MINIREVIEW
Do major species concepts support one, two or more species within
Cryptococcus neoformans?
Kyung J. Kwon-Chung & Ashok Varma
Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID/NIH Bethesda, MD, USA
Correspondence: Kyung J. Kwon-Chung,
Molecular Microbiology Section, Laboratory
of Clinical Infectious Diseases, NIAID/NIH,
Bethesda, MD 20892, USA. Tel.: 11 301 496
1602; fax: 11 301 480 3240; e-mail:
[email protected]
Received 10 November 2005; revised 3 January
2006; accepted 3 January 2006.
First published online May 2006.
doi:10.1111/j.1567-1364.2006.00088.x
Editor: Stuart Levitz
Keywords
Cryptococcus neoformans; Cryptococcus gattii;
species concept.
Abstract
Cryptococcus neoformans, the agent of cryptococcosis, had been considered a
homogeneous species until 1949 when the existence of four serotypes was revealed
based on the antigenic properties of its polysaccharide capsule. Such heterogeneity
of the species, however, remained obscure until the two morphologically distinct
teleomorphs of C. neoformans were discovered during the mid 1970s. The
teleomorph Filobasidiella neoformans was found to be produced by strains of
serotype A and D, and Filobasidiella bacillispora was found to be produced by
strains of serotype B and C. Ensuing studies revealed numerous differences
between the anamorphs of the two Filobasidiella species with regard to their
ecology, epidemiology, pathobiology, biochemistry and genetics. At present, the
etiologic agent of cryptococcosis is classified into two species, C. neoformans
(serotypes A and D) and Cryptococcus gattii (serotypes B and C). Intraspecific
genetic diversity has also been revealed as more genotyping methods have been
applied for each serotype. As a result, the number of scientifically valid species
within C. neoformans has become a controversial issue because of the differing
opinions among taxonomists as to the appropriate definition of a species. There
are three major species concepts that govern classification of organisms: phenetic
(morphologic, phenotypic), biologic (interbreeding) and cladistic (evolutionary,
phylogenetic). Classification of the two C. neoformans species has been based on
the phenetic as well as the biologic species concept, which is also supported by the
cladistic species concept. In this paper, we review and attest to the validity of the
current two-species system in light of the three major species concepts.
Introduction
The discovery of heterothallism in the pathogenic yeast
Cryptococcus neoformans has led to the recognition of
profound genetic diversity within the species. In 1975, the
first teleomorph of C. neoformans described as Filobasidiella
neoformans was found to be produced by matings between
two compatible strains of serotype D as well as between
compatible strains of serotypes A and D (Kwon-Chung,
1975, 1976). The second species of Filobasidiella, Filobasidiella bacillispora, was found to be produced by matings
between compatible strains of serotypes B and C (KwonChung, 1976). The correlation between serotypes and the
two different teleomorphs indicated that the differences
between these two sero-groups were not limited to their
antigenic properties. Subsequent studies have shown that
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the two species are distinct in their ecological niche (KwonChung & Bennett, 1978; Ellis & Pfeiffer, 1990), epidemiology (Kwon-Chung & Bennett, 1984), biochemistry (Bennett
et al., 1978; Polacheck & Kwon-Chung, 1980, 1986; Min &
Kwon-Chung, 1986; Kwon-Chung et al., 1987) and genomic
arrangement (Wickes et al., 1994; http://www.bcgsc.ca/gc/
cryptococcus/). Based on biochemical differences, the anamorph of F. bacillispora was described as the distinct species
Cryptococcus bacillisporus (Kwon-Chung et al., 1978). The
species epithet bacillisporus was chosen in accordance with
the species epithet of the teleomorph. Subsequently, the
canavanine–glycine–bromthymol blue (CGB) agar was formulated as a single-step diagnostic tool to distinguish
between C. neoformans and C. bacillisporus (Kwon-Chung
et al., 1982). D-Proline agar medium was also found to be
a reliable diagnostic tool for differentiating between the two
FEMS Yeast Res 6 (2006) 574–587
575
Major species concepts and Cryptococcus neoformans
species (Dufait et al., 1987). Kwon-Chung et al. (2002)
proposed the use of the species epithet gattii over the
epithets with priority, C. bacillisporus or C. hondurianus
(syn. Torulopsis var. hominis var. hondurianus and Cryptococcus
hominis var. hondurianus) (Castellani, 1933). Although the
name gattii had widely been used over the past 20 years, the
epithet bacillispora had seldom been used and the name
hondurianus had never been used since it was first proposed.
The present paper focuses on the validity of the twospecies system for the agent of cryptococcosis based on the
major species concepts used in the classification of the
organism.
Species concepts
Defining a biologic species is an important but difficult
subject because of the differing opinions as to the definition
of a species. Although Darwin’s work on the origin of species
has been most influential in modern biology, he focused on
the mechanisms of how new species evolved from existing
species without offering a criterion that defined the species
boundary (Darwin, 1875). How can one species be distinguished from another within the same genus? Kottelat
suggested a pragmatic definition of a species: When two or
more groups show different sets of shared characters (genetic, morphologic and physiologic) and the shared characters
for each group allow all the members of that group to be
easily and consistently distinguished from members of
another group, then they are considered a different species
(Kottelat, 1955).
There are now several competing species concepts, among
which the most widely accepted are: phenetic (morphologic,
phenotypic), biologic (interbreeding) and cladistic (phylogenetic, evolutionary) (Mayden, 1997). Each species concept
has strengths and weaknesses. For example, the phenetic
species concept defines a species as the smallest group that is
consistently and persistently distinct and can be distinguished morphologically (phenotypically) by ordinary
means (Cronquist, 1978). This system allows one to recognize differences between species easily. It works well for
sexual as well as asexual organisms and can even be applied
to extinct species. The weakness of this concept, however, is
that morphology-based concepts are not necessarily reflective of genetics. The biologic concept defines the species as a
group of actually or potentially interbreeding populations
that are reproductively isolated from other such groups
(Mayr & Ashlock, 1991). This concept fits well within
population genetics, offers an unambiguous empirical criterion and provides an important conceptual framework for
speciation. However, this system is applicable only to the
sexual organisms existing at the present time and has no
evolutionary dimension. The cladistic concept defines speFEMS Yeast Res 6 (2006) 574–587
cies as the smallest diagnosable cluster of individuals that are
interconnected by genetic relationships (Cracraft, 1983).
This concept offers a clear evolutionary dimension, is the
richest concept in paleontological studies and its branch
points make use of DNA sequences, the most reliable
characteristic. The present cladistic system, however, is
based only on a fraction of lineages that have been uncovered as a result of the details required using this approach. It
is highly subjective, depending on the DNA sequence used
for analysis of the genetic relationships and is disconnected
from population genetics.
Which of the species concepts is therefore the most
appropriate for classification of the agent that causes cryptococcosis? This subject is controversial among taxonomists,
as evidenced by the number of species suggested, which
ranges from two to eight depending on the species concept
adopted.
Is the classification of C. neoformans into
two species supported by the phenetic
species concept?
When strains of C. neoformans were classified as representing two species, C. neoformans and C. gattii (C. bacillisporus), it was based on a combination of the morphological
differences between their teleomorphs, F. neoformans and F.
bacillispora, respectively, and the biochemical differences
between the anamorphs of the two species. The differences
in size and shape of the basidiospores produced by F.
neoformans and F. bacillispora are unambiguous, as shown
in Fig. 1. Although their basidial structures are clearly
different, the morphology of their anamorphs does not offer
any markedly distinguishable traits. There are, however,
some morphological features that are specifically associated
with each of the two species. Colonies of C. gattii grown on
conventional mycological agar media are almost always
more mucoid and sticky in texture than those of C. neoformans, although this does not always reflect the size of their
capsules. The brown pigment produced by colonies of C.
gattii, grown on birdseed agar, is less intense than that of C.
neoformans. Additionally, a green hue is present around
colonies of C. gattii grown on birdseed agar whereas it is
absent around colonies of C. neoformans (Kwon-Chung
et al., 1978). Yeast cells of C. gattii are more often reported
to be elliptical or tear-shaped, whereas such cell shapes are
infrequently observed in C. neoformans (Kwon-Chung et al.,
1982). The two species, however, can be more reliably
distinguished by their growth phenotype on certain media
formulations based on their biochemical differences. Strains
of C. neoformans do not react to CGB agar whereas strains of
C. gattii react positively; this thus represents a one-step
diagnostic medium for the separation of the two species
(Fig. 2). CGB agar was formulated based on differences
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576
K.J. Kwon-Chung & A. Varma
F. neoformans (serotype A, D, AD)
F. bacillispora (serotype B, C)
Fig. 1. Phenotypic differences between
teleomorphs of Cryptococcus neoformans
(Filobasidiella neoformans) and Cryptococcus gattii (Filobasidiella bacillispora)
(bars = 1 mm).
Iatron Crypto Kit is no longer commercially available, but it
is hoped that a similar serotyping kit will become readily
available. One may, however, encounter a few untypable
strains that could still be classified as either C. neoformans
or C. gattii based on their CGB reaction. The phenotypic
differences can be ascertained readily and consistently,
thereby justifying the separation of C. neoformans from
C. gattii
C. neoformans
C. gattii
Fig. 2. Canavanine-glycine-bromthymol blue (CGB) agar reaction produced by (a) Cryptococcus neoformans and (b) Cryptococcus gattii
within 48 h, at 30 1C.
between C. neoformans and C. gattii in their ability to utilize
glycine as the sole source of nitrogen and carbon as well as
differences in their susceptibility to L-canavanine, an arginine analog (Min & Kwon-Chung, 1986; Polacheck & KwonChung, 1986). The positive reaction produced by strains of
C. gattii on CGB and the negative reaction by strains of C.
neoformans suggested a clear difference in their nitrogen
metabolisms (Polacheck & Kwon-Chung, 1986). The biochemical difference in the assimilation of nitrogen between
the two species was supported by their creatinine deaminase
activites as well as their ability to assimilate D-proline. In C.
gattii, creatinine deiminase degraded creatinine in the presence of ammonium while the same enzyme in C. neoformans was found to be inhibited (feedback inhibition) by
ammonium in the medium (Polacheck & Kwon-Chung,
1980; Min & Kwon-Chung, 1986). Cryptococcus gattii can
utilize D-proline as the sole source of nitrogen whereas C.
neoformans cannot (Dufait et al., 1987). Other differential
phenotypes included the capsular antigenic properties that
could be distinguished by the commercially available serotyping kit (Crypto Kit, Japan). At the time of writing, the
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Is the classification of C. neoformans into
two species supported by the biologic
species concept?
Heterothallism in C. neoformans was first discovered in
strains of serotype D. Single basidiospore cultures were
obtained by micromanipulation of the spores produced by
matings between a clinical strain, NIH12 (MATa), and an
environmental strain, NIH433 (MATa). Genetic recombination was confirmed by analysing the progeny of a cross
between strains B-3501(MATa) and B-3502 (MATa), the F1
generation of NIH12 NIH433. Colonies of strain B-3501
grown on malt extract agar were milky (m type) whereas
those of strain B-3502 were translucent (t type). Ninety-nine
progeny isolates exhibited independent segregation of the
two markers: 27 (a, m); 25 (a, m); 21 (a, t); 26 (a, t). This
indicated that the progeny were products of meiotic cell
division (Kwon-Chung, 1976). Segregation analysis of other
phenotypic markers, such as CAP1 (encapsulated) and
CAP– (acapsular), as well as Mel1 (melanin formation on
birdseed agar) and Mel–, further confirmed the meiotic
event (Kwon-Chung & Rhodes, 1986). Strains of serotype A
were also found to mate with serotype D strains and
produce viable basidiospores, although genetic characterization of the progeny was not reported. As the DNA sequence
of genes such as URA5 (Edman & Kwon-Chung, 1990),
LAC1 (Williamson, 1994), CAP59 (Chang & Kwon-Chung,
1994) as well as those comprising different subunits of
genomic or mitochondrial rRNA genes (Fell et al., 2000; Xu
FEMS Yeast Res 6 (2006) 574–587
577
Major species concepts and Cryptococcus neoformans
et al., 2000) became available, they were used for phylogenetic analysis. Although the extent of divergence in the DNA
sequences varied, depending on the gene used, most strains
of serotype D clustered apart from the group containing a
majority of the serotype A strains analysed (Franzot et al.,
1999; Nakamura et al., 2000; Xu et al., 2000). Based on the
observed DNA sequence diversity, a new variety, C. neoformans var. grubii, was proposed to accommodate strains of
serotype A (Franzot et al., 1999). Some investigators suggest
that the genetic differences between strains of serotypes D
and A warrant the elevation of C. neoformans var. grubii
from its current varietal status to the rank of a new species.
Such proposals can be scrutinized under the biologic species
concept. There has been genetic characterization of serotype
AD strains that react with anti-A as well as anti-D factor
sera. These strains were determined to be diploids or
aneuploids, containing serotype A- as well as serotype Dspecific sequences (Cogliati et al., 2001; Lengeler et al.,
2001). Analysis of the mating type-specific genes, such as
STE12 or STE20, in the AD serotype diploid strains indicated that a majority of them were MATa/a type with a few
strains homozygous for the MAT allele (Cogliati et al., 2001).
The ploidy and genotypes of these strains suggested that a
majority of them were products of mating between MATa
and MATa strains of serotype A and D and a few of them
were fusion products between the two strains of the same
mating type of serotype A and D.
In order to determine whether the genetic diversity
between serotype A and D strains hinders genetic recombination during meiosis, we crossed H99 with JEC20 (B-4476)
on V-8 juice agar and isolated 20 single basidiospore
cultures (F1 progeny) and analysed their genotype. The
genes used for the analysis of their genetic profiles were:
STE12, STE20 and MF on chromosome 4, URA5 and LAC1
on chromosome 7 (Edman & Kwon-Chung, 1990; Williamson, 1994), and CAP59 on chromosome 1 (Chang & KwonChung, 1994) in JEC20. Sequence-specific primers of genes
STE12, STE20, MF, CAP59 and LAC1 from strains H99 and
JEC20 (Chang et al., 2000, 2001; Lengeler et al., 2001; Petter
et al., 2001) were used to amplify PCR products from the
progeny, which were then compared with those from the
parents. The URA5 genotype of this progeny was determined by Southern blot analysis of StuI restriction patterns
of their genomic DNAs. Consistent with the restriction site
Stu1 being present in the middle of the URA5 gene in
serotype D strains, the Southern blot showed a pattern of
two barely resolvable DNA fragments. By contrast, the
serotype A strains showed two widely separated bands,
indicative of the StuI restriction site polymorphism within
the URA5 gene (Kwon-Chung et al., 1992) (Fig. 3). Serotype
of the progeny was determined using the Iatron Crypto Kit.
The progeny could be categorized into four groups: MATa
parental type, aneuploid, diploid (hybrids) and haploid
FEMS Yeast Res 6 (2006) 574–587
recombinants (Table 1). The recovery of serotype AD
diploid strains by crossing strains of serotype A and serotype
D in the laboratory had also been reported previously
(Tanaka et al., 1999). Figure 3 illustrates the genotypes of
representative strains of each group. The ploidy based on
DNA content of the progeny was confirmed by FACS
analysis (Tanaka et al., 1996) (Fig. 3). The number of
diploids and aneuploids in the progeny was significantly
higher than that of the haploids, comprising 70% of the
progeny. The ploidy of several of the progeny strains as
determined by FACS analysis did not correspond to that
based on genetic analysis. For example, the genotype of sb16 was the same as that of JEC20 but the FACS analysis
indicated it to be an aneuploid. This is not surprising as the
chromosomal markers used were only on three of the 14
chromosomes (Loftus et al., 2005). Haploid recombinants
carrying genes from both parents comprised 15% (sb-4, sb18 and sb-19) of the total progeny. These types of recombinant populations may exist in nature, although currently
available typing tools may not be adequate for the detection
of such strains. In addition, such approaches may necessitate
larger numbers of strains than thus far studied. A high
frequency of aneuploids was expected in light of differences
in the genomic organization between H99 and JEC20
(Schein et al., 2002). At the chromosomal level, for example,
the URA5 and LAC1 genes are both on chromosome 7 in
JEC20 and JEC21 (http://www.tigr.org/tdb/e2k1/cna1/
index.shtml) whereas they are on chromosome 8 in H99
(J. Kronstad, personal communication). Meiotic nondisjunction appeared to have occurred with considerable
frequency. The occurrence of stable diploids is puzzling as
laboratory diploids constructed by fusing two strains readily
segregate (Whelan & Kwon-Chung, 1986). There have been
several reports that support these conclusions. First, genetic
analysis of numerous strains of serotype AD MATa/MATa
from the environment as well as from clinical specimens
indicated that they were the products of mating between
strains of serotypes A and D (Tanaka et al., 1999; Cogliati
et al., 2001; Lengeler et al., 2001). PCR fingerprinting
revealed that the genotypes of 17 of the 68 (25%) serotype
D clinical isolates from Italy were typed as either VIN3 or
VIN4. These were presumed to be hybrids of VIN1 (serotype
D) and VIN6 (serotype A) and all were diploids (Cogliati
et al., 2001). Similarly, four of 43 (9.3%) serotype A strains
were hybrid diploids (Cogliati et al., 2001). In a more
comprehensive survey by the European Confederation of
Medical Mycology in 2002, the percentage of hybrid clinical
strains from Italy alone reached 31% (Fimua Cryptococcosis
Network, 2002). By analysis of strains using the amplified
fragment length polymorphism (AFLP) method, Boekhout
et al. also found that 9.4% of serotype A clinical isolates were
hybrids while six of 14 (60%) serotype D clinical isolates
expressed the hybrid genotype (Boekhout et al., 2001). This
c
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578
K.J. Kwon-Chung & A. Varma
(a)
n
2n
400
400
320
320
320
M1
M2
160
M1
240
Counts
240
Counts
M2
160
240
M2
160
80
80
80
0
0
0
0
200 400 600 800 1000
FL3-A
sb-16. sb-17, sb-20
sb-20
H99
sb-2
sb-1, sb-2, sb-3,
sb-6, sb-7, sb-8, sb-10,
sb-11. Sb-12, sb-13, sb-14
(b)
200 400 600 800 1000
FL3-A
sb-5
H-99, JEC-20,
sb-4, sb-5, sb-9
sb-15, sb-18, sb-19
sb-16
0
JEC20
200 400 600 800 1000
FL3-A
sb-17
Counts
M1
0
n+1
400
STE20a (D)
STE20α (A)
(c)
STE12a (D)
URA5 / Stu l
STE12α (A)
CnLAC1-A
CnLAC1-D
CAP59-A
CAP59-D
A
D
AD(2n)
MF1a
MF1α
URA5
A
D
D
D
Serotype
A
D
D
AD
D
AD
AD
AD AD/D AD/AD
may be due to instability of serotype in diploid or aneuploid
strains. Strains sb2 and sb3 (Table 1) are clear cases of
diploid progeny based on genetic as well as FACS analysis
but serotyped initially as AD and then typed as D in
subsequent testing a year later. Conversely, sb14 is an
aneuploid strain that was serotyped first as D but serotyped
as A in subsequent testing. Furthermore, some diploid and
aneuploid strains showed a significantly stronger reaction
with factor 8 (serotype D-specific) than with factor 7
(serotype A-specific) as if the expression of serotype D
epitope was dominant (e.g. sb7, sb20). These inconsistencies
in serotyping could also result from differences in the degree
of specificity and potency among the different batches of
factor sera. Regardless, our results of genetic analysis and
serotyping of the progeny from the cross between H99 and
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c
Fig. 3. Ploidy (a) and genetic analysis
(b, c) of some representative progeny
from crosses between H99 and JEC20.
JEC20 suggest that, although genetically divergent, strains of
serotype D and A interbreed and the serotype of each strain
does not always corroborate with its genotype. Therefore,
serotyping alone is not a reliable method to distinguish var.
grubii from var. neoformans.
Soon after the discovery of F. neoformans, the sexual state
of serotype B and C strains was observed by matings
between strains NIH 191 (serotype C, MATa) and NIH 444
(serotype B, MATa) (Kwon-Chung, 1976). The teleomorph
of C. gattii (C. bacillisporus) was described as a new species
based on the distinct morphology of its basidiospore as well
as by its failure to undergo meiosis when the type strains of
the two Filobasidiella species were crossed (Kwon-Chung,
1976). Prior to the description of C. bacillisporus, Vanbreuseghem and Takashio had isolated an atypical strain of C.
FEMS Yeast Res 6 (2006) 574–587
579
Major species concepts and Cryptococcus neoformans
Table 1. Genetic analysis of 20 F1 progeny from a cross between a serotype A strain, H99 and a serotype D strain, JEC20
Strain
Serotype
mating(V8)
Ploidyw
MF
URA5
CAP59
CNLAC
STE12
STE20
H-99
B-4476
sb-1
sb-2
sb-3
sb-4
sb-5
sb-6
sb-7
sb-8
sb-9
sb-10
sb-11
sb-12
sb-13
sb-14
sb-15
sb-16
sb-17
sb-18
sb-19
sb-20
A
D
AD/AD
AD/D
AD/D
D/D
AD/AD
AD/AD
AD/AD
AD/AD
D/D
AD/AD
D/D
A/A
AD/A
AD/A
AD/AD
D/D
AD/AD
AD/AD
D/D
AD/AD
a
a
aa
aa
aa
a
a
aa
–
–
a
aa
aa
aa
aa
aa
a
a
a
a
a
a
n
n
2n
2n
2n
n
n11
2n 1
n11
2n 1
n
2n 1
2n
2n 1
2n 1
2n 1
n
n11z
n11z
n
n
n11
a
a
aa
aa
aa
a
a
aa
a
aa
a
aa
aa
aa
aa
aa
a
a
a
a
a
a
A
D
AD
AD
AD
D
D
AD
D
D
D
AD
AD
AD
D
AD
D
D
D
D
D
AD
A
D
AD
AD
AD
D
D
A
AD
D
D
D
AD
D
D
A
D
D
D
D
D
A
A
D
AD
AD
AD
A
AD
AD
A
A
D
AD
AD
A
AD
AD
D
D
A
A
A
A
a
a
aa
aa
aa
a
a
aa
a
aa
a
aa
aa
aa
aa
aa
a
a
a
a
a
a
a
a
aa
aa
aa
a
a
aa
a
aa
a
aa
aa
aa
aa
aa
a
a
a
a
a
a
Font size is reflective of the reaction intensity with anti-A or anti-D factor sera.
w
FACS patterns: n = haploid; 2n = diploid; n11 = haploid by FACS, aneuploid by genetic analysis; 2n 1 = diploid by FACS, aneuploid by genetic analysis.
Haploid by genetic analysis but aneuploid by FACS pattern.
z
neoformans from a leukemic patient in Africa and described
it as C. neoformans var. gattii (Vanbreuseghem & Takashio,
1970). The description of this new variety was strictly based
on observations of elliptical and oval yeast cells in the
histopathological sections of brain tissue. When we examined the ex-type strain of C. neoformans var. gattii (CBS
6289), it showed the same serotypic, biochemical and
morphological characteristics as the serotype B strains of C.
bacillisporus (Kwon-Chung et al., 1982). Cryptococcus neoformans and C. bacillisporus were subsequently reduced to
varietal status as C. neoformans var. neoformans and C.
neoformans var. gattii. The latter was based on the observed
DNA–DNA relatedness between the two anamorphs as well
as the result of crosses between F. neoformans and F.
bacillispora. The DNA–DNA relatedness between C. neoformans and C. bacillisporus was 55–63% (Aulakh et al., 1981),
which is higher than that commonly observed between
different species of yeast. Furthermore, viable basidiospores
were produced in crosses between certain strains of F.
neoformans and F. bacillispora.
The agar medium initially used to discover heterothallism
in the two species was malt extract agar (Raper & Fennell,
1965) and although it was useful in screening for mating
type of each strain, the efficiency of mating on it was
significantly lower than on V-8 juice with 4% agar, which
had been formulated for mating of Filobasidiella species
FEMS Yeast Res 6 (2006) 574–587
(Kwon-Chung et al., 1982). V-8 juice agar allowed certain
pairs of C. neoformans and C. gattii to mate and produce
viable basidiospores. Matings between B-3502 (ATCC
34874, MATa) of C. neoformans and NIH444 (MATa) or
CBS 6289 (MATa), the ex-type strain of C. neoformans var.
gattii, produced a low percentage of viable progeny
(20–30%). Phenotypic analysis of the progeny, which included the CGB reaction, maximum growth temperature
and agglutination patterns using polyclonal antibodies for
serotyping, suggested that some of the progeny were hybrids
(Kwon-Chung et al., 1982). It was not clear whether the
hybrids were the result of genetic recombination or reassortment during meiosis, as the only genetic markers that had
been used were the MAT alleles determined by mating
reactions. It was not known whether these phenotypes were
determined by nuclear genes. Moreover, ploidy determination of the progeny had not been carried out. The hybrid
phenotype can also result from diploidy or aneuploidy
rather than from genetic recombination or reassortment.
Kwon-Chung et al. (2002) proposed raising the two varieties
to species status, C. neoformans (serotype A, D, AD) and C.
gattii (serotype B, C), based on the mounting evidence of
their phenotypic and genetic distinctiveness, despite the
mating results.
Reliability of the ploidy analysis together with the availability of gene sequences and serotype-specific factor sera
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K.J. Kwon-Chung & A. Varma
enabled us to re-examine the evidence of genetic recombination between C. neoformans and C. gattii. The F1 progeny
of CBS 6289 B-3502 maintained as glycerol stocks were
revived and subjected to FACS analysis for ploidy determination (Tanaka et al., 1996). The genetic markers used for
PCR were LAC1, URA5, CAP59 (Petter et al., 2001) and
STE12 (AY168185). Serotyping was performed using the
Crypto Check Kit (Iatron). Table 2 summarizes the antigenic, physiologic and genetic profiles of 16 progeny that
were subjected to analysis. Fifty per cent of the progeny were
haploid, serotype D, and contained alleles of genes specific
to serotype D. The other 50% were diploid or aneuploid and
contained alleles of genes from both parents. Interestingly,
five of the eight nonhaploid progeny were serotyped as D
and the remaining three switched from their initial BD
serotype to D serotype in subsequent tests. Six of the eight
diploid and aneuploid strains were weakly positive for the
CGB test by 72 h while the two remaining diploid strains
remained negative. These results indicate that CGB reaction
or serotyping alone cannot be used for the identification of
hybrids or recombinants. Unlike in the cross between
serotype A strains and serotype D strains, true haploid
recombinants were not obtained. Although the number of
progeny analysed was small due to low viability of basidiospores produced by the cross, it appears that C. gattii and C
neoformans can produce stable diploid hybrids, but not
true recombinants of haploid progeny. This suggests
that strains of C. gattii are genetically close enough to fuse
and form basidiospores of diploids and aneuploids but may
not be able to undergo meiosis and produce true recombinants. It was interesting to note that serotyping with
serotype D-specific factor was more stable and dominant in
diploid strains formed by the cross of CBS 6289 and B-3502.
An explanation of this observation awaits elucidation of
genetics on serotype-specific biochemical or antigenic
features.
Is the two-species system of
C. neoformans supported by the cladistic
(phylogenetic) species concept?
As the sequence of ribosomal DNA subunits and their
intervening regions, various housekeeping genes, inteins
and virulence-associated genes of C. neoformans became
available, phylogenetic trees or dendrograms were constructed to show the genetic relatedness of all the serotypes.
Regardless of the sequence used for comparisons of
genetic relatedness, at least two distinct large clusters, one
consisting of serotype A, D and AD and the other consisting
of serotypes B and C became clear (Fan et al., 1995;
Fell et al., 2000; Xu et al., 2000; Butler & Poulter, 2005;
Diaz et al., 2005). These studies revealed that C. neoformans
is a complex species and that the strains belong either
to C. neoformans or C. gattii. Figures 4 and 5 show examples
of trees with two major clusters: one comprised mostly of
serotype A, D and AD strains and the other of serotype B
and C strains. Monophyletic clusters of serotype A and
serotype D strains are generally well separated within
Table 2. Genetic and biochemical analysis of 16 F1 progeny from a cross between a serotype B strain, CBS6289, and a serotype D strain, B3502
CGB
Strain
Serotype
Mating(V8)
Ploidy
24 h
72 h
CAP59
CNLAC
STE12
URA5
CBS6289
B-3502
sb-17
sb-18
sb-26
sb-27
sb-28
sb-30
sb-31
sb-32
sb-37
sb-38
sb-40
sb-41
sb-52
sb-63
sb-65
sb-69
B
D
D
D
D
D
B/D;D
D
B/D;D
D
D
D
D
B/D;D
D
D
D
D
a
a
a
a
a
aa
aa
a
aa
a
aa
aa
aa
aw
a
a
a
a
n
n
n
n
n
2n
2n
n
2n
n
2n
2n
2n
2n
n
n
n11
n
1
1
B
D
D
D
D
BD
BD
D
BD
D
BD
BD
BD
BD
D
D
BD
BD
B
D
D
D
D
BD
BD
D
BD
D
BD
BD
BD
BD
D
D
BD
D
a
a
a
a
a
aa
aa
a
aa
a
aa
aa
aa
a
a
a
a
a
B
D
D
D
D
D
BD
D
BD
D
BD
BD
BD
BD
D
D
D
D
FACS patterns: n = haploid; 2n = diploid; n11 = haploid by FACS and aneuploid by genetic analysis.
w
While diploid by FACS analysis, mated as a but PCR detected only the STE12a allele.
2006 Federation of European Microbiological Societies
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c
FEMS Yeast Res 6 (2006) 574–587
581
Major species concepts and Cryptococcus neoformans
Fig. 4. The most-parsimonious tree for 34 isolates of Cryptococcus neoformans derived from
the sequence of 462 bases of the LAC1 gene
(courtesy of Dr. J. P. Xu).
C. neoformans. This suggests that strains of serotype A
and strains of serotype D can be readily classified into
two varieties: serotype A strains in the var. grubii and
serotype D strains in the var. neoformans. However, such a
clear correlation between serotype and varietal status no
longer persists when phylogenetic trees are constructed
using AFLP genotypes or IGS115S rRNA1IGSII
gene sequences. Figures 6 and 7 illustrate this point (Litvintseva et al., 2003; Diaz et al., 2005). Phylogenetic trees
constructed based on IGS115S rRNA1IGSII sequences
appear to support the three-species concept of C. neoformans. The first species contains the serotype A, D and
AD strains of genotype 1a, 1b and 1c, while the second
species comprises serotype A, D and AD strains, but their
FEMS Yeast Res 6 (2006) 574–587
genotypes are 2a, 2b and 2C. The third species comprises
serotype B and C strains of genotypes 3, 4 and 5. Thus,
the difference between var. grubii and var. neoformans can
no longer be based on the serotype of the strains but can
only be based on the genotype. A phylogenetic tree constructed on the genotype identified by AFLP also did not
support the classification of serotype D and A strains into
two different species because of the considerable number of
AD hybrids and the genetic diversity among the strains of
serotype A. The controversy regarding classification of the
var. grubii and var. neoformans into two species can be
resolved by the application of the biologic species concept
to the taxonomy of C. neoformans. However, C. gattii is
composed of serotype B and C strains, which are more
c
2006 Federation of European Microbiological Societies
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582
K.J. Kwon-Chung & A. Varma
Fig. 5. Phylogenetic tree based on an alignment of the DNA sequences of the Cryptococcus neoformans PRP8 inteins (Butler & Poulter,
2005).
divergent and can readily be differentiated from C. neoformans regardless of the sequence used to analyse the genetic
relatedness.
Discussion and concluding remarks
The nomenclature of pathogenic species such as C. neoformans affects infectious disease specialists and clinical microbiologists more so than cryptococcal taxonomists. It is
imperative, therefore, to use the most appropriate species
concept for the classification of C. neoformans in order to
achieve nomenclatural stability. This will minimize confusion in scientific communications between investigators in
2006 Federation of European Microbiological Societies
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c
different fields of cryptococcal research and physicians as
well as diagnostic mycologists. The need for recognition of
the two species within C. neoformans is clear not only due to
their biologic and ecological differences, but also due to the
differences between C. neoformans and C. gattii in their
association with host immune status. Cryptococcus gattii
infections have been documented more commonly in immunocompetent individuals, whereas a majority of the
patients infected with C. neoformans have been immunocompromised, especially those with HIV infection (Speed &
Dunt, 1995; Chen & Sorrell, 2000). Furthermore, some
reports have suggested that prognosis is worse with infections due to C. gattii (Mitchell et al., 1995). Differences in
the immunological status of the host and infections caused
FEMS Yeast Res 6 (2006) 574–587
583
Major species concepts and Cryptococcus neoformans
Fig. 6. Genetic diversity among
Cryptococcus neoformans on
the basis of 96 AFLP markers,
presented in a neighbor-joining
dendrogram with Nei-Li genetic
distances (Livintseva et al.,
2005). With Permission from
The University of Chicago Press.
by the two serotypes A and D within C. neoformans,
however, has not been documented.
The valid number of species thus far suggested for C.
neoformans has been two, three or eight as discussed at
the 6th International Conference on Cryptococcus and
Cryptococcosis (Boekhout et al., 2005; Kwon-Chung, 2005;
Meyer et al., 2005). Different numbers of species were
proposed because of the differing opinions among taxonomists as to the definition of a species. For example, those
who accept the cladistic (phylogenetic) species concept
disregard the biology of the organism and focus only on
DNA sequences chosen for the analysis of genetic relatedness, and proposed up to eight species. Fingerprinting based
on PCR amplifications using M13 (minisatellite) primers
(Kidd et al., 2003) or AFLP analysis recognized eight major
molecular types. The genotypic variation between these
types may be comparable with that seen between fungal
species established by classical methods (Meyer et al., 2005).
However, the genotypic variation between these molecular
types does not always reflect their biology as is the case with
species established by classical methods. Comparative analysis of intergenic spacer (IGS) regions of rRNA genes
revealed up to 12 different genetic lineages, although no
investigator had suggested recognition of the 12 lineages as
different species (Diaz et al., 2005). Because the IGS regions
are the most rapidly evolving regions of the rRNA gene
cluster, several more molecular types will surface as more
strains become available for genotyping from different
geographic regions where C. neoformans isolates have not
yet been studied. Should the number of C. neoformans
FEMS Yeast Res 6 (2006) 574–587
species increase as more genotypes are found without having
any clear biologic difference? When populations of C.
neoformans become isolated, either through geographic
distribution or through differences in their reproductive
biology, they will ultimately result in speciation by genetic
divergence. During this process of genetic divergence, it is
expected that there will be distinct populations on the way
to species formation. If these incipient species are described
as species or subspecies, it will create mass confusion as it is
extremely difficult and subjective to decide when a population has sufficiently diverged from other populations to
merit a subspecies or a species rank. Does the genotypic
variation between different groups represent a different
biologic species? This problem can be overcome in C.
neoformans because it has an interbreeding system. The
two-species concept employs a combination of biologic
(interbreeding), morphologic and genetic species definition.
If two different genotypic groups interbreed and produce
genetic recombinants, they are classified as the same species.
Among strains of serotype A and D, about 15% did not
belong to one particular genotypic group (Fell et al., 2000;
Boekhout et al., 2001). Investigators who accept the classification of C. neoformans in three varieties, var. neoformans,
var. grubii and var. gattii, simply designated all serotype A
strains as var. grubii as if serotype A is exclusive to that
variety. Such a simple approach in classification has created
confusion in the C. neoformans nomenclature in several
ways:
(1) The serotype is based on the antigenic property of the
strain that is not always stable. Some diploid strains have
c
2006 Federation of European Microbiological Societies
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584
K.J. Kwon-Chung & A. Varma
Fig. 7. Phylogenetic tree of
Cryptococcus neoformans derived from IGSI15S rRNAt1IGSII sequence data (Diaz et al.,
2005).
been serotyped as AD, A or D. A good example is the type
strain of C. neoformans, CBS 132T, which was originally
serotyped as D (Boekhout et al., 2001), but also reported as
serotype A (Lengeler et al., 2001) or AD (K. J. Kwon-Chung,
unpublished data) depending on the laboratory where it was
serotyped. A similar phenomenon was observed among the
diploid and aneuploid F1 strains obtained by crossing H99
and JEC20, as shown in Table 1. Although changes in
serotype from AD to A or D may be interpreted as instability
of diploid strains during prolonged maintenance (Brandt
et al., 1995), changes in the serotype of the F1 diploid strains
was seen without prolonged propagation. Molecular analysis
of these strains has shown them to contain alleles of genes
specific for both A and D.
(2) There are hybrids that contain both serotype D and A
alleles, but are serotyped as either A or D but not AD
(Boekhout et al., 2001; Cogliati et al., 2000). Such populations have been reported mostly from European countries
2006 Federation of European Microbiological Societies
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c
where strains of serotype D and A coexist in the same
environment. Our experimental results of crosses between
H99 (the type strain of var. grubii) and JEC20 ( = B-4476, the
MATa, serotype D strain isogenic to JEC21) clearly showed
that they produce diploids, aneuploids, parental types and
recombinants. The aneuploid or recombinant progeny was
unstable, in that their serotype switched between D, A or AD
in 2–5 separate serotyping tests performed a few days to a
year apart using the commercial serotyping kit. Should the
varietal status of these progeny change in accordance with
switching of their serotypes?
(3) There is confusion in the nomenclature of diploids or
aneuploids where the serotype is consistently AD. A substantial number of serotype AD strains have been isolated
from various parts of the world and they cannot simply be
ignored as rare strains. Immunofluorescence patterns using
the monoclonal antibody 13F1 suggest that strains of the AD
serotype represent a fifth C. neoformans subgroup (Cleare
FEMS Yeast Res 6 (2006) 574–587
585
Major species concepts and Cryptococcus neoformans
et al., 1999). The nomenclature of this fifth serotype will be a
problem as it is neither var. neoformans nor var. grubii.
Under the current classification of the three varieties, some
investigators have avoided classifying the AD strains in any
particular variety whereas others have included them in C.
neoformans var. grubii (Franzot et al., 1999; Diaz et al., 2005)
causing further complications.
(4) Multiple subgroups may exist within each serotype. For
example, at least two distinct subgroups of serotype A
strains have been reported from the sub-Saharan region
alone (Litvintseva et al., 2005). As further genotyping of
strains from different geographic areas becomes available, it
is bound to reveal hitherto unreported subgroups among
the serotype A strains. Genetic divergence between serotype
A strains collected from AIDS patients in Botswana and the
serotype A strains from North Carolina was just as great as
between the serotype A strains and the serotype D strains
from North Carolina (Litvintseva et al., 2003) (Fig. 6). If we
use the names var. grubii and var. neoformans to recognize
the genetic divergence between serotype A strains and
serotype D strains, which subgroup of A serotype should
represent var. grubii?
(5) Although relatively few in number, there are serotype
untypable strains that do not react with any factor sera
(Brandt et al., 1995). These strains have to be genotyped to
identify their varietal status. In laboratories where the means
either to genotype or to serotype are unavailable, these
strains can still be classified under the two-species system,
as the one-step diagnostic methods using simple media such
as D-proline agar (Dufait et al., 1987) or CGB agar are
readily available (Kwon-Chung et al., 1982).
In order to be certain about the varietal status of each C.
neoformans strain, one cannot rely on its serotype alone.
Unfortunately, the literature is full of inaccurate statements
such as ‘serotype A strains are classified in var. grubii and
strains of serotype D are classified in var. neoformans’. The
IGS data clearly show it not to be fully concordant with the
classification based on serotype (Diaz et al., 2005). The
simplest way to avoid confusion in the identity of each strain
would be to designate the strains by their serotype under the
two-species system: C. neoformans serotype A, D and AD; C.
gattii serotype B and C. In order to determine to which
subgroup the strain of interest belongs, one can further
identify the strain by PCR fingerprinting, AFLP genotyping
or other molecular typing methods. It is desirable, though,
to employ a standard molecular typing method that offers
the highest discriminatory power in order to identify the
genotype of strains. The genotype determined by a combination of the DNA sequence of IGSI15S rRNA1IGSII
appears to be an appropriate method to subtype each strain
within the two species. The rRNA gene sequence has proven
to be a stable and reliable marker for genetic relatedness
between strains as well as species. rRNA gene sequences have
FEMS Yeast Res 6 (2006) 574–587
thus been the most widely used molecular marker for the
phylogenetic study across the five kingdoms (Wainright
et al., 1993).
Acknowledgements
This work was supported by funds from the intramural
program of the National Institute of Allergy and Infectious
Diseases. Figure 5 was reprinted from Butler & Poulter
(2005), with permission from Elsevier.
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