Mycologia, 105(1), 2013, pp. 1–27. DOI: 10.3852/12-253
2013 by The Mycological Society of America, Lawrence, KS 66044-8897
Issued 8 January 2013
#
Evolution of fungal sexual reproduction
Joseph Heitman
Sheng Sun
We do not know precisely what the last common
eukaryotic ancestor looked like, but a reasonable
hypothesis is that it was a unicellular, aquatic, motile
organism with one, or perhaps two, posterior flagella.
There are extant opisthokonts that maintain some
resemblance to this mythic creature, such as the premetazoan choanoflagellates and the basal fungal Chytridiomycota (King et al. 2008, Stajich et al. 2009). Key
features that distinguish eukaryotic from prokaryotic
organisms are the presence of the nucleus and other
intracellular organelles (mitochondria, chloroplasts,
Golgi, endoplasmic reticulum), the emergence of
multicellularity and the ability to undergo true sexual
reproduction. Given that sexual reproduction is pervasive and extant in all of the major super groups of the
eukaryotic tree of life, it is hypothesized the sex emerged
once in an ancestral eukaryote and has been preserved
and conserved throughout the eukaryotic tree of life.
Thus, one can think of the ability to undergo sexual
reproduction as a synapomorphy for the eukaryotes.
Given the ubiquity of sex, it is remarkable both how
conserved the core features are and yet how plastic other
aspects appear to be, including how sex is determined
and how sexual reproduction is accomplished.
We can illustrate the conserved features of sexual
reproduction by comparing the sexual cycles for two
of our favorite systems: ourselves (Homo sapiens) and
the model yeast Saccharomyces cerevisiae (FIG. 1).
Despite having diverged , one billion years ago, the
core features of sexual reproduction are conserved.
These involve: (i) ploidy changes from diploid to
haploid to diploid states, (ii) the production of
haploid mating partners or gametes from the diploid
state via meiosis which recombines the two parental
genomes to produce novel genotypes and halves the
ploidy and (iii) cell-cell recognition between the
mating partners or gametes followed by cell-cell
fusion to generate the diploid zygote and complete
the cycle (FIG. 1). Now we typically think of sexual
reproduction as involving two genetically divergent
parents, to give rise to a diverse repertoire of progeny
in which the genetic diversity of the parents has been
admixed. And yet we also are aware that inbreeding
and selfing forms of sexual reproduction occur, and
these involve consanguineous marriages in humans
and examples such as mating-type switching in fungi
in which a mother cell can switch mating type and
mate with a daughter cell to homozygose the entire
genome (except the mating-type locus) in what
represents an extreme form of inbreeding. We will
return to the topic of inbreeding/selfing modes of
Department of Molecular Genetics and Microbiology,
Duke University Medical Center, Durham, North
Carolina 27710
Timothy Y. James
Department of Ecology and Evolutionary Biology,
University of Michigan, Ann Arbor, Michigan 48109
Abstract: We review here recent advances in our
understanding of the genetic, molecular and genomic
basis of sex determination and sexual reproduction
in the fungal kingdom as a window on the evolution
of sex in eukaryotes more generally. In particular, we
focus on the evolution of the mating-type locus and
transitions in modes of sexual reproduction using
examples from throughout the kingdom. These
examples illustrate general principles of the origins
of mating-type loci/sex chromosomes and the balance between inbreeding and outcrossing afforded by
different modes of sexual reproduction involving
tetrapolar, bipolar and unipolar sexual cycles.
Key words: evolution, fungi, mating type, meiosis,
recombination, sex, sex determination
INTRODUCTION TO SEXUAL REPRODUCTION AND THE
FUNGAL KINGDOM
The tree of life is split into two broadly successful
lineages: the prokaryotes (bacteria, archaea) and the
eukaryotes. Recent molecular phylogenetic studies
reveal that the eukaryotic tree of life can be divided
into five to eight super groups that all descend from a
central last eukaryotic common ancestor (LECA)
(Wainright et al. 1993, Baldauf and Palmer 1993,
Baldauf 2003, Simpson and Roger 2004). We are
particularly interested in one of the eukaryotic
lineages, the opisthokonts, because it is the lineage
containing both the metazoan (animal) and fungal
kingdoms. And because the animal and fungal
kingdoms last shared a common ancestor as recently
as one billion years ago, much more recently than
any of the other shared ancestral nodes among other
major eukaryotic super groups, fungi are exemplary model systems for the often more complex
biology exhibited by their multicellular metazoan
compatriots.
Submitted 12 Aug 2012; accepted for publication 18 Aug 2012.
1
Corresponding author. E-mail: sheng.sun@duke.edu
1
2
MYCOLOGIA
FIG. 1. Common sexual cycles in unicellular and multicellular eukaryotes. The sexual cycles are representative of simpler
and more complex eukaryotes, using yeast (left) and human (right) as examples. For lower eukaryotes, the haploid vegetative
cells also serve as gametes. Cells of different mating types can fuse to form the diploid zygote. Within the zygote, meiosis occurs
and haploid progeny are produced. The haploid progeny then can reproduce either asexually through mitosis or sexually by
repeating the sexual life cycle. For sexual higher eukaryotes like mammals, haploid gametes (e.g. sperm and oocyte) fuse to
form a diploid zygote. Depending on the zygote’s composition of the sex chromosomes, it can develop into either male or
female. The male and female individuals then produce gametes of different genetic compositions (at both autosomes and sex
chromosomes) through meiosis. These gametes then must fuse to complete the sexual life cycle for reproduction to occur.
sexual reproduction, and their implications, later in
this review.
Given the ubiquity of sexual reproduction, combined with the fact that the few known truly asexual
lineages appear to be of relatively recent origin and
therefore may be doomed to more rapid extinction, it
is expected that sex must confer benefits (TABLE I,
FIG. 2). For more than a century the basic tenets for
the advantages of sexual reproduction have been that
it can serve to (i) generate progeny with a diversity of
novel genotypes and (ii) purge the genome of
deleterious mutations, such as transposable elements,
which otherwise would accumulate inexorably via
Muller’s Ratchet to degrade the integrity of the
genome. These are not mutually exclusive, and sex
may confer benefits via both mechanisms. Studies in
the model yeast S. cerevisiae by Goddard et al. (2005)
provide a direct experimental test for the potential
benefits of sex. These investigators engineered an
isogenic pair of yeast strains, one a wild type diploid
and the other a mutant for two key genes required for
meiotic recombination (SPO11, SPO13) such that the
mutant strain cannot undergo meiotic recombination
but still is able to produce spores. When they grew the
two strains under a variety of different stressful
conditions they found that the sexual strain always
had the competitive edge compared to the asexual
strain, providing a direct experimental test for the
benefits of sex in a fungal model system.
More recently, an additional hypothesis has been
advanced that sex might enable organisms to escape
or outrun pathogens. This is termed the Red Queen
hypothesis, which is an allusion to Lewis Carroll’s
novel Through the Looking Glass in which the
character the Red Queen must run as fast as she
can just to stay in the same place, and in the
evolutionary analogy sexual reproduction enables
organisms to just keep ahead in the co-evolutionary
race with the pathogens that afflict them. This model
has been tested recently in two real world biological
scenarios. In one, Curt Lively and colleagues from
Indiana University studied freshwater snails that live
in lakes in New Zealand and discovered that in lakes
in which parasites are present the snails are driven to
be sexual, but in lakes in which parasites are absent
they rapidly evolve to be asexual and triploid (Jokela
et al. 2009). This example provides a real world test
and verification of the Red Queen hypothesis of
sexual reproduction. This theme recently has been
extended with a second independent validation of the
hypothesis involving studies of the role of sexual
reproduction in pathogen evasion by the model
nematode Caenorhabditis elegans (Morran et al.
2011). Specifically, different lines of Cae. elegans that
HEITMAN ET AL.: SEX IN FUNGI
TABLE I.
3
Comparison between sexual and asexual reproduction
Sexual reproductiona
Energy cost
Chance of genetic/organelle conflicts
Recombination loadb (i.e. breaking down of
co-adapted gene combinations)
Genetic diversity
Adaptation to changing/fluctuating environment
Purging deleterious mutationsb
Selection of beneficial mutations
Asexual reproductiona
High a
High
High
Low b
Low
Low
Stable or increase c
Fast e
More efficient
More efficient
Stable or decrease d
Slow f
Less efficient
Less efficient
a
a: Require energy for finding and interacting with mating partner; meiosis is more energy consuming than mitosis. b: No
energy needed to find and interact with mating partner; mitosis is more energy efficient. c: Stable if the population is in
linkage equilibrium; increase if linkage disequilibrium is present in the population. In addition, meiosis may be mutagenic,
which can introduce substitutions and ploidy changes at a higher rate than mitosis. d: Stable if there is no selection; decrease if
there is clonal expansion. e: In some microorganisms such as fungi, sexual reproduction can also produce spores that can
better resist harsh environments, as well as be better dispersed. f: The adaption of asexual population also can be fast, if a
favorable genotype emerges and takes over the population by selective sweep.
b
See FIG. 3.
differed in their patterns of sexuality were tested for
their ability to withstand infection by the bacterial
pathogen Serratia marcescens. Of interest, those lines
that were self-fertilizing rapidly became extinct,
whereas those that were capable of sexual reproduction survived, thus providing a direct experimental
validation of central tenets of the Red Queen
hypothesis.
Now sexual reproduction not only confers benefits
but also comes with costs (TABLE I, FIG. 1). This
includes the well known so-called twofold cost of sex.
In most sexual cycles it takes two parents to produce
one offspring, resulting in only 50% of any given
parent’s genes being transmitted to a progeny. This is
contrasted with asexual mitotic reproduction in which
one parent can produce one progeny, and in which
100% of the parental genes are transmitted to the
progeny, thus resulting in a twofold cost of sex vs.
mitotic asexual production of progeny. Sexual reproduction also requires an investment of time and
energy. Moreover, for some parents or mating
partners it takes time to find a partner, and for some
of us, this requires more time than others. Finally,
one of the lesser appreciated but well established
costs of sexual reproduction is that it breaks apart well
adapted genomic configurations that have run the
gauntlet of adaptive Darwinian selection. This latter
cost of sexual reproduction leads to a conundrum for
facultatively sexual organisms: Why engage in sex if
very few of your progeny might even attain the well
adapted genotype of either parent? One way to
obviate this cost is to have very many progeny, such
that at least one approximates the fitness of the most
fit parent. But under diverse or rapidly fluctuating
environmental conditions, recombinant progeny may
be more fit than either parent, which were optimized
for a different environment. We later will return to
this theme when we discuss the discovery of novel
forms of selfing/inbreeding that involve unipolar,
unisexual modes of sexual reproduction.
To introduce and frame our discussions it might
help to provide the interested reader with a roadmap
for our narration. Much of our discussion will focus
on a particular species or group of closely aligned
species in the Basidiomycota with the peculiar
predilection to cause infections in humans that are
a considerable cause of morbidity and mortality.
These are Cryptococcus neoformans and C. gattii, and
their associated varieties and molecular types (Idnurm et al. 2005, Heitman et al. 2011). We currently
recognize two extant species, but there are likely as
many as six to eight taxa in this pathogenic species
complex (FIG. 3). Now much of the emphasis on their
study has been driven by the field of medical
mycology involving studies of their interactions with
the host. And yet the groundbreaking work of June
Kwon-Chung at the NIH in the 1970s applying
classical mycological approaches led to the appreciation of their sexual cycle, demonstration of a classic
basidiomycete life cycle and recognition of the
teleomorph, Filobasidiella (Kwon-Chung 1975,
1976a, b). Our efforts similarly began with a
molecular and genetic medical mycology perspective,
but we increasingly found ourselves drawn to the
mycological. In the course of our investigations, these
species emerged as exemplary models for understanding key and general concepts on the evolution
of sex in fungi and its importance to the epidemiological structure of the species. These include the
transitions from tetrapolar to bipolar mating-type
4
MYCOLOGIA
FIG. 2. Benefits and costs of sexual reproduction. A. One of the proposed benefits of meiotic recombination during sexual
reproduction—the purging of deleterious mutations. Each line with four blocks represents an individual. For simplicity, only
four individuals from the population (represented by the brackets) are shown. The green blocks indicate wildtype genes, and
the red blocks indicate genes with deleterious mutations that have gradually accumulated within the population. (i) The
HEITMAN ET AL.: SEX IN FUNGI
systems, how gene clusters evolve and further transitions to unipolar, unisexual modes of sexual reproduction. In the process, the findings touch on
essentially all those that are interesting in the
evolution of sex: inbreeding (same-sex mating), sex
chromosome evolution and transitions between mating systems (reviewed in Lee et al. 2010, Ni et al.
2011). With this as our backdrop, let us begin our
discussions.
EVOLUTION OF THE MATING-TYPE
LOCUS
We now turn our focus to how sex or mating type is
determined in eukaryotes. We know a great deal
about how sex is determined in animals such as
humans by the X and Y sex chromosomes. Unlike the
autosomes, the sex chromosomes are very different in
size, the X spans some 200 Mb and the Y chromosome
, 50 Mb; women have two X chromosomes and men
one X and one Y. Among the dramatic differences
between these two ‘‘homologous’’ chromosomes is a
key sex-determining gene, SRY, which encodes a high
mobility group transcription factor determinant
(HMG), which is resident on the Y chromosome
and establishes the male fate. Transpositions of the
5
SRY gene from the Y to the X chromosome lead to
sex reversal phenotypes in both mice and humans,
and thus this key gene is sufficient to orchestrate
sexual identity (Koopman et al. 1991, Schiebel et al.
1997, Sharp et al. 2005). Because the X and Y
chromosomes differ dramatically in size and structure, they are referred to as heteromorphic sex
chromosomes.
Although we are all well familiar with this paradigmatic example of sex determination, this process
is remarkably plastic throughout the eukaryotes. To
provide a sense for this plasticity in other species,
including the fish medeka and the plant papaya, the
sex-specific region of their sex chromosomes is much
more restricted such that the sex chromosomes do
not differ appreciably in size from one another, and
these are therefore referred to as homomorphic sex
chromosomes to reflect that they are not size
distinguished (Fraser and Heitman 2005). In contrast
to these examples of chromosomal sex determination
(CSD) there are other species in which the temperature at which an egg is hatched determines sex
(TSD), including crocodiles and turtles, and thus is
entirely environmentally rather than genetically determined.
r
population starts with most individuals having wildtype genes. The fitness distribution of the population is approximated by
the curve underneath the population (the green background indicates a constant environment favoring green alleles). (ii)
Gradually spontaneous substitutions accumulate within the population. Because most of these substitutions tend to be
deleterious, the fitness distribution of the current population (solid curve) shifts toward the left (i.e. lower fitness) compared
to the original population (dashed curve). (iii) Without recombination and sexual reproduction, the accumulation of
deleterious mutations, and the concomitant shift of the population fitness distribution toward the left will continue. The
individual without any deleterious mutations (individual [1] in [ii]) can be lost from the population either by further mutation
or genetic drift. In additionally, because different mutations occurred in different genes in different individuals, they cannot
be purged from the population efficiently by natural selection. (iv) The ultimate fate of a non-recombining population is
decline. Eventually the population will be dominated by individuals with multiple deleterious mutations that are collectively
maladapted to the original environment. This is called Muller’s ratchet effect (i.e. the highest fitness within the population is
determined by those with the least deleterious mutations). (v) On the other hand, recombination and sexual reproduction
can restore the ‘‘perfect individual’’ that does not harbor deleterious mutations. In addition, deleterious mutations occurring
in different individuals in different genes can be brought together, thus increasing the fitness variance and increasing the
efficiency of natural selection. B. Illustrates one of the possible costs of meiotic recombination during sexual reproduction—
disruption of favorable allele combinations. Imagine there are two populations, of which most of the individuals possess blue
(I) and orange (II) alleles respectively. This scenario can arise through processes such as temporary geographic isolation as
well as in microorganisms by clonal expansion. Now populations (I) and (II) are well adapted to their respective environments
(indicated by the high fitness distribution for populations I and II in the blue, curve Ia, and orange, curve IIb, environments
respectively), while maladapted in each other’s environments (shown by the low fitness distribution of the blue population in
the orange environment (curve Ib), and vice versa (curve IIa). If individuals from these two populations engage in sexual
reproduction, meiotic recombination will generate progeny that possess mixtures of blue and orange alleles (III). These
progeny will have an overall lower fitness compared to the parental populations in the two original populations (curves IIIa,
IIIb). Thus, sexual reproduction will disrupt allele combinations that are favored in certain environments. However, on the
other hand, if the environment changes, the progeny population generated by meiosis could have a broader fitness
distribution than the two parental populations. In addition, some meiotic progeny will have much higher fitness compared to
the two parental populations (i.e. transgressive segregation), such as shown in curve IIIc. In curves IIIa–c, the dashed lines
indicated the fitness distributions of the parental populations.
6
MYCOLOGIA
FIG. 3. Phylogeny of the pathogenic Cryptococcus species complex and its closely related species. The sensu stricto and
sensu lato groups (see text) are shaded blue and green respectively. The phylogeny was adapted from (Findley et al. 2009) with
modification. On the right are microscopy images of the hyphae, basidia, as well as basidiospore chains/clusters from three
species that are closely related to the pathogenic Cryptococcus species complex: Cryptococcus amylolentus, Filobasidiella
depauperata and Cryptococcus heveanensis. The images were adapted from (Metin et al. 2010, Rodriguez-Carres et al. 2010,
Findley et al. 2012). Please refer to these original publications for detailed descriptions of the mating structures in
these species.
Two recent studies involving novel fish species
further illustrate the dramatic plasticity of sex
determination. First, in the lab model system of zebra
fish, it was well known that there are no distinct sex
chromosomes, but it was still a mystery how sex
determination was established. Bradley et al. (2011)
reveal that strikingly sex determination in zebra fish is
a quantitative trait and that as many as eight or more
distinct genes, each lying on different chromosomes,
cooperate to establish whether an individual is male
or female. Two of the genes that together account for
< 25% of the trait have been defined. One is the
homolog of the Dmrt1 sex determinant implicated in
sex reversal in humans, and the other is an enzyme
involved in sex hormone production. Other genes,
and suspected environmental factors, remain to be
identified. A second recent striking example of
plasticity in sex determination involves the cichlid
fishes from Lake Tanganyika in Africa. Remarkably,
their genomes abound in dispensable B chromosomes and individuals who inherit an abundance of B
chromosomes are more likely to be female and by
comparison; those with fewer are more likely to be
male (Yoshida et al. 2011). These vignettes serve to
illustrate that even though the origins of sex are
ancient, the molecular, genetic, and genomic mechanisms that define sexes are remarkably fluid
throughout the eukaryotic tree of life. Perhaps this
illustrates a role via which this plasticity in sex
determination may contribute to speciation or species
boundaries.
A specialized region of the genome that is
conceptually similar to homomorphic sex chromosomes, termed the mating-type locus, establishes
mating type in fungi (Fraser and Heitman 2003).
Why do we say that fungi have mating types rather
than sexes and as a consequence call these regions
mating-type loci instead of sex chromosomes? The
HEITMAN ET AL.: SEX IN FUNGI
basis for this nomenclature is that most fungi do not
have sexes as these are strictly defined, which typically
entails gross morphological differences between the
individuals of a species that produce each of the two
types of gametes (i.e. sperm vs. oocytes). We say that
fungi are isogametic (the two gametes look morphologically similar), whereas animals are typically anisogametic (the two gametes look morphologically
distinct). There are a few fungi that do have clearly
demarcated sexes, and one example is the well
established model species Neurospora crassa in which
the male is the fertilizing partner (via dispersing
conidia or hyphal fragments) and the recipient that
makes the protoperithecia is designated the female
partner (Borkovich et al. 2004). Somewhat curiously,
in this species mating type is still determined by a
mating-type locus and distinct from the sex of the
gamete (Glass et al. 1988), which has led some
investigators to conclude that mating types and sexes
must be two distinct entities. But from a practical,
pragmatic viewpoint, there are also fungi in which the
two partners produce mating entities (gametes) that
display morphological differences. This includes
Cryptococcus, in which the a mating type often makes
enlarged round cells that serve as a target for fusion
with an elongating conjugation tube produced by the
a partner, reminiscent of the sperm and the egg in
anisogametic species (Hull and Heitman 2002,
McClelland et al. 2004). Thus, to some the distinction
between sexes and mating types is more semantic
than substantive whereas to others this is dogma that
is not open to debate.
We know a great deal about the mating-type
locus from studies of the model budding yeast S.
cerevisiae, a hemiascomycetous yeast (Herskowitz
1989). In this species, a small (fewer than 1000 bp)
region of the genome that encodes only one or two
key cell identity determinants that establish mating
type. There are two haploid mating types, known as a
and a, which can fuse to produce the diploid a/a cell
type. The a mating-type locus encodes a single gene
product, a1, which is a homeodomain transcription
factor. The a mating-type locus encodes two gene
products, a2, which is a homeodomain transcription
factor heterodimeric partner of a1, and a1, which is
an a-domain transcription factor that activates a
specific cell type genes. This species is an example
of a bipolar mating-type system, because there are just
two mating types, and any given cross produces
progeny of just these two mating types. In nature,
there is a roughly equal proportion of the two mating
types produced (a majority of the population is a/
a diploid), and thus there is an , 50% chance of
inbreeding among sibling progeny of a diploid
genotype and a 50% chance of outcrossing
7
during encounters with other members of the
population.
While the bipolar mating-type system is common in
fungi, there are other fungi that have even more
exotic mating-type determination systems that result
in literally thousands of different mating types (Raper
1966). These species are found within a different
phylum of the fungal kingdom, the Basidiomycota. A
representative example is Ustilago maydis, a pathogen
of maize. In this species there are two different
mating-type loci, termed a and b, which lie on
different chromosomes and therefore are unlinked
(Schulz et al. 1990, Bölker et al. 1992). In this species,
the b locus encodes a pair of divergently oriented
homeodomain proteins, called bE and bW, which are
homologous to the a2 and a1 factors of S. cerevisiae.
The a locus encodes pheromone and pheromone
receptors. Both loci must differ for a productive,
fertile interaction to occur (the loci are not selfactivating, but are cross active with an allele of
compatible mating type). In U. maydis the b locus is
multi-allelic, whereas the a locus is bi-allelic. Recent
studies reveal that the bi-allelic version of the a locus
in U. maydis is a derived state and descends from a
cluster of smut species in which many are tri-allelic for
the a locus (Schirawski et al. 2005, Kellner et al.
2011).
Now in other model species in this phylum, such as
Schizophyllum commune and Coprinopsis cinereus, both
mating-type loci are multi-allelic, leading to literally
thousands and thousands and thousands of different
mating types, as many as . 20 000 (Pukkila 2011). In
these species, mating of an isolate of A1B1 with
another isolate of A2B2 mating type can give rise to
progeny of four different mating types: A1B1, A2B2,
A1B2 and A2B1, when we consider the pool of F1
progeny produced, which is a combination of parental
ditype, non-parental ditype, and tetratype meiotic
events. As a consequence, this is termed a tetrapolar
mating-type system. Any given progeny can only mate
with 25% of its sibling progeny, and thus this type of
mating-type system results in a relative twofold
depression of inbreeding potential from the 50%
observed for bipolar species. It is important to note
here that, when an isolate of Cop. cinereus encounters
another member of its general population, in contrast
to interactions with its siblings, almost every interaction
(. 98%) results in sexual fertility and thus the multiallelic tetrapolar mating-type system achieves a high
level of outbreeding potential, higher than the bipolar
system (Kües et al. 2011). The major differences
between these systems are the twofold increase in
inbreeding potential in bipolar systems, the , twofold
increase in outbreeding potential of tetrapolar systems,
and the genetic uncoupling of the major processes
8
MYCOLOGIA
occurring during Basidiomycete mating (nuclear
migration and synchronous nuclear division) in tetrapolar species. We posit that transitions between the
unipolar, bipolar and tetrapolar systems observed in
nature are due to the relative importance of inbreeding and outbreeding in the life cycle.
Before beginning the discussion on the evolution
of the mating-type genes we must clarify what is meant
by inbreeding and selfing and discuss evidence
supporting their occurrence in fungi. Inbreeding
can be defined simply as sexual reproduction that
brings together more related genomes than random
mating. Obvious inbreeding examples in heterothallic fungi include intra-tetrad mating in Microbotryum
violaceum, mating-type switching in Saccharomyces and
pseudo-homothallic reproduction in Agaricus bisporus, where two compatible nuclei are packaged in
the same spore. In all these examples, population
genetic analysis of these species provides evidence for
inbreeding as a form of reduction in heterozygosity
when viewed in the diploid state (Giraud 2004,
Johnson et al. 2004, Foulongne-Oriol et al. 2009).
Selfing is the most severe form of inbreeding, and
with fungi it can occur at two scales, which we may
distinguish as occurring at the level of haploid
genotypes (in the case of homothallism) or occurring
as a form of mating among the gametes produced by
a single diploid genotype.
Evolution of the mating-type locus in the Cryptococcus
pathogenic species complex.—We now turn our focus to
a pathogenic species within the Basidiomycota called
Cryptococcus neoformans (FIG. 4). This species is a
common, globally distributed pathogen of humans.
We all have been exposed by the inhalation of
desiccated yeast cells or spores, which are small enough
to penetrate the alveoli in the lung. This results in an
initial pulmonary infection, which can become latent
or disseminate to infect most prominently the central
nervous system, including both the meninges and
parenchyma of the brain to cause meningoencephalitis. The species is distributed globally in association
with pigeon guano and certain arboreal niches, and we
are exposed to airborne infectious propagules. The
magnitude of impact on human health caused by this
pathogen is considerable. Recent studies from the
Centers for Disease Control document that more than
1 000 000 infections occur annually, largely in the
context of the HIV/AIDS pandemic, resulting in
more than 600 000 attributable deaths each year, and
, one-third of all AIDS-associated deaths, now
surpassing tuberculosis as the cause of AIDS-associated
deaths in Africa (Park et al. 2009). Moreover, the
sibling species C. gattii molecular type VGII is causing
an ongoing and expanding outbreak in the Pacific
Northwest (Byrnes III et al. 2011). This outbreak began
on Vancouver Island in 1999 and has spread to both
the Canadian mainland and to the United States in
Washington and Oregon and possibly also northern
California. This molecular type lineage of C. gattii does
not typically infect HIV/AIDS patients, and roughly
half of those infected during the outbreak were
otherwise healthy before presentation. The absolute
number of cases has been modest (, 400) by
comparison to the global health burden caused by C.
neoformans. But because it is airborne, widely distributed in the environment in association with trees and
soil and immunocompetent individuals are at risk the
outbreak remains a serious public health concern.
Because sexual reproduction is the process that
produces infectious spores, there is an association of
mating type with virulence and the population is also
largely unisexual, studies in the field have focused on
both the structure, function and evolution of the
Cryptococcus mating-type locus and the detailed
mechanisms involved in sexual reproduction. We will
consider both topics here.
While this pathogenic species is a member of the
same phylum of fungi with complex tetrapolar mating
systems, it has the simpler bipolar mating-type system
like S. cerevisiae with just two mating types, a and a
(FIG. 4). We cloned and sequenced the a and a
mating-type locus from C. neoformans and found that
it is dramatically expanded compared to the small,
compact mating-type locus of S. cerevisiae, spanning
more than 100 kb and encoding more than 25 genes
(Lengeler et al. 2002, Fraser et al. 2004, Byrnes et al.
2011) (FIG. 5). Embedded within this contiguous
mating-type locus are the genes that normally reside
in the two unlinked MAT loci of tetrapolar fungi, at
the left end encoding one or the other homeodomain
protein and at the other end encoding the pheromones and pheromone receptor. Thus, to a first
approximation, it appears that a translocation event
has brought the two unlinked MAT loci into linkage,
incorporated additional genes and resulted in a
contiguous derived MAT locus that now defines a
bipolar species. It is worth stressing that this is one of
three models that John Raper originally proposed to
explain how bipolar species might be derived from
tetrapolar ones, in his classic text titled Genetics of
Sexuality of Higher Fungi (Raper 1966).
To understand how this complex gene cluster
involved in establishing cell type identity might have
been formed, we took a comparative genomics
approach. We cloned and sequenced the a and a
mating-type locus from three closely aligned varieties
or species from the Cryptococcus pathogenic species
complex (Karos et al. 2000, Lengeler et al. 2002,
Fraser et al. 2004, Byrnes et al. 2011). This analysis
HEITMAN ET AL.: SEX IN FUNGI
9
FIG. 4. Modes of reproduction of Cryptococcus neoformans. Crytococcus neoformans has three modes of reproduction:
asexual, heterosexual (a-a bipolar mating system), and unisexual (a-a monokaryotic fruiting). Asexual reproduction is similar
to mitotic reproduction of a typical budding yeast, in which the haploid cell first undergoes DNA replication, followed by
nuclear division and cell division to produce two haploid daughter cells. Heterosexual reproduction begins when two cells of
opposite mating types fuse and initiate hyphal growth. The hyphae are dikaryotic with fused clamp connections. Eventually the
tip of the hypha expands and forms a basidium. Within the basidium, the two nuclei fuse and the ensuing meiosis produces
four haploid products, which then undergo repeated rounds of mitosis to generate basidiospores that emerge on the surface
of the basidium and form four basidiospore chains. The dispersed basidiospores then can germinate under suitable conditions
and re-enter the life cycle. Unisexual reproduction so far has been observed mostly in C. neoformans cells of the a mating type.
It starts when a diploid a-a cell is formed either by cell-cell fusion or endoduplication. This diploid cell undergoes
monokaryotic hyphal growth with unfused clamp connections. Similar to heterosexual reproduction, eventually a basidium is
formed at the tip of the hypha and meiosis occurs within the basidium. The four meiotic products then undergo repeated
rounds of mitosis and produce basidiospores that emerge on the surface of the basidium and form four basidiospore chains.
The basidiospores then disperse and germinate when the environment is suitable and re-enter the life cycle. For both
heterosexual and unisexual reproduction, haploid or diploid blastospores can be generated around the clamp connection.
revealed that the size of the MAT locus in general is
conserved, spanning 100–120 kb, and is flanked by
highly syntenic regions with conserved genes and
gene order. But within the MAT locus, there have
been marked rearrangements of the gene order but
few examples of gene loss, possibly because the
presence of five essential genes constrains the
rearrangements that can occur without loss of any
of these essential genes that punctuate the locus.
While this analysis reveals that the size of the locus has
been conserved across this group of species over the
, 10, to 20 to 40 million years that separate them
from their last shared common ancestor, it doesn’t
provide insight into how this large gene cluster might
have been formed from an ancestral tetrapolar
species. We next looked in detail at the sequences
of the genes resident within the MAT locus. By
assaying the rate of synonymous substitutions (dS)
10
MYCOLOGIA
FIG. 5. Gene strata within the Cryptococcus neoformans MAT locus. On top are the illustrations of the MAT alleles of the two
opposite mating types, MATa (JEC20) and MATa (JEC21) respectively. Black highlights the flanking genes; genes within the
ancient stratum are in red; genes within the intermediate I and II strata are in blue and green respectively; genes within the
recent stratum are highlighted in pink. At the bottom are phylogenies of the genes representing each of the four strata
respectively. When C. neoformans and Cryptococcus gattii are considered together, genes within the ancient stratum all have
highly mating type-specific phylogenies, genes within the intermediate strata also have a mating type-specific phylogeny, but to
a lesser extent, while genes within the recent stratum all have a species-specific phylogeny. Below each phylogeny, the number
within the parentheses indicates the observed rate of synonymous substitutions for the genes within that specific stratum. Data
and phylogeny are adapted from (Fraser et al. 2004) with modification.
between the a and a gene alleles as a molecular clock,
we observed that four strata of genes of different
evolutionary ages are present within MAT (FIG. 5).
First, there are the most ancient genes, recognized by
their higher dS rate. Next, there are two strata of
genes of more intermediate evolutionary age. Finally,
there is a strata of the most closely related genes,
which have the lowest dS rate. These are either the
youngest genes within the MAT locus or their
evolutionary age might have been reset as a consequence of more recent gene conversions that have
punctuated the evolutionary trajectory of the MAT
locus. Generation of gene trees further corroborated
these findings. The oldest strata of genes formed very
diverged bipartite gene trees in which the a and a
alleles formed distant groups. Next, there are the two
intermediate strata, in which the genes are still clearly
mating type-specific but less diverged than the more
ancient strata of genes. Finally, there is the stratum of
the most closely related genes, which turn out to be
not mating type-specific but instead species specific,
even though they are embedded within the otherwise
mating type-specific region of the genome. In this
case the gene trees form a tripartite rather than
bipartite phylogeny, in which the a and a alleles from
each species are more closely related to each other
than to their mating-type counterparts from the other
species (FIG. 5).
This analysis provides the insight that the MAT locus
is something like a patchwork quilt sewn from different
pieces of cloth and that it evolved through a series of
steps. Based on this insight, we developed a model
(FIG. 6) in which we posited that the extant bipolar
MAT locus evolved from a simpler, standard tetrapolar
system by a series of steps that involved acquisition of
genes that function in sexual reproduction into the
ancestral A and B MAT loci, forming expanded gene
clusters. Next, the two gene clusters fused to form one
contiguous mating-type locus in one mating type,
whereas the other remained unlinked, resulting in an
intermediate transitory state we term the tripolar
system. Then, the tripolar intermediate state was
converted to a fully linked bipolar state by recombination or gene conversion, linking the remaining two
HEITMAN ET AL.: SEX IN FUNGI
11
FIG. 6. Evolution of the bipolar mating type locus of species in the pathogenic Cryptococcus species complex. I. The evolution
of the bipolar MAT locus originated from an ancestral tetrapolar mating system with unlinked HD and P/R loci. II. The two
unlinked ancestral MAT loci underwent gradual expansion by sequential rounds of gene acquisition, recruiting components
such as the pheromone signaling MAPK cascade, as well as genes that are hypothesized to be involved in dikaryon formation or
meiosis. At the same time, chromosomal rearrangements shuffled the newly acquired genes within the expanded HD and P/R
loci. III. Fusion of the expanded HD and P/R loci in one mating type, which could have occurred by processes such as
chromosomal translocation or ectopic recombination mediated by elements such as transposons or centromeric sequences
(illustrated here by the red lines), resulting in an intermediate tripolar state. IV. Intercrosses between individuals with a fused
MAT locus and individuals with separate tetrapolar MAT loci allow further recombination around the repetitive sequences
(indicated with red lines), which could result in fusion of the alleles from the unlinked MAT loci, generating a contiguous MAT
locus for the other mating type. At the same time, one of the two HD transcription factors was lost in each of the two contiguous
MAT alleles. In addition, this newly formed MAT locus configuration could have established a certain level of reproductive
isolation, because intercrosses between individuals with fused and unfused MAT loci would produce progeny with abnormal
chromosomal compositions at much higher frequencies than mating between individuals with the fused MAT locus or between
individuals with unfused MAT loci. Ongoing chromosomal rearrangements (e.g. inversions and translocations) as well as gene
conversion within the contiguous MAT locus eventually gave rise to the extant bipolar MAT alleles observed today.
mating-type alleles in the other mating type. A series of
additional recombination and gene conversion events
then occurred to give rise to the extant MAT alleles
observed today. By deleting the homeodomain genes
in MAT, and relocating them to an unlinked genomic
location (URA5), we artificially constructed strains that
mimic this proposed ancestral tetrapolar state and
intermediate tripolar state, providing direct experimental evidence for these aspects of the model (Hsueh
et al. 2008). This effort provided a plausible model by
which the MAT locus in the pathogenic species
complex might have evolved. We next sought to test
this model experimentally.
Transitions in sexual reproduction from tetrapolar to
bipolar: the mating-type locus and extant sexual cycles for
Cryptococcus heveanensis and C. amylolentus.—
To investigate the evolution of the Cryptococcus
pathogenic species complex MAT locus, we took a
molecular phylogenomic approach. First, we constructed a robust phylogenetic tree for the 15 species
that span and encompass the pathogenic species
complex (Findley et al. 2009) (FIG. 3). This effort was
greatly facilitated by key advice from Rytas Vilgalys on
which species to focus this analysis upon, and the
ongoing fungal tree of life project (AFTOL) that
provided a broader intellectual framework. In fact,
12
MYCOLOGIA
our efforts used multilocus sequence analysis of the
six genes central to the AFTOL program (James et al.
2006a). We rooted our tree with the species Tremella
mesenterica, whose genome was sequenced by the
DOE-JGI fungal genome initiative (Floudas et al.
2012). Our analysis revealed two groups of species,
which we term the sensu stricto and sensu lato
complexes (FIG. 3). The sensu stricto complex includes the three pathogenic Cryptococcus lineages (C.
neoformans var. grubii, C. neoformans var. neoformans,
C. gattii). Three other species populate this group,
Cryptococcus amylolentus, Tsuchiyaea wingfieldii and
Filobasidiella depauperata, which are most closely
related to the pathogenic species. The sensu lato
group, which is more distantly related to the
pathogenic species complex compared to the sensu
stricto species, includes several species such as
Cryptococcus heveanensis and Kwoniella mangroviensis,
as well as others. It is important to stress that other
than those species that are part of the pathogenic
species complex, none of these other species are
pathogens but instead are associated with trees or
insect debris (frass), or possibly are mycoparasitic. It
also is important to stress that none of these are
model systems and they have been subject to very little
genetic or molecular analysis beyond their mycological description as species. Moreover, for some of
these very few isolates are available, which remains a
challenge. Caveats aside, these species are molecular
windows on the evolution of the MAT locus and
sexual cycles of the pathogenic species complex, with
which they share a most recent last common ancestor.
Our next step was to focus on representatives of
these species (Rodriguez-Carres et al. 2010), and we
will focus on two of these here for which we have
cloned the mating-type locus and discovered and
characterized extant sexual cycles (Metin et al. 2010,
Findley et al. 2012). First, for the sensu lato species C.
heveanensis, we cloned and sequenced the matingtype loci (Metin et al. 2010). This analysis revealed
two gene clusters corresponding to the A and B MAT
loci, organized similarly to a canonical tetrapolar
mating-type system. The A locus encodes the pheromone and pheromone receptor with a linked STE12
gene that also is present in the MAT locus of the C.
neoformans species complex. The B locus spans two
divergently oriented homeodomain genes, similar to
other canonical fungi with tetrapolar mating systems,
such as the bE and bW genes of the b locus of U.
maydis. Second, for the sensu stricto species C.
amylolentus (and also T. wingfieldii), we cloned and
sequenced the mating-type loci (Findley et al. 2012).
We again found two distinct gene clusters corresponding to the A and B MAT loci that lie on
different chromosomes, based on pulsed field gel
electrophoresis and chromoblot analysis. In this case,
the B locus again contains two divergently oriented
homeodomain genes, similar to C. heveanensis and
other tetrapolar fungal species, while the A locus
appears to have been expanded even further compared to C. heveanensis and spans a considerable
distance (this is currently being resolved by whole
genome analysis). Thus, these two species represent
extant examples of two of the hypothesized evolutionary intermediates from an ancestral tetrapolar
state to the derived bipolar one. In both cases
acquisition of genes observed in the bipolar C.
neoformans MAT into the vicinity of the A locus of
the tetrapolar species has occurred, perhaps corresponding to the intermediate age genes type 1 and
type 2 found within the extant derived bipolar state.
This analysis supports our evolutionary model and
provides further insights into the specific nature of
the events that might have given rise to a large gene
complex linked to virulence.
To fully elucidate the nature and function of the
MAT loci-related regions in C. heveanensis and C.
amylolentus, we sought to discover extant sexual cycles
such that we might be able to correlate the molecular
MAT loci analyses with their functions in mating. For
C. heveanensis, there were three isolates available via
culture collections. We were unable to find any
evidence of mating when any of these isolates were
co-cultured, either pairwise or all three together.
Further molecular analysis suggested that while one is
the type strain of C. heveanensis (CBS569), the other
two represent isolates of two closely related but
distinct species. Through prodigious efforts and
extensive review of a broad range of primary literature
sources, Banu Metin found a chapter describing a set
of C. heveanensis isolates obtained from insect frass or
flowers in Thailand. These isolates were deposited in
a public strain repository in Thailand, from which we
were able to obtain cultures by request. Molecular
analysis revealed that all eight of these isolates were in
fact bona fide isolates of C. heveanensis. Subsequently
we discovered that several of these isolates were fertile
with the type strain CBS569, generating beautiful
dikaryotic hyphae, basidia (with cruciate septa) and
associated spores (FIG. 3). We named the teleomorph
for this species Kwoniella heveanensis. We next
characterized the molecular nature of the A and B
MAT loci in these strains and found evidence that the
A locus is at least bi-allelic, the B locus is multi-allelic
and both mating assays and population genetic
analysis provide evidence for an extant tetrapolar
mating-type system. We explicitly note that access to
well validated and accessible strains from culture
collections was integral to this effort and thus we
applaud the efforts of mycologists to collect, docu-
HEITMAN ET AL.: SEX IN FUNGI
ment and store fungal isolates that provide the key
materials for subsequent genetic, molecular and
genomic analysis.
We next turned our efforts to C. amylolentus, and
after considerable effort we also were successful in
defining an extant sexual cycle for this species
(Findley et al. 2012). Only two isolates are available
for this species, and they have been preserved at the
ATCC for several decades. We obtained these isolates,
and our molecular analysis suggested that they could
be representatives of the same species, whereas the
single isolate that is available for T. wingfieldii appears
to be a singleton isolate from a closely related but
likely sibling taxon. While C. amylolentus grows as a
yeast, both available strains produce hyphae when
grown on a variety of media that induce mating of C.
neoformans or C. gattii, complicating recognition of
mating by morphological criteria. We focused on
conditions that enhance mating of the closely aligned
pathogenic species (V8 medium pH 5, in the dark,
without parafilm, with the agar side of the Petri dish
facing up), and Keisha Findley discovered that unique
sectors arise out of the peripheral hyphae from
cocultured strains. Upon microscopic examination,
these unique sectors contain dikaryotic hyphae with
fused clamp connections, basidia, and four very long
spore chains of round spores (FIG. 3). We found that
we could readily dissect these spores following
digestion with lytic enzymes and using a standard
microdissection fiber-optic cable dissecting needle
and micromanipulator. Analysis of germinated basidiospores provided evidence that progeny of four
mating types are produced from the A1B1 3 A2B2
parental genotypes, resulting in A1B1, A2B2, A1B2
and A2B1 progeny. One caveat is that a substantial
fraction of the progeny was sterile in crosses with
either parent, or with their siblings, illustrating a
potential danger of sex with respect to fecundity.
Another interesting fact was that many of the fertile
progeny were biased toward one of the two parental
mating types, which might represent a precursor to
the unipolar mating of C. neoformans that we will
discuss below. These findings provide evidence that
this sensu stricto species exhibits key features of the
tetrapolar mating-type system. We named the teleomorph for this species Filobasidiella amylolenta.
Taking these studies together, we can now return to
our phylogenetic tree and root it with a closely
aligned species, a more distantly related species and
an outgroup species, each of which has a tetrapolar
mating-type system. This analysis lets us conclude that
the most parsimonious model is that the last common
ancestor of this species complex was tetrapolar and
that the cluster of pathogenic Cryptococcus species
that all feature a bipolar mating-type system represent
13
a derived state. We can further suggest that this
transition to bipolarity occurred once in the origins of
the pathogenic complex, given the conserved nature
of the extant mating type-locus alleles observed in the
three recognized pathogenic species/varieties. Key to
our analysis was a combination of molecular analysis
wedded with classical mycology to discover extant
sexual cycles for two species that heretofore had been
classified as asexual. It is especially gratifying to find
extant sexual cycles for both; this is not the type of
discovery that happens every day, even to the most
accomplished mycologists, and in this case was fully
attributable to the tenacity and prodigious efforts of
highly talented graduate students and fellows (Keisha
Findley, Banu Metin, Sheng Sun).
Transitions in sexual reproduction from tetrapolar to
bipolar have occurred repeatedly and independently in
the Basidiomycota.—We now appreciate that these
transitions from ancestral tetrapolar states to a
derived bipolar state have occurred repeatedly and
independently in the Basidiomycota (FIG. 7). Three
examples feature the paradigm illustrated above for
the pathogenic species complex, including Ustilago
hordei and Malassezia restricta/Malassezia globosa.
Ustilago hordei is a plant pathogen related to U.
maydis but which infects barley or rye rather than
corn. In contrast to U. maydis, which is tetrapolar, U.
hordei has a bipolar mating system. Guus Bakkeren
and Jim Kronstad demonstrated that the U. hordei
MAT locus has two alleles in which the A and B MAT
loci have been fused and are separated by a large
450–500 kb intervening region that has a relative
paucity of genes and an abundance of repetitive
sequences (Bakkeren and Kronstad 1994, Lee et al.
1999, Bakkeren et al. 2006, Bakkeren et al. 2008).
Similarly, the research team at Proctor & Gamble,
together with international collaborators, sequenced
the genomes of the human skin associated commensal Malassezia species that are linked to causing
dandruff. They discovered a MAT region, similar to
the fused locus of U. hordei in which the A and B
MAT loci lie at the ends of a contiguous region,
separated by an , 167 kb internal region (Xu et al.
2007). The fact that there is no similarity between
the intervening regions of U. hordei and Malassezia
restricta and M. globosa, combined with other
phylogenetic evidence, argues that these are independent origins of the derived bipolar state, which
are themselves independent from that which arose in
the Cryptococcus lineage (FIG. 7) (Bakkeren et al.
2008, Hsueh and Heitman 2008).
That these transitions from a tetrapolar outcrossing
system to a derived bipolar system, which could
facilitate inbreeding or have arisen due to inbreeding
14
MYCOLOGIA
FIG. 7. Phylogenetic reconstruction of the evolution of mating systems and linkage between HD and P/R loci in the
Basidiomycota. On the outer layer of the branches is the ancestral state reconstruction under parsimony of the physical linkage
between HD and P/R loci. In some cases (e.g. Melampsora larici-populina), absence of linkage is inferred by association of HD
and P/R loci to different scaffolds of draft genome sequences, and this may not be equivalent to chromosomal linkage. The
inner portion of the branches reconstructs ancestral mating systems, where known. The character states of species included in
the tree are given to the left of the names, where ‘‘Link’’ refers to linkage of HD and P/R and ‘‘MAT’’ refers to mating system.
The phylogeny is based on (Aime et al. 2006, Hibbett 2006, Matheny et al. 2006, Kellner et al. 2011).
have occurred repeatedly and independently in three
different successful pathogenic lineages of fungi that
infect plants or animals, suggests that such transitions
might be adaptive for pathogens, perhaps as they
become specialized to unique niches or hosts. A
bipolar mating system may be favored in situations
where long distance dispersal of a tetrad occurs, such
as in the case of the smut teliospore. Multiple levels of
mating system transitions serve to increase the
potential for inbreeding, including the transition
from tetrapolar to bipolar, from multi-allelic to biallelic, and the loss of one or the other paired
homeodomain that has occurred in the Cryptococcus
pathogenic species. In tetrapolar species that have
paired homeodomain genes, there are two opportunities to produce an active heterodimeric homeodomain complex, whereas in the derived bipolar
pathogenic lineages there is a single homeodomain
encoded by each MAT locus and thus these must
productively interact to form an active heterodimer or
a given cross will be infertile.
How might these recombination events between
mating-type loci on two different chromosomes have
occurred repeatedly to bring the two sex determinants into one contiguous chromosomal region?
Given that mating-type loci and sex chromosomes
HEITMAN ET AL.: SEX IN FUNGI
are sheltered from recombination, transposable
elements and other repetitive sequences tend to
accumulate and might have served to foment recombination events leading to chromosomal translocations and transition from a tetrapolar to a bipolar
system. While this is a reasonable hypothesis, recent
genetic observations by one of us (Sheng Sun) have
led us to consider models in which the A and B MAT
loci may be linked to the centromeres on their
respective chromosomes. In C. neoformans, we know
that the candidate centromeric regions of its 14 linear
chromosomes each harbor at least one copy of the
two different transposable elements, Tcn5 and Tcn6
(Loftus et al. 2005). Thus, one plausible working
hypothesis is that intercentromeric recombination
could have driven the linkage of the originally
unlinked sex determinants of the tetrapolar matingtype system. If so, then in some examples such as U.
hordei the centromere still may be apparent and even
lie between the two ends of the now contiguous
mating-type locus. Approaches to test this model will
involve dissection of spores from individual basidia of
C. amylolentus to examine the patterns of marker
segregation, which can discern centromere linkage,
and whole genome analysis to examine the chromosomal context of the A and B loci with respect to their
respective centromeres on their host chromosomes.
Unlike the earlier diverging subphyla, the Agaricomycotina is largely characterized by tetrapolar species
(Whitehouse 1949, Raper and Flexer 1971) (FIG. 3).
Because of similarity in the genetic and physiological
manifestations of the tetrapolar mating system
throughout the group, the complex genetic architecture of the tetrapolar mating-type loci and the
phylogenetically widespread distribution of tetrapolar
and bipolar species across the Agaricomycetes (mushrooms), Raper (1966) posited that the ancestor of the
Agaricomycetes was tetrapolar and that both bipolar
and homothallic species had arisen multiple times
independently from tetrapolar ancestors, much in the
way that was observed in the Ustilaginomycotina and
Tremellomycetes (Raper 1966). One major difference
between Agaricomycetes and all other basidiomycetes,
the paraphyletic group previously known as the
heterobasidiomycetes, is the presence of a high
diversity of alleles at the pheromone receptor B
mating-type locus in the former, whereas no more
than three alleles are observed in the latter. Surprisingly few studies have investigated the phylogenetic
patterns of mating system evolution in the Agaricomycetes, but one exceptional study (Hibbett and Donoghue 2001) provides support for Raper’s hypotheses
that tetrapolarity is ancestral in Agaricomycetes and
that bipolarity has arisen multiple times from tetrapolarity while the opposite transition is not observed.
15
Going beyond the Agaricomycetes, there has been no
convincing evidence that bipolarity has given rise to
tetrapolarity more than once, yet the recent demonstration of pseudo-bipolar systems (Coelho et al. 2010)
and the paucity of data in the Pucciniomycotina
suggests that this pattern ultimately will be broken.
Currently it is unclear whether the last common
ancestor of the Basidiomycota was tetrapolar or bipolar.
Phylogenetic reconstruction of the trait suggests ancestral bipolarity; however, this interpretation is again
hampered by a paucity of data at the base of the phylum
and would benefit from a closer analysis of putatively
tetrapolar rust fungi including genome sequences
(FIG. 7) (Lawrence 1980, Narisawa et al. 1994).
The single mating-type locus of a number of
bipolar Agaricomycete fungi has now been investigated and a general trend has emerged that differs from
the observations of the tetrapolar to bipolar transition
in the heterobasidiomycetes. A phylogenetically diverse set of bipolar species, Coprinellus disseminatus
(Agaricales), Pholiota nameko (Agaricales), Phanerochaete chrysosporium (polyporoid clade) and Heterobasidion annosum (Russulales), all display a similar
mating-type locus architecture, comprising either one
or two pairs of homeodomain encoding pairs (Aimi
et al. 2005; James et al. 2006b, 2011; Olson et al.
2012). The genomes of each of these species also
encode homologues of the P/R genes observed and
descended from mating-type genes of tetrapolar
species, but the genes are not genetically linked to
the HD locus or sufficiently polymorphic to encode
different mating types. In the case of Coprinellus
disseminatus, these non-mating-type specific pheromone receptors were analyzed further by transformation into a heterologous host (Coprinopsis cinerea).
We observed that the pheromone receptors of C.
disseminatus were able to confer regular B matingtype pathway development in C. cinerea, suggesting
that the pheromone receptors of C. disseminatus were
either auto-activating or auto-activated (James et al.
2006b). In another tip of the hat to John Raper’s
insights, these findings confirm a second of his
hypothesized mechanisms by which a bipolar mating
system might originate from a tetrapolar one:
through the evolution of a non-discriminating, selfcompatible B mating type. Such bipolar patterns of
mating essentially had been reconstructed in the lab
from self-compatible B mutants of Schizophyllum
commune (Parag 1962; Casselton and Olesnicky
1998; Olesnicky et al. 1999, 2000; Casselton 2002).
These examples of reversion to a bipolar system in the
Agaricomycetes might provide some insights into how
the ancestral bipolar basidiomycete mating genes
appeared because the arrangement bears similarity to
that of the Ascomycota.
16
MYCOLOGIA
UNISEXUAL (UNIPOLAR) REPRODUCTION:
CRYPTOCOCCUS PARADIGM
THE
We have discussed in detail the transitions from
tetrapolar to bipolar mating systems, and we now
consider how the Cryptococcus pathogenic species
complex has taken this one step further to result in a
unipolar mating system in which a partner of opposite
mating type is no longer obligate and unisexual
reproduction can occur. A central conundrum in the
field was that studies of both environmental and
clinical isolates revealed that the Cryptococcus population is in many aspects largely unisexual. For
example, in the predominant pathogenic lineage
(C. neoformans var. grubii serotype A) that is globally
distributed and causing . 1 000 000 infections per
year, we and others typed , 3000 isolates and found
that 2997 were of the a mating type, and only three
were of the rarer a mating type (Lengeler et al. 2000,
Keller et al. 2003, Viviani et al. 2003). And for the one
a mating-type isolate we studied most extensively, it
was only fertile with three of 150 possible a isolate
partners (Nielsen et al. 2003). Thus, it seems that
most global isolates that are predominantly a mating
type will rarely encounter an isolate of opposite a
mating type. If so, how are infectious spores produced
and how might diversity be maintained? One possible
answer to this conundrum is that they are not and
that the organism is asexual and mitotically reproducing globally. In fact, a decade or so ago it was
commonly held that not only this pathogenic
eukaryotic microbe but also many other pathogenic
fungi, and even the pathogenic parasites (Leishmania,
Giardia, Trypanosomes), were asexual and clonal,
and that this reproduction strategy had been selected
concomitant with emergence of successful pathogens
(Tibayrenc et al. 1990, 1991).
But we considered an alternative hypothesis that at
the time might have been considered somewhat
heretical, which suggested that a sexual cycle might
occur involving just one of the two mating types, a,
and in this model a cells were not required for sexual
reproduction to occur. As a fellow, Xiaorong Lin in
fact made a series of observations that revealed that
Cryptococcus has the capacity for two types of sexual
reproduction, one involving both the a and a
opposite mating types and the other involving just
cells of the a mating type (Lin et al. 2005) (FIG. 4). In
this modified sexual cycle, a cells transition from
haploid to diploid via either cell-cell fusion with
themselves or another a isolate from the population
(or undergo endoreplication), and these diploid
isolates form a monokaryotic hyphae with unfused
clamp connections, ultimately produce terminal
basidia and produce four chains of spores. In other
strain backgrounds, karyogamy may occur late, in the
basidium, similar to opposite sex mating, but the
ultimate outcome is similar: a diploid nucleus in the
basidium undergoes meiosis to produce haploid
basidiospores that are all of the a mating type
(FIG. 4). From a mycological perspective, this is at
one level simply a new form of homothallism—an
isolate in solo culture can undergo sexual reproduction all on its own. But unlike other forms of
homothallism that we already know about, there is
no mating-type switching involved here and there is
just one mating-type locus in the genome and not a
fused MAT locus or both MAT alleles, as is the case in
some other homothallic fungi (Lin and Heitman
2007).
Now, at first it might seem odd that a fungal species
could harbor both extant heterothallic, opposite
mating-type and homothallic, same mating-type sexual cycles. And yet we need look no further than the
model yeast S. cerevisiae to find a similar example.
Many natural isolates of S. cerevisiae are homothallic
because they express the Ho endonuclease that
creates the breaks that provoke mating-type switching.
However, among natural isolates about 25% are
naturally occurring ho– mutants. These isolates are
not homothallic, yet they retain their ability to
undergo heterothallic mating with suitable partners.
Thus in this well defined species, both homothallic
and heterothallic isolates are part of the naturally
occurring interbreeding set of isolates we consider
the species. Another way to consider this is that even
homothallic fungal species are capable of outcrossing
with a suitable partner and yet they are also uniquely
endowed with the ability to self. It may be that these
represent examples of a species hedging its bets,
because one or the other mating strategy might prove
more adaptive in response to different types of
environments, selective pressures or the absence of
an appropriate partner of opposite mating type. This
plasticity may be a form of environmental sex
determination/orientation similar to the earlier
examples in fishes.
We have exerted considerable effort in the laboratory comparing and contrasting the features of both
opposite-sex and same-sex mating. We found, for
example, that ploidy changes from haploid to diploid
to haploid occur in both. We also found that key
meiotic genes, including SPO11, which encodes the
endonuclease that makes DNA double strand breaks
that provoke meiotic recombination, and DMC1,
which encodes a meiotic recombinase, are both
required for the efficient production of viable, fertile
spores. Self-fertile strains lacking either produce
stunted spore chains, in some cases only two instead
of four spore chains, and the germination frequency
HEITMAN ET AL.: SEX IN FUNGI
of the fewer spores produced was dramatically
reduced. In addition, we found that the frequency
of meiotic recombination was similar and approximately equivalent in both opposite-sex and same-sex
mating (this was established by constructing a special
a/a diploid strain with distinguishable genetic markers to generate a meiotic map of its progeny).
Furthermore, we found that genes involved in
pheromone production and sensing appear to contribute to both types of sexual reproduction, perhaps
reflecting a role for cell-cell fusion or stimulated
nuclear-nuclear fusion, during same sex mating.
Finally, one key genetic difference we found was that
the homeodomain proteins that are required for
opposite-sex mating are dispensable for same-sex
mating. In one case there is an obvious explanation:
Because the SXI2a gene is specific to a cells, it is not
present in a cells undergoing same-sex mating. But it
also turns out that SXI1a is also not required for
same-sex mating in several strains in which this has
been tested. Given that the a mating type is the
numerically dominant one and thus encounters with
a cells are likely to be rare in the environment, the
fact that we have not been able to find a role for
SXI1a in unisexual mating for this conserved gene
seems puzzling. It may be that there is some other
mitotic function or a more subtle aspect of same-sex
mating we have not yet been able to score experimentally.
Recent studies have revealed two other transcription factors that function in both opposite-sex and
unisexual reproduction. One is the high mobility
group (HMG) factor called Mat2 that has been
implicated as the factor responsible for activating
pheromone responsive genes downstream of the
pheromone activated MAP kinase cascade (Lin et al.
2010, Kruzel et al. 2012). MAT2 was identified via an
Agrobacterium insertional mutagenesis screen for
genes required for unisexual reproduction. The
second factor identified is a zinc finger transcription
factor, Znf2, which cooperates with Mat2 to operate
signaling circuits that let cells engage in sexual
reproduction in the absence of the homeodomain
heterodimeric complex that normally would be
required. ZNF2 was identified via a transcriptional
profiling approach to identify genes that were
markedly induced during hyphal growth (Lin et al.
2010).
One additional question is frequently posed: If a
cells can undergo unisexual reproduction, what about
a cells? It turns out that both mating types can
undergo unisexual reproduction, but in a quantitative
trait analysis of genes that contribute to the fecundity
of unisexual reproduction we found evidence for up
to five QTL loci from a screen of , 25% of the
17
genome, and thus as many as 20 or more QTL loci
may contribute (Lin et al. 2006). Among the five QTL
loci identified in this study, the MAT locus was found
to be the one contributing the most to the trait
(enhanced hyphal growth) and the a allele of MAT
was better endowed to support unisexual reproduction than the a allele. Put another way, in a
background in which many of the QTL alleles are
those favorable for unisexual reproduction, the status
of the MAT locus will be less important and a cells are
self-fertile, whereas in backgrounds where some of the
QTL loci are those less favorable for unisexual
reproduction, the status of the MAT locus is more
important and only those that are a mating type will
be fertile. There is a Poisson distribution of strain
phenotypes, and the mean of those that are a mating
type is more skewed toward hyphal growth, whereas
the mean of the a mating-type isolates is skewed
towards less hyphal growth.
Now these are all studies of same-sex mating under
laboratory conditions, and two criticisms that can be
leveled are whether this occurs only in the laboratory
and whether it is relevant to biology of the species in
nature. Studies over the past several years have
provided a series of findings that suggest that samesex mating could be a frequent and possibly even the
predominant form of mating in nature. First, recent
studies document that spores are bona fide infectious
propagules, and given that the vast majority of isolates
in nature are a mating type, a parsimonious model is
that this may be the route via which infectious spores
are produced in nature. Second, analysis of diploid
serotype AD intervarietal hybrid strains revealed an
unusual type that, instead of descending from mating
between isolates of opposite mating type (aADa or
aADa), were produced by unisexual mating between
two a isolates and are therefore aADa hybrids (Lin et
al. 2007). Third, a series of studies applying population genetics approaches reveals that populations of
isolates from trees in India or from infected animals
in Australia (all of which are mating-type a) are
nonetheless recombining populations and in some
cases also harbor a/a diploids that appear to be
intermediates/products of same-sex mating (Bui et al.
2008, Hiremath et al. 2008, Saul et al. 2008). Fourth,
studies have revealed a novel cell type that is present
in the lungs of mice or humans infected with
Cryptococcus that are termed giant or titan cells
(Okagaki et al. 2010, Zaragoza et al. 2010). Quite
remarkably, whereas the infecting yeast cells are
, 5 mm diam, these giant cells are as large as , 100 mm.
In addition, the starting cells are haploid, but the giant
cells have been found to be octoploid. Genetic studies
with marked strains suggest they are produced via
endoreplication, although it is hard to exclude models
18
MYCOLOGIA
in which closely opposed mother and daughter cells
might be fusing. These giant octoploid cells bud to
produce daughter cells that are haploid or diploid.
Essentially nothing is known about how ploidy
increases and then reduces again in a stepwise fashion,
but we submit that this might represent a modified
form of same-sex mating occurring in the context of
infection of the mammalian host and that ploidy
reduction might involve meiotic processes also occurring in the infected lung.
WHY HAVE SEX WITH
YOURSELF?
From a broad survey of the ploidy of almost 500
isolates of C. neoformans, we found that almost 10%
were diploid (Lin et al. 2009). This was surprising
because the dogma was that this species is haploid.
And while diploids had been identified previously in
the context of intervarietal AD hybrids, the majority
of the diploids we identified were not and instead
were AA hybrids produced by intravarietal fusion.
Examination of the mating-type locus configuration
further revealed that almost all were aAAa isolates
produced via same-sex mating. One class was clearly
heterozygous for different genetic markers, and thus
the result of fusion of two genetically distinct parental
isolates, similar to opposite-sex mating. However, the
majority were apparently homozygous at all the
genetic loci we examined, suggesting they resulted
from either endoreplication or mother-daughter cell
fusion. This finding perplexed us for some time. At
the root of this is the question: Why mate with
yourself if there is no genetic diversity to admix in
your progeny? We considered a number of possible
explanations, including that this might be a route to
produce spores, which might be hardier in the
environment and also airborne and therefore more
readily dispersed. We considered that the hyphae
produced by same-sex mating and diploid isolates
might be better able to invade the growth substratum
and extract nutrients. We even considered that samesex mating might be a form of practice. In this model,
isolates that undergo periodic rounds of same-sex
mating might have enhanced fitness compared to
isolates that do not and evolve to be asexual more
quickly. Thus, same-sex mating serves to preserve
fecundity and confers an evolutionary advantage
compared to isolates that instead evolve to be asexual.
But at some level these models all seemed to be ad
hoc explanations. This led us to consider whether
there might be another underlying reason, and we
again returned to question the central premises for
the function and nature of sex: to admix pre-existing
genetic diversity of two different parental isolates. But
what if the function of sex could be to generate
genetic diversity de novo, rather than to simply admix
it. In such a model sex would be a mutagen and serve
to introduce novel genetic diversity for genetic
selection to act upon (TABLE I).
Are there precedents for this type of mutagenic
process that we know about in biology? Yes! First, you
probably are aware that B lymphocytes of the immune
system, which are the antibody producing cells, go to
great trouble to assemble an active antibody encoding
gene via a process termed VDJ recombination. And
then they do something odd: They riddle the
resulting antibody gene they have just so carefully
constructed with mutations, in a process termed
somatic hypermutation. The purpose is to diversify
the antibody repertoire, and in fact it recently has
been found that this process is able to generate high
affinity neutralizing antibodies against HIV, but this
often takes years to occur and thus is not sufficiently
efficient or rapid to control HIV earlier during
infection (Chen et al. 2010, Wu et al. 2011). A second
example involves the phenomena of mutators that
arise in bacterial pathogens (Miller 1996, Oliver et al.
2000). These are often mutations that arise in the
mismatch repair system, the genome is sprinkled with
mutations, yet ultimately the mutator is selected
against because of longer term deleterious effects.
But we can recognize the effect of the mutator by the
sequence of the starting and evolved strain and the
presence of a multitude of mutations that have been
introduced genome wide and exhibit the sequence
bias signatures of the mutator that was responsible.
Finally, a third example is the intrinsic error rate of
DNA polymerases, without which humans might not
even be here as a species, given that these errors
contribute to adaptive evolution. More accurate
polymerase mutants can be selected, but the cost
they incur is that they spend too much time
contemplating which nucleotide they have inserted,
fail to replicate the genome in time for cell division to
occur and as a consequence slow growth dramatically
and are selected against (Reha-Krantz 1998). Ultimately a balance between accuracy and efficiency is
struck, and a certain level of mutations is not only
tolerated, but in some cases adaptive. Thus, mutations, while clearly detrimental under many circumstances, also can be beneficial.
We thus considered whether unisexual reproduction might be mutagenic and serve to generate
genetic diversity de novo. One of the great advantages
of working in mycology is that, if you have an
interesting model, it can be the case that some other
investigator has tested aspects of this in a different
organism. This turned out to be the case here. Rolf
Hoekstra’s lab conducted a beautiful series of
experiments in Aspergillus nidulans that sought to
HEITMAN ET AL.: SEX IN FUNGI
address the effect of ploidy on adaptive evolution
(Schoustra et al. 2007). They constructed isogenic
strains that were haploid and diploid and subjected
them to repeated rounds of laboratory passage,
selecting for faster growing variants. They found that
these faster growing variants arose much more
frequently from the diploid compared to the haploid
background. But remarkably, with further analysis
they found that all faster growing variants that arose
from a diploid background were instead haploid.
Through a series of crosses and genetic analyses, they
deduced that the reason for this is a phenomenon
termed reverse epistasis. In essence, recessive mutations arose in the diploid background that would be
deleterious on their own but when combined are
beneficial. These mutations do not arise in the
haploid because the intermediate single mutants are
less fit. But they propose that the diploid serves as a
capacitor for evolution, letting multiple different
recessive mutations arise in the shelter of the diploid
genome. Subsequently, these mutations are released
into the haploid state via the parasexual cycle of A.
nidulans and in the haploid state together interact to
enhance growth compared to the starting haploid or
diploid wild type background.
Clearly here the parasexual cycle can serve to
generate genetic diversity de novo, and our leap of
faith was to consider that sexual cycles might operate
similarly. We have been testing this experimentally
with a strain (XL280) that undergoes robust unisexual reproduction during solo culture on V8 or MS
medium (Ni and Heitman unpubl). We generated 96
progeny by mitotic asexual growth and 96 progeny
that were derived by germinating spores produced by
unisexual reproduction. We next assessed them for
phenotypic and genotypic plasticity. The progeny
produced by mitotic growth are all boring and look
exactly like the parent in a panel of phenotypic assays.
In contrast, from a screen for variants in five different
phenotypic tests, we found that , 5% of progeny
produced by unisexual reproduction differ from the
parental isolate. Thus, unisexual reproduction can
generate considerable phenotypic diversity, even
though there is no genetic diversity to admix from
two different parents because there is only one
parent. We then subjected these variant progeny to
a series of genotypic tests, including comparative
genome hybridization (CGH), pulsed field gel analysis with band CGH for any variant chromosomes
identified and next generation sequencing. We
initially thought that we might find a high rate of
single nucleotide polymorphisms. In fact, we found
few from three progeny sequenced to high coverage
spanning the , 20 Mb genome. What we did find
were examples of intracentromeric deletions, chro-
19
mosomal translocations and high aneuploidy. In fact,
all our variant progeny were aneuploid for one of
several different chromosomes. When isolates with a
wild-type phenotype were recovered, we invariably
found that they now exhibited a euploid karyotype,
providing direct evidence that aneuploidy can significantly contribute to the altered phenotypes observed.
At this stage you are probably thinking that
aneuploidy is a bad thing, and you would be correct.
We need look no further than human biology, in
which we know that only three trisomies in humans
(2N + 1) can survive to birth and these result in
Edwards, Patau and Down syndrome, in which there
are profound health consequences. But there is both
a yin and yang to aneuploidy. Studies over the past
several years have brought aneuploidy to the fore in
studies of drug resistance and adaptive evolution in
fungi. In Candida albicans, treatment with fluconazole leads to azole resistant isolates, and many of these
turn out to harbor an unusual isochromosome 5 in
which the left arm has been duplicated via recombination involving centromeric flanking repeats (Selmecki et al. 2006). As a consequence, the genes
encoding the direct target of fluconazole (Erg11) and
the transcription factor that drives expression of drug
efflux pumps (Tac1) that are normally harbored as
single copies on the left arm of chromosome 5 were
now found in four instead of two copies in the
aneuploid strains. There is thus a direct and adaptive
benefit conferred by aneuploidy, and this is a
significant cause of drug resistance in patients treated
with the most broadly used azole in our antifungal
drug armamentarium. Recent studies reveal in C.
neoformans that an extra copy of chromosome 1
(disomy, 1N + 1 aneuploid) is common and similarly
confers resistance to fluconazole (Hu et al. 2008,
Sionov et al. 2010).
Recent studies in S. cerevisiae have documented
that aneuploidy for any chromosome results in a
common signature of phenotypes, likely by leading to
imbalances in the ratio of subunits of multiprotein
complexes (Torres et al. 2007, 2010). Moreover,
aneuploidy has been implicated in driving phenotypic
diversity and enabling adaptive evolution of strains
compromised via loss of proteins (such as molecular
motors) normally required for proper cell division
and viability (Pavelka et al. 2010, Rancati et al. 2008).
Taken together, these studies in both pathogenic and
model yeasts bring aneuploidy to the forefront of
thinking about genotypic plasticity that can rapidly
generate phenotypic diversity even in the absence of
outcrossing. And the fact that sexual reproduction
and meiosis can readily lead to aneuploidy by
promoting a variety of types of chromosome nondisjunction and occur at an elevated rate in some fungi,
20
MYCOLOGIA
such as Candida lusitaniae (Reedy et al. 2009),
suggest that sex in nature may be messier than we
typically give it credit for, given that much of the focus
in the field has been on strains that undergo robust
meiosis, producing well behaved tetrads in which all
spores germinate. It may even turn out to be the case
that genetic determinants that promote aneuploidy
during sex and meiosis remain to be identified.
UNISEXUAL REPRODUCTION:
BEYOND
CRYPTOCOCCUS
Thus far we have been considering unisexual reproduction of just one species, Cryptococcus, and a central
question is whether other fungal species exhibit a
similar mode of homothallic selfing. Recent studies
from Kevin Alby, a graduate student working with
Richard Bennett at Brown University, have shown that
unisexual reproduction also occurs in Candida
albicans, another representative of the three most
common systemic human fungal pathogens (Alby et
al. 2009). For more than a century it was thought that
Ca. albicans was asexual. But with the discovery of the
Ca. albicans mating-type locus by Christina Hull when
she was a graduate student with Sandy Johnson at
UCSF (Hull and Johnson 1999) and then the
discovery of mating under laboratory conditions
(Hull et al. 2000, Magee and Magee 2000), we now
appreciate that there is a conserved a-a opposite-sex
parasexual cycle in Ca. albicans (Miller and Johnson
2002; Bennett and Johnson 2003, 2005; Forche et al.
2008). The key advance of Alby and Bennett was to
discover conditions under which a cells can mate with
a cells in Ca. albicans. First, they and others found
that a cells transcriptionally express both mating
pheromones, and when the Bar1 protease that
destroys the a factor is mutated, a cells produce
sufficient a mating pheromone, auto-respond, form
unusual wrinkled colonies, and mate with other a
cells. This suggests that natural conditions may exist
in which Bar1 is inactivated by mutation or specific
conditions. For example, the S. cerevisiae homolog is
less active under acidic conditions, and thus in the
acidic pH of the vaginal mucosa, where the pH is , 4
due to high acetic acid, unisexual mating of Ca.
albicans might occur. Another possible way in which
Bar1 might be inhibited is by production of pseudo
substrate mimics, or even pheromones from other
related species that bind to and inhibit the protease.
The second condition found to support unisexual
mating of Ca. albicans involves special mating
conditions in which three types of partners are
present, two a strains and then a limiting number of
a cells that serve as pheromone donors that promote
like-with-like cell fusions, a so-called ménage a trios
mating reaction, which was described first in C.
neoformans (Hull et al. 2002, Hull and Heitman 2002,
Lin et al. 2005). The mechanisms via which same-sex
mating is accomplished differ between C. neoformans
and Ca. albicans, indicating that these are independent origins of unisexual reproduction in the two
species that lie in different fungal phyla and are
diverged by 500–1000 million years. It is quite striking
that two of the three most successful systemic human
fungal pathogens have not only retained sexual cycles,
but also the ability to complete two distinct types of
sexual reproduction involving heterothallic oppositesex mating and homothallic same-sex mating.
There is an expression in Japanese that things that
happen once happen once, but things that happen
twice will happen a third time. We therefore are very
interested in which species will be the third example
of unisexual reproduction. Given that there are an
estimated 1.5–5 million fungal species, we may have
a prodigious task to find the next example. On the
other hand, C. neoformans and Ca. albicans are
among the best-studied fungi of the entire kingdom
and thus the phenomenon may be universal but
universally overlooked because it does not fit into the
standard fungal genetics paradigm. But given that the
first two examples are successful human fungal
pathogens, one way to focus the search involves
examining other fungi that infect humans. One that
we are currently focusing on is Trichophyton rubrum,
the fungus that causes athletes foot and other skin
and nail infections. The population is clonal, unisexual, and thus far has no known sexual cycle. Another
approach is to scour the literature for any examples of
this type of sexual behavior. It turns out that there are
several Neurospora species (N. africana, N. lineolata,
N. galapagosensis and N. dodgei) that harbor only the
MAT1-1 idiomorph but nonetheless exhibit homothallic sexual reproduction (Glass et al. 1988, 1990;
Glass and Smith 1994; Gioti et al. 2012). While clearly
not human pathogens, these represent other candidates for the emergence of a novel form of
homothallism, unipolar reproduction, in which one
mating partner suffices for sex to occur.
While beyond the scope of this Karling Lecture
review, that by virtue of its nature and forum has a
focus on mycology, it is worth considering whether
these same types of unusual modes of sexual
reproduction may operate in other eukaryotic microorganisms. In fact, recent studies focused on an
entirely different, broad group of microbial pathogens of humans, namely the protozoan parasites, has
revealed that, like the pathogenic fungi, these
organisms have retained sexual cycles that in many
cases are cryptic and also involve selfing modes of
sexual reproduction. In contrast to fungi, in which
the mating-type locus is an extremely well established
HEITMAN ET AL.: SEX IN FUNGI
molecular paradigm, we know almost nothing about
how mating types are established at a molecular level
in any of the pathogenic protozoan parasites of
humans. But what we have come to appreciate is that
many of these organisms have unusual sexual cycles.
Let us consider Giardia as one paradigmatic
example. This organism infects the GI tract and is a
common cause of diarrheal disease globally. It was
thought to be asexual, until its genome was sequenced and genes involved in meiosis were found
to be present (Ramesh et al. 2005). Then population
geneticists found evidence that the population is
recombining (Cooper et al. 2007). Finally, Zac
Cande’s lab at UCSF discovered a novel form of
sexual reproduction involving nuclear fusion and
genetic exchange in this binucleate organism (something like the dikaryotic hyphae of the Basidiomycota) (Poxleitner et al. 2008). They also found that key
meiotic genes are induced when the trophozoite
matures into the cyst stages and these proteins also
localize to the nucleus, including homologs of Spo11
that makes DSB to stimulate meiotic recombination
and Dmc1, the meiotic recombinase (Poxleitner et al.
2008). We are still missing conditions that enable
isolates to fuse in nature, which seems a natural
prerequisite for genetic recombination in the population, but clearly there is the capacity for a selfing
genetic cycle that can generate recombinant genotypes between the two nuclear genotypes. Similar
findings have emerged for Leishmania, Trypanosoma
brucei and T. cruzi. As for Toxoplasma gondii, well
known to undergo its sexual cycle in the GI tract of
cats and other felids, recent studies revealed a
profound impact of selfing on the generation of
infectious zoospores that cause outbreaks in both
humans and other animals (Wendte et al. 2010).
These parallels between sexual reproduction in
pathogenic fungi and pathogenic parasites have been
reviewed in considerable detail, and the interested
reader is referred to these other sources (Heitman
2006, 2010).
WHY HAS UNISEXUAL REPRODUCTION ARISEN INDEPENDENTLY IN DIFFERENT LINEAGES?
Why has unisexual reproduction arisen repeatedly
and independently, both in pathogenic fungi, possibly model fungi, and pathogenic protozoans? Our
contention is that unisexual reproduction has arisen
multiple times because it can mitigate several of the
costs associated with sex that we introduced at the
beginning of this treatise. First, no longer are only
50% of any given parent’s genes transmitted to
progeny, and instead 50 to as much of 100% of
parental genes are transmitted to progeny by unisexual
21
reproduction, and even more than 100% if you
consider aneuploidy! Second, the time and energy to
find a mating partner are eliminated if you simply mate
with yourself. Third, sex no longer breaks apart well
adapted genomic configurations but instead preserves
them while still introducing a much more limited
genetic diversity, superimposed upon a well adapted
genotype that has run the gauntlet of adaptive
Darwinian selection. And this may well emerge as a
general mechanism for adaptation in pathogenic
microbes as well as those in the environment.
Let us summarize then what we have learned. First,
sex can be unisexual. Second, sex is mutagenic and
can generate diversity de novo. Third, unisexual
reproduction mitigates costs of sex and has evolved
repeatedly and independently. Fourth, pathogenic
fungi are not asexual but can be cryptically sexual or
unisexual. Finally, our musings about an unusual
form of sexual reproduction in fungi lead us to
consider that unisexual reproduction could have
been the ancestral form of sex in the eukaryotic tree
of life. Perhaps when sex first evolved, it was a selfing
mode of reproduction that was inherently mutagenic
and diversity generating. This may reflect the view
that early on, sex was likely about DNA repair, and if
serious errors occurred during replication, there was
another copy available to use to correct. And it might
be that the more complex versions involving mating
types and sexes came later and were embellishments
upon a simpler form of unisexual reproduction. If so,
then the recent transitions of some species from
opposite-sex mating to unisexual reproduction simply
recapitulate more ancient transitions in the ancestral
evolution of sex itself.
Now we have considered fungi and protozoan
parasites, but what about other eukaryotes, such as
plants and animals? Do they have anything like
unisexual reproduction? Plants are a highly successful
super group within the eukaryotic tree of life.
Moreover, we know that plants are capable of both
self-pollination and cross-pollination. In fact, transitions from cross- to self-pollination are common in
plants and are thought to underlie the transition
from provincial niches to enabling species to become
cosmopolitan. In fact, in the model plant Arabidopsis
thaliana, it is self-pollinating , 99% of the time and
only cross-pollinating 1% of the time, and this may
underlie the ability of this plant to colonize most of
the planet, given that it does not have to find a mating
partner to reproduce.
What about animals? Several years ago, a series of
reports about virgin births in sharks emerged. In
essence, astute aquaria keepers noted that females
housed in tanks in the absence of males became
pregnant and gave birth to live young. They ruled out
22
MYCOLOGIA
that these were some type of intersex individual or
that sharks store sperm for years after mating in the
ocean. Genetic analyses revealed that these are
examples of parthenogenesis (Chapman et al. 2007).
Because sharks have an XY sex-determining system, all
offspring are daughters. Concurrently reports emerged
that Komodo dragons that had not been fertilized lay
eggs capable of hatching to produce live young.
Zookeepers had known for years that Komodo dragons
lay eggs but always assumed these would be sterile. But
some curious zookeepers decided to watch these eggs
and observed they can hatch. This turns out to be
another example of parthenogenesis (Watts et al. 2006),
and because Komodo dragons have the more unusual
ZW sex-determining system (where female is ZW and
males are ZZ), they can have male offspring. One
suggestion was that a lone Komodo dragon female
could swim up on a deserted island, give birth to a live
male young, and then restart an opposite sex sexual
cycle, albeit with a genetic bottleneck as the result of
parthenogenesis. What about in mammals? We know
that imprinting normally serves as a barrier to parthenogenesis by genetically marking the sperm and egg
differently marks. But in mice with mutations in the
imprinting system, pups born via parthenogenesis can
survive to birth and beyond (Kono et al. 2004). And in
humans, while there are no reports of parthenogenetic
births, there is a case in which ES cells were derived
from embryos via parthenogenesis (Kim et al. 2007).
Thus, unusual patterns of sexual reproduction
initially discovered in kingdom Fungi turn out to
have profound implications for modes of sexual
reproduction in other eukaryotic microbes and some
of these principles even extend to plants and in some
cases animals. Moreover, fungi, especially yeast, has
served as the best models for understanding the
evolutionary importance of sex, ploidy and mate
recognition using experimental approaches (Zeyl and
Bell 1997, Anderson et al. 2004, Xu 2005, Gerstein et
al. 2011). Finally, the pheromone signaling pathway
of yeast stands as the best worked example of signal
transduction and gene interactions in all eukaryotes,
which is fortunate for mycologists because all Dikarya
appear to use the same pheromone-based mechanism
for reproduction. Yet, both outside and within
Dikarya there are many more biological questions
and mysteries that remain to be solved from the
perspective of the fungal kingdom.
ACKNOWLEDGMENTS
This review is based, in part, on the Karling lecture delivered
at the Mycological Society of America meeting in Fairbanks,
Alaska, 1–6 Aug 2011, which was dedicated to John and Cardy
Raper for their pioneering work on sex in fungi.
LITERATURE CITED
Aime MC, Matheny PB, Henk DA, Frieders EM, Nilsson RH,
Piepenbring M, McLaughlin DJ, Szabo LJ, Begerow D,
Sampaio JP, Bauer R, Weiß M, Oberwinkler F, Hibbett
D. 2006. An overview of the higher level classification of
Pucciniomycotina based on combined analyses of
nuclear large and small subunit rDNA sequences.
Mycologia 98:896–905, doi:10.3852/mycologia.98.6.896
Aimi T, Yoshida R, Ishikawa M, Bao D, Kitamoto Y. 2005.
Identification and linkage mapping of the genes for the
putative homeodomain protein (hox1) and the putative
pheromone receptor protein homologue (rcb1) in a
bipolar basidiomycete, Pholiota nameko. Curr Genet 48:
184–194, doi:10.1007/s00294-005-0012-7
Alby K, Schaefer D, Bennett RJ. 2009. Homothallic and
heterothallic mating in the opportunistic pathogen
Candida albicans. Nature 460:890–893, doi:10.1038/
nature08252
Anderson JB, Sirjusingh C, Ricker N. 2004. Haploidy,
diploidy and evolution of antifungal drug resistance
in Saccharomyces cerevisiae. Genetics 168:1915–1923,
doi:10.1534/genetics.104.033266
Bakkeren G, Jiang G, Warren RL, Butterfield Y, Shin H,
Chiu R, Linning R, Schein J, Lee N, Hu G, Kupfer DM,
Tang Y, Roe BA, Jones S, Marrac M, Kronstad JW. 2006.
Mating factor linkage and genome evolution in
basidiomycetous pathogens of cereals. Fungal Genet
Biol 43:655–66, doi:10.1016/j.fgb.2006.04.002
———, Kamper J, Schirawski J. 2008. Sex in smut fungi:
structure, function and evolution of mating-type
complexes. Fungal Genet Biol 45(Suppl 1):S15–S21,
doi:10.1016/j.fgb.2008.04.005
———, Kronstad JW. 1994. Linkage of mating-type loci
distinguishes bipolar from tetrapolar mating in basidiomycetous smut fungi. Proc Natl Acad Sci USA 91:
7085–7089, doi:10.1073/pnas.91.15.7085
Baldauf SL. 2003. The deep roots of eukaryotes. Science
300:1703–1706, doi:10.1126/science.1085544
———, Palmer JD. 1993. Animals and fungi are each
other’s closest relatives: congruent evidence from
multiple proteins. Proc Nat Acad Sci USA 90:11558–
11562, doi:10.1073/pnas.90.24.11558
Bennett RJ, Johnson AD. 2003. Completion of a parasexual
cycle in Candida albicans by induced chromosome loss
in tetraploid strains. EMBO J 22:2505–2515, doi:10.
1093/emboj/cdg235
———, ———. 2005. Mating in Candida albicans and the
search for a sexual cycle. Ann Rev Microbiol 59:233–
255, doi:10.1146/annurev.micro.59.030804.121310
Bölker M, Urban M, Kahmann R. 1992. The a mating-type
locus of U. maydis specifies cell signaling components.
Cell 68:441–450, doi:10.1016/0092-8674(92)90182-C
Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE,
Read ND, Seiler S, Bell-Pedersen D, Paietta J, Plesofsky
N, Plamann M, Goodrich-Tanrikulu M, Schulte U,
Mannhaupt G, Nargang FE, Radford A, Selitrennikoff
C, Galagan JE, Dunlap JC, Loros JJ, Catcheside D,
Inoue H, Aramayo R, Polymenis M, Selker EU, Sachs
MS, Marzluf GA, Paulsen I, Davis R, Ebbole DJ, Zelter
HEITMAN ET AL.: SEX IN FUNGI
A, Kalkman ER, O’Rourke R, Bowring F, Yeadon J, Ishii
C, Suzuki K, Sakai W, Pratt R. 2004. Lessons from the
genome sequence of Neurospora crassa: tracing the
path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108, doi:10.1128/
MMBR.68.1.1-108.2004
Bradley KM, Breyer JP, Melville DB, Broman KW, Knapik
EW, Smith JR. 2011. An SNP-based linkage map for
zebrafish reveals sex determination loci. G3 1:3–9.
Bui T, Lin X, Malik R, Heitman J, Carter D. 2008. Isolates of
Cryptococcus neoformans from infected animals reveal
genetic exchange in unisexual, alpha mating-type populations. Eukaryot Cell 7:1771–80, doi:10.1128/EC.00097-08
Byrnes EJ III, Bartlett KH, Perfect JR, Heitman J. 2011.
Cryptococcus gattii: an emerging fungal pathogen
infecting humans and animals. Microbes Infect 13:
895–907, doi:10.1016/j.micinf.2011.05.009
———, Li W, Ren P, Lewit Y, Voelz K, Fraser JA, Dietrich FS,
May RC, Chaturvedi S, Chaturvedi V, et al. 2011. A
diverse population of Cryptococcus gattii molecular type
VGIII in southern Californian HIV/AIDS patients.
PLoS Pathog 7:e1002205, doi:10.1371/journal.ppat.
1002205
Casselton LA. 2002. Mate recognition in fungi. Heredity 88:
142–147, doi:10.1038/sj.hdy.6800035
———, Olesnicky NS. 1998. Molecular genetics of mating
recognition in Basidiomycete fungi. Microbiol Molec
Biol Rev 62:55–70.
Chapman DD, Shivji MS, Louis E, Sommer J, Fletcher H,
Prodöhl PA. 2007. Virgin birth in a hammerhead shark.
Biol Lett 3:425–427, doi:10.1098/rsbl.2007.0189
Chen W, Zhu Z, Liao H, Quinnan GV, Broder CC, Haynes BF,
Dimitrov DS. 2010. Cross-reactive human IgM-derived
monoclonal antibodies that bind to HIV-1 envelope
glycoproteins. Viruses 2:547–565, doi:10.3390/v2020547
Coelho MA, Sampaio JP, Goncalves P. 2010. A deviation
from the bipolar-tetrapolar mating paradigm in an
early diverged basidiomycete. PLoS Genetics 6:
e1001052, doi:10.1371/journal.pgen.1001052
Cooper MA, Adam RD, Worobey M, Sterling CR. 2007.
Population genetics provides evidence for recombination in Giardia. Curr Biol 17:1984–1988, doi:10.1016/
j.cub.2007.10.020
Findley K, Rodriguez-Carres M, Metin B, Kroiss J, Fonseca A,
Vilgalys R, Heitman J. 2009. Phylogeny and phenotypic
characterization of pathogenic Cryptococcus species and
closely related saprobic taxa in the Tremellales. Eukaryot Cell 8:353–361, doi:10.1128/EC.00373-08
———, Sun S, Fraser JA, Hsueh Y-P, Averette AF, Li W,
Dietrich FS, Heitman J. 2012. Discovery of a modified
tetrapolar sexual cycle in Cryptococcus amylolentus and
the evolution of MAT in the Cryptococcus species
complex. PLoS Genet 8:e1002528, doi:10.1371/journal.
pgen.1002528
Floudas D, Binder M, Riley R, Barry K, Blanchette RA,
Henrissat B, Martı́nez AT, Otillar R, Spatafora JW,
Yadav JS, Andrea Aerts, Benoit I, Boyd A, Carlson A,
Copeland A, Coutinho PM, de Vries RP, Ferreira P,
Findley K, Foster B, Gaskell J, Glotzer D, Górecki P,
Heitman J, Hesse C, Hori C, Igarashi K, Jurgens JJ,
23
Kallen N, Kersten P, Kohler A, Kües U, Kumar TKA,
Kuo A, LaButti K, Larrondo LF, Lindquist E, Ling A,
Lombard V, Lucas S, Lundell T, Martin R, McLaughlin
DJ, Morgenstern I, Morin E, Murat C, Nagy LG, Nolan
M, Ohm RA, Patyshakuliyeva A, Rokas A, Ruiz-Dueñas
FJ, Sabat G, Salamov A, Samejima M, Schmutz J, Slot JC,
St John F, Stenlid J, Sun H, Sun S, Syed E, Tsang A,
Wiebenga A, Young D, Pisabarro A, Eastwood DC,
Martin F, Cullen D, Grigoriev IV, Hibbett DS. 2012.
The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science
336:1715–1719, doi:10.1126/science.1221748
Forche A, Alby K, Schaefer D, Johnson AD, Berman J,
Bennett RJ. 2008. The parasexual cycle in Candida
albicans provides an alternative pathway to meiosis for
the formation of recombinant strains. PLoS Biol 6:
e110, doi:10.1371/journal.pbio.0060110
Foulongne-Oriol M, Spataro C, Savoie J-M. 2009. Novel
microsatellite markers suitable for genetic studies in
the white button mushroom Agaricus bisporus. Appl
Microbiol Biotechnol 84:1125–1135, doi:10.1007/
s00253-009-2030-8
Fraser JA, Diezmann S, Subaran RL, Allen A, Lengeler KB,
Dietrich FS, Heitman J. 2004. Convergent evolution of
chromosomal sex-determining regions in the animal
and fungal kingdoms. PLoS Biology 2:e384, doi:10.
1371/journal.pbio.0020384
———, Heitman J. 2003. Fungal mating-type loci. Curr Biol
13:R792–5, doi:10.1016/j.cub.2003.09.046
———, ———. 2005. Chromosomal sex-determining regions in animals, plants and fungi. Curr Opin Genet
Develt 15:645–651, doi:10.1016/j.gde.2005.09.002
Gerstein AC, Cleathero LA, Mandegar MA, Otto SP. 2011.
Haploids adapt faster than diploids across a range of
environments. J Evol Biol 24:531–540, doi:10.1111/j.
1420-9101.2010.02188.x
Gioti A, Mushegian AA, Strandberg R, Stajich JE, Johannesson H. 2012. Unidirectional evolutionary transitions in
fungal mating systems and the role of transposable
elements. MolecBiol Evol 29:3215–3226.
Giraud T. 2004. Patterns of within population dispersal and
mating of the fungus Microbotryum violaceum parasitising the plant Silene latifolia. Heredity 93:559–565,
doi:10.1038/sj.hdy.6800554
Glass NL, Metzenberg RL, Raju NB. 1990. Homothallic
Sordariaceae from nature: the absence of strains
containing only the a mating-type sequence. Exp Mycol
14:274–289, doi:10.1016/0147-5975(90)90025-O
———, Smith ML. 1994. Structure and function of a matingtype gene from the homothallic species Neurospora
africana. Mol Gen Genet 244: 401–409, doi:10.
1007/BF00286692
———, Vollmer SJ, Staben C, Grotelueschen J, Metzenberg
RL, Yanofsky C. 1988. DNAs of the two mating-tye
alleles of Neurospora crassa are highly dissimilar.
Science 241:570–573, doi:10.1126/science.2840740
Goddard MR, Godfray HC, Burt A. 2005. Sex increases the
efficacy of natural selection in experimental yeast
populations. Nature 434:636–640.
Heitman J. 2006. Sexual reproduction and the evolution
24
MYCOLOGIA
of microbial pathogens. Curr Biol 16:R711–R725,
doi:10.1016/j.cub.2006.07.064
———. 2010. Evolution of eukaryotic microbial pathogens
via covert sexual reproduction. Cell Host Microbe 8:86–
99, doi:10.1016/j.chom.2010.06.011
———, Kozel TR, Kwon-Chung JK, Perfect JR, Casadevall A.
2011. Cryptococcus: from human pathogen to model
yeast. Washington DC: ASM Press. 576 p.
Herskowitz I. 1989. A regulatory hierarchy for cell specialization in yeast. Nature 342:749–757, doi:10.1038/342749a0
Hibbett DS. 2006. A phylogenetic overview of the Agaricomycotina. Mycologia 98:917–925, doi:10.3852/mycologia.
98.6.917
———, Donoghue MJ. 2001. Analysis of character correlations among wood decay mechanisms, mating systems
and substrate ranges in Homobasidiomycetes. Syst Biol
50:215–242, doi:10.1080/10635150151125879
Hiremath SS, Chowdhary A, Kowshik T, Randhawa HS, Sun
S, Xu J. 2008. Long-distance dispersal and recombination in environmental populations of Cryptococcus
neoformans var. grubii from India. Microbiology 154:
1513–1524, doi:10.1099/mic.0.2007/015594-0
Hsueh Y-P, Fraser JA, Heitman J. 2008. Transitions in
sexuality: recapitulation of an ancestral tri- and tetrapolar mating system in Cryptococcus neoformans. Eukaryot Cell 7:1847–1855, doi:10.1128/EC.00271-08
———, Heitman J. 2008. Orchestration of sexual reproduction and virulence by the fungal mating-type locus.
Curr Opin Microbiol 11:517–524, doi:10.1016/
j.mib.2008.09.014
Hu G, Liu I, Sham A, Stajich JE, Dietrich F, Kronstad JW.
2008. Comparative hybridization reveals extensive
genome variation in the AIDS-associated pathogen
Cryptococcus neoformans. Genome Biol 9:R41, doi:10.
1186/gb-2008-9-2-r41
Hull CM, Davidson RC, Heitman J. 2002. Cell identity and
sexual development in Cryptococcus neoformans are
controlled by the mating-type-specific homeodomain
protein Sxi1a. Genes Dev 16:3046–3060, doi:10.1101/
gad.1041402
———, Heitman J. 2002. Genetics of Cryptococcus neoformans. Ann Rev Genet 36:557–615, doi:10.1146/annurev.
genet.36.052402.152652
———, Johnson AD. 1999. Identification of a mating typelike locus in the asexual pathogenic yeast Candida
albicans. Science 285:1271–1275, doi:10.1126/science.
285.5431.1271
———, Raisner RM, Johnson AD. 2000. Evidence for
mating of the ‘asexual’ yeast Candida albicans in a
mammalian host. Science 289:307–310, doi:10.1126/
science.289.5477.307
Idnurm A, Bahn YS, Nielsen K, Lin X, Fraser JA, Heitman J.
2005. Deciphering the model pathogenic fungus
Cryptococcus neoformans. Nat Rev Microbiol 3:753–64,
doi:10.1038/nrmicro1245
James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V,
Cox CJ, Celio G, Gueidan C, Fraker E, Miadlikowska J,
Lumbsch HT, Rauhut A, Reeb V, Arnold AE,37, Amtoft
A, Stajich JE, Hosaka K, Sung G-H, Johnson D,
O’Rourke B, Crockett M, Binder M, Curtis JM, Slot
JC, Wang Z, Wilson AW, Schüßler A, Longcore JE,
O’Donnell K, Mozley-Standridge S, Porter D, Letcher
PM, Powell MJ, Taylor JW, White MM, Griffith GW,
Davies DR, Humber RA, Morton JB, Sugiyama J,
Rossman AY, Rogers JD, Pfister DH, Hewitt D, Hansen
K, Hambleton S, Shoemaker RA, Kohlmeyer J, Volkmann-Kohlmeyer B, Spotts RA, Serdani M, Crous PW,
Hughes KW, Matsuura K, Langer E, Langer G,
Untereiner WA, Lücking R, Büdel B, Geiser DM,
Aptroot A, Diederich P, Schmitt I, Schultz M, Yahr R,
Hibbett DS, Lutzoni F, McLaughlin DJ, Spatafora JW,
Vilgalys R. 2006a. Reconstructing the early evolution of
Fungi using a six-gene phylogeny. Nature 443:818–822,
doi:10.1038/nature05110
———, Lee M, van Diepen LTA. 2011. A single mating-type
locus composed of homeodomain genes promotes
nuclear migration and heterokaryosis in the white-rot
fungus Phanerochaete chrysosporium. Eukaryot Cell 10:
249–261, doi:10.1128/EC.00212-10
———, Srivilai P, Kues U, Vilgalys R. 2006b. Evolution of
the bipolar mating system of the mushroom Coprinellus
disseminatus from its tetrapolar ancestors involves loss
of mating-type-specific pheromone receptor function.
Genetics 172:1877–1891, doi:10.1534/genetics.105.
051128
Johnson LJ, Koufopanou V, Goddard MR, Hetherington R,
Schäfer SM, Burt A. 2004. Population genetics of the
wild yeast Saccharomyces paradoxus. Genetics 166:43–
52, doi:10.1534/genetics.166.1.43
Jokela J, Dybdahl MF, Lively CM. 2009. The maintenance of
sex, clonal dynamics, and host-parasite coevolution in a
mixed population of sexual and asexual snails. The Am
Nat 174:S43–S53.
Karos M, Chang YC, McClelland CM, Clarke DL, Fu J,
Wickes BL, Kwon-Chung KJ. 2000. Mapping of the
Cryptococcus neoformans MATa locus: presence of
mating type-specific mitogen-activated protein kinase
cascade homologs. J Bacteriol 182:6222–6227, doi:10.
1128/JB.182.21.6222-6227.2000
Keller SM, Viviani MA, Esposto MC, Cogliati M, Wickes BL.
2003. Molecular and genetic characterization of a
serotype A MATa Cryptococcus neoformans isolate.
Microbiology 149:131–142, doi:10.1099/mic.0.25921-0
Kellner R, Vollmeister E, Feldbrügge M, Begerow D. 2011.
Interspecific sex in grass smuts and the genetic diversity
of their pheromone-receptor system. PLoS Genet 7:
e1002436, doi:10.1371/journal.pgen.1002436
Kim K, Ng K, Rugg-Gunn PJ, Shieh J-H, Kirak O, Jaenisch R,
Wakayama T, Moore MA, Pedersen RA, Daley GQ.
2007. Recombination signatures distinguish embryonic
stem cells derived by parthenogenesis and somatic cell
nuclear transfer. Cell Stem Cell 1:346–352, doi:10.
1016/j.stem.2007.07.001
King N, Westbrook MJ, Young SL, Kuo A, Abedin M,
Chapman J, Fairclough S, Hellsten U, Isogai Y, Letunic
I, Marr M, Pincus D, Putnam N, Rokas A, Wright KJ,
Zuzow R, Dirks W, Good M, Goodstein D, Lemons D, Li
W, Lyons JB, Morris A, Nichols S, Richter DJ, Salamov A,
Sequencing JGI, Bork P, Lim WA, Manning G, Miller WT,
McGinnis W, Shapiro H, Tjian R, Igor V, Grigoriev IV,
HEITMAN ET AL.: SEX IN FUNGI
Rokhsar D. 2008. The genome of the choanoflagellate
Monosiga brevicollis and the origin of metazoans. Nature
451:783–788, doi:10.1038/nature06617
Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Park
ES, Seo J-S, Ogawa H. 2004. Birth of parthenogenetic
mice that can develop to adulthood. Nature 428:860–
864, doi:10.1038/nature02402
Koopman P, Gubbay J, Vivian N, Goodfellow P, LovellBadge R. 1991. Male development of chromosomally
female mice transgenic for Sry. Nature 351:117–121,
doi:10.1038/351117a0
Kruzel EK, Giles SS, Hull CM. 2012. Analysis of Cryptococcus
neoformans sexual development reveals rewiring of the
pheromone response network by a change in transcription factor identity. Genetics 191:435–449, doi:10.1534/
genetics.112.138958
Kües U, James TY, Heitman J. 2011. Mating type in
basidiomycetes: unipolar, bipolar and tetrapolar patterns of sexuality. In: Pöggeler S, Wöstemeyer J, eds.
Evolution of fungi and fungal-like organisms. Mycota
XIV:97–160, doi:10.1007/978-3-642-19974-5_6
Kwon-Chung KJ. 1975. A new genus, Filobasidiella, the
perfect state of Cryptococcus neoformans. Mycologia 67:
1197–1200, doi:10.2307/3758842
———. 1976a. Morphogenesis of Filobasidiella neoformans,
the sexual state of Cryptococcus neoformans. Mycologia
68:821–833, doi:10.2307/3758800
———. 1976b. A new species of Filobasidiella, the sexual
state of Cryptococcus neoformans B and C serotypes.
Mycologia 68:943–946.
Lawrence GJ. 1980. Multiple mating-type specificities in the
flax rust Melampsora lini. Science 209:501–503,
doi:10.1126/science.209.4455.501
Lee N, Bakkeren G, Wong K, Sherwood JE, Kronstad JW. 1999.
The mating-type and pathogenicity locus of the fungus
Ustilago hordei spans a 500 kb region. Proc Natl Acad Sci
USA 96:15026–15031, doi:10.1073/pnas.96.26.15026
Lee SC, Ni M, Li W, Shertz C, Heitman J. 2010. The
evolution of sex: a perspective from the fungal
kingdom. Microbiol Mol Biol Rev 74:298–340, doi:10.
1128/MMBR.00005-10
Lengeler KB, Fox DS, Fraser JA, Allen A, Forrester K,
Dietrich FS, Heitman J. 2002. Mating-type locus of
Cryptococcus neoformans: a step in the evolution of sex
chromosomes. Eukaryot Cell 1:704–718, doi:10.1128/
EC.1.5.704-718.2002
———, Wang P, Cox GM, Perfect JR, Heitman J. 2000.
Identification of the MATa mating-type locus of
Cryptococcus neoformans reveals a serotype A MATa
strain thought to have been extinct. Proc Natl Acad Sci
USA 97:14555–14460, doi:10.1073/pnas.97.26.14455
Lin X, Heitman J. 2007. Mechanisms of homothallism in
fungi—transitions between heterothallism and homothallism. In: Heitman J, Kronstad JW, Taylor JW,
Casselton LA, eds. Sex in Fungi: molecular determination and evolutionary implications. Washington DC:
ASM Press. p 35–58.
———, Huang J, Mitchell T, Heitman J. 2006. Virulence
attributes and hyphal growth of C. neoformans are
quantitative traits and the MATa allele enhances
25
filamentation. PLoS Genet 2:e187, doi:10.1371/journal.pgen.0020187
———, Hull CM, Heitman J. 2005. Sexual reproduction
between partners of the same mating type in Cryptococcus neoformans. Nature 434:1017–1021, doi:10.1038/
nature03448
———, Jackson JC, Feretzaki M, Xue C, Heitman J. 2010.
Transcription factors Mat2 and Znf2 operate cellular
circuits orchestrating opposite- and same-sex mating in
Cryptococcus neoformans. PLoS Genet 6:e1000953,
doi:10.1371/journal.pgen.1000953
———, Litvintseva AP, Nielsen K, Patel S, Floyd A, Mitchell
TG, Heitman J. 2007. aADa hybrids of Cryptococcus
neoformans: evidence of same-sex mating in nature and
hybrid fitness. PLoS Genet 3:1975–1990.
———, Patel S, Litvintseva AP, Floyd A, Mitchell TG,
Heitman J. 2009. Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha
mating type originate via unisexual mating. PLoS
Pathog 5:e1000283, doi:10.1371/journal.ppat.1000283
Loftus BJ, Fung E, Roncaglia P, Rowley D, Amedeo P, Bruno
D, Vamathevan J, Miranda M, Anderson IJ, Fraser JA,
Allen JE, Bosdet IE, Brent MR, Chiu R, Doering T,
Donlin MJ, D’Souza CA, Fox DS, Grinberg V, Fu J,
Fukushima M, Haas BJ, Huang JC, Janbon G, Jones
SJM, Koo HL, Krzywinski MI, Kwon-Chung KJ, Lengeler
KB, Maiti R, Marra MA, Marra RE, Mathewson CA,
Mitchell TG, Pertea M, Riggs FR, Salzberg SL, Schein
JE, Shvartsbeyn A, Shin H, Shumway M, Specht CA, Suh
BB, Tenney A, Utterback TR, Wickes BL, Wortman JR,
Wye NH, Kronstad JW, Lodge JK, Heitman J, Davis RW,
Fraser CM, Hyman RW. 2005. The genome of the
basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307:1321–1324, doi:10.1126/
science.1103773
Magee BB, Magee PT. 2000. Induction of mating in
Candida albicans by construction of MTLa and MTLa
strains. Science 289:310–313, doi:10.1126/science.289.
5477.310
Matheny PB, Curtis JM, Hofstetter V, Aime MC, Moncalvo JM, Ge Z-W, Yang Z-L, Slot JC, Ammirati JF, Baroni TJ,
Bougher NL, Hughes KW, Lodge DJ, Kerrigan RW,
Seidl MT, Aanen DK, DeNitis M, Daniele GM,
Desjardin DE, Kropp BR, Norvell LL, Parker A,
Vellinga EC, Vilgalys R, Hibbett DS. 2006. Major clades
of Agaricales: a multilocus phylogenetic overview.
Mycologia 98:982–995, doi:10.3852/mycologia.98.6.982
McClelland CM, Chang YC, Varma A, Kwon-Chung KJ. 2004.
Uniqueness of the mating system in Cryptococcus
neoformans. Trends Microbiol 12:208–212, doi:10.
1016/j.tim.2004.03.003
Metin B, Findley K, Heitman J. 2010. The mating type locus
(MAT) and sexual reproduction of Cryptococcus heveanensis: insights into the evolution of sex and sex-determining
chromosomal regions in fungi. PLoS Genetics 6:
e10000961, doi:10.1371/journal.pgen.1000961
Miller JH. 1996. Spontaneous mutators in bacteria: insights
into pathways of mutagenesis and repair. Annu Rev
Microbiol 50:625–643, doi:10.1146/annurev.micro.
50.1.625
26
MYCOLOGIA
Miller MG, Johnson AD. 2002. White-opaque switching in
Candida albicans is controlled by mating-type locus
homeodomain proteins and allows efficient mating.
Cell 110:293–302, doi:10.1016/S0092-8674(02)00837-1
Morran LT, Schmidt OG, Gelarden IA, Parrish RC, Lively
CM. 2011. Running with the red queen: Host-parasite
coevolution selects for biparental sex. Science 333:216–
218, doi:10.1126/science.1206360
Narisawa K, Yamaoka Y, Katsuya K. 1994. Mating type of
isolates derived from the spermogonial state of
Puccinia coronata var. coronata. Mycoscience 35:131–
135, doi:10.1007/BF02318489
Ni M, Feretzaki M, Sun S, Wang X, Heitman J. 2011. Sex in
fungi. Annu Rev Genet 45:405–430, doi:10.1146/
annurev-genet-110410-132536
Nielsen K, Cox GM, Wang P, Toffaletti DL, Perfect JR,
Heitman J. 2003. Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and a
isolates. Infect Immun 71:4831–4841, doi:10.1128/
IAI.71.9.4831-4841.2003
Okagaki LH, Strain AK, Nielsen JN, Charlier C, Baltes NJ,
Chrétien F, Heitman J, Dromer F, Nielsen K. 2010.
Cryptococcal cell morphology affects host cell interactions and pathogenicity. PLoS Pathog 6:e1000953,
doi:10.1371/journal.ppat.1000953
Olesnicky NS, Brown AJ, Dowell SJ, Casselton LA. 1999. A
constitutively active G-protein-coupled receptor causes
mating self-compatibility in the mushroom Coprinus.
EMBO J 18:2756–2763, doi:10.1093/emboj/18.10.2756
———, ———, Honda Y, Dyos SL, Dowell SJ, Casselton LA.
2000. Self-compatible B mutants in Coprinus with
altered pheromone-receptor specificities. Genetics
156:1025–1033.
Oliver A, Cantón R, Campo P, Baquero F, Blázquez J. 2000.
High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251–
1253, doi:10.1126/science.288.5469.1251
Olson Å, Aerts A, Asiegbu F, Belbahri L, Bouzid O, Broberg A,
Canbäck B, Coutinho PM, Cullen D, Dalman K, et al. 2012.
Insight into trade-off between wood decay and parasitism
from the genome of a fungal forest pathogen. New Phytol
194:1001–1013, doi:10.1111/j.1469-8137.2012.04128.x
Parag Y. 1962. Mutations in the B incompatibility factor of
Schizophyllum commune. Proc. Natl Acad Sci USA 48:
743–750, doi:10.1073/pnas.48.5.743
Park BJ, Wannemuehler KA, Marston BJ, Govender N,
Pappas PG, Chiller TM. 2009. Estimation of the current
global burden of cryptococcal meningitis among
persons living with HIV/AIDS. AIDS 23:525–530,
doi:10.1097/QAD.0b013e328322ffac
Pavelka N, Rancati G, Zhu J, Bradford WD, Saraf A, Florens
L, Sanderson BW, Hattem GL, Li R. 2010. Aneuploidy
confers quantitative proteome changes and phenotypic
variation in budding yeast. Nature 468:321–325,
doi:10.1038/nature09529
Poxleitner MK, Carpenter ML, Mancuso JJ, Wang CJ,
Dawson SC, Cande WZ. 2008. Evidence for karyogamy
and exchange of genetic material in the binucleate
intestinal parasite Giardia intestinalis. Science 319:
1530–1533, doi:10.1126/science.1153752
Pukkila PJ. 2011. Coprinopsis cinerea. Curr Biol 21:R616–
R617, doi:10.1016/j.cub.2011.05.042
Ramesh MA, Malik SB, Logsdon JM Jr. 2005. A phylogenomic inventory of meiotic genes: evidence for sex in
Giardia and an early eukaryotic origin of meiosis. Curr
Biol 15:185–91.
Rancati G, Pavelka N, Fleharty B, Noll A, Trimble R, Walton
K, Perera A, Staehling-Hampton K, Seidel CW, Li R.
2008. Aneuploidy underlies rapid adaptive evolution of
yeast cells deprived of a conserved cytokinesis motor.
Cell 135:879–893, doi:10.1016/j.cell.2008.09.039
Raper J, Flexer A. 1971. Mating systems and evolution of the
Basidiomycetes. In: Petersen R, ed. Evolution in the
higher basidiomycetes. Knoxville: Univ Tennessee
Press. p 149–167.
Raper JR. 1966. Genetics of sexuality in higher fungi. New
York: Ronald Press Co. 283 p.
Reedy JL, Floyd AM, Heitman J. 2009. Mechanistic plasticity
of sexual reproduction and meiosis in the Candida
pathogenic species complex. Curr Biol 19:891–899,
doi:10.1016/j.cub.2009.04.058
Reha-Krantz LJ. 1998. Regulation of DNA polymerase
exonucleolytic proofreading activity: studies of bacteriophage T4 ‘antimutator’ DNA polymerases. Genetics
148:1551–1557.
Rodriguez-Carres M, Findley K, Sun S, Dietrich FS, Heitman
J. 2010. Morphological and genomic characterization
of Filobasidiella depauperata: a homothallic sibling
species of the pathogenic Cryptococcus species complex.
PLoS ONE 5:e9620, doi:10.1371/journal.pone.0009620
Saul N, Krockenberger M, Carter D. 2008. Evidence of
recombination in mixed-mating-type and alpha-only
populations of Cryptococcus gattii sourced from single
eucalyptus tree hollows. Eukaryot Cell 7:727–734,
doi:10.1128/EC.00020-08
Schiebel K, Winkelmann M, Mertz A, Xu X, Page DC, Weil
D, Petit C, Rappold GA. 1997. Abnormal XY interchange between a novel isolated protein kinase gene,
PRKY, and its homolog, PRKX, accounts for one third
of all (Y+)XX males and (Y2)XY females. Human Mol
Genet 6:1985–1989, doi:10.1093/hmg/6.11.1985
Schirawski J, Heinze B, Wagenknecht M, Kahmann R. 2005.
Mating type loci of Sporisorium reilianum: novel pattern
with three a and multiple b specificities. Eukaryot Cell
4:1317–1327, doi:10.1128/EC.4.8.1317-1327.2005
Schoustra SE, Debets AJ, Slakhorst M, Hoekstra RF. 2007.
Mitotic recombination accelerates adaptation in the
fungus Aspergillus nidulans. PLoS Genet 3:e68,
doi:10.1371/journal.pgen.0030068
Schulz B, Banuett F, Dahl M, Schlesinger R, Schafer W,
Martin T, Herskowitz I, Kahmann R. 1990. The b alleles
of U. maydis, whose combinations program pathogenic
development, code for polypeptides containing a
homeodomain-related motif. Cell 60: 295– 306,
doi:10.1016/0092-8674(90)90744-Y
Selmecki A, Forche A, Berman J. 2006. Aneuploidy and
isochromosome formation in drug-resistant Candida
albicans. Science 313:367–70, doi:10.1126/science.1128242
Sharp A, Kusz K, Jaruzelska J, Tapper W, Szarras-Czapnik M,
Wolski J, Jacobs P. 2005. Variability of sexual phenotype
HEITMAN ET AL.: SEX IN FUNGI
in 46,XX(SRY+) patients: the influence of spreading X
inactivation versus position effects. J Med Genet 42:
420–427, doi:10.1136/jmg.2004.022053
Simpson AGB, Roger AJ. 2004. The real ‘kingdoms’ of
eukaryotes. Curr Biol 14:R693–R696, doi:10.1016/
j.cub.2004.08.038
Sionov E, Lee H, Chang YC, Kwon-Chung KJ. 2010. Cryptococcus
neoformans overcomes stress of azole drugs by formation of
disomy in specific multiple chromosomes. PLoS Pathog 6:
e1000848, doi:10.1371/journal.ppat.1000848
Stajich JE, Berbee ML, Blackwell M, Hibbett DS, James TY,
Spatafora JW, Taylor JW. 2009. The Fungi. Curr Biol 19:
R840–R845, doi:10.1016/j.cub.2009.07.004
Tibayrenc M, Kjellberg F, Arnaud J, Oury B, Breniere SF,
Darde ML, Ayala FJ. 1991. Are eukaryotic microorganisms clonal or sexual? A population genetics vantage.
Proc Natl Acad Sci USA 88:5129–5133, doi:10.1073/
pnas.88.12.5129
———, ———, Ayala FJ. 1990. A clonal theory of parasitic
protozoa: the population structures of Entamoeba,
Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas and Trypanosoma and their medical and taxonomical consequences. Proc Natl Acad Sci USA 87:2414–
2418, doi:10.1073/pnas.87.7.2414
Torres EM, Dephoure N, Panneerselvam A, Tucker CM,
Whittaker CA, Gygi SP, Dunham MJ, Amon A. 2010.
Identification of aneuploidy-tolerating mutations. Cell
143:71–83, doi:10.1016/j.cell.2010.08.038
———, Sokolsky T, Tucker CM, Chan LY, Boselli M,
Dunham MJ, Amon A. 2007. Effects of aneuploidy on
cellular physiology and cell division in haploid yeast.
Science 317:916–924, doi:10.1126/science.1142210
Viviani MA, Nikolova R, Esposto MC, Prinz G, Cogliati M.
2003. First European case of serotype A MATa
Cryptococcus neoformans infection. Emerg Infect Dis 9:
1179–1180, doi:10.3201/eid0909.020770
Wainright P, Hinkle G, Sogin M, Stickel S. 1993. Monophyletic
origins of the metazoa: an evolutionary link with fungi.
Science 260:340–342, doi:10.1126/science.8469985
Watts PC, Buley KR, Sanderson S, Boardman W, Ciofi C,
27
Gibson R. 2006. Parthenogenesis in Komodo dragons.
Nature 444:1021–1022, doi:10.1038/4441021a
Wendte JM, Miller MA, Lambourn DM, Magargal SL, Jessup
DA, Grigg ME. 2010. Self-mating in the definitive host
potentiates clonal outbreaks of the apicomplexan
parasites Sarcocystis neurona and Toxoplasma gondii.
PLoS Genet 6:e1001261, doi:10.1371/journal.pgen.
1001261
Whitehouse HLK. 1949. Multiple-allelomorph heterothallism in the fungi. New Phytol 48:212–244, doi:10.1111/
j.1469-8137.1949.tb05120.x
Wu X, Zhou T, Zhu J, Zhang B, Georgiev I, Wang C, Chen
X, Longo NS, Louder M, McKee K, et al. 2011. Focused
evolution of HIV-1 neutralizing antibodies revealed by
structures and deep sequencing. Science 333:1593–
1602, doi:10.1126/science.1207532
Xu J. 2005. Cost of interacting with sexual partners in a
facultative sexual microbe. Genetics 171:1597–1604,
doi:10.1534/genetics.105.045302
Xu J, Saunders CW, Hu P, Grant RA, Boekhout T, Kuramae
EE, Kronstad JW, DeAngelis YM, Reeder NL, Johnstone
KR, et al. 2007. Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits
shared with plant and human fungal pathogens. Proc
Natl Acad Sci USA 104:18730–18735, doi:10.1073/
pnas.0706756104
Yoshida K, Terai Y, Mizoiri S, Aibara M, Nishihara H,
Watanabe M, Kuroiwa A, Hirai H, Hirai Y, Matsuda Y,
et al. 2011. B chromosomes have a functional effect on
female sex determination in Lake Victoria Cichlid fishes.
PLoS Genet 7:e1002203, doi:10.1371/journal.pgen.
1002203
Zaragoza O, Garcı́a-Rodas R, Nosanchuk JD, CuencaEstrella M, Rodrı́guez-Tudela JL, Casadevall A. 2010.
Fungal cell gigantism during mammalian infection.
PLoS Pathog 6:e1000945, doi:10.1371/journal.ppat.
1000945
Zeyl C, Bell G. 1997. The advantage of sex in evolving
yeast populations. Nature 388:465–468, doi:10.1038/
41312