Why are we interested in understanding the mode of reproduction being used by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii? Empirical evidence has finally supported the long-held assumption that, by increasing the rate of adaptive evolution, sex increases the chances of long-term survival. Understanding the ability of pathogenic organisms to adapt to diagnostic and treatment regimes is also important in the fight against the diseases caused by these organisms. This review looks at the different approaches used to identify population structure in C. neoformans and C. gattii. These are sexual species; however, recombination in natural populations has only recently been found. We highlight the importance of population selection and the value of both indirect molecular analysis and direct biological evidence for sexual recombination, when looking for the mode of reproduction in these fungal pathogens.
Sex is an expensive process. Undergoing sexual recombination costs time and energy, and favorable gene combinations can be disrupted during sexual recombination (Bell, 1982; Xu, 2005a). However, although all microorganisms can reproduce asexually, often producing prolific numbers of clonal propagules, the majority also undertake some form of sexual reproduction (Xu, 2004a). Sex has been hypothesized to increase the rate of adaptive evolution, potentially allowing offspring to be more robust than either parent in a harsh or changing environment (Butlin, 2002), and sexual populations of yeasts have been found to adapt more rapidly to harsh environmental conditions than asexual clones, which can rapidly lose fitness (Xu, 2004b; Goddard, 2005). An increased rate of adaptive evolution is of particular importance when considering pathogenic microorganisms such as Cryptococcus neoformans and Cryptococcus gattii. Knowing whether or not sexual recombination is occurring in pathogens is key to the development of diagnostic and treatment procedures (Tibayrenc, 1990, 1991). In recombining organisms, genes governing virulence or antimicrobial resistance traits are passed between strains and reassorted to produce novel combinations, which may lead to increased pathogenicity or resistance (Grigg, 2001). Determining whether recombination occurs can also yield information about the ecology of primary fungal pathogens such as C. neoformans and C. gattii, which are almost always acquired by the host animal directly from the environment. Ecological disruptions due to anthropological factors such as monoculture of agricultural species, habitat fragmentation, or importation of new species into favorable, naïve habitats, may cause otherwise sexual species to undergo rapid clonal expansion, whereas species maintained in a natural undisturbed habitat are likely to undergo at least periodic recombination. Finding clonal behavior in sexually competent species may therefore point to recent disturbances; conversely, finding recombination may help define the natural ecological niche of the organism (Fisher, 2001).
Tools used to look for sex in microorganisms
There are two main approaches used in looking for evidence of sex or recombination: the direct observation of biological mating ability; and an indirect assessment of recombination based on detecting reassortment in molecular multilocus genotypes. The observation of mating is the only real biological proof of fertility; however, appropriate mating conditions and mating partners must be identified for crosses to be successful. An inability to mate may be due to non-optimal conditions rather than lack of fertility. Also, the observation of mating under laboratory conditions may not reflect behaviour in the natural environment, and in organisms that reproduce both sexually and asexually, laboratory matings give no indication of the proportion of a natural population that is sexually recombining vs. that which is propagating clonally.
The application of molecular analyses to natural populations eliminates the need for laboratory tester strains and allows the degree of sexual recombination or clonal propagation occurring in a population to be assessed (Kasuga, 1999). In ecological terms, populations are defined as a collection of organisms belonging to a single species, present in one place at one time (Callow, 1998). In practice, the ‘place’ occupied by a population may not be clear cut and may be set by the biological question under study, for example, it might be a wide geographic region, a certain ecological niche or a single body site, or even a body site present only in a certain demographic sector of the host population. The choice of population is critical for the analysis of recombination. If isolates are sampled too widely they may belong to isolated subpopulations that never have the opportunity to meet and mate; if the focus is too narrow in an organism that can reproduce asexually there may be an oversampling of clonemates. In both cases, clonality may be erroneously concluded, and it can be difficult to prove that a population is genuinely, and not artifactually, clonal. Phylogenetic analysis of the population structure (see below) and a continual refining of the sampling strategy may be required to identify clonal and recombining subgroups within a larger population set.
The molecular analysis of a population requires the development of molecular markers and an analysis of their distribution among members of the population. A variety of molecular techniques have been used in fungal population genetics (see for example Xu, 2005b). For recombination analysis, the major requirement is that all loci assort independently. This rules out profiling strategies based on arbitrarily primed PCR reactions, such as random amplification of polymorphic DNA (RAPD) or arbitrarily primed PCR (AP-PCR), or those that perform PCR amplification using primers with only partial homology to their target sequences, such as DNA fingerprinting (Carter, 2004). In profiles generated using these techniques, bands can be ‘co-dependent’, such that some small amplified fragments are generated from within larger fragments (Hadrys, 1992). Co-dependent bands will be lost or gained as a group, and will therefore appear linked to one another, which will skew the analysis in favour of clonality (see below). Methods that produce discrete loci that can be assumed to be independent include multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) analysis, and multilocus sequence typing (MLST), and these have been used to generate loci for recombination studies. With the reduction in sequencing costs and increased availability of genome data, MLST, in which independent, polymorphic loci are sequenced, is likely to become the method of choice. This method has the advantage that it is completely reproducible between laboratories, allowing data from different studies to be compiled in large MLST databases.
There are numerous different techniques that have been used to assess population structure in fungi, and these have been comprehensively reviewed by Burnett (2003) and Xu (2005b). For the purpose of this review we will concentrate on techniques that have been applied in studies of sex in the pathogenic cryptococci.
Phylogenetic approaches to determining population structure and mode of reproduction
A useful preliminary step when determining the structure of a population is to first map the multilocus genotype data to a phylogenetic tree, with each isolate treated as an individual taxonomic unit. Tree structure can give an immediate indication of the genetic structure of the population, with clonal populations forming well-resolved, highly structured trees (Fig. 1a) and sexually recombining populations resulting in an undifferentiated ‘bush-like’ appearance (Fig. 1b). Phylogenetic analysis can also resolve substructure within populations, such as genetic partitions and the presence of clonal and recombining subgroups (Fig. 2a), and clonal blooms (Fig. 2b). In these cases, recombination analysis should be targeted to the appropriate subgroups.
Models for the transfer of genetic markers in a clonal vs. sexually recombining population. In this highly simplified diagram, multilocus genotypes consist of three loci designated a, b and c, with two possible alleles, 1 or 0, at each locus. The ancestral strain for both populations is initially 1 1 1, and random mutations over time at alleles b and c give rise to genotypes 101 and 110, respectively. In a clonal population (a) these altered alleles become fixed in the descendents with no recombinants and few genotypes. The resulting phylogenetic tree is well structured and the relationships between genotypes are clear. In a sexually recombining population (b), alleles are reassorted among isolates and all possible genotypes are apparent. The relationship between genotypes is less predictable and many steps are required to fit all individuals, so that the phylogenetic tree is less structured.
Phylogenetic trees showing genetic structure within populations that can complicate recombination analysis. (a) A genetically subdivided population. Even if there is recombination in the two subgroups, if this population is analysed as a whole it will appear clonal, as any loci that are fixed for one allele only in either subgroup will appear to be genetically linked. (b) A recombining population in which clonal expansion of one genotype (isolates a – f) has occurred. If all clonemates are included, this will bias the analysis in favor of clonality.
The parsimony tree length permutation test (PTLPT) exploits this difference in the structure of clonal and recombining trees to assess population structure (Archie, 1989; Burt, 1996). In this test, the length of a phylogenetic tree fitted to the multilocus genotype data for the population under study is compared to the tree lengths generated from the same population in which recombination has been simulated by shuffling alleles at each locus among the members of the population. As clonal populations result in well-resolved trees they are expected to have a shorter length than recombining trees, in which many instances of branch swapping must be accommodated. Trees derived from the population under study that are significantly shorter than the set of tree lengths produced by the simulated recombining trees can therefore be concluded to represent clonal populations (Burt, 1996; Carter, 1996); those that lie within the range of recombining tree lengths indicate a history of recombination.
Phylogenetic assessment of gene genealogies can also indicate whether a population results from clonal or sexual reproduction. Individual unlinked genes are sequenced from all members of the population of interest and a phylogenetic tree is derived from each sequence. In clonal organisms, all genes will share a common evolutionary history and would be expected to produce highly similar trees. In contrast, genes in recombining organisms will be swapped between individuals and each gene tree might indicate quite different evolutionary relationships. A combined phylogeny produced from different genes in a recombining population will need to resolve conflicts produced from the divergent evolutionary histories represented by each gene, and will have a longer overall tree length than that produced from a clonal population (Geiser, 1998; Kasuga, 1999, 2003).
Population genetic approaches to determining mode of reproduction
Population genetic approaches examine the distribution of alleles among members of a population to assess whether or not recombination has occurred. In both haploid and diploid organisms, the over-representation of identical multilocus genotypes, the absence of recombinant genotypes, and the non-random association of alleles at different loci (linkage disequilibrium), all indicate a clonal population structure. In diploid organisms the observation of fixed heterozygosity at one or more loci is additional evidence of deviation from random recombination (Tibayrenc, 1996). As most fungi are haploid, recombination analyses are generally based on looking for linkage disequilibrium between alleles at independent loci.
The Index of Association (IA) is a statistical measure of the non-random association among alleles at different loci (Brown, 1980; Maynard-Smith, 1993). This test performs a pairwise comparison of loci within a multilocus genotype to answer the question: does the fact that two individuals are the same at one locus make them more (or less) likely to be the same at another? In a clonal population if two individuals are identical at one locus there is a greater probability they will be identical (or in repulsion) at a second locus. A sexual population, by undergoing meiotic recombination, will reassort loci among its members and alleles at each locus will be randomly associated (McDonald, 1995; Carter, 1996). IA examines whether the variance of these associations in an observed population differs from that expected if there is no linkage disequilibrium (i.e. all loci are independent), and approaches zero in a randomly recombining population. In clonal populations, identity between locus pairs will be more (when isolates belong to the same clonal group) or less (when isolates belong to different clonal groups) likely to occur than by chance. This will result in a bimodal distribution of associations and a higher variance than expected for independent alleles, to give an IA that is significantly greater than zero. As with the PTLPT analysis outlined above, the significance of IA in the observed population can be computed by comparing it with a range of IAs calculated for artificially recombined datasets. IA becomes less reliable when the number of loci being analysed is low, and a correction, rBarD, has been introduced to remove this dependency (Apagow & Burt, 2001).
Detecting barriers to sex–genetic differentiation and cryptic speciation
Frequently, a set of molecular markers designed to detect polymorphism in a given, defined population may be monomorphic, or have very different allele frequencies in a second population (Burt, 1997; Carter, 2001). The two differentiated populations may be separated in time or space and may or may not retain the ability to interbreed, or they may reside together but be genetically differentiated such that mating is no longer possible. This difference in allele frequencies can be exploited to give a measure of the degree to which the two populations are differentiated. Wright's Fst is the classic measure of population subdivision, and can be estimated in haploid organisms using Weir and Cockerham's Theta (θ). If two populations have the same allele frequencies, θ=0, whereas if they are fixed for different alleles at all loci, θ=1 (Weir, 1996). Even relatively low values for θ can indicate significant differentiation, which is calculated by comparing the observed θ with values generated when individuals are randomly exchanged among populations (Carter, 2001).
A significant level of differentiation between populations of a single species can indicate that genetic exchange is no longer possible, and the populations have diverged into independent, ‘cryptic species’ that cannot be discriminated by standard morphological criteria. Cryptic speciation can be confirmed by examining the genealogies of unlinked genes for all isolates within the different populations. Under the phylogenetic species concept, isolates belonging to different species will group together on well-supported branches in every informative gene that is analysed (Taylor, 2000). Cryptic species have been identified in a number of fungal species, as phenotypic characters are often too limited to enable speciation by conventional taxonomy (e.g. Coccidioides immitis, Koufopanou, 1997; Aspergillus flavus, Geiser, 1998; Histoplasma capsulatum, Kasuga, 2003).
Gene genealogies can also reveal rare hybridisation events, whereby isolates belonging to one species have the occasional gene sequence that is characteristic of a second species. Given the complexity and diversity of fungal lifecycles, speciation and hybridisation are likely to be ongoing, dynamic processes, influenced by temporal, geographic and genetic factors (Taylor, 1999).
Sex, fertility and clonality in the pathogenic cryptococci
Cryptococcus neoformans and C. gattii are basidiomycetous yeasts and are the causal agents of cryptococcosis, an important mycotic infection in humans and animals (Casadevall & Perfect, 1998). Recent changes have occurred in the taxonomy of the cryptococcal species that cause cryptococcosis. Initially, C. neoformans was divided into C. neoformans var. neoformans and C. neoformans var. gattii, with a third variety, C. neoformans var. grubii, subsequently proposed (Franzot, 1999). Cryptococcus neoformans var. gattii had a very limited capacity for mating with the other two varieties (Kwon-Chung, 1982), and observed differences in physiology, genetics, ecology and associated disease manifestations argued for its elevation to the separate species, C. gattii (Kwon-Chung, 2002). Cryptococcus neoformans var. neoformans and C. neoformans var. grubii have been retained, as although they differ in serotype and genetic makeup, it is not possible to reliably differentiate them by morphology or physiology. As such they are classic examples of cryptic species, and the subject of ongoing debate (Kwon-Chung, 2005).
Cryptococcosis cannot be readily transferred among infected humans and animals, and is almost always acquired directly from the environment. This makes the study of the ecology and lifestyle of these fungi particularly important for understanding disease transmission and infectivity. Cryptococcus neoformans and C. gattii have a known sexual phase with two mating types, MATα and MATa (Kwon-Chung, 1975, 1976). Mating type is defined by the MAT locus, which governs sexual identity. In C. neoformans and C. gattii this locus is unusually large (>100 kb) and contains more than 20 genes. This is a non-recombining chromosomal region and each meiotic segregant receives a complete and intact MATα or MATa allele (Hull & Heitman, 2002; Lengeler, 2002). The product of sexual mating between two isolates of opposite mating type is the basidiospore, which is likely to be an important infectious propagule (Sukroongreung, 1998).
Cryptococcus neoformans and C. gattii: sexual fungi with diverse lifestyles
Despite being shown to be capable of undergoing sexual recombination, the first major population studies, which used MLEE, RAPD, RFLP and DNA sequencing, indicated that C. neoformans had a clonal population structure (Brandt, 1993, 1995, 1996; Chen, 1995; Franzot, 1997). However, parsimony analysis of the URA5 gene returned many different most-parsimonious trees, which was not consistent with a clonally reproducing population (Franzot, 1997). These initial studies identified extensive sub-grouping within the cryptococcal species complex, and C. neoformans was subsequently divided into C. neoformans var. neoformans and C. neoformans var. grubii (Franzot, 1999). Upon reanalysis of the multilocus genotype data, in which var. grubii and var. neoformans had been grouped as a single species, Taylor (1999) found that within the var. grubii population the null hypothesis of recombination could not be firmly rejected. This was the first indication that determining whether genetic differentiation occurred within the collection isolates under study would be of critical importance in understanding the population structure of C. neoformans and C. gattii. A subsequent study by Xu & Mitchell (2003) on var. grubii/var. neoformans hybrids found that phylogenetic trees based on the LAC and URA5 genes derived from each variety had significantly different genealogies, suggesting a past history of recombination in both varieties.
PCR-fingerprinting of mini and microsatellites, RAPD, AFLP and RFLP analyses have since identified eight major molecular genotypes within the pathogenic cryptococci. The C. neoformans molecular types correlate with serotype and have been designated VNI to IV, or AFLP 1-3. VNI (AFLP 1) and VNII (AFLP 1A) correspond to var. grubii (serotype A), VNIII (AFLP 3) comprises hybrid var. grubii/var. neoformans strains (serotype AD), and VNIV (AFLP 2) corresponds to var. neoformans (serotype D). VGI to VGIV (AFLP 4–7) encompass the C. gattii strains, but do not correlate with serotype (Meyer, 1999, 2003; Boekhout, 2001). These molecular genotypes have been found to cluster somewhat according to their country of origin, and as such have an uneven global distribution (Kidd, 2003). Each molecular genotype is genetically distinct, and it is likely that gene flow between these types is infrequent, although evidence for occasional hybridization between genotypes, serotypes and even varieties has been found (Xu, 2000, 2002; Boekhout, 2001). For population analysis, each molecular genotype should be considered independently when looking for evidence of recombination, as genetic partitioning between them will skew the analysis in favor of clonality (see Fig. 2).
Understanding the diversity of the C. neoformans/C. gattii species complex allowed cryptococcal population genetic studies to redress the issues of sex and clonality in these pathogens. The identification of a MATa var. grubii strain, a type previously thought to be extinct, led to the demonstration of the sexual cycle of this variety (Lengeler, 2000; Viviani, 2001; Nielsen, 2003). This was followed by the identification of a clinical C. neoformans var. grubii population in Botswana with an unprecedented number of MATa isolates (Litvintseva, 2003). AFLP analysis of this population generated 29 polymorphic markers, which identified 34 distinct genotypes. These genotypes clustered into two distinct groups, which the authors designated I and II, with group I further subdivided into IA and IB. When all 34 genotypes were analyzed together the population appeared clonal. However, when groups I and II were analyzed independently the null hypothesis of recombination could not be rejected, and the results for group II were even more statistically significant when subgroups IA and IB were analyzed independently. Multigene sequence analysis conducted on a 722-bp portion of the IGS region of the ribosomal DNA and a 516-bp region of the LAC1 gene in a representative of each AFLP genotype supported the groupings identified via AFLP analysis. This, together with the genealogical study by Xu & Mitchell (2003), provided clear evidence of sexual recombination in at least some natural populations of C. neoformans.
The first study of recombination in C. gattii population structure focussed on environmental isolates collected from hollows in thirteen Eucalyptus camaldulensis trees (Halliday & Carter, 2003). This tree species had previously been identified as a reliable source of C. gattii (Ellis & Pfeiffer, 1990; Krockenberger, 2002) and it was hypothesised that it served as the primary ecological niche in which the fungus completed its lifecycle, to be dispersed as sexual basidiospores. A survey of the mating types of C. gattii present in various regions of Australia identified the first approximately 1 : 1 distribution of MATα : MATa strains, as would be expected in a sexually recombining population (Halliday, 1999). Located in Renmark, South Australia, this population was obtained from colonised E. camaldulensis trees growing within an approximately 250-m length of riverbank (Fig. 3). AFLP analysis generated unique multilocus genotypes for each of 30 isolates included in a population analysis. There was no evidence of genetic exchange within this population, which was concluded to be clonal. However, statistical analysis revealed an association of genotype with the tree of origin, suggesting that subpopulations associated with individual trees might occur (Halliday & Carter, 2003). It remains to be seen whether recombination might occur within individual tree hollows, and the extent to which the Eucalyptus influences the production and dispersal of infectious C. gattii propagules (Saul, 2005).
Riverbank in Renmark, South Australia, where a Cryptococcus gattii population consisting of c. 1 : 1 MATa : MATα was found. Numbers indicate individual trees colonised by C. gattii. Scale bar represents c. 100 m.
In Australia, the area of highest C. gattii cryptococcosis is the Northern Territory, where the ‘normal’ host Eucalyptus trees do not occur (Chen, 1997). Likewise, Papua New Guinea (PNG), which has one of the highest rates of C. gattii cryptococcosis in the world, has no endemic Eucalyptus species commonly colonised by C. gattii, and the fungus has never been isolated from the PNG environment (Laurenson, 1997). In an attempt to understand C. gattii ecology in the absence of the Eucalyptus, we have targeted isolates from these areas for population analysis (Campbell, 2005a). A third population was included, which consisted of veterinary isolates collected from the greater Sydney metropolitan area. This group was analysed to avoid a problem inherent in all human clinical populations, which is the propensity of humans to travel, potentially acquiring the cryptococcal infection outside the region in which cryptococcosis is later diagnosed. This study used AFLP analysis to identify 55 polymorphic loci, which generated unique multilocus genotypes for each of the 81 isolates included. Phylogenetic analysis of the multilocus data found isolates mostly partitioned according to VG genotype, and grouped largely according to their geographic source (Figs 4, 5a). VGI isolates were generally closely related, and each cluster identified on the phylogram was highly clonal. Each geographic VGI population was highly differentiated from the other two, indicating no gene flow between regions (Fig. 5b). VGII isolates were generally more genetically diverse, and there was not a complete segregation of the Sydney veterinary isolates and the Northern Territory human clinical isolates. Two small populations, consisting of five Northern Territory isolates and the six Sydney veterinary isolates, were recombining. In addition, the significance of θ for the Sydney VGII isolates and four Northern Territory VGII isolates that branched with them was borderline (P=0.039; Figs 4, 5c). Mating crosses with appropriate tester strains revealed a high degree of association between fertility and molecular genotype VGII, whereas all VGI isolates were either infertile or only weakly fertile (Campbell, 2005b). It was concluded that asexual propagation of VGI isolates as yeast cells was preventing long-range dispersal and might be causing a gradual loss of sexual function. Long-term, serial subculture in the laboratory has been found to attenuate sexual function in C. neoformans (Xu, 2002). VGII isolates, however, were recombining in the environment and retaining a high level of fertility, and might be dispersed over long distances as sexual basidiospores.
Unrooted phylogenetic tree showing genetic relationships among isolates from Australia and Papua New Guinea (PNG) and associated recombination and mating data. PNG isolates are shown in red, Northern Territory isolates are blue, and Sydney isolates are green. Recombining populations are circled. MATa isolates labelled a. Isolates with a red cross appeared infertile, those with a green tick were fertile and those with a green four-pointed star were robustly fertile (adapted from Campbell, 2005a).
Molecular type distribution, fertility and genetic differentiation in Australian Cryptococcus gattii populations. Regional map showing areas sampled; (a) proportion of molecular types in each region; (b) VGI populations are largely infertile and genetically isolated; (c) VGII populations are highly fertile and may be genetically connected (F = fertile, I = infertile).
Sex outside the norm: single sex and cross- species unions in Cryptococcus
Although Cryptococcus appears to have a relatively straightforward mating system, recent evidence suggests it may not limit sexual reproduction to the normal union of two sexes within a single species. In a bipolar mating system, an equal ratio of α : a mating type offspring is expected, and this has been confirmed in Cryptococcus following the dissection and analysis of basidiospores (Wickes, 1996). However, a significant bias of the α mating type is observed in clinical and environmental populations of C. neoformans and C. gattii (Kwon-Chung & Bennett, 1978; Madrenys, 1993). It had been difficult to explain this bias in nature with no apparent physiological difference between a and α cells, until Wickes (1996) reported the ability of haploid C. neoformans cells, under nitrogen starvation conditions and in the absence of a mating partner, to undergo haploid fruiting with the production of basidiospores (Wickes, 1996). Haploid, monokaryotic or homokaryotic fruiting, although common in higher basidiomycetes, was previously unknown in Cryptococcus (Stahl & Esser, 1976; Esser & Meinhardt, 1977; Esser & Graw, 1980). This ability appeared to be specific to MATα strains, providing a feasible explanation for the preponderance of MATα in environmental and clinical populations (Wickes, 1996).
The relative scarcity of MATa strains meant that only four had been included in the analysis; however, with the subsequent identification of new MATa isolates from Brazil, Tscharke (2003) observed two MATa strains that could also undergo haploid fruiting. This study tested whether there was a genetic association between mating and fruiting ability by assessing the progeny of a cross between a fruiting MATα strain and a non-fruiting MATa strain. Of the 39 basidiospores dissected from successful mating crosses (20 MATα and 19 MATa), 20 demonstrated haploid fruiting ability (14 MATα and 6 MATa). Statistical analysis allowed the null hypothesis, that the ability or inability to haploid fruiting was independent of mating type, to be accepted. Thus, although haploid fruiting appeared to be a potential mechanism for survival of C. neoformans under harsh environmental conditions, it could not adequately explain the dominance of MATα strains in natural and clinical populations (Tscharke, 2003).
A further twist to understanding the role of haploid fruiting in C. neoformans came with the finding that hallmark features of mating, including diploidization and meiosis, occurred during haploid fruiting. This led to the novel finding that sexual reproduction could occur in C. neoformans between partners of α mating type only (Lin, 2005). Blastospores are vegetative yeast cell that bud off hyphae during the fruiting process. When these were analyzed for ploidy, the majority were found to be diploid, uninucleate and MATα, indicating that nuclear diploidization had occurred. Furthermore, when an α/α diploid produced from two genetically marked strains was induced to fruit, the progeny were found to inherit markers in a Mendelian fashion. The authors concluded that, during fruiting, α/α diploid cells undergo independent chromosome assortment and complete genome reduction into haploid basidiospores. These progeny also demonstrated a high rate of recombination, with all progeny having unique genotypes, none of which matched the parental strains.
Same-sex mating might provide a long-term survival advantage for MATα cells, and could be either the cause or the consequence of the α mating-type bias. However, demonstrating its occurrence in the laboratory may not necessarily mean it takes place in the environment. Evidence for the latter came from the analysis of C. gattii strains responsible for the ongoing outbreak of cryptococcosis on Vancouver Island, Canada (Fraser, 2005). Multilocus sequence typing of 202 clinical and environmental isolates from the outbreak found these divided into two groups: a major group, which included both clinical and environmental isolates, and a minor group, which consisted of a single clinical isolate and several environmental isolates. All isolates were MATα and fertile, but diversity in each group was low, and each appeared to be a clonal population. The major group genotype matched a clinical strain isolated in Seattle 30 years ago, whereas the minor genotype was identical to isolates from recombining Australian populations (Campbell, 2005a). The major and minor genotypes were more closely related to one another than to other C. gattii genotypes, suggesting that these groups were either siblings or parent and offspring. As the minor genotype was present at multiple locations in Australia, but the major genotype was only found in the Pacific-northwest, it was proposed that the minor group was a parental strain, which had combined with a second unknown parent to produce the major group responsible for the Vancouver Island outbreak (Fraser, 2005).
Sequence analysis of the MAT locus of a major group type strain indicated that this strain might be the result of an α-α sexual cycle. Traditionally, α progeny inherit an identical MATα allele from the MATα parent (Loftus, 2005). However, sequence comparison across a large region of the MAT locus from the major and minor group type strains identified widespread, low levels of polymorphism (0.37% divergence), indicating that these were related but different MATα alleles. This supported the hypothesis of an α-α mating event in the recent past, giving rise to a new, hypervirulent genotype. The authors suggested that this ability to sexually recombine with a partner of the same mating type might confer an evolutionary advantage, in the absence of the opposite mating type, allowing the production of recombinant basidiospores which are easily aerosolized and dispersed, and leading to geographic range expansion into this new region.
Finally, hybridization, or the union of isolates from different serotypes, varieties and species, has been observed in Cryptococcus. Boekhout (2001) identified clusters on AFLP dendrograms that lay intermediate to serotype groups but within C. neoformans and C. gattii, which they classified as hybrid genotypes (Boekhout, 2001). More definitive evidence came from a gene genealogical approach, employed by Xu and colleagues (2000), who sequenced four unlinked genes from 34 strains of C. neoformans var. neoformans, C. neoformans var. grubii and C. gattii. They identified five strains, belonging to serotypes AD, C, D and B/A, that had inconsistent genealogical placements of one or more genes, indicating that these had arisen due to intervariety and interspecies hybridization events. The relatively common occurrence of these apparent hybrids suggests that this may occur with some frequency. The authors suggested that hybridization in Cryptococcus was linked to recent dispersion events, with dispersal of the yeast assisted by humans and other vectors bringing divergent strains into close proximity to enable subsequent mating (Xu, 2000).
Cryptococcus neoformans and C. gattii are sexual yeast species with a complex population biology. They are capable of employing a variety of mechanisms for both sexual and asexual propagation, ranging from recombination through mating between partners of the opposite or the same mating type, both within and among varieties and species, to extensive clonal propagation via haploid fruiting or budding. This suite of reproductive strategies may have enabled Cryptococcus to establish itself as the ubiquitous environmental organism it is today, and, as seen with the outbreak of C. gattii on Vancouver Island, to continue to expand its ecological range. These findings point to an ongoing and dynamic process driving the evolution of Cryptococcus, which has important ramifications for the transmission, diagnosis, and treatment of this pathogen.
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