ABSTRACT
The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are closely related sibling species that cause respiratory and neurological disease in humans and animals. Within these two recognized species, phylogenetic analysis reveals at least six cryptic species defined as molecular types (VNI/II/B, VNIV, VGI, VGII, VGIII, and VGIV) that comprise the pathogenic Cryptococcus species complex. These pathogenic species are clustered in the Filobasidiella clade within the order Tremellales. Previous studies have shown that the Filobasidiella clade also includes several saprobic fungi isolated from insect frass, but information evaluating the relatedness of the saprobes and pathogens within this cluster is limited. Here, the phylogeny encompassing a subset of species in the Tremellales lineage that clusters closely with the pathogenic Cryptococcus species complex was resolved by employing a multilocus sequencing approach for phylogenetic analysis. Six highly conserved genomic loci from 15 related basidiomycete species were sequenced, and the alignments from the concatenated gene sequences were evaluated with different tree-building criteria. Furthermore, these 15 species were subjected to virulence and phenotype assays to evaluate their pathogenic potential. These studies revealed that Cryptococcus amylolentus and Tsuchiyaea wingfieldii, two nonpathogenic sibling species, are the taxa most closely related to the pathogens C. neoformans and C. gattii and together with Filobasidiella depauperata form a Cryptococcus sensu stricto group. Five other saprobic yeast species form the Kwoniella clade, which appears to be a part of a more distantly related sensu lato group. This study establishes a foundation for future comparative genomic approaches that will provide insight into the structure, function, and evolution of the mating type locus, the transitions in modes of sexual reproduction, and the emergence of human pathogenic species from related or ancestral saprobic species.
Recent phylogenetic and genomic studies of the fungal kingdom have illustrated that analysis of both distantly and closely related species provides insight into the evolutionary trajectories of fungal species (16, 53). The Fungal Tree of Life (AFTOL) project applied a high-resolution multilocus sequencing (MLS) approach to 170 species, elucidating broad and specific evolutionary relationships among species (16). Yet, the entire fungal kingdom encompasses an estimated 1.5 million species (14), many more than can be analyzed by this approach. More than 100 fungi have been subjected to whole-genome analysis, including distantly related but also, in some cases, closely aligned species (53). Taken together, these approaches illustrate that comparisons of closely related species, first by MLS and then by whole-genome studies, can impact our understanding of how closely related pathogenic and saprobic fungi have evolved to occupy specialized niches in nature.
The human fungal pathogen and basidiomycete Cryptococcus neoformans and its closest relative Cryptococcus gattii cause both respiratory and neurological diseases in immunocompromised and immunocompetent patients (7). The sexual states of those species, which form on artificial culture media, were named Filobasidiella neoformans and Filobasidiella bacillispora, respectively (21, 22). C. neoformans and C. gattii are members of the pathogenic Cryptococcus species cluster, which contains two currently recognized varieties, C. neoformans var. neoformans and C. neoformans var. grubii, and the sibling species C. gattii (7, 23). Recent studies based on MLS typing analysis provide evidence that C. neoformans var. neoformans and C. neoformans var. grubii are distinct species and that a few subpopulations are found in C. neoformans var. grubii (VNI, VNII, and VNB) (6, 28, 29). Furthermore, C. gattii can be further subdivided into at least four cryptic species (VGI, VGII, VGIII, and VGIV) (6, 10, 34). Hence, as many as six species define the pathogenic Cryptococcus species complex. All six of these pathogenic Cryptococcus species cluster together in the Filobasidiella clade, which appears to have emerged from the Tremella lineage (8, 48). Nonetheless, the genus Cryptococcus is polyphyletic, and species cluster within the Tremellales, the Trichosporonales, the Filobasidiales, and the Cystofilobasidiales clades (8, 45, 48), including the less common human pathogens Cryptococcus albidus, Cryptococcus laurentii, and Cryptococcus adeliensis (43, 49) and the saprobe Cryptotrichosporon anacardii (39), which shares phenotypic traits (melanin production and capsule formation) similar to C. neoformans.
Previous taxonomic studies based on ultrastructural features and basidial morphology separated the hymenomycetous yeasts (now classified in the order Tremellomycetes [15]) into two orders: Tremellales, with cruciately septate basidia, and Filobasidiales, with aseptate basidia (19). Because Filobasidiella species have aseptate holobasidia, they were included in the Filobasidiales together with the Filobasidium and Cystofilobasidium species. However, molecular data later showed that Filobasidiella species are more closely related to those in Tremellales (48). Most of the species with defined sexual cycles in the Tremellales have septate two- to four-celled basidia (5, 18, 19, 27, 45, 47, 54). The holobasidia of Filobasidiella species are thus unique in the Tremellales.
Mating systems also differ among the Tremellales. All known heterothallic Tremella species have been reported to have a tetrapolar (bifactorial) mating system with a multiallelic A locus and a biallelic B locus involved in production and sensing of pheromones (13, 65). Fibulobasidium inconspicuum has also been shown to have the same type of mating system (3). However, other teleomorphic species in the Tremellales, such as Auriculibuller fuscus and Kwoniella mangroviensis, are reported to have bipolar mating type systems (5, 46, 54).
Tremellales generally have haustorial branches, which are short branches of hyphae with a basal clamp connection (33). Haustorial filaments of the mycoparasitic Tremella species were observed attached to host cells, enabling penetration and parasitic interactions (22, 54, 66). Haustorial branches have also been described in C. neoformans (19, 20), suggesting an ancestral mycoparasitism (2).
Previous studies examining the genetic relatedness of the species in the Filobasidiella clade, namely, the homothallic filamentous fungus Filobasidiella depauperata and the heterothallic yeasts C. neoformans and C. gattii, are based on internal transcribed spacer (ITS) analysis and/or ribosomal DNA sequence divergence and basidial morphology (10, 20, 24, 25, 57). Results from these studies strongly support the possibility that these three species form a monophyletic clade. Additional studies have shown that these fungi also align closely with Cryptococcus amylolentus and Tsuchiyaea wingfieldii (8, 12, 23, 36). Because of their proximity to the pathogenic Cryptococcus species cluster, these species all constituted the focus of our phylogenetic study. Several additional saprobic anamorphic yeasts, Cryptococcus bestiolae (59), Cryptococcus dejecticola (59), Cryptococcus heveanensis, and Bullera dendrophila (61, 63), also appear to be closely related to the Filobasidiella clade and to form a monophyletic lineage related to the dimorphic, heterothallic basidiomycetous yeast Kwoniella mangroviensis (formerly Cryptococcus sp. strain CBS 8507), recently discovered to have an extant sexual cycle (54). Therefore, for a more robust comparison, several members of the Kwoniella lineage were also included in our analyses.
Many of the species nested in the Tremellales can be found as mycoparasites and saprobes associated with wood, plants, soil, and arthropod frass (66). Similar habitats and environmental associations have been found for C. neoformans and C. gattii. These two species have been isolated from a variety of tree species, pigeon guano, and insects. For example, C. neoformans has been isolated from beetles (55a), and C. gattii has been detected in honeybee hives and insect frass (7a, 17a, 26). This accumulating circumstantial evidence might imply a possible arthropod-associated habitat for the pathogenic Cryptococcus species. Previous studies have used several invertebrate model hosts such as Acanthamoeba spp., Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, and Galleria mellonella to model bacterial or fungal infections in mammals (37). These host systems are in some cases genetically tractable, relatively simple to manipulate, and inexpensive (37). Interestingly, the virulence potential of the pathogenic Cryptococcus species correlates with the ability of the fungus to cause lethal infections in the heterologous insect host G. mellonella (wax moth) (38). Moreover, C. neoformans pathogenicity has been studied extensively in G. mellonella, and genes required for infection of wax moth larvae are necessary during C. neoformans and C. gattii infection of mammalian hosts (38). Results from these studies reveal insights into the ecological niche and the evolution of pathogenesis of C. neoformans and C. gattii.
The fungal species in the Tremellales are numerous, representing over 120 species, and many of their phylogenetic relationships are weakly supported due to the lack of multilocus phylogenetic and phenotypic analyses. Here, we use molecular and phenotypic methods to examine the species surrounding the monophyletic pathogenic Cryptococcus complex clustered within the Tremellales. We employed an MLS approach encompassing six highly conserved genomic loci present in the fungal kingdom. The RPB1, RPB2, EF1α, and mitochondrial small subunit (mitSSU) rRNA genes, the nuclear large subunit (nucLSU) rRNA (D1/D2 domains), and the ITS regions were amplified and sequenced to determine the divergence among the 15 fungal isolates tested. The virulence potential of all 15 isolates was also evaluated in G. mellonella. These studies provide insight into the genotypic and phenotypic trajectory of a highly successful pathogenic clade that likely emerged from saprobic fungi associated with insects in the environment.
MATERIALS AND METHODS
Fungal isolates.The isolates used in this study are listed in Table 1. All were grown and maintained on yeast-peptone-dextrose (YPD) medium at 24°C.
Description of species studied
DNA isolation, PCR, and sequencing.To isolate fungal DNA, cells were harvested after shaking at 24°C in YPD liquid medium overnight, followed by lyophilization. DNA was isolated using the CTAB (cetyltrimethylammonium bromide) method (64). PCR amplification was performed with the following six fungal-specific, highly conserved genes encoding the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), elongation factor 1 alpha (EF1α), the mitochondrial small-subunit (mitSSU) rRNA, the D1/D2 domains of the nucLSU rRNA (nucLSU [D1/D2]), and the ITS region of the ribosomal DNA unit, which includes the ITS1 and ITS4 regions and the 5.8S rRNA gene. Individual PCRs were performed for each of the six genes. Primer information can be found at http://www.aftol.org/primers.php (the primers used in this study were RPB1Af and RPB1Cr, RPB2-5f and RPB1-11bR, EF1α1F and EF1α1R, mitSSU1F and mitSSU3R, nucLSU LrDNA and nucLSU LR3, and ITS1 and ITS4). PCR products were separated on an agarose gel and purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA). Sequencing reactions were performed using BigDye chemistry version 3.1 (Applied Biosystems, Foster City, CA) and analyzed with an Applied Biosystems 3730xl capillary sequencer. Sequence reads were trimmed and assembled with a DNA sequence assembly software program, Sequencher (version 4.8; Gene Codes Corporation, Ann Arbor, MI). Individual contigs were generated with Sequencher, and a BLAST (1) analysis was performed to confirm the identity of the sequenced products (see Table S1 in the supplemental material for GenBank accession numbers.).
Phylogenetic analysis.A total of 92 out of 96 DNA sequences (4% of the molecular data was missing) were aligned using ClustalW version 1.81 (60). The FASTA alignment files for each of the six loci were imported into MacClade version 4.08 (32) for manual editing and to identify and correct ambiguously aligned regions (732 characters). Edited files were concatenated, resulting in the inclusion of 3,422 characters for phylogenetic analyses. Heuristic searches for maximum parsimony (MP) (Fig. 1A) and maximum likelihood (ML) (Fig. 1B) criteria were conducted using PAUP version 4.0 (56). MP searches were also conducted with the concatenated data set, with a user-defined step matrix generated in STEP3 matrix for conserved regions (31), and using a matrix generated by INAASE for ambiguously aligned regions (35). Model parameter estimates for ML analysis were obtained using MODELTEST (42). Statistical support was calculated using 1,000 bootstrap replicates from MP and ML criteria, and all trees were rooted using Cryptococcus humicola as the outgroup (a member of the order Trichosporonales [8, 48]) (Table 1). MacClade software was also used to trace the habitat preferences (Fig. 2) of the fungi in the current study (32).
Phylogenetic relationships among members of the Tremellales. A combined data set of concatenated gene sequences in a parsimony bootstrap phylogenetic tree representing six fungal-specific genomic loci (RPB1, RPB2, EF1α, mitSSU, nucLSU (D1/D2), and ITS) reveals the phylogeny of the Cryptococcus pathogenic species complex with the insect-associated species. Single topology for the MP tree is shown in panel A, and an ML tree is shown in panel B. Nucleotide sequences for each gene were aligned and analyzed using Mesquite and PAUP. Numbers on branches are bootstrap values from 1,000 replicates. The outgroup in the analysis is C. humicola.
Phylogram showing the preferred habitats of the fungal species used in this study. The tree diagram is based on data from Fig. 1 and indicates evolutionary lineages and natural habitats of the isolates examined in the study. These species are possibly derived from a common insect-associated ancestor.
Phenotypic analysis of isolates.The phenotypic properties of each isolate were examined using several assays. First the ability of each strain to grow on YPD and yeast nitrogen base (YNB) media at 24°C, 30°C, and 37°C was tested. Each isolate was analyzed for melanin production on Niger Seed, Rose Bengal, and low-glucose (0.1%) media supplemented with the diphenolic molecule l-DOPA (dihydroxyphenylalanine) (100 mg/ml). To assay isolates for capsule production, isolates were grown in capsule induction medium (5 g glucose, 5 g asparagine, 400 mg K2HPO4, 80 mg MgSO4 · 7H2O, and 250 mg CaCl2 · 2H2O, and 1,000× vitamin-mineral mixture [0.4 g thiamine, 0.057 g B(OH)3, 0.004 g CuSO4 · 5H2O, 0.01 g MnCl2 · 4H2O, 2 g ZnSO4 · 6H2O, and 0.46 g sodium molybdate in 1 liter of water]) with low iron (20 mg/liter of the iron chelator, EDDHA [ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid)]) at 24°C for 2 days, followed by India ink staining. Isolates were also tested at 24°C for urease activity, using Christensen's agar (Beckton Dickinson, Cockeysville, MD). For these assays, all isolates were grown to exponential phase in liquid YPD medium, serially diluted in spots onto YPD, YNB, Niger Seed, Rose Bengal, l-DOPA, and Christensen's agar, and incubated for 4 to 5 days. The phenotypic assays were performed in triplicate. We note that some images of growth patches on the same medium were cropped to produce the composite image shown in Fig. 3.
Phenotype and morphology of species grown under different conditions. Spot assays of individual isolates were grown for 4 to 5 days on different media at 24°C, 30°C, and 37°C. Each isolate was individually tested for the ability to grow on the nutrient-rich medium, YPD, and the minimal medium, YNB. For the melanin assay, strains were grown for 4 to 5 days at 24°C on l-DOPA medium. F. depauperata grows more slowly than any of the other isolates tested (approximately 10 days were required to achieve the same colony size shown in Fig. 3) and is therefore not included here.
Galleria mellonella survival assay.To analyze virulence in the heterologous host G. mellonella, a previously described protocol (38) was followed with minor modifications. Wax moth larvae (∼12) were injected in the last pseudopod with 5 × 105 cells of each isolate, and wax moths were incubated at 24°C. The number of surviving wax moths was monitored and recorded daily. Survival curves were plotted using Prism software (version 4.0a; Prism Computational Sciences, Incorporated, Madison, WI). A P value of <0.05 was considered significant. P values were calculated and compared to those of the reference phosphate-buffered saline (PBS) (or mock inoculation) control and to those of the pathogenic control C. neoformans var. grubii strain H99 (Fig. 4 and 5). The experiment was performed three times.
Infection of the heterologous host G. mellonella with species closely related to C. neoformans. Survival of G. mellonella after inoculation with species in the Filobasidiella clade was assayed. At least 12 larvae were injected with 5 × 105 cells for each isolate. After inoculation, larvae were incubated at 24°C and survival was monitored for 17 days postinoculation. The experiment was repeated three times, and results from one representative experiment are presented here. In one of three replicates, C. amylolentus exhibited an intermediate virulence level (data not shown). The mock inoculation is injection of a PBS control.
Infection of the heterologous host G. mellonella with species more distantly related to C. neoformans. Survival of G. mellonella larvae after inoculation with species in the Kwoniella clade and outgroup species representing Trichosporon and Tremella was assayed. At least 12 larvae were injected with 5 × 105 cells of each isolate. After inoculation, larvae were incubated at 24°C, survival was monitored daily, and the experiment was terminated 12 days postinoculation. The experiment was repeated three times, and one representative result is presented here. The mock inoculation is injection of a PBS control.
RESULTS
Cryptococcus amylolentus and Tsuchiyaea wingfieldii are the species most closely related to C. neoformans.To establish evolutionary relationships among the pathogenic Cryptococcus species cluster and the related saprobic yeasts in the Tremellales, we performed a multilocus phylogenetic analysis of the 15 species listed in Table 1. Six highly conserved, fungal-specific genomic loci were sequenced and analyzed, as follows: RPB1, RPB2, EF1α, mitSSU, nucLSU (D1/D2), and ITS. Phylogenetic trees from concatenated sequences were generated with PAUP (56), and the parsimony heuristic searches in PAUP (56) generated only one tree topology (Fig. 1A). The same topology for the Filobasidiella clade was obtained under ML criteria searches (Fig. 1B) when the substitution parameters estimated by MODELTEST were used (42).
This MLS analysis resolved the phylogeny of the pathogenic Cryptococcus species complex and its closest relatives. In agreement with previous studies (57), two closely related monophyletic sister clades, the Filobasidiella and Kwoniella lineages, were identified and showed over 96% bootstrap support (Fig. 1). The Filobasidiella clade represents a sensu stricto clade of Cryptococcus species (it contains the type species of the genus, C. neoformans), while the Kwoniella clade contains the majority of the more distantly related insect-associated yeasts, named the sensu lato group (Fig. 1 and 2). These two clades are reminiscent of the sensu stricto and the sensu lato groups used to describe the phylogeny of Saccharomyces cerevisiae and related species (62). Our results show that the two closest relatives of C. neoformans are the sibling species C. amylolentus and T. wingfieldii. These findings do not support previous phylogenetic studies (based on a single gene) that placed F. depauperata as the closest relative of the pathogenic Cryptococcus species (25, 39, 51). However, the current and previous results agree that F. depauperata is a member of the monophyletic Filobasidiella clade, which also includes C. amylolentus, T. wingfieldii, and the pathogenic species C. neoformans and C. gattii.
The Kwoniella lineage includes several arthropod-associated species, Bullera dendrophila, Cryptococcus dejecticola, Cryptococcus bestiolae, and C. heveanensis (the C. heveanensis strain used in this study was isolated from sheet rubber), and one species isolated from mangrove areas, Kwoniella mangroviensis (Table 1 and Fig. 2). Our findings confirm previous studies that suggested the Kwoniella clade is split into two monophyletic clusters, one lineage represented by C. heveanensis and the other including B. dendrophila, C. dejecticola, C. bestiolae, and K. mangroviensis (Fig. 1 and 2).
The three Tremella strains used in the analysis represent more distantly related taxa within the Tremellales. Interestingly, the two strains of Tremella mesenterica appear to be as divergent from each other as the two C. neoformans varieties are and also as diverged as the two C. gattii VG groups, suggesting the existence of subpopulations, varieties, or cryptic species within T. mesenterica (Fig. 1 and 2). The monophyletic clades that are defined based on this phylogenetic analysis were rooted with C. humicola, a member of the Trichosporonales (8, 48, 58).
Phenotypic and morphological differences in close and distant relatives of C. neoformans.The virulence potential of the isolates studied was assessed by examining three well-established virulence attributes: growth at 37°C, capsule formation, and melanin production. To assay growth at different temperatures, serial dilutions of individual species were spotted at 24°C, 30°C, and 37°C on both nutrient-rich and minimal media. The three species belonging to the pathogenic Cryptococcus species complex, C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii, all grew at the highest temperature, 37°C (Fig. 3). Additional species clustering in the sensu stricto Filobasidiella lineage, C. amylolentus, T. wingfieldii, and F. depauperata, all failed to grow at 37°C. Five of the six species in the sensu stricto Filobasidiella lineage were able to grow at 30°C (Fig. 3). The only strain in this group that did not grow at 30°C was T. wingfieldii. We speculate that because T. wingfieldii is a cryptic or sibling species of C. amylolentus (Fig. 1 and 2), the lower thermotolerance of the former might be a distinct trait between the two species; alternatively, this property could vary between isolates of the same species. At present, no other T. wingfieldii or C. amylolentus isolates are available to explore this point further.
None of the species in the sensu lato Kwoniella clade, including K. mangroviensis, B. dendrophila, C. bestiolae, C. dejecticola, and C. heveanensis, or the species representing the paraphyletic lineages, Tremella spp. and C. humicola, grew at 37°C (Fig. 3). Only two isolates, B. dendrophila and Tremella globispora, could not grow at 30°C. A previous report showed that B. dendrophila displays little to no growth at 30°C or at 35°C (4). In this case, lack of growth at 30°C seems to be a phenotypic difference that could be of assistance in distinguishing B. dendrophila from the rest of the species in the Kwoniella lineage (Fig. 3), since the majority of their close relatives are able to grow at 30°C (for a current study, see reference 41). In summary, the saprobic yeasts are distinguished from closely aligned pathogenic yeasts by their inability to grow at 37°C.
To examine capsule production, all strains were grown under conditions (low-iron media) known to induce capsule formation in C. neoformans and C. gattii. Under the conditions tested, none of the saprobic yeasts produced capsules that could be visualized by India ink exclusion, in contrast to those observed for both C. neoformans and C. gattii (data not shown). Melanin production was tested using three different melanin-inducing media (l-DOPA, Rose Bengal, and Niger Seed). The species in the pathogenic Cryptococcus clade all produced melanin (Fig. 3). Of the remaining species in the study, C. heveanensis and C. humicola also showed faint coloration that might have been attributable to melanin production (Fig. 3). The ability to hydrolyze urea was also tested with Christensen's agar (40). All 15 species tested hydrolyzed urea, confirming that they are all basidiomycetes. Based on these findings, we hypothesize that the environmental cues triggering capsule production might differ for C. neoformans and C. gattii compared to that of their saprobic relatives. Furthermore, both melanin production and growth at 37°C are characteristic of human pathogenic species, whereas, other traits, such as the ability to hydrolyze urea, are plesiomorphic.
Virulence potential of Cryptococcus and neighboring taxa using G. mellonella.To further assess the virulence potential of each isolate in the heterologous host G. mellonella, larvae were inoculated with 5 × 105 yeasts and incubated at 24°C for the duration of the experiment. Of the species in the Filobasidiella lineage, the two C. neoformans varieties and C. gattii exhibited the highest virulence potential (P < 0.0001, compared to PBS) during infection assays in G. mellonella (a sample survival curve is shown in Fig. 3). Of the remaining species in the Filobasidiella sensu stricto lineage, T. wingfieldii (P = 0.285), F. depauperata (P = 0.3458), and C. amylolentus (P = 1.0) (Fig. 3) were avirulent in G. mellonella compared to the negative control, PBS. Three of the five species clustering in the Kwoniella clade, C. bestiolae (P = 0.0128, PBS; and P = 0.0160, compared to strain H99), B. dendrophila (P = 0.0382, PBS; and P = 0.0128, H99), and C. heveanensis (P = 0.0181, PBS; and P = 0.0160, H99) displayed intermediate virulence. The other two species in the Kwoniella clade, C. dejecticola (P = 0.1473, PBS; and P < 0.0001, H99) and K. mangroviensis (P = 0.023, PBS; and P < 0.0001, H99) were consistently severely attenuated or avirulent compared to the pathogenic H99 control. Furthermore, the Tremella outgroup species T. mesenterica and T. globispora (P = 1, PBS; and P < 0.0001, H99) were clearly avirulent in this assay. However, the Trichosporon outgroup species C. humicola displayed an intermediate level of virulence (P = 0.030, PBS; and P = 0.02, H99). In summary, the pathogenic Cryptococcus species displayed the highest virulence in the greater wax moth, whereas the majority of their close relatives were attenuated or avirulent.
DISCUSSION
Previous phylogenetic studies evaluating the phylogeny of the Filobasidiella clade used single genomic loci, such as 5.8S, SSU, nucLSU (D1/D2), or the ITS region (8, 12, 45, 48). Results from these analyses were conflicting and failed to accurately resolve the phylogeny of the pathogenic Cryptococcus species within the Tremellales. To generate a robust data set to resolve the phylogenetic relationships surrounding this clade, 15 species representing the Filobasidiella, Kwoniella, and Tremella lineages were examined by using a multilocus approach.
The MP and ML trees concordantly defined two monophyletic groups: the sensu stricto Filobasidiella and the sensu lato Kwoniella lineage, as rooted with C. humicola (Fig. 1 and 2). The monophyletic clades defined based on this phylogenetic analysis are analogous to the sensu stricto and the sensu lato groups in Saccharomycetaceae. The sensu stricto species include T. wingfieldii, C. amylolentus, and F. depauperata, which form a monophyletic cluster with the pathogenic Cryptococcus complex. The previously accepted Filobasidiella clade is composed of members with known sexual cycles. Therefore, only Filobasidiella neoformans (anamorph C. neoformans), Filobasidiella bacillispora (anamorph C. gattii), and Filobasidiella depauperata were included (24, 51). Considerably less attention has focused on T. wingfieldii and C. amylolentus, although previous molecular evidence suggested they were closely related to the Filobasidiella clade (8, 45, 48). The holobasidium of the sexual taxa in the Filobasidiella clade (sensu stricto species) is clearly a synapomorphy (shared derived character) for the clade. The phragmobasidium of the remaining taxa in the Tremellales seems to be a simplesiomorphy (ancestral character).
In the sister Kwoniella clade, the sensu lato species include B. dendrophila, C. bestiolae, C. dejecticola, C. heveanensis, and K. mangroviensis. The basidia of the only sexual species in the clade K. mangroviensis are similar to those of the remaining members of the Tremellales (54), and we could not identify a synapomorphy for the Kwoniella clade. Single-gene phylogenies depicting the relationships between C. bestiolae, C. dejecticola, and neighboring species, using nucLSU (D1/D2), revealed conflicting views, especially compared to the neighbor-joining analyses of the nucLSU (D1/D2) rRNA gene in Kwoniella and closely related species (54, 59). Our multilocus approach confirmed the relatedness among members of the Filobasidiella and Kwoniella clades as two distinct monophyletic clades, which had been suggested previously based on SSU (57) and ITS (48) sequence analyses. The six genomic loci employed in this study did not completely resolve the species within the Kwoniella clade, given the lower bootstrap values (Fig. 1). We attempted to add additional resolution to this clade by including ambiguously aligned regions in the analyses and, although MP (Fig. 1) and neighbor-joining trees (not shown) had identical topologies, results from bootstrapping and the ML topology strongly indicated that these relationships were not well supported. Future studies including additional species, such as recently discovered yeasts that cluster near these fungi or other species in the Tremellales, should increase the phylogenetic resolution of the Kwoniella clade. For example, Cryptococcus cuniculi (not included in this study), a previously described yeast isolated from rabbit feces in Korea, is phylogenetically related to C. heveanensis (50). Additionally, a novel Cryptococcus species, Cryptococcus pinus, was recently described (11). C. pinus was isolated from dead needles of Pinus sylvestris, and ribosomal DNA sequence data reveal that C. pinus is also a member of the Kwoniella clade related to C. dejecticola (11). In summary, the robust data set generated in our study provides a platform for future studies exploring the biology, genetics, and genomics of defined species in the Tremellales and those that remain to be discovered.
Unlike many ascomycetous yeasts, which are commonly found in insect communities, basidiomycetes are less frequently isolated from living or dead insects (48). Moreover, these ascomycetes are not limited solely to insect communities but can also be found in association with other habitats like plants and aquatic environments. Although basidiomycetes are rarely associated with insects, most of the sensu stricto and sensu lato species in this study are saprobic yeasts frequently associated with decaying insects and arthropod frass. The data presented here suggest that the fungi in the Filobasidiella and Kwoniella lineages likely represent a group of phylogenetically related fungi that inhabit similar ecological niches (Fig. 2). Moreover, another possibility is that the successful human pathogenic fungi emerged from an insect-frass-associated ancestor.
Phenotypic assays were performed to identify features shared with or distinct from the extensively studied and well-characterized human pathogen C. neoformans. Melanin production and growth at 37°C were observed only in the pathogenic Cryptococcus species, whereas growth at 30°C appears to be fairly common in all lineages (Fig. 3). Under the conditions of our tests (low-iron media to induce capsule formation in the pathogenic Cryptococcus), visible capsule production was not observed in any of the close or more distant relatives. Previous results have provided evidence that C. humicola produces laccase, the enzyme required for melanin production, and a capsule composed of polysaccharides similar to those found in Cryptococcus (41). Additional studies have indicated the presence of a capsule in many of the Tremella species (9, 52). Like C. neoformans, the Trichosporonales species C. anacardii (not included in this study) also produces both capsule and melanin. Biochemical studies have reported that T. mesenterica produces extracellular polysaccharides, such as those often found in the capsules of other Tremellales (9), as does the pathogenic Cryptococcus (41), in which growth on YPD medium was used to assay capsule production. We speculate that these contradictory findings might be the results of different environmental cues triggering capsule production among the different lineages or the sensitivity of the assays used to detect capsule production. Taken together, the results may imply that shared ancestral traits (capsule formation, melanin production, or growth at high temperatures) and also novel traits (or more recently evolved phenotypes) play a role in the pathogenic life styles of the Cryptococcus species.
C. neoformans and C. gattii displayed the highest virulence in G. mellonella (Fig. 4). The species in the Kwoniella clade were also able to infect G. mellonella larvae, and several exhibited intermediate virulence compared to that of the pathogenic C. neoformans and C. gattii species (Fig. 5). The outgroup in this study, C. humicola, has been previously isolated from immunocompromised patients (44) and insect frass or the gastrointestinal tracts of insects (55). Although the specific strain used in this study was isolated from soil (Table 1), it also displayed intermediate virulence in G. mellonella. All of the Tremella isolates (T. globispora and the two T. mesenterica strains) were found to be avirulent in G. mellonella (Fig. 4). Thus, we hypothesize that ancestral characteristics found in both the Kwoniella and the Filobasidiella lineages may play a role in the pathogenic potential (Fig. 3) of the pathogenic Cryptococcus species.
Moreover, several traits associated with C. neoformans pathogenic potential, for example, virulence in G. mellonella and melanin production, appear to be present in other closely and distantly related species. Therefore, these shared traits might be examples of plesiomorphic, i.e., ancestral, traits retained in these species due to their selection by the environment. Alternatively, some of these characteristics and their distribution, such as the faint melanin-like pigment produced by C. humicola, could be the product of convergent evolution. Current advances, such as the T. mesenterica genome recently sequenced by the Joint Genome Institute at the U.S. Department of Energy (isolate ATCC 24925), in addition to the sequenced genomes available for several strains of C. neoformans and C. gattii (30), set the stage for future comparative genomic analyses to differentiate between these divergent evolutionary trajectories.
Within the Saccharomycotina, the human-associated and sometimes pathogenic Candida species are clustered within lineages of endosymbiotic and commensal Candida species found in the gastrointestinal tracts of insects (55). In a complementary study, four novel anamorphic yeast species were isolated from the gastrointestinal tracts of flower-visiting beetles in China (17). These novel insect-associated Candida species closely cluster with the human fungal pathogen Candida albicans/Lodderomyces elongisporus clade (17). The emergence of human pathogenic Cryptococcus species from an ancestral lineage of mycoparasitic and insect frass-associated fungi might be similar to these previously illustrated examples in the ascomycetes. Consequently, strong evolutionary selection might drive the emergence of successful human pathogens from ancestral yeasts associated with insects and/or other insect habitats or selection for isolates that survive at higher temperatures.
ACKNOWLEDGMENTS
We thank Tim James for MLS typing primers, information, and advice and Robyn Hicks for technical assistance.
This work was supported by NIAID RO1 grants AI50113 and AI063443.
Keisha Findley was supported by NIH Minority Supplement grant 5R01-AI063443-04 S1 Sub no. 1-P30. Marianela Rodriguez-Carres was supported by Molecular Mycology Pathogenesis training program grant 2T32-AI052080-06A1. Álvaro Fonseca received a grant from Fundação Luso-Americana para o Desenvolvimento, Portugal (project no. 600-09/2006).
FOOTNOTES
- Received 24 November 2008.
- Accepted 10 January 2009.
- Copyright © 2009 American Society for Microbiology
REFERENCES
