gattii appears to invade the tion [46] and can be induced by high CO2 concen- brain parenchyma more commonly than C. neoformans. in vitro Furthermore, in C. gattii infected patients, pulmonary tration [59], iron deprivation [163], and serum [178]. infections are more likely and pulmonary mass-like le- Although some properties of the capsule enable the host sions are more common than in C. neoformans infected to clear C. neoformans more effectively, others protect patients [111,147]. C. neoformans against host defenses. Overall, the effect of Although C. neoformans is a human pathogen, the the capsule is beneficial: encapsulated cryptococcal cells human host is probably an accidental encounter and not are not phagocytized or killed by neutrophils, monocytes its primary niche. Infection of macrophages and amoebae or macrophages to the same degree as acapsular cells [9]. by C. neoformans is similar, and it was therefore postula- The production of melanin is another important virulence ted that mammalian virulence factors of C. neoformans factor (Fig. 2). Melanin synthesis is catalyzed by a laccase evolved as a defense mechanism to environmental preda- and may occur when phenolic compounds, e.g. catechola- tors [148,149]. There are several virulence factors that mines, are present [127,130,172,173]. Dopamine, a cate- contribute to the virulence of C. neoformans in mammals. cholamine which is present in the brain, is a substrate for Obviously, the ability to grow at 37 °C is essential. C. neo- melanin synthesis [10,172]. The ability of C. neoformans formans and C. gattii are the only Tremellales that can and C. gattii to form melanin has been used to develop S6 Rev Iberoam Micol 2008; 25: S4-S12 diagnostic selective media, such as L-DOPA or nor-epi- Epidemiology and ecology nephrine media. It is important to note that also non-neofor- mans cryptococci are able to form melanin, such as C. pod- C. neoformans and C. gattii can be distinguished by zolicus and Cryptotrichosporon anacardii [124,129]. In serotype (A, D and AD for the former, B and C for the lat- C. neoformans and C. gattii, melanin is synthesized during ter) [80,82], karyotype [6,171], RAPD patterns [6,40,137], infection [122,134] and mutants that do not produce mela- PCR fingerprints [110], RFLP patterns [89,108], AFLP nin are less virulent [133,138]. Melanin protects C. neofor- fingerprint [7] and DNA sequencing [5,15,28,37,38,69, mans from oxidative damage by scavenging host-produced 151,175,176]. Most of these methods have shown that two anti-oxidants [9,167]. In addition, laccase decreases the genotypic groups can be recognized within C. neoformans amount of hydroxyl radicals directly, thereby protecting and these groups have been described as separate varieties, C. neoformans [101]. Given the notion that C. neoformans namely var. grubii (serotype A) and var. neoformans may represent a secondary (or dormant) pathogen contrary (serotype D) [51] (see Table 1). However, although several to C. gattii (or at least some of the genotypes occurring in molecular methods distinguish four genotypic groups this species) it would be particularly interesting to study within C. gattii [5,15,28,37,40,53,89,108,137], these the main virulence traits in both in order to understand the groups have not yet been interpreted as distinct taxa (see molecular basis of these differences in pathogenicity. This Table 1). Interestingly, C. gattii serotypes B and C do not is not only important from a clinical point of view, but may correspond to a specific group, but appear to occur in all also largely contribute to our understanding of speciation genotypic groups [108]. within this complex of important human and animal patho- C. neoformans and C. gattii differ in epidemiology gens. Recently, in addition to vertebrates, such as the mou- and in their ecological niche. C. neoformans var. grubii se, rat and rabbit, a number of invertebrate model orga- occurs throughout the world and causes 99% of the crypto- nisms have been explored for their potential contributions coccal infections in HIV-infected patients [111]. In Europe to our understanding of cryptococcal pathogenesis [103]. this variety occurred in 51% of the isolates analyzed in a Experiments with Caenorhabditis elegans, a widely used 30-month survey [165]. C. neoformans var. neoformans model organisms in molecular biology, showed that cap- occurs worldwide as well, but appears to be more common sule and G-␣ protein-cAMP-protein kinase A (PKA) pla- in Europe, where 30% of the reported infections are cau- yed a role in cryptococcal virulence [116]. Also Droso- sed by this variety [39,79,158,159,165]. In France, it was phila melanogaster and Galloria mellonella have been found that infection by this variety occurred more fre- used for cryptococcal virulence assays. Because each of quently among patients from certain geographical areas, these model organisms have their advantages and disad- among patients receiving corticoid steroid treatment, and vantages [103], it may well be that an integrated approach in patients with skin lesions [39]. In contrast, the risk of using multiple vertebrate and invertebrate hosts is needed being infected was smaller among female patients, those to understand the full extent of cryptococcal pathogenicity. originating from Africa, and for patients suffering from

Table 1. Names in current and old use in the C. neoformans / C. gattii species complex and with reference to the different typing nomenclatures in current use.

1. Cryptococcus neoformans, type strain CBS 1321 Teleomorph Filobasidiella neoformans, holotype BPI 71843 (= CBS 6885 [serotype D, AFLP 2] ϫ CBS 6886 [serotype D, AFLP 2]). – Cryptococcus neoformans var. neoformans, type strain CBS 1321 = serotype D = AFLP genotype 2 = genotype VNIV [reference strain WM629 = CBS 10079]. Synonyms for which type is available: Saccharomyces neoformans, type CBS 1321; Torula nasalis, type strain CBS 882. – Cryptococcus neoformans var. grubii, type strain CBS 8710 = serotype A = AFLP genotype 1/1A/1B = genotype VNI/VNII [reference strains WM148/WM626 = CBS 10085/10083]. Synonyms for which type or authentic strain is available: Candida psicrophylicus2, type CBS 996; Saccharomyces hominis2, authentic strain CBS 879. 2. Intervarietal grubii ϫ neoformans hybrids – Serotype AD Hybrids = AFLP genotype 31 = genotype VNIII [reference strain WM 628 = CBS 10080]. Note that CBS 132, the type strain of C. neoformans variety neoformans is a serotype AD hybrid.

3. Cryptococcus gattii3, type strain CBS 6289 = RV 20186. Teleomorph Filobasidiella bacillispora, holotype BPI 71855 (= CBS 6956 [serotype B, AFLP 6] ϫ CBS 6993 [serotype C, AFLP 5])4 No infraspecific taxa are described, but four different genotypes do occur. – AFLP genotype 4 = serotype B (or C)6 = genotype VGI [reference strain WM179 = CBS 10078]. Synonyms for which type or authentic strains are available: Cryptococcus neoformans var. gattii, type strain CBS 6289; Candida hondurianus3, syntype strain CBS 883; Torulopsis neoformans var. sheppei, type strain CBS 919; Cryptococcus neoformans var. shanghaiensis, type strain CBS 7229, Saccharomyces subcutaneous tumefaciens5, authentic strain CBS 1622. – AFLP genotype 5 = serotype B or C = genotype VGIII [reference strain WM161= CBS 10081]. Synonym for which a type strain is available: Cryptococcus bacillisporus, type strain CBS 6955. – AFLP genotype 6 = serotype B (or C)6 = genotype VGII [reference strain WM 178 = CBS 10082]. – AFLP genotype 7 = serotype B or C = genotype VGIV [reference strain WM779 = CBS 10101]. 4. Interspecific gattii ϫ neoformans hybrids – Serotype BD hybrid = AFLP genotype 8 [reference strain CBS 10488]. – Serotype AB hybrid = AFLP genotype 9 [reference strain CBS 10496].

1 The type strain of Cryptococcus (=Saccharomyces) neoformans CBS 132 is a serotype AD hybrid. When both serotypes A and D will be recognized as separate species, this type strain may need to be replaced in order to establish nomenclatural stability. 2 These names have priority at the species level. 3 Cryptococcus gattii is conserved against Cryptococcus hondurianus. 4 These two strains belong to different genotypes within C. gattii as follows: CBS 6956 (NIH 444) is AFLP6/VGII, whereas CBS 6993 is AFLP5/VGIII. Given the many genomic differences between these two genotypes, it may be that they represent different infraspecific taxa or even microspecies. 5 This name is an illegitimate trinomial. 6 Although serotype C has been reported to occur in these genotypes, this could not be confirmed by the present authors. This, however, may be due to the different strain collections used in the various studies. Diversity of Cryptococcus neoformans Bovers M, et al. S7 meningeal cryptococosis [39]. In Europe, 30% of the isolates into the conjugation tube. Simultaneously, the MATa were found to represent this variety, and in Italy it nucleus divides and the MATa cell initiates hyphal forma- was most frequently isolated in the Northern part of the tion. Nuclei from both mating-types then migrate into the country [165]. In contrast to C. neoformans, C. gattii mainly hyphae. The now dikaryotic hyphae are linked by fused infects otherwise healthy individuals [31,111,135,147] and clamp connections. A is formed on the tip of occurs predominantly in subtropical areas [79,100,108]. hyphae and subsequently karyogamy and meiosis occur C. gattii has, however, also been isolated in Europe in within the basidium [75]. The four resulting nuclei remain areas with a temperate or Mediterranean climate [3,34,35, in the basidium and repeated post-meiotic mitosis genera- 114,164] and it is also known from temperate climate tes four long chains of spores [75,77]. A single spore chain zones in Colombia [45]. Furthermore, C. gattii is responsi- may contain parental spore types as well as recombinants, ble for the ongoing outbreak of cryptococcosis on Vancou- indicating that the nuclei in the basidium are randomly dis- ver Island, Canada [65,72,150], and has recently been de- tributed and that mitosis of these nuclei occurs randomly tected in other areas in the Pacific Northwest, USA [105]. prior to spore formation [77]. C. neoformans is often isolated from avian excreta, C. neoformans cells may also reproduce by haploid mainly from pigeon excreta [20,44]. In addition, it has fruiting, which occurs in response to nitrogen starvation been isolated from soil [43] and decaying wood [91]. Zoo- and/or desiccation [170]. Haploid fruiting resembles notic transmission of C. neoformans from bird’s excreta to mating, but there are some differences. Mating involves immunocompromised or immunocompetent humans has partners of opposite mating-types, whereas haploid frui- been demonstrated in a few cases [88,121] or has been ting occurs in strains of the same mating-type [170]. In seriously considered in others [145]. addition, nuclear fusion occurs early during haploid frui- C. gattii has been isolated from several tree species ting [95], but late during mating [75]. Furthermore, clamp since the initial finding of C. gattii on Eucalyptus camal- connections of filaments produced during mating are dulensis [16,41,42,45,49,54,57,72,73,90,92,131,132]. Re- fused, whereas clamp connections produced during frui- cently, however, it has been suggested that soil may in fact ting are unfused [170]. During haploid fruiting basidia be the principal reservoir for this species [71]. In a detai- with viable basidiospores are formed, albeit at a lower fre- led analysis of Colombian isolates it was observed that quency than in a regular MATa ϫ MAT␣ cross [170]. climatic conditions were related to the distribution of both Although haploid fruiting has first been described in C. neoformans var. grubii and serotypes B and C of C. gat- MAT␣ isolates of all serotypes [170], it has also been tii. It seems likely therefore that differential tolerances of observed in a few MATa isolates [161]. Interestingly, one the genotypes/serotypes to environmental conditions, such of the environmental C. gattii isolates from Vancouver as climate, may effect their geographic and ecologic distri- Island is a diploid homozygous MAT␣ strain [53], which butions [58]. may have been generated by aberrant ‘haploid fruiting’, i.e. same sex mating [95]. Several purposes have been suggested for haploid Reproduction fruiting, or more specifically for the resulting filamenta- tion. Haploid fruiting may increase the chance of finding a C. neoformans and C. gattii are usually haploid mating partner [67,168]. An indication for this is the sti- that predominantly reproduce asexually, i.e. by bud- mulation of haploid fruiting of MAT␣ cells in response to ding. However, they also possess a bipolar mating system, MFa pheromone [144]. Haploid fruiting may also increase with mating-types a and ␣ [74,75]. The mating-type (MAT) the foraging capacity under low nutrient conditions, as locus is the region of a fungal genome that regulates the is indicated by the observation that haploid fruiting of sexual cycle and which is different between cells of oppo- MAT␣ cells is enhanced by overexpression of MF␣ phero- site mating-type. Mating may occur if cells of opposite mone genes, which are induced during starvation condi- mating-types meet [74-76]. possesses a sin- tions [144]. Recently, a phenomenon called same-sex C. neoformans ␣ gle MAT locus, which is unusually large, i.e. more than mating, i.e. mating between two non-isogenic MAT cells, 100 kb for both C. neoformans and C. gattii [52,53,94]. It has been described [95]. The isolation of serotype A encodes more than twenty genes, including homeodomain MAT␣-serotype D MAT␣ environmental isolates [97] sug- genes which establish cell type identity, genes involved in gests that same-sex mating occurs in the environment. pheromone production and sensing, components of a MAP Although mating of C. neoformans or C. gattii was kinase cascade, essential genes, and genes which do not shown to occur under laboratory conditions [74, 76], it seem to have a function in mating [52,94,115]. Evidence has never been found in the environment. In addition, suggests that the ancestor of C. neoformans had two unlin- past studies have found evidence for clonal population ked sex-determining regions, which expanded by acquisi- structure [12,13,29,50]. However, when C. neoformans tion of genes of related function. A chromosomal translo- var. grubii and var. neoformans were studied separately cation fused the two regions, which resulted in a tripolar the null hypothesis of recombination could no longer intermediate mating system that collapsed into a bipolar be rejected [157]. In addition, analysis of the LAC1 and system. In this bipolar system inversions, that suppressed URA5 genes for AD hybrid isolates showed that recombi- recombination, occurred, which resulted in the currently nation occurred within each variety [175]. Furthermore, re- known MAT loci [52]. combination may occur within subpopulations of var. gru- MATa cells produce MFa pheromone, e.g. in res- bii [97,99] and within populations of var. neoformans [97]. ponse to nitrogen starvation, and in response to this phero- In addition, recombination was found in subpopulations of mone MAT␣ cells form a conjugation tube [26,168]. In res- C. gattii AFLP6 [18]. In summary, C. neoformans and ponse to MF␣ pheromone, which is induced by starvation C. gattii reproduce through diverse sexual and asexual conditions as well as by the presence of MATa cells [144], strategies, in which both recombination and clonal disper- the MATa cells dramatically enlarge to form large swollen sal do occur [17]. cells that can fuse with the conjugation tubes of the MAT␣ The majority of environmental and clinical isolates cells [36]. McClelland et al. [107] hypothesized about the belongs to MAT␣. [1,18,19,21,29,45,54,61,64,66,68,78,97, cascade of events following conjugation tube formation by 99,100,106,123,125,126,132,140,142,154,160,165,177]. the MAT␣ cell. The MAT␣ nucleus divides and migrates However, C. gattii MATa and MAT␣ isolates have been S8 Rev Iberoam Micol 2008; 25: S4-S12 found in an 1:1 ratio and MATa isolates sometimes even Genetic and molecular studies performed on AD outnumbered the MAT␣ isolates [45,61]. Unequal inheri- hybrid isolates showed that these were formed multiple tance of the MAT locus could provide an explanation for times [174,177] and AD as well as BD hybrids have been the excess of MAT␣ isolates. However, in laboratory cros- generated in the lab [33,87,93,146,155]. When serotype A ses MATa and MAT␣ isolates were either obtained in an and serotype D strains were mated, 24% to 70% of the 1:1 ratio [70,75,77,80,99,118,161,170] or an excess of progeny were diploid or aneuploid [33,87,155]. In addi- MATa progeny was obtained [80,161]. When haploid frui- tion, mating of a serotype B with a serotype D strain resul- ting in MAT␣ isolates was first described [170] this pheno- ted in 50% diploid or aneuploid progeny [87]. These menon was thought to explain the high number of MAT␣ results show that AD and BD hybrids can be generated isolates in environmental and clinical isolates. However, quite easily. In some areas of Europe, 16 to 30% of the Tscharke et al. [161] showed that MATa isolates are also clinical isolates were found to be AD hybrids [32,79,165], capable of haploid fruiting. In addition, the poor haploid with Spain, Greece and Portugal showing the highest inci- fruiting capability of C. neoformans var. grubii isolates, dence, namely 45%, 48% and 50%, respectively [165]. which are responsible for the majority of cryptoccal infec- These observations indicate that in these areas hybrids are tions [111,161,170], indicated that haploid fruiting does responsible for a significant part of the cryptococcal infec- not provide an explanation for the high number of MAT␣. tions. It has also been suggested that MAT␣ isolates might Serotype AD hybrids may contain both MATa and be more virulent than those belonging to MATa. Several MAT␣ alleles for both serotypes A and D, and, hence, both approaches have been used to test this hypothesis. Conge- AaD␣ and A␣Da genotypes occur [33,97, M. Bovers & nic pairs of both serotype A and D were created and T. Boekhout, unpublished observations]. Recently it was although virulence was associated with MAT␣ in the se- proposed that the AaD␣ genotype may have originated rotype D congenic pair [84], no clear association between from a mating population in Botswana, which, due to bet- virulence and mating-type was found when progeny of a ter fitness characteristics could disseminate globally [98]. serotype D mating was compared [85]. Furthermore, the serotype A congenic pair did not differ in virulence [118]. Interestingly, during C. neoformans var. grubii MATa and Conclusions and perspective MAT␣ coinfection MAT␣ cells out-competed MATa in entry to the central nervous system. Nielsen et al. [117] C. neoformans and its sibling species C. gattii and Barchiesi et al. [2] found that the presence of a form a species complex that has been studied from many serotype A-MAT␣ allele is associated with virulence. different perspectives. The results suggest that these two Finally, virulence studies carried out using serotype D con- species are distinct. Accumulating evidence, however, genic pairs of different genetic background showed that suggests that the varieties grubii and neoformans, and pro- the genetic background plays a significant role in determi- bably the four genotypes in C. gattii may also represent ning the effect of mating-type on virulence [119]. separate microspecies. Further molecular, genetic and ecological investigations are needed to confirm this hypo- thesis. The existence of both intervarietal (serotype AD) Hybrids and interspecific (serotype BD and AB) hybrids, which are diploid or aneuploid, indicate that species boundaries may Occasionally diploid or aneuploid isolates, such as be maintained by postzygotic isolation mechanisms. The AD, BD or AB hybrids, are found [4,8,14,32,33,53,64,79, continued sampling of clinical and environmental isolates, 97,100,109,120,140,153,154,156,158-160,165, M. Bovers, together with adequate genotyping, may further strengthen F. Hagen & T. Boekhout, unpublished observations], the role of each genotype in the various diseases caused by although the diploid phase of C. neoformans and C. gattii representatives of the species complex, as well as the is normally associated with the filamentous sexual stage various patient categories in which cryptococcosis occurs. rather than the yeast stage. Hybrids usually possess both mating-type loci which indicates that they most likely result from mating between isolates of opposite mating- types [8,32,93]. Hybrids can result either from pre-meio- tic sporulation or from fusion of haploid meiotic nuclei followed by the packing of a diploid nucleus in a basidios- pore [146]. Indeed, Kwon-Chung [75] has observed atypi- cal basidia producing two normal spores and one spore which was twice as large and may have contained a diploid nucleus. These observations are consistent with the forma- tion of hybrids by fusion of haploid meiotic nuclei. Diversity of Cryptococcus neoformans Bovers M, et al. S9

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