doi: 10.1111/j.1420-9101.2012.02495.x
REVIEW
Sex, outcrossing and mating types: unsolved questions
in fungi and beyond
S. BILLIARD*, M. LÓPEZ-VILLAVICENCIO , M. E. HOODà & T. GIRAUD§–
*Laboratoire de Génétique et Evolution des Populations Végétales, UMR CNRS 8016, Université des Sciences et Technologies de Lille – Lille1, Villeneuve d’Ascq Cedex,
France
Origine, Structure, Evolution de la Biodiversité, UMR 7205 CNRS-MNHN, Muséum National d’Histoire Naturelle, Paris Cedex, France
àDepartment of Biology, McGuire Life Sciences Building, Amherst College, Amherst, MA, USA
§Ecologie, Systématique et Evolution, Université Paris-Sud, Orsay cedex, France
–Ecologie, Systématique et Evolution, CNRS, Orsay cedex, France
Keywords:
Abstract
ascomycete;
asexual reproduction;
basidiomycete;
breeding systems;
diploid selfing;
gametophytic selfing;
haploid selfing;
mating systems;
oomycetes;
sexual reproduction.
Variability in the way organisms reproduce raises numerous, and still
unsolved, questions in evolutionary biology. In this study, we emphasize that
fungi deserve a much greater emphasis in efforts to address these questions
because of their multiple advantages as model eukaryotes. A tremendous
diversity of reproductive modes and mating systems can be found in fungi,
with many evolutionary transitions among closely related species. In addition,
fungi show some peculiarities in their mating systems that have received little
attention so far, despite the potential for providing insights into important
evolutionary questions. In particular, selfing can occur at the haploid stage in
addition to the diploid stage in many fungi, which is generally not possible in
animals and plants but has a dramatic influence upon the structure of genetic
systems. Fungi also present several advantages that make them tractable
models for studies in experimental evolution. Here, we briefly review the
unsolved questions and extant hypotheses about the evolution and maintenance of asexual vs. sexual reproduction and of selfing vs. outcrossing,
focusing on fungal life cycles. We then propose how fungi can be used to
address these long-standing questions and advance our understanding of
sexual reproduction and mating systems across all eukaryotes.
Introduction
The process of reproduction is one of the most variable
traits in the living world, with organisms giving rise to
progeny either clonally or sexually, and in the latter case
by selfing or outcrossing (see the Glossary for definitions). Understanding what factors shape an organism’s
reproductive mode is of fundamental importance because
patterns of inheritance drastically affect major evolutionary and ecological processes (adaptation, e.g. Charlesworth & Charlesworth, 1995; Otto, 2009; colonization,
e.g. Busch, 2011; Richards, 2003), as well as the applied
Correspondence: Sylvain Billiard, Laboratoire de Génétique et Evolution des
Populations Végétales, UMR CNRS 8016, Université des Sciences et
Technologies de Lille – Lille1, F-59655 Villeneuve d’Ascq Cedex, France.
Tel.: (+33)3 20 33 59 23; fax: (+33)3 20 43 69 79;
e-mail: [email protected]
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fields of biotechnology, breeding and artificial selection
(Whitton et al., 2008), conservation biology (e.g. Fréville
et al., 2007), species invasion (Barrett, 2011) and pathogen evolution and control (Shea et al., 2000; Zuk,
2009).
The prevalence of sexual or asexual reproduction is
highly variable among eukaryotes, with the vast majority
of taxa being able to perform sexual reproduction either
exclusively (e.g. mammals) or alternatively with clonality
(e.g. aphids, ciliates, many angiosperms and fungi).
Sexual reproduction implies the succession of haploid
and diploid phases, transitions occurring by meiosis,
where recombination and chromosomal segregation
occur, and syngamy, where two haploids fuse. The
long-term persistence of eukaryotes relying exclusively
on asexual reproduction is rare and is thought to include
the famous bdelloid rotifers (Welch et al., 2000),
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Fig. 1 Synthetic view of the different possible modes of reproduction in homothallic vs. heterothallic fungi and oomycetes. *Some diploid
selfing may be possible for heterothallic oomycetes but only when in presence of a different mating partner, i.e. when also performing
outcrossing.
Glomeromycota fungi (Kuhn et al., 2001), some insects
and some plants (Judson & Normark, 1996). Even in
these textbook examples of asexuality, however, recent
studies have suggested the occurrence of cryptic sex, in
particular, because all the genes required for the meiotic
machinery are maintained in the genomes (Schurko
et al., 2009; Halary et al., 2011).
Mating systems, governing which haploids fuse at
syngamy, are also highly variable: most species undergo predominantly selfing or outcrossing, whereas a few
species show a mixed-mating system in both animals
and plants (e.g. Jarne & Auld, 2006; Igic & Kohn,
2006). Outcrossing results from the syngamy between
haploid cells produced by separate diploid individuals,
whereas selfing results from the syngamy between
haploid cells produced by the same diploid individual
(Figs 1 and 2). Selfing occurs in plants and animals
through the fusion of gametes produced from meioses
of a single diploid individual, referred to as diploid
selfing. In some eukaryotes, in particular those with an
extended haploid life stage such as ascomycete fungi,
mosses, ferns and some algae, selfing is sometimes
possible through the fusion of two mitotic descendants
of the same haploid cell, which is called intrahaploid
mating, intragametophytic selfing (Hedrick, 1987), sameclone mating (Perrin, 2012), gametophytic selfing (Epinat
& Lenormand, 2009) or haploid selfing (Billiard et al.,
2011). Diploid selfing and haploid selfing can have
very different consequences for genetic structure, and
care should be taken in the application of different
terms to describe their occurrence in eukaryotes.
The proximate mechanisms controlling mating systems
are also highly diverse (sometimes called ‘breeding
systems’, Neal & Anderson, 2005); obligate outcrossing
can result, for instance, from the existence of (i) separate
sexes (even though outcrossing may not be the main
force responsible for the evolution of separate sexes),
(ii) sexual morphs (e.g. heterostyly in angiosperms,
where two morphs with different stigma and pistil
lengths coexist in populations, mating being only possible
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S . B I L L I A R D E T A L.
Fig. 2 Illustration of the different modes of
reproduction and mating systems in fungi.
between individuals of different morphs), (iii) molecular
recognition mechanisms (e.g. self-incompatibility systems in angiosperms), or (iv) asynchrony in the production of male and female gametes in an individual (see
Barrett, 2010 for a review). The tendency for selfing can
result, for instance, from nonopening hermaphroditic
flowers (cleistogamy) or developmental proximity between gametes from the same individual (Giraud et al.,
2008).
Biologists have struggled for more than a century to
identify factors responsible for the evolution of such
preference for outcrossing vs. selfing or for sexual vs.
asexual reproduction. Sexual reproduction has apparently been lost independently in diverse eukaryotes
(reviewed in fungi, Billiard et al., 2011; Lobuglio et al.,
1993; López-Villavicencio et al., 2010; Kuhn et al., 2001;
in microsporidia, Ironside, 2007; in animals, Simon et al.,
2003; and in plants, Whitton et al., 2008). However,
there is growing evidence that some species long thought
to be asexual undergo cryptic sex (Burt et al., 1996;
Schurko et al., 2009; Lee et al., 2010). Nevertheless,
anciently asexual lineages undoubtedly exist in many
different taxonomic groups, as revealed, for instance, by
the Meselson effect, in which the divergence between
alleles in the two nuclei of each cell is so great that it can
only be explained by a long divergence without recombination (Kuhn et al., 2001; Enjalbert et al., 2002; RooseAmsaleg et al., 2002; Schurko et al., 2009). Similarly,
transitions between outcrossing and selfing preferences
have occurred independently in the evolution of many
plant groups (for instance, the gain or loss of heterostyly,
e.g. Barrett & Shore, 2008; Busch, 2011; and in animals,
Jarne & Auld, 2006).
Evolutionary biologists attempt to explain the derivation of particular reproductive modes in terms of fitness
benefits and costs. Sexual reproduction can induce
fitness costs through energetic, genetic transmission
and demographic constraints compared to asexual reproduction (see Otto, 2009; Lehtonen et al., 2012, for
reviews). When sex requires males that contribute only
with their genes and do not directly produce offspring, a
mutation allowing females to reproduce independently
of males, either asexually or through various forms of
self-fertilization, is expected to invade because its progeny grows at twice the rate of the ancestral lineage (the
famous ‘two-fold cost of males’; Maynard Smith, 1978).
Recombination can furthermore break locally adapted
combinations of alleles at multiple loci, which is called
the recombination load. Even in organisms without
either separate sexes or outcrossing, however, meiosis
itself can be costly because it takes more time and energy
than mitotic cell divisions. Other costs of sex may apply
in specific systems: cost of finding and courting a mate,
risk of predation or contracting sexually transmitted
diseases or parasitic genetic elements. On the other hand,
sex is considered to provide benefits, both proximate (e.g.
DNA repair) and ultimate, by limiting the accumulation
of deleterious mutations and by creating novel and
advantageous genetic combinations, especially in environments varying in space and time. Lineages that
undergo only asexual reproduction or obligate selfing
in fact tend to have high extinction rates (Beck et al.,
2011; Goldberg et al., 2010; Igic et al., 2008; Simon et al.,
2003).
Biological groups exhibiting a large diversity of reproductive strategies provide unique opportunities to iden-
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tify the factors underlying the evolution of sex and
mating systems. The majority of theories and observations have been based on vertebrate, insect and plant
models, whereas other groups of eukaryotes, with even
more diverse reproductive strategies, have too often been
excluded from the debate (Birky, 1999). Groups such as
fungi are excellent models to study these topics (Lee
et al., 2010; Billiard et al., 2011; Whittle et al., 2011).
Fungi include species with obligate sexual species (e.g.
Microbotryum, Giraud et al., 2008), species exhibiting both
sexual and asexual reproduction and others appearing
strictly asexual (Taylor et al., 1999; Roose-Amsaleg et al.,
2002; Schurko et al., 2009); there seem to have been
multiple transitions from sexuality to asexuality (Lobuglio et al., 1993; López-Villavicencio et al., 2010; Coelho
et al., 2011; Schurko et al., 2009). Fungi are also potentially informative because they present a huge diversity
in the degree of haploid and diploid selfing rates (Billiard
et al., 2011), and they provide significant advantages over
other eukaryotic models for empirical studies (Goddard
et al., 2005, Bruggeman et al., 2003, 2004). For instance,
many can easily be cultivated under laboratory conditions, can be cloned, have relatively short generation
times and can be revived after long-term frozen storage,
which is then useful in studies of experimental evolution.
Here, we thus would like to argue that fungi have
much to bring to questions on the evolution of sex and
mating systems, being tractable experimental models for
addressing the advantages and costs of the various
reproductive systems, and would benefit from being
studied further within an evolutionary context. Several
concepts referring to the mating system of fungi, however, need clarification because the same terms (e.g.
selfing and outcrossing) are used for different phenomena with completely different evolutionary consequences
(Giraud et al., 2008; Neal & Anderson, 2005). Furthermore, knowledge on the advantages and costs of modes
of reproduction and mating systems in fungi can have
direct applications, for instance, to the numerous fungi
used in industry or that threaten health and agricultural
production.
We first describe the different modes of reproduction
and mating systems in fungi, highlighting frequent
misconceptions, and we review their respective evolutionary benefits and costs. We will also consider
oomycetes, which are protists, but have long been
studied by mycologists and share with fungi many
morphological and genetic peculiarities. We argue that
understanding the evolution of modes and systems of
reproduction, especially in fungi, requires (i) assessing
their frequencies in natural populations, and we will
review the (scarce) evidence available in the literature,
(ii) mapping these, as well as other life-history traits
onto phylogenies, and (iii) measuring experimentally
the advantages gained by the different modes of
reproduction and mating systems. We will propose
some possible experimental settings to compare the
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fitness of the progeny resulting from different modes
and systems of reproduction.
1. The different modes of reproduction
and mating systems in fungi
Fungi exhibit a huge variety of life cycles, but we tried to
draw in Figs 3 and 4 some typical sexual cycles of
filamentous ascomycetes and mushrooms (homobasidiomycetes), respectively. Most fungi are able to undergo
both asexual reproduction and sexual reproduction
(Figs 1 and 2), where, as in other sexual eukaryotes,
tightly regulated mechanisms determine which haploid
cells can fuse at syngamy. However, additional possibilities of syngamy exist in fungi as compared to plants and
animals. In fungi considered to be ‘heterothallic’, haploid
selfing is prevented because syngamy can only occur
between haploid cells carrying different alleles at the
mating type locus ⁄ loci (Fig. 1, Billiard et al., 2011).
However, in homothallic fungi, syngamy can occur
between genetically identical haploid cells, that is, clones,
resulting in haploid selfing (Figs 1 and 2, Billiard et al.,
2011). Several proximal mechanisms may confer homothallism in fungi: most often each haploid carries two
active mating type alleles (Coppin et al., 1997), whereas
in some species haploid individuals can ‘switch’ which
mating type allele is expressed (e.g. in Saccharomyces
cerevisiae, Haber, 1998). In other species, syngamy can
simply occur between haploid cells carrying and expressing the same single mating type allele (i.e. ‘same-sex
mating’, Alby et al., 2009; Fraser et al., 2005; Lin et al.,
2005; Metzenberg & Glass, 1990). In homothallic fungi
where mating type switching occurs, haploid selfing
results from syngamy between cells genetically identical
except at the expressed mating type locus.
In both heterothallic and homothallic fungi, two other
types of syngamy, which are also common in plants and
animals, can occur: (i) syngamy between two different
meiotic products originating from a single diploid individual, that is, diploid selfing (more commonly referred
to simply as selfing in plants and animals, but the term
‘diploid selfing’ here allows the distinction from haploid
selfing) and (ii) syngamy between meiotic products
originating from two different diploid individuals, which
is called outcrossing (Figs 1 and 2).
It should be noted that molecular and developmental
systems of self-incompatibility exist in plants and animals
preventing diploid selfing (such as dioecy or self-incompatibility alleles systems in angiosperms), but these are
absent in true fungi where mating compatibility is
determined by the genotype of haploid nuclei. Fungal
species are always able to undergo both outcrossing and
diploid selfing (Giraud et al., 2008); mating types in fungi
can only prevent haploid selfing in heterothallic species.
Nevertheless, some life-history traits may increase the
probability of outcrossing in fungi. For example, many
species of ascomycetes and rust fungi exhibit a long-
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S . B I L L I A R D E T A L.
Fig. 3 Typical life cycle of filamentous ascomycetes.
Typical ascomycete
sexual life cycle
Fig. 4 Typical life cycle of homobasidiomycetes (mushrooms).
distance dispersal and persistence of the haploid stage.
The random encounter of haploid genotypes should
cause outcrossing to be more prevalent than diploid
selfing, although this remains to be investigated by
population genetics analyses in natural populations.
Quite to the opposite extreme, cases exist where haploid
dispersal is very limited or nonexistent and mating occurs
among the immediate products of meiosis, a mating
system called automixis (Mogie, 1986), that is, intrate-
trad mating (see Fig. 2; e.g. the anther smut fungi
Microbotryum, Giraud et al., 2008). The genetic consequences of automixis differ from diploid selfing, due to
the restriction of the effective population of mating
partners and the tendency to retain heterozygosity
(Kirby, 1984; Hood & Antonovics, 2004).
Several ascomycete and basiomycete fungi have been
characterized as being ‘pseudo-homothallic’, due to their
apparent ability to complete the sexual cycle without the
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need for a mate that is achieved by the presence of two
haploid nuclei of opposite mating types and from a single
meiosis in the dispersed spore (e.g. Neurospora tetrasperma,
Merino et al., 1996; Raju & Perkins, 1994; Agaricus bisporus,
Callac et al., 2006; Saccharomycodes ludwigii, Zakharov,
2005). Here, a developmental process allows, and even
seems to promote, the automictic combination of postmeiotic nuclei. However, here again one can wonder
whether the cosegregation of two haploid nuclei of
opposite mating types has been selected to favour
automixis or to allow universal compatibility under
outcrossing. The presence of nuclei of the two mating
types within dispersing spores can indeed ensure that the
spore will be able to mate with any first encountered
haploid. Interestingly, recent studies have suggested that
pseudo-homothallic fungi may exhibit a mechanism
favouring outcrossing over automixis. In A. bisporus, a
choice experiment in seminatural conditions has shown
that outcrossing was indeed more frequent than automixis in this pseudo-homothallic mushroom (Callac et al.,
2006). In this case, the cosegregation of two meiotic
products of opposite mating types in a spore may have
actually evolved for allowing universal mating compatibility that achieves outcrossing rather than to favour
automixis (Billiard et al., 2011). It should be noted,
however, that the choice experiment was performed in
conditions where spore inoculum was maybe higher than
in natural conditions (Callac et al., 2006) and pseudohomothallism may also allow intertetrad mating when no
other mating is available. In contrast, a few basidiomycetes do undergo preferentially automixis, even when
gametes from other diploid individuals are available, via a
developmental specificity promoting automixis (e.g. the
anther smut fungi Microbotryum, Giraud et al., 2005).
The terms ‘homothallism’ and ‘heterothallism’ are also
used to describe the phenomena in other groups as
fungal-like oomycetes, which have been studied for long
by mycologists. Nevertheless, the genetic basis and the
evolutionary consequences are strikingly different from
true fungi (Fig. 1). In oomycetes, which are phylogenetically closer to brown algae and diatoms, ‘heterothallism’
and ‘homothallism’ are used to describe how sexual
reproduction is started: heterothallic oomycetes cannot
undergo gamete production and sexual reproduction
unless an individual of the opposite mating type is
present, while homothallic oomycetes can (Fig. 1, see
also Judelson, 2007). Once gametes are produced, diploid
selfing and outcrossing are possible in both, homothallic
and heterothallic oomycetes. Haploid selfing (a kind of
syngamy possible in homothallic fungi) is prevented in
oomycetes due to the lack of mitotic multiplication of
gametes, as in plants and animals. The terms ‘homothallism’ and ‘heterothallism’ thus correspond to different
phenomena in different organisms. This is probably
because of the potential to isolate haploid cell lineages
(therefore considered as the ‘thallus’) in fungi, but this is
not possible in oomycetes, where the diploid stage was
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considered the ‘thallus’. This situation is confusing,
especially as the terms ‘homothallism’ and ‘heterothallism’ are also used interchangeably with ‘selfing’ and
‘outcrossing’ in both oomycetes and fungi. It is essential
to distinguish the types of syngamy illustrated in Figs 1
and 2 with the consistent use of terminology, because
there are different consequences in evolutionary terms,
as explained in the section 2 below.
More generally, the confusion over the different forms
of the term ‘selfing’ as described above stems from the
fact that ascomycetes have a long-lasting haploid stage,
so that the ‘individual’ is considered as the haploid
mycelium or cells. Thus, haploid selfing is called selfing
in ascomycetes, and it remains essential to distinguish
diploid selfing from haploid selfing because of the
different effects on genetic structure, in particular the
genome-wide distribution of heterozygosity. We therefore argue that, even in ascomycetes, the syngamy
between identical haploids should be consistently
described by the term ‘haploid selfing’. For the same
reason, outcrossing for ascomycetes often refers to mating
with a nonidentical haploid (i.e. ‘nonhaploid selfing’),
which can actually be either diploid selfing or true
outcrossing in the classical sense for plants and animals
(i.e. syngamy between products of meiosis of different
diploid individuals). The use of nonambiguous terms is
important for understanding the underlying concepts and
evolutionary consequences, and for rendering the literature on fungi useful, without misunderstandings, for
evolutionary biologists less familiar with fungal life cycles.
2. Evolutionary benefits and costs
of the different modes of reproduction,
breeding systems (homothallism vs.
heterothallism) and mating systems
(selfing vs. outcrossing)
2.1. Costs and benefits of sex
The costs and benefits of sex have been briefly presented
in introduction and have been extensively reviewed
elsewhere (e.g. Otto, 2009; Lehtonen et al., 2012).
However, some classically assumed costs of sex cannot
be applied directly to fungi because many species are
isogamous and determine mating compatibility only
during the haploid stage through molecular means of
nonself-recognition. Furthermore, many sexual fungi
undergo multiple rounds of asexual reproduction for
each sexual cycle, thus benefiting both from the recombinational advantages of sex and from the demographic
advantages of clonality.
2.2. Costs and benefits of homothallism
Although it has been traditionally accepted in mycology
that homothallism (i.e. the possibility for an haploid
individual to mate with any other haploid individual,
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even itself, Fig. 1) evolved to favour haploid selfing, it is
important to recognize that the potential for homothallism as determined by haploid selfing in the laboratory
does not imply that this is the prominent mating system
of the species in nature. The prevalence of haploid
selfing in nature is difficult to assess. Indeed, most
homothallic species can also reproduce asexually and
these two modes of reproduction cannot be distinguished by population genetics analyses. In order to
measure the actual frequency of haploid selfing in
nature, genetic analyses of progeny in sexual structures
collected in nature are required. Moreover, it is not
clear why having sex with an identical haploid would
be favoured. Two benefits are possible: benefits from
segregation and those from recombination. Concerning
the segregation advantage, Epinat & Lenormand (2009)
showed, in a model with a modifier of haploid selfing
partially linked to a locus under diploid selection, that
haploid selfing may be favoured when the decrease in
the mean fitness of offspring due to inbreeding depression is low relative to the advantages of the transmission of selfing and of the increase in the fitness variance
in offspring. Concerning the recombination advantage,
there are no models to our knowledge investigating the
possible recombination advantage or costs of homothallism but we can expect a priori that haploid selfing
should confer no recombinational advantages over
asexual reproduction, and it should still incur some of
the costs of sex, such as the physiological costs involved
in utilizing the meiotic machinery and costly sexual
structures. Other costs of sex should, however, be
minimal under haploid selfing, in particular avoiding
the costs of finding a mate and of risking parasite
transmission.
Instead of having evolved to favour haploid selfing,
homothallism could alternatively be viewed as a lack of
discrimination at syngamy: in homothallic species, each
haploid is compatible with all other haploids in the
population (including incidentally, genetically identical
haploids). In fact, several models suggest that such a
universally compatible mutant should easily invade
populations when there is a cost for waiting for a
compatible mate (Iwasa & Sasaki, 1987). If we consider
a fungal species experiencing random encounters of
haploids because its gametes widely disperse, homothallism as a mutant form should be selected for because it
allows compatibility with all other gametes in the
population. An interesting observation is that some
species have recently been reported to exhibit polymorphism for homothallism that may reflect the invasion of
universally compatible mutants (homothallic mutants):
in the pathogens Candida albicans and in Cryptococcus
neoformans, both heterothallism and homothallism via
‘same-sex mating’ are possible (Heitman, 2010 and refs
therein; Alby et al., 2009). These species are human
pathogens, especially in immunocompromised patients.
A possible explanation could be the incipient invasion of
universally compatible mutants (homothallic mutants)
from an ancestral heterothallic population.
Homothallism, whatever the proximal mechanism,
could thus have evolved for universal compatibility of
gametes under outcrossing when there is a cost in finding
a mating partner and little risk of engaging in haploid
selfing, for instance, due to gamete dispersal. In case the
gametes remain near their mother for mating, homothallism may be selected against to avoid haploid selfing
that brings no recombinational advantage but still incurs
some costs of sexual reproduction (section 2.5 below;
Giraud et al., 2008). If this is the case, a prediction would
then be that homothallic fungi are predominantly outcrossing. This prediction may seem paradoxical at first
sight because it is generally believed that homothallism
evolved to promote selfing. Studies on the population
genetics analyses in homothallic species are rare, but
several in fact have found evidence of outcrossing (see
section 3.2). In the homothallic Aspergillus nidulans, it has
been suggested that mating occurs preferentially between
genetically different individuals (which has unfortunately and confusingly been called ‘relative heterothallism’, Pontecorvo et al., 1953).
Nevertheless, some homothallic fungi (e.g. homothallic
species of Neurospora) have been suggested to rely mainly
on haploid selfing for reproduction because of the lack of
conidia (male gametes) production (Glass & Kuldau,
1992). In some other ascomycetes, such as the sexual
Talaromyces species (Stolk & Samson, 1972), the male
structures surround the female organs, which may in fact
promote haploid selfing. In these homothallic species that
seem to reproduce sexually only via haploid selfing, it is
intriguing why sex is retained instead of asexuality, which
would allow bypassing the physiological costs of meiosis
and syngamy. If sex via haploid selfing is disadvantageous
over the short term compared to asexual reproduction,
because it incurs costs without providing recombinational
advantages, it should be lost. It has been proposed that
haploid selfing could, nevertheless, be maintained if it
increased the chance to engage in occasional outcrossing
(Lee et al., 2010); this would work only if outcrossing
occurs frequently enough to counter the expected rapid
invasion by asexuals, in which case we should detect such
events of outcrossing in natural populations. Unfortunately, too few studies exist that investigate mating
systems in natural populations (see section 3).
Some advantages of sex that are not related to recombination have been proposed to exist in fungi, and they
could provide advantages to haploid selfing over asexuality, as explained in the section 2.3 below.
2.3. Advantages of haploid selfing vs. asexual
reproduction
A possible advantage of haploid selfing over asexual
reproduction relies on the advantage of segregation.
Indeed, haploid selfing immediately produces diploid
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Fig. 5 Life cycle of the anther smut fungus
Microbotryum violaceum (adapted from LopezVillavicencio, et al. 2007). Diploid teliospores
are produced in the anthers (a) and are
transmitted by pollinators (b) onto a healthy
plant (c). The teliospores germinate, undergo
meiosis and produce yeasy-like sporidia (d).
Conjugation takes place on the plant
between sporidia of opposite mating types
(e). Dicaryotic hyphae grow in the plant (f)
and overwinter in vegetative tissues (g). The
following year, infection is systemic (h) and
all flowers produce teliospores (a).
offspring with homozygosity across the whole genome,
which can increase the efficacy of selection for deleterious alleles, especially for recessive mutations (e.g. Haag &
Roze, 2007). However, this mechanism might be of
evolutionary importance only in species with a significant diploid phase under which strong selection can act.
This is likely not the case in most ascomycetes, the group
of fungi in which homothallism is most frequent: the
diploid stage of the life cycle is most often transient in this
group, although some exceptions exist, such as in some
hemiascomycetous yeasts (Knop, 2006).
Yet, some empirical studies suggest that there indeed
might be advantages of haploid selfing vs. asexual
reproduction associated with the elimination of newly
arisen deleterious mutations. Compared to asexual reproduction, haploid selfing seems to reduce the accumulation of slightly deleterious mutations in the homothallic
A. nidulans (Bruggeman et al., 2004). Haploid selfing does
not allow the classical purging of deleterious alleles by
outcrossing between individuals carrying deleterious
mutations at different loci (which is needed to prevent
Müller’s ratchet). It has, however, been proposed that
haploid selfing would be more efficient at purging newly
arisen deleterious mutations from the population than
asexual reproduction in A. nidulans. In a mycelium, all
the nuclei can form spores because no special germ line
exists. Nuclei can be packed to form asexual spores or can
fuse to form sexual spores after meiosis. The concept of a
selection arena is proposed such that when deleterious
mutations appear, they could be eliminated by a selective
maternal diversion of resources to the nonmutant fittest
progeny, which would develop better while getting rid of
lower fitness offspring. This selective resource allocation
has been shown to be more stringent in the more costly
sexual pathway (Bruggeman et al., 2004). Theoretical
work is required to investigate the extent to which
deleterious mutations rates are sufficiently high to create
an advantage for haploid selfing that balances the cost of
sex. The costs of sex are likely much lower in the case of
haploid selfing than for outcrossing, being restricted to
those related to physiological costs of meiosis and sexual
structures. These physiological costs can, however, be
significant (Aanen & Hoekstra, 2007).
Sexual reproduction by haploid selfing may also be
advantageous compared to asexual reproduction when it
allows for the purging of parasites. In some ascomycete
species, DNA and RNA viruses are only transmitted via
asexual spores. Sexual spores produced by either outcrossing or haploid selfing are free of these parasites
(Coenen et al., 1997; van Diepeningen et al., 2008).
Similarly, repeat-induced point (RIP mutation) is a
mechanism restricting the proliferation of transposable
elements, occurs only in association with the dikaryotic
stage that follows mating and precedes karyogamy and
meiosis (Galagan & Selker, 2004). Also, the sexual cycle is
also associated with cell rejuvenation through the resetting epigenetic signalling and the avoidance of senescence. In senescent cultures of Podospora anserina, sexual
reproduction restores healthy mitochondria, which is
called ‘rejuvenation’ (Silliker et al., 1996). These benefits
that are not associated with recombination have likely
been linked with sex secondarily, but they can now
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contribute to rendering haploid selfing advantageous over
asexual reproduction.
Haploid selfing may also occur in nature because sex is
associated with a variety of beneficial functions other
than recombination (reviewed in Burt, 2000). For example,
there may be ecological or developmental differences
between offspring produced by sexual or asexual reproduction, which then contribute to the maintenance of
sex. In fact, this hypothesis is supported by numerous
observations in fungi. In most fungi, sexual spores are,
for instance, considered to be more environmentally
resistant than asexual ones (Aanen & Hoekstra, 2007), so
that haploid selfing may permit to produce resistant
structures even in the absence of a mating partner. Some
sexual structures, such as basidiocarps, also allow efficient spore dispersal by wind, water or animals. Another
example is when sex is required before the colonization
of nutrient source, such as a host: even if sex does not
provide recombinational advantages, haploid selfing
would allow producing infectious spores when no
genetically different mating partner is available (Byrnes
et al., 2010; Fraser et al., 2005; Heitman, 2010). Third, it
has been suggested that sexual reproduction and asexual
reproduction coexist in many species because the cost of
sex varies in time (Burt, 2000). For instance, individuals
undergo sex rather than asexual reproduction when the
population is crowded because there is no difficulty to
find a mate. It would be interesting to assess whether this
hypothesis is supported in fungi. However, assessing the
relative frequency of haploid selfing and asexual reproduction remains difficult in nature because they leave
little distinguishing impacts upon genetic structure.
Actually, these evolutionary forces preventing the loss
of sex beyond the advantages of recombination have
been proposed to be the main general evolutionary force
maintaining sex over the short term (Gouyon, 1999;
Nunney, 1989). A long-term species selection level would
favour species not able to lose sex over the short term
because it is linked to other essential functions; only
those species retaining sexual reproduction would be
able to persist over the long term because the associated
recombination allows more rapid adaptation and purging
deleterious mutations. One would only be able to
observe only the species in which sex has been evolutionarily linked to other essential functions.
2.4. Advantages of sexual reproduction over
asexual reproduction, other than those regarding
haploid selfing
Most of the advantages of sex other than haploid selfing
have been already mentioned above. One of the most
important advantages of sexual reproduction between
two different genomes is to create new adaptive allelic
combinations, often called ‘Fisher–Muller hypothesis’, or
to get rid of deleterious alleles, that is, avoiding Muller’s
ratchet (Otto, 2009). Most theoretical papers show that
this genetic advantage of sex and recombination can
explain the evolution and the maintenance of a small
rate of sexual reproduction (e.g. Roze, 2009). This is
consistent with the observation that many species alternate asexual reproduction and sexual reproduction,
especially in fungi. However, it is still not clear why so
few species are exclusively asexual given the costs of sex.
An explanation may be higher extinction rates in asexual
species, which would be less able to adapt to changing
environments or are subject to mutational decay of
Muller’s ratchet over the long term (Gouyon, 1999). This
hypothesis is supported by the observation that exclusively asexual taxa seemed to have appeared relatively
recently (Beck et al., 2011).
2.5. Advantages of heterothallism over homothallism
An interesting question is why heterothallism is sometimes favoured over homothallism, whereas heterothallism restricts the number of potentially compatible
partners for any gamete. Some authors proposed that
gamete classes such as mating types evolved as a means to
avoid cytoplasmic conflicts between organelles by enforcing their uniparental inheritance (Hurst & Hamilton,
1992). The idea behind this hypothesis is that uniparental
inheritance should discourage the evolution of selfish
cytoplasmic elements, which would replicate faster but
would be less efficient for providing energy to the
organism. Indeed, it seems highly advantageous for
nuclear genes to limit the opportunity to be associated
with selfish organelles. Uniparental inheritance of organelles is efficient for this and in fact most often seems
controlled by a mechanism associated with gamete
classes, such as the size of the gametes or mating types
(Hurst & Hamilton, 1992). However, the association
between uniparental inheritance of organelles and gamete classes is not necessarily evidence for gamete classes
having evolved for controlling uniparental inheritance.
The evolution of uniparental inheritance should be easier
if it can use a pre-existing system of gamete classes that
may have evolved for other reasons. In fact, we showed in
a previous review (Billiard et al., 2011) that evidence
exists in fungi suggesting that gamete classes may have
evolved independently of the system regulating uniparental inheritance of organelles. Some species do exhibit
mating types, but nevertheless show biparental inheritance of organelles (e.g. Coprinus cinereus, May & Taylor,
1988; Neurospora crassa, Yang & Griffiths, 1993), or show
uniparental inheritance but with a system independent of
mating types (e.g. A. bisporus, Jin & Horgen, 1994). On
the other hand, in some other fungal species without
mating types, organelles are inherited uniparentally,
which means that mechanisms other than mating types
exist that ensure uniparental inheritance (e.g. in yeasts
with mating type switching mitochondria actively segregate during the first few rounds of cell division; Berger &
Yaffe, 2000). Also in many ciliates, mating types exist
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despite the lack of any cytoplasmic exchange during
syngamy, and thus the impossibility for the evolution of
selfish organelles (Phadke & Zufall, 2009). Therefore, the
absence of strict association between organelle uniparental transmission and gamete classes as would have been
predicted by the hypothesis of Hurst & Hamilton (1992)
suggests that other evolutionary pressures are involved in
the appearance and maintenance of gamete classes, such
as mating types. The most likely evolutionary sequence
thus seems that gamete classes evolved first and that they
allowed the evolution towards uniparental transmission
of the organelles (Maynard Smith & Szathmáry, 1995).
A number of other hypotheses have been proposed to
explain the evolution of mating types, defined here as a
molecular mechanism of the gametes allowing discrimination for syngamy, independent of size dimorphism.
We summarize below only the main hypotheses and
their limits (see Billiard et al., 2011, for more details).
The advantages put forward to explain the evolution of
mating types depend on the specificities of mating types
in different organisms. Mating types indeed do not
restrict the possibility of syngamy in similar ways in all
organisms. In plants, for instance, mating types are called
self-incompatibility systems and they prevent diploid
selfing (i.e. syngamy between gametes produced by the
same diploid individual; Figs 1 and 2). In such cases
where mating types prevent diploid selfing, it is generally
assumed that they evolved precisely to avoid diploid
selfing, and thus promote outcrossing to limit inbreeding
depression. This probably also applies to oomycetes.
In fungi however, mating types are effective at the
haploid stage and therefore do not prevent diploid selfing:
any diploid stage of a heterothallic species is heterozygous
for the mating type and can therefore produce haploids
that can undergo diploid selfing (Fig. 2). Mating types in
fungi will only prevent haploid selfing. Besides, inbreeding depression is not expected to act in predominantly
haploid organisms, such as ascomycetes, because deleterious mutations are not sheltered and can be purged from
the population as they appear. Inbreeding depression
therefore has likely played much less of a role in the
evolution of fungal mating types as it does in plants. A
possible explanation for the evolution of mating types in
fungi would therefore be to prevent haploid selfing, thus
affording the benefits of sex associated with recombination (Czaran & Hoekstra, 2004). Indeed, if recombination
has an advantage, whatever it is, individuals gain this
advantage only when the haploid genomes that are
recombining are not strictly identical. For example, the
prevention of Müller’s ratchet (i.e. the restoration of
haplotypes free of deleterious mutations in a population
with no individual carrying zero mutations) requires
mating between nonidentical haploids. This hypothesis
remains poorly investigated, but if valid, then the fitness
of progeny produced by haploid selfing should be lower
than the fitness of individuals produced by diploid selfing
(see section 2.6 below). We see here that the distinction
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between diploid selfing and haploid selfing contributes to
understand why mating types exist in fungi.
2.6. Advantages of diploid selfing over haploid
selfing
As we have seen in the previous sections, we expect little
or no advantage of sex when it occurs as haploid selfing,
especially regarding those advantages of sex that rely on
recombination. In the case of diploid selfing, genetic
differences exist between the two haploid genomes
undergoing syngamy and recombination, allowing some
of the recombinational advantages of sex to be realized.
In this case, the diploid or haploid progeny produced by
diploid selfing should have a higher fitness than that
produced by haploid selfing.
2.7. Advantages and drawbacks of diploid selfing
over outcrossing
A long-standing question is why so many species
undergo outcrossing instead of diploid selfing. Indeed,
there is an automatic advantage of diploid selfing
relative to outcrossing: an individual undergoing diploid
selfing transmits two copies of its haploid genome in
selfed progeny and in many species they can in addition
sire offspring by fertilizing outcrossers, whereas these
outcrossers cannot fertilize selfers (Fisher, 1941). Furthermore, in species with a predominant diploid phase,
the ability to undergo diploid selfing avoids mating
partner limitation, that is, selfing provides reproductive
assurance. Diploid selfing may, however, be disadvantageous relative to outcrossing because of inbreeding
depression, that is, when recessive deleterious mutations are present in a population and are exposed to
selection in the homozygous condition due to mating
with close relatives. However, in fungi with a predominantly haploid life cycle, such as most ascomycetes, the
importance of inbreeding depression should be
restricted to loci with expression limited to the dikaryotic or diploid stages. This has been observed, for
example, in N. crassa, the only ascomycete species
where inbreeding depression has been investigated:
crosses between highly related individuals exhibited
reduced fertility; inbred lines produced deficient perithecia with no or few viable ascospores or presented
ascospore maturation defects (Leslie & Raju, 1985). In
basidiomycetes in contrast, the dikaryotic stage is
predominant in the life cycle; inbreeding depression
may therefore have important consequences and be a
significant evolutionary force. Inbreeding depression has
in fact been found in the basidiomycetes A. bisporus
where outcrossed populations showed higher fitness
than inbred ones in several fitness components (Xu,
1995). Some mechanisms in fungi may have in fact
evolved to control the mating system by limiting diploid
selfing or promoting outcrossing (see section 3 below).
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2.8. Conclusions and prospects
We argue that answering the questions raised above
about the evolution of asexual vs. sexual reproduction,
homothallism vs. heterothallism, haploid selfing vs.
diploid selfing and diploid selfing vs. outcrossing requires
(i) measuring what types of syngamy occur in nature (see
section 3 below), (ii) retracing their evolution using
phylogenies and identifying associated life-history traits
(see section 4) and (iii) experimentally assessing the
benefits and costs of the different possible modes of
reproduction, asexual or sexual, and the mating systems
of haploid selfing, diploid selfing and outcrossing (see
section 5).
3. Modes of reproduction and mating
systems in nature
In order to understand why particular breeding systems
(homothallism vs. heterothallism), mating systems and
modes of reproduction have evolved, we need to assess
what types of reproduction and syngamy are actually
performed in natura. Indeed, in vitro observations do not
allow for strong inferences of the reproductive mode in
natural conditions. For instance, homothallism have
been suggested to evolve either for allowing haploid
selfing or, on the contrary, for allowing higher mate
availability under outcrossing (Billiard et al., 2011).
Disentangling between these hypotheses requires
assessing how often in nature homothallic fungi undergo
haploid selfing, diploid selfing or outcrossing. Similarly,
the frequency of sexual reproduction in nature cannot be
inferred from in vitro observations. Below, we briefly
review the modes of reproduction and mating systems of
fungi (sensu lato, i.e. including oomycetes, and the two
main groups of true fungi, ascomycetes and basidiomycetes), as revealed in natura using population genetics
approaches, although such studies remain scarce.
3.1. Modes of reproduction: sexual vs. asexual
reproduction
As a measure of relative time or growth, most ascomycetes are haploid during almost their entire life cycle, as
mycelium or yeast, and often they are capable of both
asexual and sexual reproduction. Ascomycetes include
yeasts, filamentous species and many species responsible
for crop or tree diseases. Basidiomycetes include mushrooms, smuts, yeasts and rusts, the latter being also
responsible for plant diseases and producing large
numbers of asexual spores, as do ascomycetes. Linkage
disequilibrium among genetic markers and the number
of repeated haplotypes isolated from natural populations
have been used to infer the occurrence and degree of
clonality, although this latter phenomenon is not
distinguishable from haploid selfing by these methods
(Giraud et al., 2008; Gladieux et al., 2010). A great
diversity of reproductive systems has been revealed
(Giraud et al., 2008; Taylor et al., 1999), from exclusively clonal propagation, at least in some introduced
ranges, to a high prevalence of sex, with many
surprising results compared to what was expected from
in vitro observations. In the ascomycete Botrytis cinerea,
responsible for grey mould, the human pathogen
Coccidioides immitis or the Macrotermes natalensis fungus
grown by termites, sexual structures have never been
observed and these species were long thought to be
strictly clonal. However, molecular markers revealed
pervasive recombination (Burt et al., 1996; Fournier &
Giraud, 2008; Giraud et al., 1997; De Fine Licht et al.,
2006). In contrast, sexual forms have been observed in
the wheat leaf rust Puccinia triticina (Wahl et al., 1984),
yet genetic analyses of French populations revealed a
completely clonal structure, with no or very little
evidence of genetic recombination (Goyeau et al.,
2007). In the fungus responsible for the yellow rust of
wheat, Puccinia striiformis f.sp. tritici, studies measuring
linkage disequilibrium in European populations similarly indicated prominent clonality (Hovmøller et al.,
2002). Moreover, the high degrees of heterozygosity
observed at microsatellite markers (Enjalbert et al., 2002)
and in sequences associated with ribosomal RNA genes
(Roose-Amsaleg et al., 2002) provided evidence for a
Meselson Effect, where ancient asexual lineages exhibit
high divergence between their homologous chromosomes
due to the accumulation of independent mutations on
different alleles (Halkett et al., 2005). A Meselson effect
has also been detected in Glomales (Kuhn et al., 2001;
Schurko et al., 2009), similarly indicating very ancient
clonality, but recent data showing recombination events
and that the meiotic machinery is conserved might
suggest that these fungi undergo cryptic sex (Halary et al.,
2011). Sex seems to have been lost several times
independently in certain groups, such as in the Penicillium
genus (Lobuglio et al., 1993; López-Villavicencio et al.,
2010).
However, exclusively clonal fungi are scarce, molecular markers having most often revealed the occurrence of
at least some degree of recombination, even in species
where no sexual stages are known (Burt et al., 1996; Lee
et al., 2010). Besides footprints of recombination based
on population genetics data, another type of evidence
indicating that sex occurs regularly in most fungi comes
from the apparent functionality of the mating types
genes, even in species without known sexual structures.
Virtually, all the mating type genes analysed so far indeed
present apparent functional sequences (Debuchy et al.,
2010). Most ascomycetes actually exhibit mixed reproductive systems, with indications of both sexual and
asexual reproduction (e.g. Dutech et al., 2008; Kiss et al.,
2011). However, as mentioned above, a problem is that
haploid selfing cannot be distinguished from clonality in
homothallic fungi using most common approaches of
population genetics.
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In contrast with ascomycetes, mushrooms (homobasidiomycetes) do not produce asexual spores, so that
clonal propagation is restricted to dispersal via mycelia.
Clonality appears therefore of minor importance in most
mushrooms at the population level (Prospero et al.,
2008), although clone growth can occur over quite large
distances (Anderson & Kohn, 1995; Smith et al., 1992).
3.2. Mating systems in fungi: Haploid selfing vs.
diploid selfing vs. outcrossing
In the following sections, we review briefly the evidence
for the occurrence of the different types of syngamy in
natural populations.
Diploid selfing vs. outcrossing
The relative importance of diploid selfing vs. outcrossing
is most easily examined in basidomycetes and oomycetes
because, with their main state being respectively dikaryotic and diploid, the FIS index estimated using codominant genetic markers can give direct indications about
the degree of diploid selfing. Surprisingly however, such
studies are still quite rare compared to those in plants or
animals.
Most basidiomycetes disperse primarily as haploid
basidiospores just before mating, which greatly favours
outcrossing. In fact, heterozygote deficiencies are quite
rare in the few studies having measured FIS in nature in
sexual basidiomycetes (Barrès et al., 2008; Bergemann &
Miller, 2002; Engh et al., 2010; Franzen et al., 2007;
Kauserud & Schumacher, 2002; Kretzer et al., 2004;
Rosewich et al., 1999; Roy et al., 2008). Some sexual
basidiomycetes showed heterozygote excess (Amend
et al., 2010; Engh et al., 2010; Rosewich et al., 1999),
which has been interpreted as due to disassortative
mating (preferential mating with individuals genetically
more different than the average of the population) or
linkage of markers with the MAT or vic (vegetative
incompatibility) loci (which maintains high heterozygosity as fusion can only occur between cells carrying
different alleles at these loci, Hood & Antonovics, 2000).
In contrast to most basidiomycetes, the heterothallic
Microbotryum violaceum, population genetics studies have
shown strong heterozygote deficiency (Delmotte et al.,
1999; Giraud, 2004), indicating high rates of diploid
selfing. Diploid selfing may be common in natural
populations because of a lack of available mating partners
or because of mechanisms favouring diploid selfing
compared with outcrossing. In M. violaceum, diploid
spores are dispersed and only one may arrive at a time
on a new resource for growth (i.e. a host plant) (Fig. 3),
and because further dispersal of the haploid products of
spore germination and meiosis is limited, diploid selfing
may often be the primary option available. Some data,
however, also indicate that a preference for diploid
selfing vs. outcrossing exists (Giraud et al., 2005; Hood &
Antonovics, 2000) and that this is mediated through
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developmental patterns of the promycelium where meiosis takes place (Hood & Antonovics, 2000).
In oomycetes, an example of contrasted mating systems
in two closely related species is given by Plasmopara viticola
and Plasmopara halstedii, respectively, responsible for the
downy mildews of grapevine and sunflower. Molecular
markers showed that the two species displayed similar
levels of genetic diversity (Chen et al., 2007; Delmotte
et al., 2006). The homothallic (sensu oomycetes) species
P. halstedii showed considerable heterozygote deficiency
(FIS = 0.95), probably due to the limited dispersal ability
before mating and thus lack of available sexual partners in
the field (Giresse et al., 2007), whereas the populations of
the heterothallic (sensu oomycetes) species P. viticola only
slightly deviated from Hardy–Weinberg proportions
(Chen et al., 2007; Delmotte et al., 2006).
Haploid selfing vs. clonality vs. outcrossing
Haploid selfing is difficult to distinguish from clonality
using molecular markers as both yield progeny not
segregating for any marker and yield linkage disequilibrium over the long term at the population level.
Sampling and genotyping the progeny within sexual
structures in natural populations should allow determining whether haploid selfing actually occurs in nature.
The literature on mating systems in fungi is, however,
unfortunately blurred by the pervasive confusion between haploid selfing and diploid selfing. For instance, a
study on the ascomycete pathogen Cryphonectria parasitica, responsible for the chestnut blight, claims to have
shown that this ascomycete has a predominantly outcrossing mating system (Marra et al., 2004). In fact, the
authors looked at whether the progeny within perithecia
segregated for some highly polymorphic markers and
could thus only infer the occurrence of haploid selfing vs.
either diploid selfing or outcrossing. Their results interestingly showed that sampling progeny in natural populations can reveal whether haploid selfing occurs, and it
was in fact rare in this species (although not completely
absent, which is surprising for a supposedly heterothallic
species). Such studies estimating the rate of haploid
selfing are, however, still too rare, and the questions are
confused by ambiguous terms like ‘selfing’ and ‘outcrossing’.
Another example is the study by Pérez et al. (2010)
which also called ‘selfing’ the phenomenon of haploid
selfing, and estimated that ‘outcrossing’ (i.e. in fact either
diploid selfing or outcrossing) was frequent in the
homothallic plant pathogen Mycosphaerella nubilosa.
Recent population genetics studies in yeasts have
managed to estimate the rates of haploid selfing, diploid
selfing and outcrossing at the same time by using linkage
disequilibrium with the mating type locus in these
species capable of mating type switching. Tsai et al.
(2008) estimated that a sexual cycle occurred every
1000 asexual cycles in Saccharomyces paradoxus. The sex
events were estimated to be 94% from within the same
tetrad (i.e. the automixis for diploid selfing), 5% with a
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clonemate after switching the mating type (i.e. haploid
selfing) and 1% outcrossed. This was consistent with
another study of natural populations estimating outcrossing at 1.1% in this species (Johnson et al., 2004).
Murphy & Zeyl (2010) have shown, using an experimental approach, that S. cerevisiae and S. paradoxus
undergo even higher rates of outcrossing (ca. 10–25%)
when mates are available, much higher than what was
long expected based on the existence of a capsuleprotecting whole ascus. Earlier experimental studies had
also estimated intratetrad mating rates at about 75–80%
in vitro (Knop, 2006).
Several other studies have showed high rates of
outcrossing in homothallic or pseudo-homothallic species, in particular in Cryptococcus and Agaricus (Bui et al.,
2008; Callac et al., 2006; Heitman, 2010 Hiremath et al.,
2008; Saul et al., 2008), supporting the idea that homothallism may have evolved for higher mate availability,
instead of and ⁄ or in addition to promoting haploid selfing.
Another interesting possibility offered by phylogenies
would be to assess whether associations exist between
the evolution of certain mating or breeding systems and
other traits. For instance, if homothallism evolves for
universal compatibility at syngamy, whereas heterothallism evolves for preventing haploid selfing, we may
expect that homothallism evolved in outcrossing species
in which other mechanisms prevent haploid selfing, such
as rapid gamete dispersal after meiosis or lack of clonal
multiplication of gametes. If selfing ensures reproductive
success, we expect that it evolves in species where
finding a mate is challenging. These approaches can be
applied with great power to diverse organisms (e.g. in
corals Kerr et al., 2011) but we first need to acquire data
on fungal mating systems in nature (haploid selfing vs.
diploid selfing vs. outcrossing) and on multiple lifehistory traits, in order to analyse them on phylogenies
together with the mode of reproduction and breeding
system (heterothallism vs. homothallism).
4. Utility of phylogenies in the study
of sex, breeding systems and mating
systems
5. Experimental designs to distinguish
between hypotheses
Phylogenetic studies can enlighten the evolutionary history of mating systems and modes of reproduction in fungi.
Some studies have shown, for example, that sex has been
independently lost several times in fungal groups such as
Penicillium (Lobuglio et al., 1993; López-Villavicencio
et al., 2010). Phylogenetic analysis is also a powerful tool
to study the relative ages of clades depending on the type of
reproduction and thereby to assess whether different
reproductive systems are advantaged over the long term.
For example, the advantage of sex and outcrossing would
be supported if asexual clades and selfing clades appeared
younger than sexual outcrossing clades.
Phylogenies also allow estimating the ancestral and
derivate modes of reproduction in given clades. Phylogenies have shown that homothallism has probably appeared
from a heterothallic ancestor in some groups of fungi
(Fusarium, O’Donnell et al., 2004; Neurospora, Nygren et al.,
2011; Penicillium, López-Villavicencio et al., 2010). In other
specific groups such as Aspergillus, sequencing and analyses
of mating type genes suggest that heterothallism may have
reappeared from homothallism via gene loss from a homothallic ancestor (Galagan et al., 2005; Geiser et al., 1998;
Ramirez-Prado et al., 2008). Phylogenetic studies also
revealed that homothallism has evolved in some cases by
lateral transfer of mating type genes: both mating type
idiomorphs were acquired by horizontal gene transfer in
Stemphylium (Inderbitzin et al., 2005), and clonal populations of the Dutch elm disease agent Ophiostoma novo-ulmi,
presenting a single mating type for many years, recovered a
sexual cycle after acquiring the MAT1-2 idiomorph from
the closely related species O. ulmi (Paoletti et al., 2006).
Phylogenies have also revealed the history of the acquisition of mating type switching in yeast (Butler et al., 2004).
Most of the models proposed to explain the origin and
maintenance of sexual reproduction and mating types
assume fitness differences between the progeny depending on the mode of reproduction and the mating system
(see section 2). Experimental approaches may therefore
allow testing for the existence and the amount of these
differences using fungi.
The experimental designs we propose are based on the
comparison of the fitness of the diploid or haploid
individuals produced by different modes of reproduction
and by different types of syngamy. We note WAR, WHS,
WDS and WOC the fitnesses of progeny produced by
asexual reproduction, haploid selfing, diploid selfing and
outcrossing, respectively. Different fungal models may be
used to test these different hypotheses. It should be noted
that these fitnesses can be measured either in haploid or
in diploid individuals, depending on the biology of the
species under investigation.
Does haploid selfing afford the advantages of sex not
related to recombination? WAR < WHS would then be
expected
Testing the existence of fitness differences between progeny produced by haploid selfing vs. asexual reproduction
can provide evidence of advantages of sex that are
unrelated with recombination. By definition, haploid
selfing is possible in homothallic ascomycetes and it is not
clear why some species would only undergo haploid
selfing, because it has no recombinatorial advantages over
asexual reproduction and is at the same time more costly.
However, nonrecombinatorial advantages may also exist
(section 2). This question can be investigated using a
homothallic species able to reproduce both sexually and
ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1020–1038
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Sexy fungi
asexually. Some experimental work has already been
carried out on these aspects in A. nidulans (Bruggeman
et al., 2004): several lineages were started, some transferred between plates exclusively by sexual spores resulting from haploid selfing, and others transferred between
plates exclusively by asexual spores. After a certain number
of replating events (i.e. several generations), fitness comparisons were made based on sizes of the colonies. Results
indicated that haploid selfing slowed down the accumulation of deleterious mutations. Similar experiments are
needed on additional species, and other traits can be used to
estimate the fitness depending on the species, such as
asexual spore production, sexual reproduction, viability of
the progeny or competitive ability. Other features unrelated to the recombinational advantages of sex could also
be compared in the progeny resulting from asexual
reproduction vs. haploid selfing, such as the protection
from the proliferation of parasites or transposable elements
in sexual and asexual structures, or senescence.
Is haploid selfing only an artefactual by-product of
the evolution of homothallism for higher mate
availability? WAR ‡ WHS would then be expected
Testing the existence of fitness differences between
progeny produced by haploid selfing vs. asexual
reproduction as above can also allow testing the hypothesis that homothallism evolved not to allow haploid
selfing but to allow universal mating compatibility in
outcrossing species. In this case, one does not expect any
advantage of haploid selfing over asexual reproduction,
and even some costs of meiosis under haploid selfing as
compared to asexual reproduction. We would also expect
that homothallic species outcross in nature (see section 3).
Does diploid selfing provide the recombinatorial
advantage of sex? WHS < WDS would then be
expected
We hypothesize, with others (Czaran & Hoekstra, 2004),
that the origin and maintenance of mating types might
be explained because they allow benefiting from the
recombinatorial advantages of mating between genetically different gametes. This can be tested in haploid
organisms using homothallic species, by testing fitness
differences between lines from sexual reproduction by
haploid selfing vs. diploid selfing. These experiments can
be performed in a fixed or variable controlled environment, in order to distinguish whether recombination
benefits rely on the decrease in mutation accumulation
or on the increase in adaptation speed. A fitness comparison between offspring produced by haploid vs. diploid
selfing is possible only if the parents show a reasonable
level of heterozygosity. Hence, in both types of experiments, a first generation of outcrossing, preferentially
between individuals from different populations, should
be performed.
1033
Deleterious mutation accumulation experiments
Recombination is considered to be fundamental to
prevent the accumulation of deleterious mutations in
sexual populations. Haploid selfing does not allow
recombination between different genetic haploid genomes; it therefore does not allow the elimination of
deleterious mutations, except in the meiosis where they
appear (Bruggeman et al., 2004). Experimental protocols
using fungi can allow testing the importance of recombination for the fitness of the progeny after several
generations of mutation accumulation. Different lineages
can be created with individuals grown in a controlled
environment for a given number of generations.
A generation begins with a single individual produced
by haploid or diploid selfing. Lineages can be maintained
for enough generations to permit mutations to appear or
mutational load could be artificially increased. To detect
advantages of recombination, the fitness of the progeny
formed by haploid vs. diploid selfing could be compared.
If differences exist (WHS < WDS), this could support the
idea that sex is advantageous only when recombination
exists.
Adaptation speed experiments
Sexual recombination between genetically different genomes is also considered to increase the rate of adaptation in populations. In yeast, sex has been shown to
increase adaptation compared to asexual reproduction in
nonfavourable environments (Goddard et al., 2005). This
advantage is expected only under diploid selfing,
whereas no differences should be expected between
asexual reproduction and haploid selfing under which no
effective recombination occurs. Different lineages formed
via either haploid or diploid selfing can be created. These
different lineages can be submitted to different environmental conditions, in harsh or mild environments (by
modifying nutrients, temperature or by adding antifungal
compounds that could make environmental conditions
more stringent and require adaptations). The fitness of
these two lineages can be measured at some time points
comparatively in the different environments.
Do progeny produced by diploid selfing suffer from
inbreeding depression? WDS < WOC would then be
expected in diploid progeny
Inbreeding depression is a frequent phenomenon in
plants and animals (Charlesworth & Willis, 2009).
Inbreeding depression is easily measured in controlled
experiments, by comparing the fitness of outcrossed vs.
selfed offspring, and can be estimated in natural
populations using genetic markers, by estimating the
relatedness between individuals and the fitness of their
offspring. Surprisingly, although numerous measures of
inbreeding depression have been performed for decades
in plants and animals, it has rarely been measured
ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1020–1038
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1034
S . B I L L I A R D E T A L.
in fungi. Yet, estimating inbreeding depression in
basidiomycetes and oomycetes would be straightforward, as they have a life cycle with a predominant
dikaryotic and diploid stage, respectively. Inbreeding
depression indeed results from the homozygosity of
deleterious rare mutations in inbred offspring, and can
be estimated by comparing the fitness between outcrossed dikaryotic or diploid progeny and progeny
produced by diploid selfing. Traits that can be measured
to estimate fitness in basidiomycetes and oomycetes
include heterokaryon growth rate, primordium formation, number of fertile fruiting bodies and average
weight per fruiting body.
Conclusion
This study had two main goals: first, clarifying the
concepts and terminology used to describe sexual
reproduction in fungi by placing them in an evolutionary perspective, and explaining the theoretical costs and
benefits of each mode of reproduction and mating
system, and second, proposing some general experimental considerations and population genetics analyses
in fungi to help disentangle hypotheses about the
evolution of the mode of reproduction and mating
systems. In the evolutionary biology literature, many
models and experiments have focused on the question
of why many species undergo outcrossing instead of
diploid selfing. So far however, very few studies have
used fungi as models, and even worse, the mating
systems and modes of reproduction of fungi in nature
have been poorly investigated. Yet, fungi have particularities that can give valuable insights into long-standing
questions in evolutionary biology, especially the ability
of most fungi to undergo both asexual and sexual
reproduction, and to undergo sexual reproduction via
three mating systems: haploid selfing, diploid selfing and
outcrossing. Original questions can even be addressed in
fungi: do many species undergo sexual reproduction
through haploid selfing? And in this case, why is it so,
while they are able of performing asexual reproduction?
Are benefits of recombination sufficient to explain the
evolution of heterothallism, with the evolution of
complex molecular mechanisms restricting syngamy
between identical haploids? We argue that these questions can be reasonably addressed using population
genetics, phylogenetics and experimental approaches
and would give invaluable insights into the evolution of
sex and mating systems.
Acknowledgments
We thank Robert Debuchy, Philippe Silar, Jacqui Shykoff,
Xavier Vekemans, Phillipe Callac, Pascal Frey, Laurence
Hurst, Thomas Lenormand, Bart Nieuwenhuis, Didier
Andrivon and all the scientists who participated in the
workshop funded by the GDR ComEvol for useful and
stimulating discussions. We also thank Hanna Kokko,
Duur Aanen and two anonymous reviewers for helpful
comments. We acknowledge grants FungiSex ANR-090064-01 and NSF-DEB 0747222. S.B. acknowledges postdoctoral grants from the CNRS. We apologize to all those
colleagues whose work we may have failed to cite in this
article. The authors declare no conflict of interest.
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Glossary
Anisogamy: a feature of species where the two fusing
cells at syngamy are of different sizes or morphologies
and often do not provide the same amount of resources
to the zygote (e.g. large and small gametes, or as in some
fungi where syngamy involves a spore fusing with a
hypha or mycelium).
Automixis: syngamy between haploid cells or haploid
nuclei derived from the same meiosis, also referred to as
intratetrad mating when involving syngamy.
Breeding system: the cellular or developmental mechanisms that determine how syngamy can be achieved for
an organism (e.g. homothallism vs. heterothallism in
fungi, hermaphroditism vs. dioecy in plants).
Diploid selfing: syngamy between haploid cells derived
from meioses of a single diploid individual.
Haploid selfing (or same-clone mating, intragametophytic mating, gametophytic selfing): syngamy
between haploid cells that are mitotic descendants of a
common haploid progenitor, often the cells are genetically identical except for mutation and at the mating type
locus in the case of species with mating type switching.
Homothallism: for fungi, the potential to undergo
haploid selfing; for oomycetes, the potential to undergo
diploid selfing without the presence of another individual
with another mating type.
Heterothallism: for fungi, the requirement for genetic
differences between haploid cells to undergo syngamy;
for oomycetes, the requirement of hormonal signals from
a diploid individual carrying a different mating type to
produce gamete and undergo either diploid selfing or
outcrossing.
Idiomorph: sequences present at the same locus but that
are not derived from a common ancestral sequence (i.e.
in contrast to alleles); idiomorphs at the mating type
locus in ascomycete fungi determine the compatibility for
syngamy.
Isogamy: a feature of species where the two fusing cells
at syngamy are of the same size and morphology, often
yielding the same amount of resources to the zygote (e.g.
in basidiomycete fungi, syngamy can be between two
mycelia).
Mating system: the realized type of syngamy in nature
in terms of the relatedness of the cells involved (e.g.
inbreeding or outcrossing).
Mating type: molecular recognition systems determining the compatibility between cells for syngamy where
haploid cells expressing identical mating types at syngamy cannot fuse.
Mating type switching: a system found in some
ascomycete yeasts where the determinants of alternate
mating types are present but the determination of which
mating type is expressed can change by localized genetic
rearrangements.
Modes of reproduction: a basic distinction between
asexual and sexual reproduction.
Outcrossing: syngamy between haploid cells derived
from meioses that occurred in different diploid individuals.
Pseudo-homothallism: ability of some fungi to complete the sexual cycle without the apparent need for a
syngamy, which is achieved by the presence of two
haploid nuclei of opposite mating types and from a single
meiosis in the dispersed spore.
Syngamy: fusion of haploid cells (often gametes, but
sometimes mitotically replicating haploid cell or hyphae,
as in homobasidiomycetes) for fertilization, which leads
to zygote formation after karyogamy; karyogamy can
occur long after syngamy in fungi, for example, in
basidiomycetes.
Received 26 November 2011; revised 21 February 2012; accepted 22
February 2012
ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1020–1038
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY