Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia.

Abstract

Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.
A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a "smouldering" outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.
The detection of sexual recombination in MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.

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Clonality and a-a Recombination in the Australian
Cryptococcus gattii VGII Population - An Emerging
Outbreak in Australia
Fabian Carriconde1, Fe ´lix Gilgado1, Ian Arthur2, David Ellis3, Richard Malik4, Nathalie van de Wiele1,5,
Vincent Robert6, Bart J. Currie7, Wieland Meyer1*
1Molecular Mycology Research Laboratory, Sydney Medical School - Westmead Hospital, Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead
Millennium Institute, Sydney Emerging Infections and Biosecurity Institute, The University of Sydney, Sydney, New South Wales, Australia, 2PathWest Laboratory Medicine
WA, QEII Medical Centre, Nedlands, Western Australia, Australia, 3SA Pathology at Women’s and Children’s Hospital, North Adelaide, South Australia, Australia, 4Centre
for Veterinary Education, The University of Sydney, Sydney, New South Wales, Australia, 5Hogeschool, Leiden, The Netherlands, 6CBS-Fungal Biodiversity Center, Utrecht,
The Netherlands, 7Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Northern Territory Clinical School and Infectious Diseases
Department, Royal Darwin Hospital, Casuarina, Northern Territory, Australia
Abstract
Background: Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals.
Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight
since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia,
Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is
nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian
VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population
and determine processes shaping its population structure.
Methodology/Principal Findings: A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the
Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America,
Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international
consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5
and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the
first time in association with recombination in MATa-MATa populations. Our results also support the hypothesis of a
‘‘smouldering’’ outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is
possibly caused by a founder effect followed by a clonal expansion.
Conclusions/Significance: The detection of sexual recombination in MATa-MATa population in Australia is in accordance
with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating
strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active
surveillance procedures.
Citation: Carriconde F, Gilgado F, Arthur I, Ellis D, Malik R, et al. (2011) Clonality and a-a Recombination in the Australian Cryptococcus gattii VGII Population - An
Emerging Outbreak in Australia. PLoS ONE 6(2): e16936. doi:10.1371/journal.pone.0016936
Editor: Kirsten Nielsen, University of Minnesota, United States of America
Received October 25, 2010; Accepted January 6, 2011; Published February 24, 2011
Copyright: ? 2011 Carriconde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Richard Malik is supported by the Valentine Charlton Bequest administered by the Centre for Veterinary Education of the University of Sydney. This
work was supported by a University of Sydney Bridging grant #100124681 to WM. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: w.meyer@usyd.edu.au
Introduction
Life-threatening infections due to fungi have increased signif-
icantly over recent decades, posing new challenges for public
health [1–3]. Fungal emergence appears to be driven by various
factors, including rising numbers of immunocompromised patients
and the development of antimicrobial resistance [2,4]. In the
context of a worldwide expansion of fungal pathogens, it is
essential to understand the taxonomy, epidemiology, ecology and
population biology of the fungi involved.
The two basidiomycetous haploid yeasts, Cryptococcus neoformans
and Cryptococcus gattii are causative agents of cryptococcosis, a
serious disease that manifests as meningitis and meningoenceph-
alitis in humans [5]. C. neoformans has a worldwide distribution and
infects predominantly patients with impaired immunity. In
contrast, C. gattii infection has been mostly associated with
immunocompetent hosts and was originally designated as a
tropical and subtropical pathogen [6]. Infections due to C. gattii
have been reported from human and a wide range of animal
species [7–9]. Cryptococcosis is initially caused by the inhalation
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of airborne infectious propagules released from environmental
niches. Because of the well-known behaviour of aerosolized
particles after inhalation it is presumed that suitable inocula are
either basidiospores or desiccated yeast cells [10]. Determination
of the primary ecological niches of C. gattii is of great importance to
better understand its life cycle and thereby determine exposure
risks and implement preventive strategies, as required.
In Australia, numerous studies have revealed an association
between C. gattii VGI and eucalyptus trees, particularly with the
native species Eucalyptus camaldulensis (river red gum) [11]. Viable
yeast cells have been commonly isolated from woody debris and
detritus in hollows of mature trees and sometimes in nearby soil
[11–13]. It was initially postulated that exposure to eucalyptus
trees may account for the high incidence of cryptococcosis within
Australia [10]. Subsequently, associations between C. gattii and
trees have been reported from other host plants in various
countries [7,12,14,15], indicating the existence of additional
ecological niches. Nevertheless, the high prevalence of C. gattii in
Australia and its association with native eucalypt trees and the
extensive exportation of those trees led to the hypothesis that C.
gattii originated from Australia and was subsequently dispersed into
other parts of the world through man-made horticulture
[10,16,17].
Besides studying the environmental niche of a potential human
pathogen, the determination of the relative importance of sexual
versus asexual reproductions in the life cycle of a fungus is a crucial
biological issue. Sex allows for new genetic recombination and
increases the potential for adaptation to new environments [18].
Furthermore, it potentially results in the emergence of new
virulent genotypes [19]. On the other hand, asexual reproduction
enables the propagation of well-adapted clones to certain
environmental conditions without disrupting favourable gene
combinations [20]. Cryptococcus can reproduce both sexually and
asexually. Sexual reproduction involves a bipolar mating system
with two mating type alleles, MATa and MATa. Mating occurs
between opposite mating types, resulting in the formation of
basidiospores. Asexual reproduction occurs via budding [10].
Recently, same sex mating between two a cells has been suggested
to occur naturally in C. gattii [16]. However, for its sibling species
C. neoformans, this has only been observed under laboratory
conditions [21].
Within C. gattii, four molecular types are recognized: VGI,
VGII, VGIII and VGIV [22], which may in fact represent
different varieties or phylogenetic species [23]. VGI is the major
molecular type recovered from clinical, veterinary and environ-
mental samples from eastern Australia where human populations
are most concentrated [24]. In addition, numerous VGII
infections have been reported in Australia from the eastern states,
from the southwest of Western Australia (WA) and the Northern
Territory [25,26]. The Australian VGII population has been in
the spotlight since 2005, when it was suggested as the possible
origin of an on-going outbreak of cryptococcosis at Vancouver
Island, BC, Canada [16]. Two genotypes have been delineated as
the causative agents of this outbreak, the major genotype VGIIa
and the minor genotype VGIIb [16,27]. Based on the fact that
some Australian isolates had an identical genotype to VGIIb,
which might represent a potential parental strain for the highly
virulent VGIIa genotype, it was postulated that this genotype
originated from Australia and subsequently was dispersed to the
North Pacific coast. The association between eucalyptus trees and
C. gattii, in concert with the large-scale exportation of these trees to
other parts of the world over the last century supports the notion of
an Australian origin for this fungus [10,16]. Despite extensive
environmental sampling only a mating type isolates have been
observed from Vancouver Island, leading to the suggestion that
same-sex mating between two a cells is the driving force for the
emergence of the outbreak. Previous population genetic studies
carried out on VGII populations from two Australian regions, the
Northern Territory and the greater Sydney area have detected
statistical evidence of recombination only when tests were
performed between genetically closely related isolates, in the
absence of any MATa VGII isolates [17,25,28]. This finding
supports the same-sex mating hypothesis [16].
To shed further light on the low virulent VGIIb Vancouver
Island outbreak strain and its relationship with Australian isolates,
the current study focused on (i) characterizing the genetic
variability within the Australian C. gattii VGII population on a
large geographical scale investigating 54 clinical, veterinary and
environmental isolates from Queensland (QLD), New South
Wales (NSW), Northern Territory (NT) and Western Australia
(WA), using multilocus sequence typing (MLST); and (ii)
determining the processes shaping its population structure, in
particular the reproductive modes (sexual vs asexual).
Results
Genetic variability
The 7 sequenced loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5
and IGS1) of the Cryptococcus consensus multilocus sequence typing
(MLST) scheme adopted by the International Society of Human
and Animal Mycology (ISHAM), resulted in 4166 bp nucleotide
positions when aligned with the two reference strains of the
Vancouver Island outbreak, CDC R265 (VGIIa) and CDC R272
(VGIIb), from which 47 polymorphic sites were identified
(Table 1). When only Australian isolates were considered,
4165 bp were in the total aligned and 46 polymorphic sites were
observed. This difference was due to the strain CDC R265
(VGIIa) presenting one additional nucleotide polymorphism
compared to the Australian dataset at position 318 in the GPD1
locus (Table 1). Thus, regarding the Australian population, among
the 7 MLST loci studied, the number of polymorphic sites ranged
from 12 for SOD1 to 3 for GPD1 and URA5 (Table 1 and Table 2).
From these polymorphisms, the highest number of alleles was
observed for IGS1 (6 alleles), followed by CAP59, PLB1 and SOD1
(5 alleles each), GPD1 and LAC1 (4 alleles each) and URA5 (3
alleles) (Table 2).
The allele combinations (Table S1) and the phylogenetic
relationships (Figure 1) revealed six distinct sequence types among
the 54 Australian isolates: ST5, ST7, ST21, ST33, ST38 and
ST48. Thirty-nine isolates belonged to ST7, six to ST48, five to
ST33, two to ST38 and one each to ST5 and ST21.
The majority of the Australian isolates (,72%) belonged to a
single sequence type (ST7), which was identical to the allelic profile
of the reference strain CDC R272 corresponding to the VGIIb
low virulent genotype from Vancouver Island (British Columbia,
Canada) (Table 1 and Table S1). None of the Australian isolates
had an allelic profile corresponding with the reference strain CDC
R265 of the high virulent VGIIa genotype of the Vancouver
Island outbreak. A different sequence type number was thus given
to this strain - ST20 (Table S1 and Figure 1). The ST48 was the
most closely related genotype to ST20 (Figure 1), with only 5
nucleotides differences over the 7 investigated loci (Table 1).
To determine whether scoring more loci would or wouldn’t
have increased the genetic diversity, the detected genotypic
diversity was plotted against the number of loci analysed
(Figure 2). This analysis clearly revealed that the genotypic
diversity reached a plateau at 3 loci. Thus, the 7 loci used were
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sufficient to discriminate all observed sequence types within the
Australian VGII population.
To place the Australian population in a global context
additional isolates from North America, Europe, Asia and South
America were studied (Table S1 and Figure 3). This analysis
reemphasised the low genetic diversity found in the Australian
VGII population. The highest genetic diversity within the global
VGII population was seen in South American isolates, as shown
by representative isolates (Table S1 and Figure 3) selected from an
ongoing global VGII MLST study.
Repartition of the genetic variability
Looking at the geographical regions within Australia, WA
showed the highest genetic diversity, with five sequence types
detected out of the six present throughout Australia (Figure 4).
Among the five genotypes detected in this state three were so far
unique to WA (ST5, ST38 and ST48) (Figure 1 and Figure 4). In
the NT and NSW, two sequence types were observed, while only
one was delineated in QLD.
The ST7 was present in all regions investigated and was by far
the most common sequence type (Figure 1 and Figure 4). Indeed,
in NSW and WA, 90.0% (n=9) and 68.8% (n=22) of the isolates,
respectively, belonged to this ubiquitous MLST type. Likewise 2/2
isolates genotyped in QLD were identified as ST7. In the NT, ST7
was also the main sequence type (60.0%; n=6), with ST33 being
the only other sequence type detected (40.0%; n=4). The ST33
genotype appeared to have a large distribution, being present in
the NT and WA. However, it was found only once in WA. In
NSW, in addition to ST7, a sequence type unique to this region
was delineated (ST21).
The distribution of the genetic diversity was also investigated in
relation to the source of isolation (Figure 5). Amongst the human
clinical isolates, four sequence types were delineated: ST7 (n=5;
35.7%), ST21 (n=1; 7.1%), ST33 (n=5; 35.7%) and ST48 (n=3;
21.4%). Isolates from veterinary cases were distributed among four
distinct MLST types: ST5 (n=1; 3.6%), ST7 (n=24; 85.7%),
ST38 (n=1; 3.6%) and ST48 (n=2; 7.1%). From environmental
isolates, three sequence types were observed, ST7 (n=10; 83.3%),
ST38 (n=1; 8.3%) and ST48 (n=1; 8.3%). ST7 and ST48 were
therefore obtained from all three sources of isolation (clinical,
veterinary and environmental) (Figure 1 and Figure 5). Finally,
three genotypes present in the environment were also recovered
from animals (ST7, ST38 and ST48). In contrast, ST21 and
especially ST33 were only obtained from human clinical isolates
and ST5 was only isolated from a dog with cryptococcosis (Table
S1). The lack of these sequence types from environmental samples
highlights the need for further extensive sampling of C. gattii VGII
isolates in Australia.
Mating types and multilocus linkage disequilibrium
Mating type PCR revealed that 52 out of the 54 C. gattii VGII
isolates studied were mating type a (Table S1). The remaining two
isolates were of mating type a: WM 09.165 and WM 09.94 (Table
S1). Both isolates belonged to ST38 and originated from WA
(Table S1 and Figure 1). Isolate WM 09.94 was obtained from a 3-
year-old dog from Geraldton with meningitis, while the isolate
WM09.165 was recovered from a eucalyptus tree trunk (species
not determined) at Caversham Wildlife Park (13 km from Perth).
In order to test for linkage disequilibrium among the seven loci
and consequently investigate the presence of recombination, the IA
and rBard association indexes were calculated. Both indexes were
computed on the complete and clone-corrected datasets for
populations having more than 3 sequence types, thus, for the
overall Australian and the restricted WA populations. The clone-
Table 1. Nucleotide polymorphism of the seven MLST loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1) for the six sequence types (STs) delineated in this study.
MLST Locus
CAP59
GPD1
LAC1
PLB1
SOD1
URA5
IGS1
Position
(bp)
7
79
220
424
547
86
106
318
330
283
371
406
63
157
168
277
382
484
486
511
35
97
211
387
396
430
435
464
496
527
536
550
144
146
263
37
127
294
351
376
412
429
481
542
597
604
Sequence
Type
ST7 (n=39)
C T G C A
A T G T
C C C G
A G T C A A G A
A T G C G T C G G C A A
G C A
A G A A G G T T C T T
ST5 (n=1)
. C A . .
. C . .
. . . .
. . . . . . . G
G C C . . . T A A . . .
. . .
. . G . . . . . T . .
ST38 (n=2)
. . . . G
. . . .
. . . A
. . . . . . C G
G C . . T C T . A A G G
. . .
. . . . . A . . T C .
ST21 (n=1)
. C . T .
T . . .
. . G .
G G C T C G . G
G C . . T C T . A A G G
. T .
. . . C . . . . T C .
ST33 (n=5)
. C A . .
. . . A
T T . .
G C . T C G . G
G C . T . . T . A . . .
T . G
G T . . C . A G T . C
ST48 (n=6)
T C . . .
. . . .
. . . .
G C . T C G . G
G C . T . . . . A . . .
. T .
. . . . . . . . T . .
VGIIa=ST20
. C . T .
. C A .
. . . .
G C . T C G . G
G . . T . . . . A . . .
. T .
. . . . . . . . T . .
VGIIb=ST7
. . . . .
. . . .
. . . .
. . . . . . . .
. . . . . . . . . . . .
. . .
. . . . . . . . . . .
Positions of the polymorphic nucleotides have been determined after alignment with the two reference strains CDC R265 and CDC R272 corresponding to VGIIa and VGIIb genotypes, respectively. Number of strains belonging to
each sequence type is indicated between brackets.
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corrected analysis was performed by removing replicates of the
same sequence type, as repetition of the same sequence type, due
to clonality, can lead to the detection of linkage disequilibrium and
consequently could affect the ability to detect recombination
among genotypes. Indeed, when all isolates were included in the
analyses, both the IAand rBard tests strongly rejected the null
hypothesis of no linkage disequilibrium, which would indicate the
absence of recombination (Table 3). However, after clone
correction, the null hypothesis was not rejected (Table 3),
indicating the absence of linkage disequilibrium and therefore
suggesting the potential existence of recombination. Furthermore,
the presence of a and a mating type isolates in these populations
suggests that recombination may be occurring between the two
opposite mating types.
Demographic history
The historical demography of the Australian C. gattii VGII
population was investigated by analysing the pairwise sequence
differences via mismatch distribution and neutrality tests. Mis-
match distributions for four of the seven loci were adjusted to the
distribution predicted under the sudden expansion model and
were L-shaped (Figure 6). The genetic loci CAP59, GPD1, LAC1,
and URA5 showed no significant differences between observed and
expected mismatch distributions (SSD p-values.0.05), and overall
high pairwise frequency comparisons were obtained from 0 to 2
nucleotide differences. This goodness-of-fit between observed and
expected pairwise difference distributions is likely to indicate an
historical population expansion. The three neutrality tests,
Tajima’s D, Fu & Li’s F* and Fu’s Fs, failed to reveal a departure
from the null hypothesis of neutral selection and/or population at
equilibrium for all MLST loci (Table 4). However, slightly
negative values were observed, which are expected when there is
an excess of singletons (substitutions present in only one sampled
sequence). The lack of significant values could be due to the large
number of isolates belonging to the same sequence type, leading to
high frequencies of 0 pairwise differences.
Discussion
Life-threatening fungal infections represent a major contem-
porary challenge owing to their increasing occurrence and the
emergence and re-emergence of outbreaks [2,16,29,30]. It is
therefore critical to investigate the population biology and the
epidemiology of these organisms in order to better understand the
associated risks of expansion to new environments, where
indigenous human and animal populations are immunologically
naı ¨ve and therefore at increased risk of infection. Indeed, the
epidemiology of disease is closely related to the life history of
fungi, including their reproductive strategies and dispersal
abilities. In nature, there is a large continuum of breeding
behaviour, ranging from exclusively asexual to fully sexual
organisms [31].
The present study revealed a relatively low genetic diversity
within the Australian C. gattii VGII population, with only six
MLST types delineated amongst the 54 clinical, veterinary and
environmental isolates studied. The majority (,72%) of all isolates
belonged to a single widely distributed sequence type, namely
ST7, corresponding to VGIIb, the minor/less virulent genotype
involved in the Vancouver Island outbreak [16,27]. The over-
representation of one sequence type suggests a clonal structure for
the Australian C. gattii VGII population. Among the 75 C. gattii
VGII isolates typed in a study by Fraser et al. [16] using a different
MLST scheme, 24 were from Australia. Of these 24 Australian
isolates, 50% (n=12) showed an identical allelic profile,
corresponding to the VGIIb genotype. Thus, their results are
consistent with our findings of a low-level genetic diversity and an
asexual reproduction structure of the Australian C. gattii VGII
population. Such a low genetic diversity is supported by
comparisons with other regions, especially South America, where
of the seven MLST loci investigated, each observed sequence type
corresponded to a distinct genotype (Table S1).
ST7 is numerically abundant and geographically widespread in
Australia. Indeed, it was present in all four different regions
investigated, i.e. QLD, NSW, NT and WA. Furthermore, it was
the preponderant sequence type isolated. These large numerical
and spatial representations of ST7 suggest that this genotype
harbours intrinsic abilities to survive and flourish under different
environmental conditions, from a tropical climate in Arnhemland
at the ‘‘top-end’’ of Australia to much more temperate climates in
the Sydney area of NSW and Perth in WA [32]. It thus could be
categorized as a generalist genotype with the capacity to colonize
various habitats. This observation is further reinforced by the
detection of this genotype in other parts of the world (Table S1)
[16,27,33]. In contrast to the widespread genotype ST7, five other
sequence types encountered showed a more restricted spatial
distribution. ST5, ST38 and ST48 were only found in WA, while
the ST21 was found only in NSW. ST33 was mostly present in the
NT, although, one isolate of this MLST type was also isolated
from WA (Figure 4). This raises the question whether these
sequence types are largely endemic to a restricted geographical
region, and as such, are they more ecologically specialized
compared to the more cosmopolitan ST7?
Table 2. Sequence polymorphism summary of the 7 MLST loci used in this study for the 54 Australian isolates.
Locus No. of aligned bases (bp)No. of polymorphic sites Number of alleles defined
CAP59557a,b
5a,b
5a,b
GPD1549a; 548b
4a; 3b
5a; 4b
LAC1475a,b
4a,b
4a,b
PLB1 534a,b
8a,b
5a,b
SOD1 712a,b
12a,b
5a,b
URA5638a,b
3a,b
3a,b
IGS1701a,b
11a,b
6a,b
Total4166a; 4165b
47a; 46b
33a; 32b
aValues when CDC R265 (VGIIa = ST20) was included.
bValues without CDC R265 sequences (VGIIa = ST20).
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The occurrence of several MLST types throughout Australia,
especially of ST7, is a striking finding and may indicate the ability
of C. gattii VGII to disperse over long geographical distances. A
previous study, investigating the genetic diversity and the
associated population structure using AFLP markers on VGII
isolates from the NT and the Sydney area, revealed a genetic
differentiation between both defined populations [25]. However,
when the authors restricted the analyses to closely related samples,
a decreased statistical significance of the test of genetic
differentiation was observed, leading the authors to conclude that
there are potential genetic exchanges over a large spatial range
[25]. This notion of long-distance dispersal within the C. gattii
VGII population is somehow supported by the detection of the
sequence type ST20 (VGIIa) from European patients, which had
previously travelled to Vancouver Island and those had physically
transposed this genotype to Europe (Table S1) [34,35]. Several
examples for the occurrence of long-distance dispersal events in
the fungal kingdom have been well documented; particularly
among plant pathogenic fungi [36]. Dissemination of fungi might
be due to various vectors, for instance, transport of infected plant
material [16], airborne dispersal [36,37] and/or animal activities,
such as migrating birds [37]. Dispersal of asexual spores and/or
yeast cells of ST7 by different and undetermined biological and/or
mechanical vectors might account for its abundance and
widespread distribution throughout the Australian continent, and
indeed, around the planet.
Sexual recombination has been suggested as a major force for
the natural evolution of virulence [19]. For C. gattii VGII, same-sex
mating between a-partners has been suggested, as the mechanism
underlying the emergence of the Vancouver Island outbreak in
British Columbia (Canada) based largely on circumstantial
evidence [16,30]. One of the scenarios postulated is that the
hypervirulent VGIIa genotype (ST20 in the current study)
originated from a mating event between two MATa parents,
namely the low virulent genotype VGIIb (ST7 in the current
study) and an unknown mating partner. This speculation draws on
the observation that, to date, only a-isolates have been detected on
Vancouver Island and the notion that recombination has been
detected within Australian populations constituted exclusively of
VGII MATa - isolates [25]. Indeed, prior to the present study,
MATa isolates had never been detected in Australia [16,25]. As
suggested by Hiremath et al. [38], MATa strains may contribute
critically to breeding, but are in such low overall abundance that
they are difficult to isolate via routine environmental sampling.
Population genetic studies generally require two main aspects, (i)
the use of polymorphic molecular markers, such as the seven
MLST-ISHAM-adopted loci and (ii) access to a sampling size as
large as possible. The extensive molecular analyses realized in this
study have demonstrated for the first time the presence of the
mating type a in Australia, more precisely in south-western WA.
Two isolates belonging to the same MLST genotype (ST38) were
characterized as MATa. One isolate was isolated from a veterinary
case, a Dalmatian dog from the Geraldton area, WA, in 2001, and
the second one from a eucalyptus tree trunk (species not
determined) from the Caversham Wildlife Park to the north of
Perth, WA, in 2009, approximately 400 km apart. This clearly
indicates that MATa-strains are present in the Australian
environment. The current study further suggests sexual recombi-
nation among VGII MATa and MATa strains in Australia, an
observation in accordance with the natural life cycle of C. gattii.
Sexuality among a- and a-mating partners has also been suggested
in the related C. gattii molecular type VGI [12,39].
In this context, it is important to note that when a pattern of
recombination is detected, it is hard to differentiate whether it
corresponds to a past or a contemporary event [31]. Regarding the
Vancouver Island outbreak, same-sex mating could be the driving
force, but alternative processes might be involved, such as long
distance dispersal events and multiple introduction phenomena.
Figure 2. Relationships between the number of loci and the genotypic diversity in the Australian C. gattii VGII population. Each data
point corresponds to the mean genotypic diversity and its standard error from 1000 permutations.
doi:10.1371/journal.pone.0016936.g002
Figure 1. (A) Unrooted Neighbor-Joining consensus tree of the 54 Crytococcus gattii VGII Australian isolates and the two reference
strains CDC R265 (VGIIa = ST20) and CDC R272 (VGIIb = ST7) based on the 7 concatenated MLST loci. The two ST38 isolates are MATa,
all others are MATa. For each sequence type the isolate proportions regarding their (B) geographical origin (QLD: Queensland, NSW: New South
Wales, NT: Northern Territory and WA: Western Australia) and their (C) source of isolation (CLIN: clinical, VET: veterinary and ENV: environmental) are
presented. Pie charts are proportional to the sampling size.
doi:10.1371/journal.pone.0016936.g001
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To answer this question, further population genetic investigations
based on an extensive sampling on a global scale are currently
underway by our research team using the international ISHAM
consensus MLST scheme.
The findings presented here suggest that asexual reproduction
and sexual recombination both contribute to the genetic diversity
and structure of the Australian C. gattii VGII population. Clonality
and sexual reproduction are not mutually exclusive. Evidence of
both modes has already been highlighted [38]. The relative
importance of the breeding system, i.e. asexual versus sexual
reproduction, has important evolutionary implications. Asexual
reproduction promotes the colonization of new habitats and
infections by one or a few clones [20], whereas sexual
reproduction favours genetic re-assortment with increased prob-
ability of survival in changing and/or competitive environments
[18]. Indeed, it has been shown in Thailand that genotypes of
Penicillium marneffei, an opportunistic fungus capable of infecting
HIV/AIDS patients, may be clustered according to ecological
conditions. This suggests that clonality has led to the evolution of
niche-adapted genotypes [20]. Based on experiments in the
laboratory using populations of the yeast Saccharomyces cerevisiae,
Goddard et al. [18] demonstrated that sex can provide a selective
advantage for adaptation to new environmental conditions.
Combination of both modes – sexual and asexual – could greatly
facilitate the population expansion of microorganisms.
Mismatch distributions for four of the seven loci were consistent
with the distribution obtained under the sudden expansion model
and thus may indicate an historical population expansion.
Furthermore, repartitions of pairwise differences were typically
L-shaped, a pattern consistent with a bottleneck phenomenon
followed by a demographic expansion [40]. A possible scenario
would involve the Australian C. gattii VGII population having
undergone a reduction of its population size resulting in a historic
founder effect (colonization of a new habitat by few individuals, in
Figure 3. Unrooted Neighbor-Joining tree of the sequence types delineated for C. gattii VGII in this study and originated from
different parts of the world. Bootstrap values over 50% are given at the nodes.
doi:10.1371/journal.pone.0016936.g003
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Page 8

this case yeast cells or spores). This presumptive founder effect was
subsequently followed by asexual population growth. It has been
postulated that C. gattii originating from Australia was subsequent-
ly exported to other regions of the world by the transport of
eucalyptus trees [10]. This hypothesis is to some extent, supported
by Fraser and colleagues [16], who argue that Australia is the
source of the Vancouver Island outbreak through an introduction
of the VGIIb genotype to North West America. In contradiction,
the present results of low genetic diversity, clonal structure and
founder effect taken together suggest an alternative hypothesis,
that the molecular type VGII has been introduced to Australia in
the past, while persisting in its natural environment within other
geographical regions.
Considering the behaviour of emerging and re-emerging
infectious diseases [29,30,41], the ST7 (VGIIb), that is widely
dispersed around Australia and accounts for numerous human and
animal cases, could potentially be responsible for triggering an
ongoing outbreak on a continental scale in Australia. It could be
argued that, together with ST33, it already is responsible for an
outbreak in Arnhemland, NT, whose scale is diminished only by
the low population density of indigenous aboriginals in this
location. The incidence rate of cryptococcosis in Arnhemland
certainly rivals the one of Vancouver Island, Canada [42].
Although isolates of VGIIb (ST7) have been characterized as
being of low virulence when compared to genotype VGIIa (ST20)
in mice models [16,43], a retrospective survey from 1999 to 2007
from Vancouver Island revealed that human death is actually
more likely to be attributable to VGIIb infections. Furthermore,
VGIIa infections apparently do not cause more severe illness than
those caused by VGIIb strains [44]. According to our records, the
Figure 4. Spatial distribution of the different sequence types delineated in the Australian C. gattii VGII population. Pie charts are
proportional to the number of samples. The symbols n and nST corresponds to the number of samples and the number of sequence types observed,
respectively.
doi:10.1371/journal.pone.0016936.g004
Figure 5. Repartition of the sequence types according to their source of isolation.
doi:10.1371/journal.pone.0016936.g005
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first human infections attributed to C. gattii VGII occurred in 1983
and 1985, in the NT and WA, respectively (Table S1). The first
veterinary evidence of VGII infection was reported from a horse in
1988 (Table S1) [9]. Recently, it has been proposed that canine
and feline cryptococcosis due to VGII isolates may be increasing in
WA [8]. This is further supported by the detection of an outbreak
affecting simultaneously over 100 sheep near Busselton, WA, that
was investigated in 1993 (Table S1) [24]. In addition, Caversham
Wildlife Park continues to have a very high environmental
presence of VGII (including isolates of several different sequence
types types, one being of the a mating type), with a high
prevalence of asymptomatic nasal colonisation, subclinical infec-
tion and clinical disease in exhibited animals (koalas and
wombats), which persists despite attempted environmental control
measures (Mark Krockenberger and Karen Payne, personal
communication). Overall, these observations are consistent with
the potential for more widespread outbreaks due to C. gattii VGII
strains in Australia, in either south-western WA and/or Arnhem-
land, NT. Despite the fact that this study contains the largest set of
VGII isolates ever collected from Australia, the limited sample size
can only point to the possibility of an outbreak at a continental
scale in Australia. However, the existing data without doubt
emphasizes the need for an on-going surveillance of environmen-
tal, clinical and veterinary cryptococcal isolates from Australia to
identify the extent of clonal outbreaks that might account for cases
in high incidence areas. A problem concerns the question of how
to monitor regions where the population density of humans and
domesticated animals is low [26,45–47].
A secondary outcome of the current study has been the
demonstration that MLST genotyping results in stable, robust and
reproducible data [48], which permits comparisons between
different research groups and an exchange of typing data via
web-based databases (e.g. MLST home page: http://www.mlst.
net). As a result of the current study an online database for C. gattii
has been established (http://mlst.mycologylab.org) on the basis of
the seven loci adopted by the ISHAM Working group for
genotyping of C. neofromans and C. gattii [48]. The database enables
online single or multiple loci assignments using polyphasic
sequence alignment algorithms. In addition the database allows
online depositing of interesting strains, associated data and
sequences, allowing the cryptococcal research community to
contribute to a better understanding of the global C. gattii
population diversity. Considering intra-MLST comparisons, this
study demonstrated unambiguously that the seven chosen loci
were sufficient to analyze the genetic variability within the
Australian C. gattii population, with the genotypic diversity
reaching a plateau for a total of only three loci (Figure 2).
Conclusion
The investigation of the molecular epidemiology of C. gattii
VGII on a large geographical scale in Australia has led to two key
Table 3. Multilocus linkage disequilibrium analyses
performed on C. gattii VGII Australian populations.
Population (n)IA(p-value)rBarD (p-value)
Australian population
All samples (54)4.683 (,0.001) 0.782 (,0.001)
Clone-corrected (6)
20.098 (0.725)
20.027 (0.725)
Western Australian population
All samples (32)4.070 (,0.001)0.685 (,0.001)
Clone-corrected (5)
20.290 (0.863)
20.077 (0.863)
doi:10.1371/journal.pone.0016936.t003
Figure 6. Observed (white bars) and expected (solid line and triangles) mismatch distributions under the sudden expansion model
for the seven loci used in this study. The abscissa corresponds to the number of nucleotide differences between pairwise of sequences and the
ordinate to the frequency. Pairwise nucleotide differences were realized on the global C. gattii VGII Australian population. Dashed lines represent the
90% confident interval of the expected mismatch distribution. Goodness-of-fit between the observed and expected mismatch distributions were
tested using the sum of square deviation index (SSD) and loci for which a good match has been detected are highlighted by dashed squares.
doi:10.1371/journal.pone.0016936.g006
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findings. Firstly, the presence of both MATa and MATa strains
and the detection of potential recombination suggest the presence
of sexual breeding between opposite mating types. Secondly, the
data have revealed evidence of a potential on-going outbreak
throughout Australia due to a limited number of VGII genotypes,
possibly caused by a founder effect followed by clonal expansion.
Finally, to understand the underlying mechanisms of fungal
emergence and spread in Australia, Vancouver Island and
elsewhere, a global population genetics approach using the
internationally adopted MLST scheme and web-based databases
is required.
Materials and Methods
VGII C. gattii isolates studied
Fifty-four Australian C. gattii VGII isolates were retrieved from
the Molecular Mycology Research Laboratory culture collection
(Westmead Hospital, University of Sydney, Westmead, NSW,
Australia) (Table S1), representing the major areas from which C.
gattii VGII has been isolated (2 from QLD, 10 from NSW, 10 from
NT and 32 from WA). These isolates reflect also all possible
isolation sources (14 clinical, 28 veterinary and 12 environmental)
(Table S1).
The strains CDC R265 (representing the VGIIa, major
Vancouver Island outbreak genotype) and CDC R272 (represent-
ing the VGIIb, minor Vancouver Island genotype) [16,27] were
included as reference strains (Table 1 and Table S1). To place the
Australian VGII population in the context of the worldwide
population, 6 isolates from North America, 3 from Europe, 1 from
Asia and 7 from South America were also included (Table S1).
Therefore a total of 71 C. gattii VGII isolates were studied.
DNA extraction
Isolates were subcultured onto Sabouraud Dextrose Agar (SDA)
at 37uC for 72 h prior to DNA extraction. High molecular weight
DNA was than extracted according to Ferrer et al. [49] with minor
modifications. Half an inoculation loop of the culture was
transferred to a microcentrifuge tube and kept at 220uC
overnight. Thereafter, the fungal material was incubated at
65uC for 1 h with 500 ml of lysis buffer (17.3 mM SDS, 0.25 M
NaCl, 25 mM EDTA, 0.2 M Tris-HCl) and 5 ml of 2-mercapto-
ethanol. After incubation, 500 ml of phenol-chloroform-isoamyl
alcohol (25:24:1), vol/vol/vol) were added to the tube and the
mixture centrifuged at 14,000 rpm for 15 min. The upper phase
was taken and mixed with an equal volume of isopropanol and the
DNA was precipitated at 220uC overnight. After washing with
70% ethanol, the DNA pellet was resuspended in sterile deionized
water. DNA concentration was determined by reading the UV
absorbance at 260 nm (BioPhotometer, Eppendorf) and diluted to
10 ng/ml.
Molecular typing
Restriction fragment length polymorphism (RFLP) analysis of
the URA5 gene via double digestion with the enzymes HhaI and
Sau96I was performed to determine the molecular types, as
previously described [22].
Mating type identification
To determine the mating type of all studied isolates, a mating
type specific polymerase chain reaction (PCR) was carried out
using the a mating type specific primer pair MFaU and MFaL
[12], and the a mating type specific primer pair JOHE9787 and
JOHE9788 [50] (Table S2). Amplifications were performed as
previously published [12,50]. PCR reactions were repeated
independently, three times, for the two samples identified as
mating type a (see Results).
Multilocus sequence typing (MLST)
The genetic variation within the Australian VGII population
was studied using the ISHAM consensus MLST scheme for the C.
neoformans/C. gattii species complex [48]. The typing scheme
consists of seven unlinked genetic loci, including six housekeeping
genes, namely the capsular associated protein (CAP59), glyceral-
dehydes-3-phosphate dehydrogenase (GPD1), laccase (LAC1),
phospholipase B (PLB1), Cu, Zn superoxide dismutase (SOD1)
and orotidine monophosphate pyrophosphorylase (URA5), and a
non-coding region, the intergenic spacer region of the rDNA
(IGS1).
Amplifications were carried out in a 50 ml reaction volume,
containing: 100 ng of template DNA, 0.2 mM of deoxynucleoside
triphosphate each, 7.5 pmol of the appropriate primers [48]
(Table S2), 2 mM of MgCl2, 2.5 U of taq polymerase (BIO-
TAQTMDNA polymerase, BIOLINE), together with the buffer
recommended by the manufacturer (10x NH4Buffer, BIOLINE),
following the published amplification conditions [48]. Purified
PCR products were sent to MACROGEN (Seoul, Korea) for
commercial sequencing. Sequences were edited using Sequencer
version 4.7 (Gene Codes, Ann Arbor, MI).
Genetic variability
Each sequence was assigned a unique MLST allele number.
Allele numbers were assigned for the following five loci: CAP59,
GPD1, PLB1, LAC1 and IGS1, according to Fraser et al. [16] and
Byrnes et al. [29,30]. For each new allele identified, a new allele
number was given in order of discovery. For the URA5 and SOD1
loci, allele identification was undertaken by comparison with our
own global cryptococcal sequence database. Due to the lack of a C.
gattii MLST database, an MLST database based on BioloMICS
software (BioAware, Belgium) was constructed for the 7 ISHAM
consensus loci at the Molecular Mycology Research Laboratory
and can be accessed at http://mlst.mycologylab.org. For allele
identification, sequences were aligned using CLUSTAL X version
2.0 [51]. GenBank accession numbers for all MLST sequences
used in this study are listed in Table S3. The allele numbers of the
7 genetic loci sequenced gave allelic profiles and allowed the
designation of Sequence Types (STs). For example, the strain
CDC R272 presents the following profile: CAP59-2, GPD1-6,
LAC1-4, PLB1-2, SOD1-15, URA5-2, IGS1-10, which corresponds
to the sequence type 7 (ST7). Each discrete sequence type was also
Table 4. Neutrality tests (Tajima’s D, Fu & Li’s F* and Fu’s FS)
performed on the seven MLST loci. None of the statistics gave
significant p-values.
Locus Tajima’s DFu & Li’s F*Fu’s Fs
CAP59
20.462
20.077
20.381
GPD1
21.274
21.873
22.446
LAC1
21.082
20.486
21.012
PLB1 0.8030.761 2.751
SOD1
20.205 0.214 3.079
URA5
20.265 0.6240.715
IGS1
20.903
20.1440.600
doi:10.1371/journal.pone.0016936.t004
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assigned an arbitrary number in the order of detection. The
sequence type numbers were given in accordance with a global
study currently undertaken in our laboratory.
An unrooted Neighbor-Joining tree was constructed from the
concatenated DNA sequences (a combination of the sequences
from the 7 loci) of all Australian isolates using MEGA version 4
[52]. The genetic distance between isolates was computed using
the p-distance and all positions containing alignment gaps were
eliminated in the pairwise sequence comparisons. The significance
of nodes was tested by bootstrapping with 1000 replications. The
two strains CDC R265 and CDC R272 were included as reference
strains in this analysis. An unrooted Neighbor-Joining tree has also
been constructed considering all the sequence types delineated in
Australia and the representative sequence types from other parts of
the world. The isolates used to represent sequence types from
other regions then Australia were selected for sequencing
according to previous studies [16,29,30].
Nucleotide polymorphism positions for the 7 loci were
determined using the software DnaSP version 5 [53] and checked
manually with CLUSTAL X version 2.0 [51]. Polymorphic
positions were obtained after alignment of the 54 Australian
isolates with the two references strains CDC R265 (VGIIa) and
CDC R272 (VGIIb). Gaps variations were not considered.
To determine whether the number of loci used was sufficient to
access the genetic diversity in the Australian C. gattii VGII
population we plotted genotypic diversity against the number of
loci using Multilocus version 1.3 software [54]. Genotypic diversity
is given as n/n – 1(1 – Spi2) where n is the total number of
individuals sampled and pi the relative frequency of the ith
genotype [54]. The standard error was determined by 1000
randomizations.
Tests for multilocus linkage disequilibrium
To test for multilocus linkage disequilibrium (i.e. non random
association) among the 7 MLST loci the index of association IA
and the slightly modified statistic rBard were computed using the
Multilocus version 1.3 software [54]. Calculation of the rBard
statistic has been performed in order to complement the IAindex.
Indeed, the IAvalue obtained is dependent of the number of loci
included in the analyses, whereas rBard removes this dependency
and would allow comparisons among studies [54]. The observed
dataset is compared to 1000 datasets in which alleles have been
randomly shuffled across isolates for each locus separately. The
1000 artificially produced datasets will thus simulate complete
panmixia, i.e. infinite recombination. Thus, the null hypothesis of
no linkage disequilibrium, consequently of recombination, will not
be rejected if the observed values of both statistics are not
significantly different from the distribution of the values obtained
with the 1000 artificially recombining datasets. IA and rBard
indexes were computed on (i) the complete dataset and (ii) the
clone-corrected dataset from which replicates from the same
sequence type were removed. Both complete and clone-corrected
analyses were carried out on populations having a number of
sequence types greater than 3, and consequently on the global and
the WA populations.
Demographic history
Historical demography of the Australian population was exam-
ined using two approaches. First, the distribution of the pairwise
sequence differences, called mismatch distribution, [40,55,56] was
generated for each locus and compared to the expected distribution
under the sudden expansion model using Arlequinversion 3.11 [57].
Goodness-of-fit between the observed and the expected mismatch
distribution was tested using the sum of square deviation (SSD)
approach. The 90% confidence interval of the expected mismatch
distribution was also computed. Second, several statistical neutrality
testswereused,includingTajima’sD[58],Fu&Li’sF*[59]andFu’s
FS[60] statistics. These tests were computed independently for each
locus using DnaSP version 5 [53]. Departure from the null
hypothesis of neutral selection and/or constant population size was
determined by generating 1000 permutations.
Supporting Information
Table S1
information: location, source of isolation (CLIN: clinical, VET:
veterinary and ENV: environmental), specific source and date of
isolation. Mating types, allele numbers for the seven MLST loci
and the corresponding sequence type (ST) are also presented.
(DOC)
List of isolates used in this study and their related
Table S2
(DOC)
List of primers used in this study.
Table S3
the current study of the seven MLST loci studied.
(DOC)
GenBank accession numbers for the alleles obtained in
Acknowledgments
We thank David and Pat Thorne, and their children David junior and Deb
for helping with sample collection and veterinary data on koalas at
Caversham Wildlife Park. We thank Teun Boekhout and Ferry Hagen
(CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands,
Tania Sorrell and Sharon Chen (Westmead Hospital, Westmead,
Australia), Murray Fyfe (British Columbia Centre for Disease Control,
Vancouver, BC, Canada), June Kwon-Chung (National Institute of Health,
Bethesda, MA, USA), Marcia Lazera, Luciana Trilles and Bodo Wanke
(Instituto de Pesqusa Clinica Evandro Chages, Fundacao Qswaldo Cruz,
Rio de Janeiro, Brazil), Katrin Tintelnot (Robert Koch Institut, Berlin,
Germany), Aristea Velegraki (University of Athens, Athens, Greece) for
providing cryptococcal isolates. We thank Shawn Lockhart (CDC, Atlanta,
GA, USA) for providing the MLST sequences for strain B7432 (VGIIc).
We thank the Institut Agronomique ne ´o-Cale ´donien (IAC; New Caledo-
nia) for hosting Fabian Carriconde. We thank Laurent Maggia (IAC-
CIRAD, New Caledonia) for his useful comments on the analyses. We
thank Sylvain Merlot (IRD, New Caledonia) for his useful comments on
the manuscript.
Author Contributions
Conceived and designed the experiments: WM FC FG. Performed the
experiments: FC FG. Analyzed the data: FC FG WM. Contributed
reagents/materials/analysis tools: WM IA DE RM BJC VR. Wrote the
paper: FC FG IA DE RM VR BJC WM. Supplied strains: IA DE RM BJC
WM. Designed the MLST database software: NvdW VR.
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PLoS ONE | www.plosone.org12 February 2011 | Volume 6 | Issue 2 | e16936