f u n g a l b i o l o g y r e v i e w s 2 9 ( 2 0 1 5 ) 1 0 8 e1 1 7

journal homepage: www.elsevier.com/locate/fbr

Review

Evolution of sexual reproduction: A view from the

fungal kingdom supports an evolutionary epoch
with sex before sexes

Joseph HEITMAN*
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA

article info abstract

Article history: Sexual reproduction is conserved throughout each supergroup within the eukaryotic tree
Received 16 February 2015 of life, and therefore thought to have evolved once and to have been present in the last eu-
Received in revised form karyotic common ancestor (LECA). Given the antiquity of sex, there are features of sexual
10 June 2015 reproduction that are ancient and ancestral, and thus shared in diverse extant organisms.
Accepted 17 August 2015 On the other hand, the vast evolutionary distance that separates any given extant species
from the LECA necessarily implies that other features of sex will be derived. While most
Keywords: types of sex we are familiar with involve two opposite sexes or mating types, recent studies
Evolution in the fungal kingdom have revealed novel and unusual patterns of sexual reproduction,
Heterothallic including unisexual reproduction. In this mode of reproduction a single mating type can
Homothallic on its own undergo self-fertile/homothallic reproduction, either with itself or with other
Inbreeding members of the population of the same mating type. Unisexual reproduction has arisen
MAT independently as a derived feature in several different lineages. That a myriad of different
Mating type types of sex determination and sex determinants abound in animals, plants, protists, and
Meiosis fungi suggests that sex specification itself may not be ancestral and instead may be a
Outcrossing derived trait. If so, then the original form of sexual reproduction may have been unisexual,
Selfing onto which sexes were superimposed as a later feature. In this model, unisexual reproduc-
Sex chromosomes tion is both an ancestral and a derived trait. In this review, we consider what is new and
Sex determination what is old about sexual reproduction from the unique vantage point of the fungal
Sexual reproduction kingdom.
ª 2015 The Author. Published by Elsevier Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction nucleus, mitochondria, secretory apparatus, RNAi, and repro-

No one knows the exact nature of the LECA, but we think that
this ancestor was a unicellular, aquatic, motile creature with
one or two flagella. Thus, in some respects the LECA was sim-
ple. But in other ways, it was already quite complex, with a
ducing both asexually and sexually. Thus, when we think of
where sex first evolved, it was in the water, involving swim-
ming cells (Levin and King, 2013; Umen and Heitman, 2013).
And when we think of how sex first evolved, this involved
changes in ploidy and the process of meiosis, given their

* Tel.: þ1 919 684 2824; fax: þ1 919 684 5458.

E-mail address: heitm001@duke.edu
http://dx.doi.org/10.1016/j.fbr.2015.08.002
1749-4613/ ª 2015 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Evolution of sexual reproduction
conserved nature throughout eukaryotes. And while cellecell
and nuclearenuclear fusion play prominent roles in sexual
reproduction today, there may have been an era in which
endoreplication cycles followed by meiosis drove the pro-
cesses of ploidy change during ancestral modes of sexual
reproduction. In this view, cellecell fusion may be ancient,
but perhaps not as ancient as other features of sexual
reproduction.
Why sex is so pervasive is thought to result from potential
benefits conferred by sexual reproduction. These include
purging the genome of deleterious mutations and shuffling
the genome via independent chromosomal assortment and
recombination to give rise to a diverse repertoire of meiotic
progeny. Sex may also enable organisms to keep pace with
or outrun pathogens, including those both external and those
internal (such as transposons). There is sound experimental
evidence from studies in Caenorhabditis elegans and in natu-
rally occurring snails in New Zealand for this last hypothesis
in which sex allows species to keep pace with their pathogens
(King et al., 2009, 2011; Morran et al., 2011; Vergara et al., 2013).
However, these potential benefits of sex are pitted against
well-known costs of sexual reproduction: that only 50 % of a
parental genome is transmitted to any given progeny, the
time and energy required to locate mates, and the breaking
apart of well adapted genomic configurations.
The core features of sexual reproduction are conserved in
organisms as diverse as the model budding yeast Saccharo-
myces cerevisiae and humans, despite a billion years or more
of evolution separating us from our last common shared
ancestor. These conserved features include: 1) ploidy changes
from haploid to diploid to haploid (or diploid to haploid to
diploid), 2) the process of meiosis that enables meiotic recom-
bination and halves the ploidy of the genome, and 3) cellecell
fusion between mating partners (a and a cells) or gametes (the
sperm and the egg). This ubiquity of the conserved features of
sex again speaks to the antiquity of the process.
Beyond the commonalities in the mechanisms of sex, there
are also shared features to the modes of sexual reproduction.
This includes outbreeding between genetically divergent
members of the population, but also types of inbreeding that
can involve the ability of the yeast S. cerevisiae to undergo mat-
ing type switching that allows mother cells to mate with their
daughter cells. And in humans there are the examples of
consanguineous marriages, resulting for example from cous-
inecousin pairings, which lead to considerable inbreeding
with the risk of exposure of recessive alleles in a homozygous
configuration. We will return to this theme of the balance be-
tween outbreeding and inbreeding.
2. Mechanisms of sex determination
Sex in humans and many other animals is determined by the
X and Y sex chromosomes, in which individuals with XX kar-
yotype are female and those with XY are male. The two sex
chromosomes are dramatically different in size, and are
referred to as heteromorphic sex chromosomes. A single
gene resident on the Y chromosome, SRY, is sufficient to
direct male fate and transferring this single gene from the Y
to the X chromosome suffices to cause sex reversal in both
109
humans and in mice. But in other plants and animals, there
are different mechanisms of sex determination. Some species,
such as the plant Papaya and the fish Medaka, have sex chro-
mosomes in which the sex specific region is small and the two
sex chromosomes are the same size, so called homomorphic
sex chromosomes (Kondo et al., 2004; Liu et al., 2004; Myosho
et al., 2012). Chickens and other birds have a completely
different type of sex chromosome, called Z and W, and in
these lineages it is the heterogametic ZW pattern that spec-
ifies female and the homogametic ZZ the male (Zhou et al.,
2014). In some animals, including turtles and crocodiles, the
temperature at which an egg hatches determines sexual iden-
tity and this is called Environmental Sex Determination (ESD)
to distinguish it from Chromosomal Sex Determination (CSD)
(Barske and Capel, 2008). Yet other species appear to be hy-
brids of the two with features of both environmental and
chromosomal sex determination. Finally in some lines of the
zebra fish Danio rerio sex appears to be a quantitative trait, in
which genes on multiple different chromosomes come
together in allelic combinations that favor either female or
male fate (Anderson et al., 2012; Bradley et al., 2011; Liew
et al., 2012; Liew and Orban, 2014). This quantitative sex deter-
mining system has been termed polygenic sex determination
(PSD). Recent studies of wild D. rerio reveal a sex determining
region on one end of chromosome 4 that may be consistent
with a WZ/ZZ sex chromosome system, suggesting loss of a
sex determinant or recent origin of a novel one during domes-
tication (Wilson et al., 2014). To summarize, in simple terms
the ways in which sex is determined are plastic and diverse.
What about fungi? Relatively few fungi have large size
dimorphic sex chromosomes, but there are a few well studied
examples such as Neurospora tetrasperma, Podospora, and Micro-
botryum (Ellison et al., 2011; Fraser et al., 2004; Fraser and
Heitman, 2004; Grognet et al., 2014; Hood et al., 2013; Menkis
et al., 2008; Whittle et al., 2015). Most fungi have relatively
smaller regions of their genome, called mating-type loci, or
MAT for short, that dictate their mating type (Fraser and
Heitman, 2003). The paradigmatic example is S. cerevisiae in
which a relatively small region of the genome, less than a
thousand base pairs, expresses in the alternate mating types
one or two key cell fate determinants, all of which are tran-
scription factors responsible for orchestrating both haploid
cell type specificity (a or a) and the diploid zygote fate (a/a).
Two are homeodomain proteins of the HD1 and HD2 class
that form a heterodimer, a1/a2, which is necessary for the
diploid zygote fate. The other factor, a1 from MATa, encodes
an alpha domain transcription factor necessary for turning
on genes required for the a cell fate, while a2 represses a genes
to further enforce the a cell haploid fate. The a haploid cell
type is the default, and is not actively specified by the MAT lo-
cus. This type of mating-type system is called bipolar to reflect
the two mating types, a and a. When the two mating types are
in balance in the population, bipolar mating systems enable
50 % outcrossing and 50 % inbreeding.
But other fungi have much more exotic sex lives, and have
a more complex mating-type determining system in which
there are literally thousands and thousands and thousands
of different mating types (Brown and Casselton, 2001;
Casselton, 2002, 2008; Heitman et al., 2007; Raper, 1966). In
these species there are two loci that lie unlinked on different
110
chromosomes that specify mating type. These are called the A
and B MAT loci, and one encodes the homeodomain factors,
and the other encodes pheromones and pheromone receptors
(which locus encodes which genes depends on the species,
because these loci were named historically as they were
discovered genetically long before their molecular basis was
elucidated). In many species, both loci are multiallelic, and
as a result there are many different mating types. Both loci
must differ for productive mating and thus an isolate of
A1B1 mating type can mate with an isolate of A2B2 mating
type, but not with isolates that are A1B2 or A2B1. Because
there are thousands of different mating types, most encoun-
ters between isolates in nature will be fertile, and this drives
the frequency of outbreeding to >99 %. On the other hand,
from any given cross, say A1B1 by A2B2, because two MAT
loci segregate independently four different types of progeny
are produced (A1B1, A2B2, A1B2, and A2B1) and these systems
are therefore called tetrapolar mating systems. Because any
given progeny is only interfertile with 25 % of its siblings
(A1B2 can mate with A2B1 but not with A1B1 or A2B2), the tet-
rapolar configuration not only promotes outcrossing but also
leads to inbreeding depression. It is thought to be these differ-
ences in the frequency of outcrossing and inbreeding that pro-
vided the evolutionary pressure for transitions between
bipolar and tetrapolar mating systems.
Phylogenetic reconstructions across the fungal kingdom
support the conclusion that bipolar mating type is an ances-
tral state and the tetrapolar configuration is a derived state.
If we examine species in the Ascomycota and Zygomycota/
Mucorales, all species with known mating type systems are bi-
polar. This includes a myriad of species throughout the Asco-
mycota including S. cerevisiae (a and a), Schizosaccharomyces
pombe (P and M), Candida albicans (a/a and a/a), Neurospora
crassa (A and a), and Aspergillus fumigatus (MAT1-1 and
MAT1-2) just to name a few (Glass et al., 1988; Hull et al.,
2000; Magee and Magee, 2000; O’Gorman et al., 2009). In the
Mucorales, we again find just two mating types, typically
called P and M for plus (þ) and minus (), in Phycomyces blake-
sleeanus, Mucor circinelloides, and Rhizopus oryzae (Gryganskyi
et al., 2010; Idnurm et al., 2008; Lee et al., 2008). This is in
marked contrast to the Basidiomycota branch of the dikarya
in which a majority of species have the tetrapolar mating sys-
tem configuration. But thus far, no species with the tetrapolar
mating system have been found outside of the Basidiomycota.
Thus, we can root the phylogenetic tree with multiple out-
groups that are all bipolar, and conclude that the tetrapolar
system is a derived state, possibly with a single origin at the
base of the Basidiomycota. In essence, an ancestral system
with just one MAT locus encoding homeodomain factors
evolved into a system with a second sex determinant on
another chromosome encoding pheromones and pheromone
receptors. How such a transition might have occurred could
have first involved an auto-stimulated pheromone, phero-
mone receptor gene pair, which underwent genetic drift to
form more than one self-activating allele, followed by recom-
bination between the two to generate two self-sterile/cross-
fertile gene combinations that became a second MAT locus.
Some aspects of this have been modeled, conceptualizing
how such an event might have transpired (Fraser et al., 2007).
J. Heitman
Interestingly, there are examples of species with bipolar
mating type within the Basidiomycota phylum. Are these
remnants of the bipolar ancestral state, like the microwave
echoes of the Big Bang that formed our universe, or rather
derived from the tetrapolar configuration? This question has
been addressed in some detail in the Cryptococcus pathogenic
species complex (Heitman et al., 2011). All of the pathogenic
species and lineages, including Cryptococcus neoformans var.
grubii (serotype A), C. neoformans var. neoformans (serotype D),
and Cryptococcus gattii (VGI, VGII, VGIII, and VGIV) have just
two mating types and are bipolar species. Analysis of a series
of closely aligned and related species reveals that multiple
outgroup species have tetrapolar mating systems. This in-
cludes Cryptococcus amylolentus and Cryptococcus heveanensis,
for which our group discovered extant sexual cycles, cloned
and characterized their mating-type loci, and showed defini-
tively that both are tetrapolar (Findley et al., 2012; Metin
et al., 2010). Related studies for Kwoniella mangrovensis charac-
terized its mating type locus and clarified that it is tetrapolar
(Guerreiro et al., 2013), in contrast to a previous report from
others that had concluded it was bipolar (Statzell-Tallman
et al., 2008). Taken from this vantage point of multiple tetrapo-
lar outgroup closely aligned species, we can conclude that the
bipolar configuration of the pathogenic species complex is a
derived state and has a monophyletic origin. E pluribus duo!
The longer evolutionary pathway was thus bipolar to tetrapo-
lar and then a return to bipolar. In this view then the bipolar
state is both an ancestral state and a derived one.
The transition from tetrapolar to bipolar has occurred
several times independently in the Basidiomycota, and other
examples include Ustilago hordei and Malassezia species
(Bakkeren et al., 2006; Bakkeren and Kronstad, 1994; Gioti
et al., 2013; Hsueh and Heitman, 2008; Lee et al., 1999). That
the cases that are known involve pathogens of either humans
or plants suggests there may be a causal association between
restricted outbreeding and enhanced inbreeding and patho-
genesis. Perhaps as microbial pathogens become well adapted
to their hosts, less rather than more genetic exchange is adap-
tive. They clearly do conserve the ability to undergo sex, and
thus this may ensure their longer term evolutionary success,
but restricting genetic exchange may allow them to vary just
enough to keep pace with their hosts.
We return then to a more general consideration of MAT loci
and several recent poignant examples prove illuminating.
Recent studies have defined the MAT loci for two ciliates in
the Alveolate supergroup (Tetrahymena and Paramecium), for
two representative algae in the Planta supergroup (Chlamydo-
monas and Volvox), and for the slime mold Dictyostelium in
the Amoebozoa supergroup (Bloomfield et al., 2010;
Cervantes et al., 2013; Ferris et al., 2010; Singh et al., 2014).
While there are fascinating features, such as the three mating
types of the slime mold, and the dramatic expansion of MAT
loci into true sex chromosomes from Chlamydomonas to
Volvox, and the roulette type mating-type switching of Tetra-
hymena which has seven mating types (one for each day of
the week, or seven brides for seven brothers), there is essen-
tially no underlying conserved feature. Taken from the
perspective of diverse and plastic sex chromosomes and
mating-type loci from at least 5 of the 8 supergroups of the
Evolution of sexual reproduction
eukaryotes, there seems to be nothing ancestral or static
about sex determination.
These considerations lead us to conclude that mating type
and sex determination are not ancestral, but rather derived
features. If so, then what was the ancestral pattern of sexual
reproduction? Put another way, we know that the LECA was
sexy but how sexuality was manifested is the central ques-
tion. As we will consider in the section that follows, our cen-
tral premise is that LECA was unisexual (Fig. 1).
3. Modes of sexual reproduction
We now turn our considerations from mechanisms of sexual
reproduction to modes of sexual reproduction. Consideration
of the sexual nature of the Cryptococcus pathogenic species
complex provides further illumination. We have discussed
how they have undergone a transition from an enhanced out-
crossing/restricted inbreeding ancestral tetrapolar state with
thousands of mating types to a derived bipolar state with
just two mating types with enhanced inbreeding and
restricted outbreeding. We will now consider how they have
taken this transition one step further from a bipolar to a uni-
polar sexual state with just one mating type that reproduces
unisexually, dispensing with the need for a partner of oppo-
site mating type. E pluribus unum!
A central conundrum in the field was that while there was
a defined bipolar mating-type system with two mating types
that could occur under lab conditions (Kwon-Chung, 1976a),
the vast majority of clinical and environmental isolates were
all of just one mating type, a. In many populations no isolates
of a mating type could be identified. This led to the conclusion
that the organism might be largely clonal and asexual. Similar
reasoning was applied to the vast majority of fungal patho-
gens and eukaryotic parasite pathogens of humans, and as
recently as a decade ago it was thought that the majority
were asexual and clonal. We now appreciate that they are sex-
ual, but in unusual ways, including unisexual and parasexual
(Heitman, 2006, 2010).
The discovery of unisexual reproduction of Cryptococcus
came from revisiting older observations (Erke, 1976; Wickes
et al., 1996) about the production of hyphae, basidia, and
spores by certain isolates cultured solo on a variety of media
that support mating (V8, MS, SLAD, FA). Several lines of evi-
dence converged to show that this developmental process,
which had been called haploid or monokaryotic fruiting and
was thought to be strictly asexual, is in fact an unusual form
of self-fertile homothallic sexual reproduction (Feretzaki and
Heitman, 2013a, b; Lin et al., 2005). The process involves ploidy
changes which, in some cases, can involve cellecell fusion,
but likely also occur via endoreplication. Key meiotic genes,
including SPO11 and DMC1, which encode the proteins that
make and repair the DSB DNA breaks that provoke meiotic
recombination, are required for this developmental sexual
process. Our group has recently reported a detailed analysis
of meiotic recombination features occurring during bisexual
and unisexual reproduction, further underscoring that core
features of sexual reproduction occur during both modes of
sexual reproduction (Sun et al., 2014). Thus, under laboratory
conditions unisexual reproduction has all of the hallmark
111
features associated with bisexual reproduction, with the
exception that it dispenses with the obligate need for a part-
ner of opposite mating type. There are situations, so called
me
nage a  trois matings, in which three partners are present
(two a and one a) and a small number of limiting a cells serve
as pheromone donors to stimulate the fusion and unisexual
reproduction of two a partners present as the majority in the
population. In fact in mixed populations, both a-a bisexual
and aea unisexual reproduction can occur concomitantly
and the balance depends upon the ratio of the mating types
and who encounters whom.
While early reports focused on unisexual reproduction/
monokaryotic fruiting of isolates of a mating type (Wickes
et al., 1996), we now appreciate that isolates of a mating type
can also undergo unisexual reproduction (Hull and Heitman,
2002; Tscharke et al., 2003). Subsequently, by detailed genetic
analysis of progeny from a cross of a more and a less fertile
parental strain, it was found that unisexual reproduction is
a quantitative trait, to which multiple quantitative trait loci
(QTL) scattered around the genome contribute (Lin et al.,
2006). The MAT locus was found to be the most significant of
the QTLs mapped, with the a allele promoting unisexual hy-
phal development to a greater extent than the a allele (Lin
et al., 2006). To put this another way, isolates with the a allele
are skewed towards being of higher fecundity compared to
isolates with the a allele, but if the other QTLs are those that
promote unisexual reproduction they can elicit development
in an a mating type background. Why a isolates predominate
in nature is not known, and could result from a bottleneck
when C. neoformans emerged from Africa to become global-
ized, or may result from enhanced fitness under conditions
that have not yet been found.
Population genetic evidence also supports that unisexual
reproduction may be a predominant mode of sexual reproduc-
tion in nature. This includes detailed analyses of aADa hybrids
produced via unisex (Lin et al., 2007), and also aAAa diploids
that may be intermediates or products of the unisexual cycle
(Lin et al., 2009). Finally a series of studies have revealed evi-
dence of recombination, even in populations that are exclu-
sively of one mating type (Bui et al., 2008; Hiremath et al.,
2008; Saul et al., 2008).
The analysis of aAAa diploids reveals two distinct types.
One class is clearly the progeny of two genetically different
parents, and thus appears to have been generated via aea
cellecell fusion. The second class has two seemingly identical
genomes, and thus may have arisen from either endoreplica-
tion or cellecell fusion between a mother and her daughter.
Our group is currently identifying and studying genes
involved in cellecell fusion and nuclear fusion to test these
and other models.
The fact that some unisexual reproduction appears to
involve two genetically identical genomes or isolates chal-
lenges conventional models for the evolution and impact of
sexual reproduction. Put another way, if there is no pre-
existing genetic diversity to admix, why go to the trouble of
expending so much energy undergoing sexual reproduction?
Are these just teenagers spinning their wheels in a parking
lot, or is there some potential benefit? It is important to
consider that the null hypothesis may be that this mode of
sexual reproduction is not of benefit, rather that it may have
112
Fig. 1 e Unisexual reproduction as an ancestral and derived trait. A phylogenetic depiction of the major supergroups of the
Eukaryotic tree of life depicting the LECA as sexual/unisexual in the ancestral state, with a diverse pattern of derived sexual
states including predominantly bisexual extant species but also those that are unisexual, trisexual, septasexual, and mul-
tisexual. Modified from Baldauf (2003) with permission of the author.
little cost and therefore is just tolerated (Lynch, 2007). But this
said, our group has been exploring experimentally if and how
it might confer some type of evolutionary benefit, and have
published three studies thus far supporting the contention
that it may.
First, our group has presented evidence that unisexual
reproduction allows a transition from yeast to hyphae and
thereby promotes more efficient foraging for nutrients and
also the generation of spores that can be disseminated to
more distant locales by wind currents (Phadke et al., 2013).
Second, our research has shown that unisexual reproduction
can generate genetic diversity de novo, and that much of this
diversity results from aneuploidy as a consequence of
meiosis, and that this occurs during both unisexual and
bisexual reproduction (Ni et al., 2013). Third, our studies
recently reported indicate that unisex has the capacity to
turn back the hands of time and reverse Muller’s ratchet and
prevent the otherwise inexorable accumulation of deleterious
mutations (Roach and Heitman, 2014), the process that is
thought to be that which dooms asexual species to inevitable
extinction. There are a variety of possible benefits that are in
the process of being tested experimentally, some of which
have been reviewed (Roach et al., 2014).
Things that are found once in biology are interesting, but
those that are found more than once are more interesting
because they may be generalizable. Thus, the discovery that
J. Heitman
C. albicans has the capacity to undergo both bisexual mating
and unisexual mating as a prelude to its parasexual cycle
was an important advance (Alby et al., 2009). Thus, we now
know that two of the most common systemic human fungal
pathogens have extant sexual/parasexual cycles and the ca-
pacity for unisexual reproduction. Interestingly, there are a
group of four Neurospora species that have extant homothallic
(self-fertile) sexual cycles but only one mating type, and thus
likely another independent origin and example of unisexual
reproduction (Gioti et al., 2012; Glass et al., 1990; Glass and
Smith, 1994). Similarly, a group of Stemphylium isolates repre-
senting a novel self-fertile group of isolates with only the
MAT1-1 mating type information at the MAT locus and no iso-
lates with the MAT1-2 information represents another inde-
pendent example and origin of unisexual reproduction
(Inderbitzin et al., 2005). A very recent study reports that Pneu-
mocystis species, which are obligate fungal pathogens that
infect and only live in animal lungs, have a fusion of the two
opposite mating types related to the S. pombe MAT loci, sug-
gesting that Pneumocystis is homothallic/self-fertile and may
be undergoing its sexual cycle in the lungs of infected animals
(Almeida et al., 2015). This is not strictly a case of unisexual
reproduction because both MAT loci are present in a fused
configuration rather than just one MAT locus, but it does serve
to further illustrate how commonly pathogenic microbes have
undergone the transition from outcrossing heterothallic
Evolution of sexual reproduction
sexual reproduction to homothallic self-fertile inbreeding. It is
important to note that these types of fused mating-type loci
leading to self-fertile homothallic fungal species had been
described more than a decade earlier by Gillian Turgeon and
colleagues in now classic studies of plant fungal pathogens,
including Cochliobolus sp. and Gibberella sp., illustrating
convergent evolution to homothallism in diverse fungal line-
ages (Yun et al., 2000, 1999).
This review is dedicated to considering sexual reproduc-
tion in the fungal kingdom, so we will just note in passing
that it is also the case that multiple eukaryotic parasitic path-
ogens including Toxoplasma, Giardia, Trypanosoma, and Leish-
mania appear to undergo selfing forms of sexual
reproduction (Heitman, 2010; Poxleitner et al., 2008; Wendte
et al., 2010). Nothing is as yet known about whether they
have mating types and if so how these are configured, but it
does serve to illustrate common features shared between
fungal and parasite eukaryotic pathogens.
I wish to return then to our question of what the sexuality
of the LECA ancestor of all eukaryotes looked like and propose
that there was an evolutionary epoch in which there was sex
before sexes and in which unisexual reproduction was the
mode of sexual reproduction. In this view, sexes and mating
types were then added later, superimposed on an earlier
ancestral system that was unisexual. In this model, the uni-
sexual state is then both an ancestral state and a derived
one. And in this view, the pathogenic microbes have not
invented an entirely new mode of sexual reproduction out of
whole cloth. Rather they have returned to an ancestral model
of reproduction in an evolutionary pathway involving transi-
tions from unisexual to bisexual and back again to unisexual.
It seems fitting to close this section with a quote:
“We shall not cease from exploration
And the end of all our exploring
Will be to arrive where we started
And know the place for the first time.”
T.S. Eliot, from “Little Gidding”, Four Quartets
4. What questions remain to be addressed by
studies of fungal sex?
I Asexuality?
Given that there are an estimated 5 million species of fungi,
and maybe more, much remains to be learned about their sex-
ual nature (Blackwell 2011). Many fungi are currently classified
as asexual, and yet many fungi long thought to be asexual
have been subsequently found to be sexual, or in some cases
at least parasexual (i.e. C. albicans). Are there any truly asexual
fungi? Candida glabrata is one potential candidate, but also a
mystery in that all known isolates are haploids of one or the
other of two opposite mating types and much of the machin-
ery associated with mating and meiosis are present in the
genome (Muller et al., 2008; Ramirez-Zavaleta et al., 2010; Sri-
kantha et al., 2003; Wong et al., 2003). There is even evidence
113
for recombination in the population. Despite prodigious effort,
no extant sexual cycle has as yet been found, but it seems
likely that some clever genetic trick, or specific environmental
condition, or combination of fertile strains, remains to be
discovered.
If a fungus were asexual, how would we recognize it? It is
often posited that when a pathway is lost that the genes asso-
ciated with it rapidly decay into pseudogenes and then are
lost. And yet recent studies from Greg Lang and David Botstein
reveal that sterile variants of S. cerevisiae rapidly arise during
asexual vegetative growth (Lang et al., 2011, 2009). They
discovered that 23 genes activated by the mating pheromone
response pathway are expressed at some level even during
asexual growth. Mutants that inactivate any step in the pher-
omone response pathway grow w2 % faster than their compa-
triots because they no longer express these 23 messages and
the encoded proteins. And relieved of this gene expression
deadweight, they are more fit as asexual competitors.
Sequence of their genomes reveals a variety of simple muta-
tions in many steps in the pheromone response pathway.
But the pathway has not yet had time to decay away, and
once the first mutation has occurred to relieve the burden of
futile gene expression, there is very little additional benefit
conferred by loss of other genes in the pathway. Thus after a
very quick coup de gra ^ ce, the decline of the rest of the
pathway is likely to be anything but swift. Put another way,
the rate of decay of the pathway is not constant, and there
is a very rapid first step followed by a series of much slower
steps. This is interesting for several reasons. First, it appears
to be very easy and even beneficial to evolve asexuality, at
least for some clones in a population. Second, genome inspec-
tion of an unknown species may lull us into complacency
when we find the mating and meiotic genes seem to be largely
intact; it seems likely that a single nucleotide variant (SNV) in
one gene in either pathway might be easily missed. Finally,
evolutionary paths are often thought to be unidirectional.
Losing sex and becoming asexual is thought to be irreversible.
But if the loss of sex is due to one SNV in one gene, this is likely
to be fully reversible given sufficient selective pressure for
mating or recombination.
Other ways in which an asexual fungal species might man-
ifest could involve gross rearrangements of the genome. Reg-
ular repeated rounds of meiosis serve to cull from the
population those members whose genomes have strayed
from the canonical karyotype. But if a species were to be
asexual, one might find a highly variable karyotype from
isolate to isolate in the population. And examples of just this
pattern have been observed in Verticillium species and sug-
gested to reflect asexuality (de Jonge et al., 2013).
What other asexual hallmarks might we search for? It has
been hypothesized that to survive a transition to asexuality,
species might need to have shed their entire transposon
load in order to survive. So searching for fungal species whose
genomes lack transposons might be a starting place.
Alternatively, we might explore for fungi that lack one or
more key meiotic genes, such as SPO11 or other genes in the
meiotic toolkit (Schurko and Logsdon, 2008). Thus far, the
only known examples of eukaryotes lacking SPO11 are the Dic-
tyostelium species. In those cases, meiosis and meiotic recom-
bination are known to occur and it seems plausible and even
114
likely that some other enzyme has stepped into the shoes of
SPO11 and serves to produce the lesions that provoke meiotic
recombination (Goodenough and Heitman, 2014).
II Why two sexes?
Why are there most commonly two sexes? We do not
know the answer to this seemingly simple question, but hy-
potheses abound (Haag, 2007; Schaffer 2007). 1) It may be to
increase the efficiency of mate recognition and fusion. 2) It
may serve to restrict outbreeding compared to populations
or species in which there is one universal mating type that
can mate with anyone. 3) Having two sexes may be also to
restrict inbreeding compared to one universal mater that
can mate with itself as well as with anyone else. 4) Having
two mating types or sexes may have evolved as a response
to conflicts between nuclear and mitochondrial genomes, or
between mitochondrial genomes, and thereby given rise to
uniparental inheritance of mitochondria and chloroplasts.
5) Having two defined mating types or sexes may have
enabled specialization of gametes and differential contribu-
tion of resources, giving rise to certain development out-
comes that we observe such as the transition from
isogametic species to anisogametic ones. 6) Two sexes might
have served to enable the development of sex specific and
sex antagonistic traits. 7) Finally, having two mating types
or sexes may serve to enable organisms to know when they
are diploid, as they will be heterozygous rather than homozy-
gous for sex determinants contributed by each haploid
parent. These possible functions are also not mutually exclu-
sive, either with each other or with other possible functions
not considered here.
In the context of considering why there are most
commonly two sexes, it is worth remembering that there are
examples in which there are more than two mating types or
sexes. These include the basidiomycete fungi we have dis-
cussed that have literally thousands and thousands and thou-
sands of different mating types (multisexual) including
Coprinopsis cinerea, the slime mold Dictyostelium with its three
mating types (trisexual), and the ciliate Tetrahymena with
seven (septasexual) (see Fig. 1). These may seem exceptions,
but considering what is shared and what is distinct between
closely aligned species with two sexes vs. those with more,
or those with only one (unisexual, to mean “uni” as in one
sex, or “universal” as in pansexual and capable of mating
with any other member of the population as well as itself),
may prove enlightening.
III Why obligate sexuality?
Many animals are obligately sexual. Why? This is in
marked and striking contrast to the vast majority of microbial
eukaryotes which are facultatively sexual, and happily
capable of vegetative mitotic clonal reproduction interspersed
with bouts of sexual reproduction. Why this is not so for
humans, mice, rabbits, is an interesting conundrum. At the
root it suggests that recombinant progeny may be always bet-
ter than the parental. If so, what selective force might have
driven the transition from a facultatively sexual last common
ancestor to a derived obligately sexual state? One driving force
J. Heitman
may have been a need to outrun pathogens, either exogenous
(bacteria, viruses, parasites, fungi, prions) or endogenous ones
(transposons).
Another way to frame this key evolutionary transition and
question about the facultative to obligately sexual state is to
consider a simple question? Are there any fungi who are obli-
gately sexual? One might make the case that some fungi such
as basidiomycetous mushrooms spend a significant fraction
of their lifecycle as a dikaryotic mycelium and fruiting bodies,
and these might be examples of either very frequently sexual
or even obligately sexual fungi.
One interesting species that our group has been studying
as one of the outgroups to the Cryptococcus pathogenic species
complex is Filobasidiella depauperata (Rodriguez-Carres et al.,
2010). It is one of three species that are most closely aligned
with the pathogenic species (Findley et al., 2009), but it itself
is not a pathogen of animals, but it may be a mycoparasite
(a parasite of other fungi). This is a particularly fascinating
species. It grows remarkably slowly, is easily contaminated,
and is quite heat intolerant and must be cultured at tempera-
tures of 24  C or lower. But what is fascinating is that this spe-
cies has no yeast form, and it is strictly hyphal. Microscopic
examination of its hyphae reveals that it shares features
with the hyphae produced during the sexual cycle of Crypto-
coccus, and the hyphae are decorated with basidia and abun-
dant, long, glorious chains of spores. Thus, it appears that F.
depauperata may be a close relative of Cryptococcus that has
become locked in a permanent sexual state. If so, it may be
a case of an obligately sexual fungus. That there are only
two isolates known, and their growth is limited and restricted
under lab conditions, does not provide a ready explanation for
why an obligate sexual state would be more beneficial than a
facultative one, but given further study, explanations may
emerge. It is at least one vantage point from which to begin
to address this question.
Coda Some things are new, and others old, and sometimes
what is new is also old.
Acknowledgments
Our research on fungal mating-type loci is supported by NIH/
NIAID R01 grant AI50113-11, and our studies on fungal unisex-
ual reproduction are supported by NIH/NIAID R37 MERIT
award AI39115-18. I thank Soo Chan Lee, Ci Fu, Kevin Roach,
and Sheng Sun for comments and suggestions. This review
is dedicated to Lorna Casselton for inspiration and for always
asking questions that forced one to think, and to think deeply
and with clarity and conviction.
references
Alby, K., Schaefer, D., Bennett, R.J., 2009. Homothallic and het-
erothallic mating in the opportunistic pathogen Candida albi-
cans. Nature 460, 890e893.
Almeida, J.M., Cisse, O.H., Fonseca, A., Pagni, M., Hauser, P.M.,
2015. Comparative genomics suggests primary homothallism
of pneumocystis species. mBio 6 e02250e02214.
Evolution of sexual reproduction
Anderson, J.L., Rodriguez Mari, A., Braasch, I., Amores, A.,
Hohenlohe, P., Batzel, P., Postlethwait, J.H., 2012. Multiple sex-
associated regions and a putative sex chromosome in zebra-
fish revealed by RAD mapping and population genomics. PLoS
One 7, e40701.
Bakkeren, G., Jiang, G., Warren, R.L., Butterfield, Y., Shin, H.,
Chiu, R., Linning, R., Schein, J., Lee, N., Hu, G., Kupfer, D.M.,
Tang, Y., Roe, B.A., Jones, S., Marra, M., Kronstad, J.W., 2006.
Mating factor linkage and genome evolution in basidiomyce-
tous pathogens of cereals. Fungal Genet. Biol. 43, 655e666.
Bakkeren, G., Kronstad, J.W., 1994. Linkage of mating-type loci
distinguishes bipolar from tetrapolar mating in basidiomyce-
tous smut fungi. Proc. Natl. Acad. Sci. U. S. A. 91, 7085e7089.
Baldauf, S.L., 2003. The deep roots of eukaryotes. Science 300,
1703e1706.
Barske, L.A., Capel, B., 2008. Blurring the edges in vertebrate sex
determination. Curr. Opin. Genet. Dev. 18, 499e505.
Blackwell, M., 2011. The fungi: 1,2,3.5.1 million species? Am. J.
Bot. 98, 426e438.
Bloomfield, G., Skelton, J., Ivens, A., Tanaka, Y., Kay, R.R., 2010.
Sex determination in the social amoeba Dictyostelium discoi-
deum. Science 330, 1533e1536.
Bradley, K.M., Breyer, J.P., Melville, D.B., Broman, K.W.,
Knapik, E.W., Smith, J.R., 2011. An SNP-based linkage map for
zebrafish reveals sex determination loci. G3 (Bethesda) 1, 3e9.
Brown, A.J., Casselton, L.A., 2001. Mating in mushrooms:
increasing the chances but prolonging the affair. Trends
Genet. 17, 393e400.
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. Eu-
karyot. Cell 7, 1771e1780.
Casselton, L.A., 2002. Mate recognition in fungi. Heredity 88,
142e147.
Casselton, L.A., 2008. Fungal sex genes-searching for the ances-
tors. Bioessays 30, 711e714.
Cervantes, M.D., Hamilton, E.P., Xiong, J., Lawson, M.J., Yuan, D.,
Hadjithomas, M., Miao, W., Orias, E., 2013. Selecting one of
several mating types through gene segment joining and
deletion in Tetrahymena thermophila. PLoS Biol. 11, e1001518.
de Jonge, R., Bolton, M.D., Kombrink, A., van den Berg, G.C.,
Yadeta, K.A., Thomma, B.P., 2013. Extensive chromosomal
reshuffling drives evolution of virulence in an asexual path-
ogen. Genome Res. 23, 1271e1282.
Ellison, C.E., Stajich, J.E., Jacobson, D.J., Natvig, D.O., Lapidus, A.,
Foster, B., Aerts, A., Riley, R., Lindquist, E.A., Grigoriev, I.V.,
Taylor, J.W., 2011. Massive changes in genome architecture
accompany the transition to self-fertility in the filamentous
fungus Neurospora tetrasperma. Genetics 189, 55e69.
Erke, K.H., 1976. Light microscopy of basidia, basidiospores, and
nuclei in spores and hyphae of Filobasidiella neoformans (Cryp-
tococcus neoformans). J. Bacteriol. 128, 445e455.
Feretzaki, M., Heitman, J., 2013a. Genetic circuits that govern
bisexual and unisexual reproduction in Cryptococcus neofor-
mans. PLoS Genet. 9, e1003688.
Feretzaki, M., Heitman, J., 2013b. Unisexual reproduction drives
evolution of eukaryotic microbial pathogens. PLoS Pathog. 9,
e1003674.
Ferris, P., Olson, B.J., De Hoff, P.L., Douglass, S., Casero, D.,
Prochnik, S., Geng, S., Rai, R., Grimwood, J., Schmutz, J.,
Nishii, I., Hamaji, T., Nozaki, H., Pellegrini, M., Umen, J.G.,
2010. Evolution of an expanded sex-determining locus in
Volvox. Science 328, 351e354.
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, 353e361.
115
Findley, K., Sun, S., Fraser, J.A., Hsueh, Y.P., Averette, A.F., Li, W.,
Dietrich, F.S., Heitman, J., 2012. Discovery of a modified tet-
rapolar sexual cycle in Cryptococcus amylolentus and the evo-
lution of MAT in the Cryptococcus species complex. PLoS Genet.
8, e1002528.
Fraser, J.A., Heitman, J., 2003. Fungal mating-type loci. Curr. Biol.
13, R792eR795.
Fraser, J.A., Diezmann, S., Subaran, R.L., Allen, A., Lengeler, K.B.,
Dietrich, F.S., Heitman, J., 2004. Convergent evolution of
chromosomal sex-determining regions in the animal and
fungal kingdoms. PLoS Biol. 2, e384.
Fraser, J.A., Heitman, J., 2004. Evolution of fungal sex chromo-
somes. Mol. Microbiol. 51, 299e306.
Fraser, J.A., Hsueh, Y.P., Findley, K., Heitman, J., 2007. Evolution of
the mating-type locus: the basidiomycetes. In: Heitman, J.,
Kronstad, J., Taylor, J.W., Casselton, L.A. (Eds), Sex in Fungi:
Molecular Determination and Evolutionary Implications. ASM
Press, Washington, D.C., pp. 19e34
Gioti, A., Mushegian, A.A., Strandberg, R., Stajich, J.E.,
Johannesson, H., 2012. Unidirectional evolutionary transitions
in fungal mating systems and the role of transposable ele-
ments. Mol. Biol. Evol. 29, 3215e3226.
Gioti, A., Nystedt, B., Li, W., Xu, J., Andersson, A., Averette, A.F.,
Munch, K., Wang, X., Kappauf, C., Kingsbury, J.M., Kraak, B.,
Walker, L.A., Johansson, H.J., Holm, T., Lehtio, J., Stajich, J.E.,
Mieczkowski, P., Kahmann, R., Kennell, J.C., Cardenas, M.E.,
Lundeberg, J., Saunders, C.W., Boekhout, T., Dawson, T.L.,
Munro, C.A., de Groot, P.W., Butler, G., Heitman, J.,
Scheynius, A., 2013. Genomic insights into the atopic eczema-
associated skin commensal yeast Malassezia sympodialis. mBio
4 e00572e00512.
Glass, N.L., Vollmer, S.J., Staben, C., Grotelueschen, J.,
Metzenberg, R.L., Yanofsky, C., 1988. DNAs of the two mating-
type alleles of Neurospora crassa are highly dissimilar. Science
241, 570e573.
Glass, N.L., Metzenberg, R.L., Raju, N.B., 1990. Homothallic Sor-
dariaceae from nature: The absence of strains containing only
the a mating type sequence. Exp. Mycol. 14, 274e289.
Glass, N.L., Smith, M.L., 1994. Structure and function of a mating-
type gene from the homothallic species Neurospora africana.
Mol. Gen. Genet. 244, 401e409.
Goodenough, U., Heitman, J., 2014. Origins of eukaryotic sexual
reproduction. Cold Spring Harb. Perspect. Biol. 6 a016154.
Grognet, P., Bidard, F., Kuchly, C., Tong, L.C., Coppin, E.,
Benkhali, J.A., Couloux, A., Wincker, P., Debuchy, R., Silar, P.,
2014. Maintaining two mating types: structure of the mating
type locus and its role in heterokaryosis in Podospora anserina.
Genetics 197, 421e432.
Gryganskyi, A.P., Lee, S.C., Litvintseva, A.P., Smith, M.E.,
Bonito, G.M., Porter, T.M., Anishchenko, I.M., Heitman, J.,
Vilgalys, R., 2010. Structure, function, and phylogeny of the
mating locus in the Rhizopus oryzae complex. PLoS One 5,
1e12.
Guerreiro, M.A., Springer, D.J., Rodrigues, J.A., Rusche, L.N.,
Findley, K., Heitman, J., Fonseca, A., 2013. Molecular and ge-
netic evidence for a tetrapolar mating system in the basidio-
mycetous yeast Kwoniella mangrovensis and two novel sibling
species. Eukaryot. Cell 12, 746e760.
Haag, E.S., 2007. Why two sexes? Sex determination in multicel-
lular organisms and protistan mating types. Semin. Cell Dev.
Biol. 18, 348e349.
Heitman, J., 2006. Sexual reproduction and the evolution of mi-
crobial pathogens. Curr. Biol. 16, R711eR725.
Heitman, J., Kronstad, J.W., Taylor, J.W., Casselton, L.A., 2007. Sex
in Fungi: Molecular Determination and Evolutionary Implica-
tions. ASM Press, Washington, D.C.
Heitman, J., 2010. Evolution of eukaryotic microbial pathogens via
covert sexual reproduction. Cell Host Microbe 8, 86e99.
116
Heitman, J., Hsueh, Y.P., Metin, B., Findley, K., Rodriguez-
Carres, M., 2011. The mating-type locus of Cryptococcus: evo-
lution of gene clusters governing sex determination and sex-
ual reproduction from the phylogenomic perspective. In:
Heitman, J., Kozel, T.R., Kwon-Chung, K.J., Perfect, J.R.,
Casadevall, A. (Eds), Cryptococcus: From Human Pathogen to
Model Yeast. ASM Press, Washington, D.C., pp. 139e149
Hiremath, S.S., Chowdhary, A., Kowshik, T., Randhawa, H.S.,
Sun, S., Xu, J., 2008. Long-distance dispersal and recombina-
tion in environmental populations of Cryptococcus neoformans
var. grubii from India. Microbiology 154, 1513e1524.
Hood, M.E., Petit, E., Giraud, T., 2013. Extensive divergence be-
tween mating-type chromosomes of the anther-smut fungus.
Genetics 193, 309e315.
Hsueh, Y.-P., Heitman, J., 2008. Orchestration of sexual repro-
duction and virulence by the fungal mating-type locus. Curr.
Opin. Microbiol. 11, 517e524.
Hull, C.M., Heitman, J., 2002. Genetics of Cryptococcus neoformans.
Annu. Rev. Genet. 36, 557e615.
Hull, C.M., Raisner, R.M., Johnson, A.D., 2000. Evidence for mating
of the “asexual” yeast Candida albicans in a mammalian host.
Science 289, 307e310.
Idnurm, A., Walton, F.J., Floyd, A., Heitman, J., 2008. Identification
of the sex genes in an early diverged fungus. Nature 451,
193e196.
Inderbitzin, P., Harkness, J., Turgeon, B.G., Berbee, M.L., 2005.
Lateral transfer of mating system in Stemphylium. Proc. Natl.
Acad. Sci. U. S. A. 102, 11390e11395.
King, K.C., Delph, L.F., Jokela, J., Lively, C.M., 2009. The geographic
mosaic of sex and the Red Queen. Curr. Biol. 19, 1438e1441.
King, K.C., Jokela, J., Lively, C.M., 2011. Parasites, sex, and clonal
diversity in natural snail populations. Evolution 65, 1474e1481.
Kondo, M., Nanda, I., Hornung, U., Schmid, M., Schartl, M., 2004.
Evolutionary origin of the medaka Y chromosome. Curr. Biol.
14, 1664e1669.
Kwon-Chung, K.J., 1976a. Morphogenesis of Filobasidiella neofor-
mans, the sexual state of Cryptococcus neoformans. Mycologia
68, 821e833.
Lang, G.I., Murray, A.W., Botstein, D., 2009. The cost of gene
expression underlies a fitness trade-off in yeast. Proc. Natl.
Acad. Sci. U. S. A. 106, 5755e5760.
Lang, G.I., Botstein, D., Desai, M.M., 2011. Genetic variation and
the fate of beneficial mutations in asexual populations. Ge-
netics 188, 647e661.
Lee, N., Bakkeren, G., Wong, K., Sherwood, J.E., Kronstad, J.W.,
1999. The mating-type and pathogenicity locus of the fungus
Ustilago hordei spans a 500-kb region. Proc. Natl. Acad. Sci. U. S.
A. 96, 15026e15031.
Lee, S.C., Corradi, N., Byrnes 3rd, E.J., Torres-Martinez, S.,
Dietrich, F.S., Keeling, P.J., Heitman, J., 2008. Microsporidia
evolved from ancestral sexual fungi. Curr. Biol. 18, 1675e1679.
Levin, T.C., King, N., 2013. Evidence for sex and recombination in
the choanoflagellate Salpingoeca rosetta. Curr. Biol. 23,
2176e2180.
Liew, W.C., Bartfai, R., Lim, Z., Sreenivasan, R., Siegfried, K.R.,
Orban, L., 2012. Polygenic sex determination system in ze-
brafish. PLoS One 7, e34397.
Liew, W.C., Orban, L., 2014. Zebrafish sex: a complicated affair.
Brief Funct. Genomics 13, 172e187.
Lin, X., Hull, C.M., Heitman, J., 2005. Sexual reproduction between
partners of the same mating type in Cryptococcus neoformans.
Nature 434, 1017e1021.
Lin, X., Huang, J., Mitchell, T., Heitman, J., 2006. Virulence attri-
butes and hyphal growth of C. neoformans are quantitative
traits and the MATa allele enhances filamentation. PLoS
Genet. 2, e187.
Lin, X., Litvintseva, A.P., Nielsen, K., Patel, S., Floyd, A.,
Mitchell, T.G., Heitman, J., 2007. aADa hybrids of Cryptococcus
J. Heitman
neoformans: evidence of same-sex mating in nature and hybrid
fitness. PLoS Genet. 3, 1975e1990.
Lin, X., Patel, S., Litvintseva, A.P., Floyd, A., Mitchell, T.G.,
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.
Liu, Z., Moore, P.H., Ma, H., Ackerman, C.M., Ragiba, M., Yu, Q.,
Pearl, H.M., Kim, M.S., Charlton, J.W., Stiles, J.I., Zee, F.T.,
Paterson, A.H., Ming, R., 2004. A primitive Y chromosome in
papaya marks incipient sex chromosome evolution. Nature
427, 348e352.
Lynch, M., 2007. The frailty of adaptive hypotheses for the origins
of organismal complexity. Proc. Natl. Acad. Sci. U. S. A. 104
(Suppl 1), 8597e8604.
Magee, B.B., Magee, P.T., 2000. Induction of mating in Candida al-
bicans by construction of MTLa and MTLa strains. Science 289,
310e313.
Menkis, A., Jacobson, D.J., Gustafsson, T., Johannesson, H., 2008.
The mating-type chromosome in the filamentous ascomycete
Neurospora tetrasperma represents a model for early evolution
of sex chromosomes. PLoS Genet. 4, e1000030.
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 chro-
mosomal regions in fungi. PLoS Genet. 6, e1000961.
Morran, L.T., Schmidt, O.G., Gelarden, I.A., Parrish 2nd, R.C.,
Lively, C.M., 2011. Running with the Red Queen: host-parasite
coevolution selects for biparental sex. Science 333, 216e218.
Muller, H., Hennequin, C., Gallaud, J., Dujon, B., Fairhead, C., 2008.
The asexual yeast Candida glabrata maintains distinct a and
alpha haploid mating types. Eukaryot. Cell 7, 848e858.
Myosho, T., Otake, H., Masuyama, H., Matsuda, M., Kuroki, Y.,
Fujiyama, A., Naruse, K., Hamaguchi, S., Sakaizumi, M., 2012.
Tracing the emergence of a novel sex-determining gene in
medaka, Oryzias luzonensis. Genetics 191, 163e170.
Ni, M., Feretzaki, M., Li, W., Floyd-Averette, A., Mieczkowski, P.,
Dietrich, F.S., Heitman, J., 2013. Unisexual and heterosexual
meiotic reproduction generate aneuploidy and phenotypic
diversity de novo in the yeast Cryptococcus neoformans. PLoS
Biol. 11, e1001653.
O’Gorman, C.M., Fuller, H., Dyer, P.S., 2009. Discovery of a sexual
cycle in the opportunistic fungal pathogen Aspergillus fumiga-
tus. Nature 457, 471e474.
Phadke, S.S., Feretzaki, M., Heitman, J., 2013. Unisexual repro-
duction enhances fungal competitiveness by promoting
habitat exploration via hyphal growth and sporulation. Eu-
karyot. Cell 12, 1155e1159.
Poxleitner, M.K., Carpenter, M.L., Mancuso, J.J., Wang, C.J.,
Dawson, S.C., Cande, W.Z., 2008. Evidence for karyogamy and
exchange of genetic material in the binucleate intestinal
parasite Giardia intestinalis. Science 319, 1530e1533.
Ramirez-Zavaleta, C.Y., Salas-Delgado, G.E., De Las Penas, A.,
Castano, I., 2010. Subtelomeric silencing of the MTL3 locus of
Candida glabrata requires yKu70, yKu80, and Rif1 proteins.
Eukaryot. Cell 9, 1602e1611.
Raper, J.R., 1966. Genetics of Sexuality in Higher Fungi. The Ro-
nald Press Company, New York.
Roach, K.C., Feretzaki, M., Sun, S., Heitman, J., 2014. Unisexual
reproduction. Adv. Genet. 85, 255e305.
Roach, K.C., Heitman, J., 2014. Unisexual reproduction reverses
Muller’s ratchet. Genetics 198, 1059e1069.
Rodriguez-Carres, M., Findley, K., Sun, S., Dietrich, F.S.,
Heitman, J., 2010. Morphological and genomic characteriza-
tion of Filobasidiella depauperata: a homothallic sibling species
of the pathogenic cryptococcus species complex. PLoS One 5,
e9620.
Saul, N., Krockenberger, M., Carter, D., 2008. Evidence of recom-
bination in mixed-mating-type and a-only populations of
Evolution of sexual reproduction
Cryptococcus gattii sourced from single eucalyptus tree hol-
lows. Eukaryot. Cell 7, 727e734.
Schaffer, A., 2007. Pas de deux: Why are There Only Two Sexes?
http://www.slate.com/articles/life/the_sex_issue/2007/2009/
pas_de_deux.html.
Schurko, A.M., Logsdon Jr., J.M., 2008. Using a meiosis detection
toolkit to investigate ancient asexual “scandals” and the evo-
lution of sex. BioEssays 30, 579e589.
Singh, D.P., Saudemont, B., Guglielmi, G., Arnaiz, O., Gout, J.F.,
Prajer, M., Potekhin, A., Przybos, E., Aubusson-Fleury, A.,
Bhullar, S., Bouhouche, K., Lhuillier-Akakpo, M., Tanty, V.,
Blugeon, C., Alberti, A., Labadie, K., Aury, J.M., Sperling, L.,
Duharcourt, S., Meyer, E., 2014. Genome-defence small RNAs
exapted for epigenetic mating-type inheritance. Nature 509,
447e452.
Srikantha, T., Lachke, S.A., Soll, D.R., 2003. Three mating type-like
loci in Candida glabrata. Eukaryot. Cell 2, 328e340.
Statzell-Tallman, A., Belloch, C., Fell, J.W., 2008. Kwoniella man-
groviensis gen. nov., sp.nov. (Tremellales, Basidiomycota), a tele-
omorphic yeast from mangrove habitats in the Florida
Everglades and Bahamas. FEMS Yeast Res. 8, 103e113.
Sun, S., Billmyre, R.B., Mieczkowski, P.A., Heitman, J., 2014. Uni-
sexual reproduction drives meiotic recombination and
phenotypic and karyotypic plasticity in Cryptococcus neofor-
mans. PLoS Genet. 10, e1004849.
Tscharke, R.L., Lazera, M., Chang, Y.C., Wickes, B.L., Kwon-
Chung, K.J., 2003. Haploid fruiting in Cryptococcus neoformans is
not mating type a-specific. Fungal Genet. Biol. 39, 230e237.
Umen, J., Heitman, J., 2013. Evolution of sex: mating rituals of a
pre-metazoan. Curr. Biol. 23, R1006eR1008.
Vergara, D., Lively, C.M., King, K.C., Jokela, J., 2013. The
geographic mosaic of sex and infection in lake populations of
a New Zealand snail at multiple spatial scales. Am. Nat. 182,
484e493.
117
Wendte, J.M., Miller, M.A., Lambourn, D.M., Magargal, S.L.,
Jessup, D.A., Grigg, M.E., 2010. Self-mating in the definitive
host potentiates clonal outbreaks of the apicomplexan para-
sites Sarcocystis neurona and Toxoplasma gondii. PLoS Genet. 6,
e1001261.
Whittle, C.A., Votintseva, A., Ridout, K., Filatov, D.A., 2015. Recent
and massive expansion of the mating-type specific region in
the smut fungus Microbotryum. Genetics 199, 809e816.
Wickes, B.L., Mayorga, M.E., Edman, U., Edman, J.C., 1996.
Dimorphism and haploid fruiting in Cryptococcus neoformans:
association with the a-mating type. Proc. Natl. Acad. Sci. U. S.
A. 93, 7327e7331.
Wilson, C.A., High, S.K., McCluskey, B.M., Amores, A., Yan, Y.L.,
Titus, T.A., Anderson, J.L., Batzel, P., Carvan 3rd, M.J.,
Schartl, M., Postlethwait, J.H., 2014. Wild sex in zebrafish: loss
of the natural sex determinant in domesticated strains. Ge-
netics 198, 1291e1308.
Wong, S., Fares, M.A., Zimmermann, W., Butler, G., Wolfe, K.H.,
2003. Evidence from comparative genomics for a complete
sexual cycle in the ’asexual’ pathogenic yeast Candida glabrata.
Genome Biol. 4, R10.
Yun, S.H., Arie, T., Kaneko, I., Yoder, O.C., Turgeon, B.G., 2000.
Molecular organization of mating type loci in heterothallic,
homothallic, and asexual Gibberella/Fusarium species. Fungal
Genet. Biol. 31, 7e20.
Yun, S.H., Berbee, M.L., Yoder, O.C., Turgeon, B.G., 1999. Evolution
of the fungal self-fertile reproductive life style from self-sterile
ancestors. Proc. Natl. Acad. Sci. U. S. A. 96, 5592e5597.
Zhou, Q., Zhang, J., Bachtrog, D., An, N., Huang, Q., Jarvis, E.D.,
Gilbert, M.T., Zhang, G., 2014. Complex evolutionary trajec-
tories of sex chromosomes across bird taxa. Science 346, 1332.
http://dx.doi.org/10.1126/science.1246338.