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Fungal Genetics and Biology 45 (2008) 791–802
www.elsevier.com/locate/yfgbi
Review
Speciation in fungi
Tatiana Giraud a,*, Guislaine Refrégier a, Mickaël Le Gac b,
Damien M. de Vienne a, Michael E. Hood c
a
Ecologie, Systématique et Evolution, UMR 8079 CNRS, ESE, Bâtiment 360; Université Paris-Sud, 91405 Orsay cedex, France
b
Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T1Z4
c
Department of Biology, Amherst College, Amherst, USA
Received 4 February 2008; accepted 7 February 2008
Available online 14 February 2008
Abstract
In this review on fungal speciation, we first contrast the issues of species definition and species criteria and show that by distinguishing
the two concepts the approaches to studying the speciation can be clarified. We then review recent developments in the understanding of
modes of speciation in fungi. Allopatric speciation raises no theoretical problem and numerous fungal examples exist from nature. We
explain the theoretical difficulties raised by sympatric speciation, review the most recent models, and provide some natural examples consistent with speciation in sympatry. We describe the nature of prezygotic and postzygotic reproductive isolation in fungi and examine
their evolution as functions of temporal and of the geographical distributions. We then review the theory and evidence for roles of cospeciation, host shifts, hybridization, karyotypic rearrangement, and epigenetic mechanisms in fungal speciation. Finally, we review the
available data on the genetics of speciation in fungi and address the issue of speciation in asexual species.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Reproductive character displacement; Reinforcement; Time course of speciation; Tempo of speciation; Reproductive isolation; Genetic
divergence; Specialization; Intersterility; Species concept; Cryptic species
1. Introduction
Speciation, the splitting of one species into two, is one of
the most fundamental problems of biology, being the process by which biodiversity is generated. Understanding how
the 1.5 millions of fungal species (Hawksworth, 1991) have
arisen is of fundamental interest and has tremendous
applied consequences in the cases of agricultural pathogens, emerging human diseases, or fungal species used in
industry and biotechnology. Although much progress on
the origin of species has been made since the book of Darwin (1859), the subject remains heavily debated.
Fungi are excellent models for the study of eukaryotic
speciation in general (Burnett, 2003; Kohn, 2005), although
they are still rarely included in general reviews on this sub-
*
Corresponding author. Fax: +33 1 69 15 73 53.
E-mail address: Tatiana.Giraud@u-psud.fr (T. Giraud).
1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2008.02.001
ject (e.g. Coyne and Orr, 2004). First, many fungi can be
cultured and crossed under laboratory conditions, and
mycologists have long reported numerous mating experiments among fungal species (reviewed in Le Gac and Giraud, in press). Second, fungi display a huge variety of life
cycles and geographical distributions, allowing the study
of which parameters most significantly influence the speciation processes. Third, numerous species complexes are
known in fungi, encompassing multiple recently diverged
sibling species (e.g. Dettman et al., 2003a; Fournier et al.,
2005; Johnson et al., 2005; Le Gac and Giraud, in press;
Le Gac et al., 2007a; Pringle et al., 2005), which allows
investigations on the early stages of speciation.
Excellent reviews on the modes of speciation in fungi
have already been published (Burnett, 2003; Kohn, 2005;
Natvig and May, 1996), and we therefore focus mostly
on recent developments. We first address the question of
species definitions and species criteria and then review the
patterns of speciation in fungi, situating them in the general
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theory as applied to eukaryotes. We focus particularly on
the aspects that have not been extensively reviewed previously and/or that have seen recent and significant developments, such as sympatric speciation, cospeciation, hosts
shifts, reproductive character displacement, and the time
course of speciation. For other aspects, readers should
refer to the previous reviews to have more exhaustive discussions and references (Burnett, 2003; Kohn, 2005; Natvig
and May, 1996; Olson and Stenlid, 2002; Taylor et al. 2000;
Schardl and Craven, 2003).
2. Species definition vs species criteria
To study speciation, it seems necessary to first define
species. The continual proposal of new species concepts
may lead one to think that there is no general agreement
about what species are. To the contrary, it has been argued
that all modern biologists agree that species correspond to
segments of evolutionary lineages that evolve independently from one another (de Queiroz, 1998). The apparently endless dispute about species concepts stems from
the confusion between a species definition (describing the
kind of entity that is a species) and species criteria (standard for judging or recognizing whether individuals should
be considered members of the same species). Many socalled ‘‘species concepts” actually correspond to species criteria, i.e., practical means to recognize and delimit species
(De Queiroz, 2007; Hey, 2006; Taylor et al., 2000). The
Biological Species Concept (BSC) for instance emphasizes
reproductive isolation, the Morphological Species Concept
(MSC) emphasizes morphological divergence, the Ecological Species Concept (ESC) emphasizes adaptation to a particular ecological niche, and the Phylogenetic Species
Concept (PSC) emphasizes nucleotide divergence. These
species criteria correspond to the different events that occur
during lineage separation and divergence, rather than to
fundamental differences in what is considered to represent
a species. One may wonder why there are conflicts over
which species criterion we adopt. There are three main reasons why such criteria cannot be universal: (i) speciation is
a temporally extended process, but one which varies tremendously in its pace among different types of organisms,
(ii) several modes of speciation can occur, during which the
phenomena used for species recognition do not necessarily
appear in the same chronological order, (iii) characteristics
of certain organisms render some criteria difficult to apply.
Let us take as example the most popular yet the most
challenged species criterion, the BSC. For proponents of
the BSC, the capacity to interbreed delimits the infraspecies
level, and ‘‘Biological Species” are intersterile groups
(Mayr, 1942). This criterion is based on reproductive isolation, but this is only one of the many stages of speciation.
Depending on the mode of speciation, intersterility can
occur at early or late stages of speciation, and can constitute the critical stage (in sympatric speciation), or it may
be only a by-product of genetic divergence (in allopatric
speciation). Obviously, the BSC will be most useful in the
first case (sympatric speciation), whereas species criteria
based on evidence for lack of gene flow using molecular
markers will be more discriminating in the latter case.
Intersterility is the stage at which the process has become
irreversible, but this stage may take very long to reach.
Many well-recognized species of plants and animals are still
interfertile.
Until quite recently, the most commonly used species
criterion for fungi has been the MSC. However, many
cryptic species have been discovered within morphological
species, using the BSC (e.g. Anderson and Ullrich, 1978),
or the GCPSR (Genealogical Concordance Phylogenetic
Species Recognition, Taylor et al., 2000), an extension of
the PSC. This latter species criterion uses the phylogenetic
concordance of multiple unlinked genes to indicate a lack
of genetic exchange and thus evolutionary independence
of lineages. Species can thus be identified that cannot be
recognized using other species criteria due to the lack of
morphological characters or incomplete prezygotic isolation. The GCPSR criterion has proved immensely useful
in fungi, because it is more finely discriminating than the
other criteria in many cases, or more convenient (e.g. for
species that we are not able to cross), and is currently the
most widely used within the fungal kingdom (e.g. Dettman
et al., 2003a; Fournier et al., 2005; Johnson et al., 2005;
Koufopanou et al., 2001; Le Gac and Giraud, in press;
Le Gac et al., 2007a; Pringle et al., 2005).
3. Allopatric speciation
How new species arise in nature is still a highly active
field of research. It has long been believed that species originate mostly through allopatric divergence (Mayr, 1963),
because extrinsic geographic barriers seemed obvious
impediments to gene flow. Fungi could appear as exceptions because eukaryotic micro-organisms have long been
considered to have global geographic ranges (ubiquitous
dispersal hypothesis; Finlay, 2002), at least for those not
dependent on a host having a restricted range. This was
in particular true for airborne fungal pathogens because
their spores can be dispersed over very long distance
(Brown and Hovmoller, 2002). Among the numerous complexes of sibling species recently uncovered using the
GCPSR criterion, many however appear consistent with
allopatric divergence, because the cryptic species occupy
non-overlapping areas separated by geographic barriers
(Taylor et al. 2006).
Among the recent examples, a multiple gene genealogies
approach revealed the existence of cryptic species among
the morphological species Neurospora crassa (Dettman
et al., 2003a). They had non-overlapping geographical
ranges, suggesting allopatric speciation: one phylogenetic
species was located in the Congo, another in the Caribbean
and Africa (but not Congo), and a third one was restricted
to India. In yeasts, Kuehne et al. (2007) showed, also using
a multiple gene genealogies approach, that Saccharomyces
paradoxus, a close relative of Saccharomyces cerevisiae
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
present in temperate woodlands in the northern hemisphere, was composed of two distinct genetic groups, A
and B. The majority of isolates from group A were from
Eurasia whereas all isolates from group B had been collected in North America, suggesting a differentiation of
these incipient species in separate continents. Another
example comes from Fusarium graminearum, a fungus
responsible for scab on wheat and barley, which had long
been considered as a panmictic species with a broad distribution. Recent studies however identified at least nine phylogenetically distinct and geographically separated species
(O’Donnell et al., 2004). Four of them were clearly endemic
to South America, one was found only in Central America,
one in India and one in Australia (O’Donnell et al., 2000;
O’Donnell et al., 2004). Yet another example is Coccidioides immitis, responsible for coccidioidomycosis (Valley
fever) in humans and other mammals in America. An analysis conducted after a burst of infected patients in California revealed that this pathogen was composed of two
cryptic species. One was located in California and the other
all around in America (Koufopanou et al., 1997,1998).
These groups were estimated to have been genetically isolated from one another for 11–12.8 million years and had
largely distinct geographical distributions, suggesting that
their genetic isolation had a biogeographic origin. Examples can also be found among basidiomycetes (Kohn,
2005; Le Gac and Giraud in press), for instance in Armillaria mellea, where North American and European strains
have been shown to belong to different species (Anderson
et al., 1989, 1980).
4. Theoretical issues of sympatric speciation
In contrast to the wide acceptance of allopatric speciation, the possibility of sympatric speciation in sexual populations had long been dismissed. This is because
recombination between different subsets of a population
that are adapting to different resources or habitats counteracts natural selection for locally adapted gene combinations (e.g. Felsensein, 1981; Rice, 1984). Recombination
indeed prevents both the building of linkage disequilibrium
between adaptive alleles at different loci and divergence at
loci not under disruptive selection. Population genetics has
shown that a very low level of gene flow, such as one
migrant per generation, is sufficient to prevent differentiation (Slatkin, 1987). This is true regardless of population
size, because while one migrant has a more diluted effect
in a large population, the effect of drift is also smaller.
Theoretical models have shown that the simplest way to
eliminate the role of recombination in breaking down the
effects of selection, and thereby allow sympatric speciation,
is to have the same gene(s) controlling pleiotropically both
enhanced fitness in a specialized habitat and assortative
mating (mate choice, i.e., prezygotic isolation) or both fitness and habitat choice if mating takes place within habitats (Rice, 1984). Such ‘‘magic traits” (Gavrilets, 2004)
have however proved difficult to find in nature. Another
793
way to reduce recombination between two populations specialized on different niches is to build up an association
(linkage disequilibrium) between habitat-based fitness
genes and either assortative mating genes and/or habitat
choice genes if mating is restricted within habitats (e. g.
Dickinson and Antonovics, 1973; Johnson et al., 1996).
Theoretical models have shown that this is plausible under
certain conditions, although the limitations to the process
are far from trivial.
Fungi cannot actively choose the habitat in which they
will grow, but for many fungal species sex must occur in
the habitat after mycelial development (e.g. on or within
the host for fungal parasites). A recent model has shown
that, due to this important characteristic of the life style
(inability of disperse between development on the host or
habitat and mating), mutations providing adaptation on
a new habitat can affect pleiotropically both the fitness
on the habitat and the ability to mate in this habitat. Adaptation to a new habitat can thus be sufficient to restrict gene
flow in sympatry in fungi for which mating occurs within
their specialized host or habitat, without requiring active
assortative mating, i.e., prezygotic intersterility (Giraud,
2006a; Giraud et al., 2006). Specialization would act in
these fungi as a ‘‘magic trait” (Gavrilets, 2004), pleiotropically allowing both adaptation to the new host or habitat
and reproductive isolation, thus facilitating sympatric
speciation.
Sympatry is often said to be difficult to define for
micro-organisms and parasites. For instance, parasites
specialized on different sympatric hosts are sometimes
considered allopatric (e.g. Huyse et al., 2005). A simple,
widely applicable definition of sympatry is however available: ‘‘in sympatry, the probability of mating between two
individuals depends only on their genotypes” and not on
extrinsic barriers (Kondrashov, 1986). Following this definition, parasite individuals infecting two different host
species will only be in allopatry when: (i) the host species
are allopatric, i.e., there is an extrinsic, geographic barrier
to gene flow that prevents dispersal for both hosts and
parasites; or (ii) the host species are sympatric but their
parasites cannot disperse to other host because of extrinsic barriers other than geographic ones; examples are rare,
but include cases where parasite vectors are strictly specific to the different host species. Because the probability
of mating among parasites then depends on host genotypes and not on parasite genotypes (the vector chooses
the host species), the reproductive barriers can be considered as extrinsic to the parasites. In all other cases, the
problem of speciation in sympatry remains: mechanisms
must evolve that will prevent crosses between individuals
specialized on different niches (Giraud, 2006b; Le Gac
and Giraud, 2004). These can be assortative mating or
specialization if mating occurs within hosts or habitats
(Giraud et al., 2006). This latter case is still sympatric speciation because the probability of mating only depends on
the genotypes of fungal individuals (at specialization loci)
and not on extrinsic barriers.
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The possibility of sympatric speciation in fungi thus
heavily relies on the specialization of individuals to different ecological niches, possibly causing directly reproductive
isolation or selecting for assortative mating. An important
issue is then what are the selective pressures that determine
whether fungi become niche specialists or generalists. At
first sight, being able to exploit a variety of ecological
niches, or hosts, seems a better strategy (Felsensein,
1981). The most commonly invoked explanation for specialization is the existence of trade-offs. In parasites,
trade-offs imply that an allele allowing greater exploitation
of a given host reduces the ability or benefit of infecting
other hosts (van Tienderen, 1991). However, despite
numerous works looking for such trade-offs, they have
been difficult to find in nature. An alternative and interesting hypothesis is that specialist parasites can evolve faster
in response to their single host, which provides them a
great advantage in the endless coevolution associated with
antagonistic systems (Kawecki, 1998; Whitlock, 1996).
5. Possible cases of sympatric speciation
Compelling evidence for the sympatric divergence is
extremely difficult to provide, because excluding a past period of allopatry is almost always impossible (Coyne and
Orr, 2004, p. 142). Even the most famous candidate cases
are still debated, such as the phytophagous insect Rhagoletis pommonella (Coyne and Orr, 2004, pp. 159–162) or the
cichlid fishes in African lakes (Coyne and Orr, 2004, pp.
145–154). Evidence consistent with sympatric divergence
of fungal populations driven by parasitic adaptation to different hosts has however been reported.
An example is provided by Ascochyta pathogens, where
recent multilocus phylogenetic analyses of a worldwide
sample of Ascochyta fungi causing blights of chickpea, faba
bean, lentil, and pea have revealed that fungi causing disease on each of these hosts form distinct species (Peever,
2007). Experimental inoculations demonstrated that infection was highly host-specific, yet in vitro crosses showed
that the species were completely interfertile. The host specificity of these fungi may therefore constitute a strong
reproductive barrier, and the sole one (Peever, 2007), following a mechanism of sympatric divergence by host usage
(Giraud, 2006a; Giraud et al., 2006). The coexistence in
sympatry of interfertile populations specialized on different
hosts that remain reproductively isolated cannot indeed be
explained currently by models other than the reduced viability of immigrants (Giraud, 2006a; Giraud et al., 2006;
Nosil et al., 2005). This mechanism seems to be able to
maintain the species differentiated in sympatry and could
similarly have created the divergence in sympatry. It is
however difficult to exclude a period of allopatry in the past
that would have facilitated specialization, i.e., the accumulation of different alleles beneficial on alternate hosts, as
has been proposed for the well-studied case of the phytophagous insect Rhagoletis pommonella (Coyne and Orr, 2004,
pp. 159–162).
An elegant way to demonstrate the sympatric occurrence of speciation is to show that gene flow has occurred
after initial divergence (Wu and Ting, 2004). This approach
is very promising and has been used so far in fungi only on
Mycosphaerella graminicola, showing that this wheat pathogen arose recently, most probably during wheat domestication in the fertile crescent, by sympatric differentiation
from Mycosphaerella species pathogens of natural grasses
(Stukenbrock et al., 2007).
6. Nature of reproductive isolation
As seen above, a sine qua non of speciation in sexually
reproducing organisms is the decrease of gene flow between
incipient species due to the development of reproductive
barriers. Two types of reproductive barriers are usually distinguished, prezygotic and postzygotic, depending on their
time of occurrence, before or after fertilization. In fungi
having a long dikaryotic stage, nuclear fusion occurs long
after individual or gamete fusion, which may render the
term postzygotic ambiguous. We will therefore here use
the terms pre- and postmating for fungi, which qualifies
time before or after cell fusion.
Premating isolation may include different kinds of barriers: (1) for organisms depending on biotic vectors, specialization of these vectors can prevent contact between two
populations even if they lie close to one another, yielding
ecological isolation. For example, the endophyte Epichloë
typhina is preferentially chosen by their fly vectors Botanophila as opposed to Epichloë clarkia (Bultman and Leuchtmann, 2003), which may promote a certain degree of
reproductive isolation. Another example is the complex
Microbotryum violaceum, where the insect vectors are different to some extent between host species, leading to a reduction in mating opportunities among strains from different
plants, although the barrier is not complete (van Putten
et al., 2007). (2) Specialization may also allow for ecological
premating isolation if mating occurs within habitats (hosts
for parasites), as discussed above (Giraud, 2006a; Giraud
et al., 2006). (3) Allochrony, i.e., differences in the time of
reproduction, may also be efficient to promote premating
isolation. The sister species Saccharomyces cerevisiae and
S. paradoxus exhibit for instance different cell growth kinetics; this allows most individuals of one species to undergo
homospecific crosses before or after reproduction of the
individuals of the other species. Proportion of interspecific
matings can therefore be significantly reduced without the
need of incompatibility factors (Murphy et al., 2006). (4)
As has been invoked in plants (Fishman and Wyatt, 1999),
a high rate of selfing may be efficient in limiting interspecific
matings. Selfing has been suggested to act as a reproductive
barrier in the anther smut fungus M. violaceum (Giraud
et al., in press). (5) Assortative mating due to mate recognition occurs if individuals or gametes are able to discriminate
between conspecifics and heterospecifics. Assortative mating seems to be especially important in the reproductive isolation of Homobasidiomycota, where clamp connections
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
between mycelia of opposite types are almost exclusively
observed when the tested mycelia belong to the same species
(Le Gac and Giraud, in press).
Postmating isolation refers to barriers associated with
hybrid inviability and sterility and is expected to arise as
a result of the divergence of incipient species. In the case
of postmating isolation, heterospecific crosses occur and
lead to the production of unfit offspring. Hybrids may be
inviable or sterile due to genetic incompatibilities if mutations fixed independently in the diverging lineages display
negative epistatic interactions when brought together in
the same individual, a phenomenon known as Dobzansky–Müller incompatibilities (Orr and Turelli, 2001). This
kind of intrinsic postmating reproductive isolation is
responsible for the numerous reported cases in fungi of
crosses that initiate and subsequently abort during
in vitro experiments. For instance, heterospecific crosses
among Microbotryum species produce in vitro fewer viable
mycelia than conspecific ones (Le Gac et al., 2007b), and
crosses among Neurospora species lead to few or abnormal
perithecia or to few viable ascospores (Dettman et al.,
2003b). Postmating isolation may also be linked to ecological factors. Hybrids are then perfectly viable and fertile in
a benign environment, such as in vitro conditions, but unfit
in a natural environment. This can be the case if hybrids
display intermediate traits between parental phenotypes
and, as a result, are poor competitors in either parental
environment. Despite its potential importance to reduce
gene flow, such ecological, postmating barriers have rarely
been investigated in fungi. In the species complex M. violaceum, hybrids between two close species were inoculated
onto both parental host species. In one of the host species,
hybrids performed as well as the parental species specialized for this host, indicating that there are no genetic
incompatibilities in hybrids. However, when inoculated in
the reciprocal host, hybrids did not perform as well as
the parental species specialized for this host, showing that
in other environmental conditions the same hybrids had a
lower viability (Le Gac et al., 2007b).
Mycologists have extensively studied the pre and postmating reproductive barriers that are accessible via
in vitro crosses, namely intrinsic premating mate recognition and intrinsic postmating barriers. Despite the potential
importance of ecological barriers to gene flow, they are still
understudied in fungi. Using fungal systems to investigate
reproductive isolation both in the lab and in nature would
be a great approach to the virtually unexplored question of
the relative contributions of the various reproductive barriers to the decrease of gene flow between sibling species (see
Ramsey et al., 2003, for one of the rare examples of such an
approach, in plants) and to understand which barriers arise
first during speciation.
7. Evolution of reproductive isolation
How does reproductive isolation evolve with time? This
question of primary importance for the understanding of
795
the speciation process has been investigated both theoretically and experimentally during the last decades. In the few
biological models studied so far (mainly animals), some
trends start to emerge (Coyne and Orr, 2004): (1) preand postmating isolation evolve gradually; (2) premating
isolation evolves faster or at the same rate as postmating
isolation; (3) postmating hybrid sterility evolves faster than
postmating inviability; (4) reproductive isolation evolves at
different rates in different groups. Despite their great potential as models for studying speciation, only few studies have
investigated the evolution of reproductive isolation in
fungi. Dettman et al. (2003b) investigated reproductive isolation among five phylogenetic species of Neurospora.
Reproductive isolation was scored according to a scale of
seven categories ranging from the ability to produce
numerous viable ascospores to the inability to produce
any perithecia, and thus encompassed both pre- and postzygotic isolation. Liti et al. (2006) investigated the evolution of postmating isolation among the six species of the
genus S. sensus stricto by measuring hybrid sterility. In
these two studies on Ascomycota, reproductive isolation
increased with genetic distance among species, in agreement with findings in other organisms. Another study
investigating the evolution of reproductive isolation in fungal organisms used the species complex M. violaceum.
Reproductive isolation was measured among 10 species
as the ability to mate, to produce hybrid mycelium, and
to infect plants. Postmating isolation increased with genetic
divergence, but premating isolation did not (Le Gac et al.,
2007b). The absence of correlation between premating isolation and genetic distance in the M. violaceum complex
strongly contrasts with results obtained in other biological
systems, and might be explained by a high selfing rate (Giraud et al., in press). Indeed, if selfing is the rule, the proportion of interspecific matings may be too low for a selection
pressure for assortative mating to have an exist.
Evolution of reproductive isolation is expected to differ
according to the geographic distribution of incipient species. Those evolving in allopatry have no opportunity to
mate with each other, so reproductive isolation is expected
to arise gradually and slowly as a result of independent
mutation, genetic drift, and indirect effects of natural selection driving local adaptation (Coyne and Orr, 2004, pp.
83–110). In contrast, close species in sympatry may have
the opportunity to mate with each other and a strong selection for avoiding interspecific crosses is expected (Coyne
and Orr, 2004, chapters 4 and 10). Altogether, stronger
reproductive isolation is therefore expected between sympatric than allopatric sibling species. Such reproductive
character displacement (i.e., the pattern of enhanced reproductive isolation in sympatry) is expected to mainly affect
premating reproductive isolation and has extensively been
studied in the theoretical literature dealing with reinforcement (Servedio, 2000). This pattern has been detected in
many natural cases, for instance among Drosophila, damselflies, frogs, fish, crickets, toads, birds, marine organisms
and rodents (Coyne and Orr, 2004, pp. 357–360). In a
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T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
comparative study, Le Gac and Giraud (in press) analyzed
published works reporting crossing experiments within 16
species complexes of Ascomycota and 16 of Homobasidiomycota, representing a total of 431 species pairs. In
Homobasidiomycota, sympatric species were virtually
always isolated by very strong premating isolation, as
shown by the absence of clamp connections between sympatric species during in vitro assays, while allopatric species
pairs often display a higher compatibility levels. Homobasidiomycota fungi thus show a pattern of reproductive
character displacement and are a potential empirical model
to study the details of this phenomenon in nature. In Ascomycota in contrast, both sympatric and allopatric species
pairs display a similarly low level of reproductive isolation,
mainly postmating (i.e., reproductive isolation linked to the
inability to produce normal perithecia containing viable
and fertile ascospores). The lack of consistent premating
isolation due to mate recognition among sympatric pairs
of Ascomycota may be due to the presence of species in this
group that mate within their hosts or habitat and for which
specialization could act as a premating barrier (Giraud,
2006a; Giraud et al., 2006).
8. Speciation by hybridization
Many fungal species do not exhibit complete intersterility (Le Gac and Giraud, in press), which gives the opportunity for hybridization. Hybrid speciation is classified
according to the ploidy level of the resulting individuals:
when hybrids have a chromosomal number that sums that
of the parental species, the process is called allopolyploid
speciation, whereas hybrids with ploidy identical to that
of the parents are referred to as allodiploids or
homoploids.
Allopolyploids have a higher ploidy level than the
parental lines, but their karyotype is interestingly often
not the exact addition of the two parental genomes, due
to losses of chromosomes (Leitch and Bennett, 2004). As
a consequence, many ancient polyploidy speciation events
may have been overlooked. Recent allopolyploid hybrids
have however, been identified in diverse genera: Botrytis
allii, the agent of gray mold neck rot of onion and garlic
(Staats et al., 2005), several Neotyphodium species, symbiotic endophytes of grasses (Moon et al., 2004), and several
Saccharomyces species empirically selected for brewing
(Masneuf et al., 1998); see Olson and Stenlid (2002) for
more examples.
The presence of multiple hybrids in some taxa suggests
that hybrids could have selective advantages over parental
species, at least in some cases. Indeed, by combining traits,
allopolyploids of animals and plants exhibit a wider range
of phenotypes making them able to exploit vacant ecological niches (Mallet, 2007; Rieseberg and Willis, 2007a).
Alloploidy would provide simultaneously instant reproductive isolation, due to triploidy in backcrosses, and a new
ecological niche. Examples can be found among grass
endophytes, whose perfect forms belong to the Epichloe
genus and the imperfect stage to the Neotyphodium genus,
which is constituted by hybrids between Epichloe species.
Most of the Neotyphodium hybrid species parasitize grass
species that are not hosts for Epichloe species. Some Neotyphodium hybrid species exploit the same host as one of their
Epichloe parental species, but exhibit different life-history
traits. Indeed, Neotyphodium are mutualistic and asexual,
while Epichloë species are sexual and parasitic, sterilizing
their host during their reproduction (Schardl et al., 1997).
The asexual form of reproduction in Neotyphodium, which
occurs only as vertical seed-borne transmission, is thought
to be a direct result of the hybridization (Kuldau et al.,
1999). These asexual hybrids may thus have been selected
for mutualism: they avoid sterilizing their host, which is
the only way for them to be transmitted (Selosse and
Schardl, 2007). Neotyphodium hybrids further contribute
to host fitness through the combination of several genes
involved in alkaloid production, as these compounds protect the host plants from herbivores (Tanaka et al., 2005).
Neotyphodium grass endophytes may thus have come to
occupy a different ecological niche as the result of hybridinduced asexuality, even when they infect the same host
as their ancestors. Another example of fungal allopolyploids can be found in Cryptococcus neoformans, a human
pathogen causing meningoencephalitis. Diploid hybrids
between two different serotypes have been found to be
highly prevalent in nature and to show higher fitness than
the parental haploid forms, both in vivo and in vitro (Lin
et al., 2007).
Evidence for homoploid speciation comes from a ploidy
level identical to that of its parents and a broad heterozygosity. Contrary to allopolyploids that are reproductively
isolated from their parents, homoploid hybrids are in competition not only with their parents but also with backcrossed individuals, which renders stable allodiploid
species much more unlikely than polyploid ones. A welldescribed case of homoploid speciation is that of the rust
Melampsora columbiana that emerged from hybridization of M. medusa, parasite of Populus deltoides, and M.
occidentalis, parasite of P. trichocarpa (Newcombe et al.,
2000). This hybrid emerged in 1997 when a poplar hybrid
resistant to the two parental rust species was widely grown
in California, the hybrid rust being able to infect the hybrid
poplar. In this case, the homoploid hybrid clearly had a
novel ecological niche, a new host. Another question is
why many loci actually stay heterozygous despite potential
recombination among F1 hybrids? This may be due to a
selective advantage of simultaneous heterozygosity at
many loci.
Interestingly, recent focus on the gene expression in
hybrids provides a potential mechanism for such an advantage. Studies on hybrid strains of Drosophila and Arabidopsis showed that most of the genes exhibiting different
expression levels in the two parental species were not
expressed at an intermediate level in the hybrid. In average,
half of them were underexpressed as compared to the
expression in each of the two species, and one fourth was
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
overexpressed (Landry et al., 2007). Hence, hybridization
may allow exploring fitness landscapes outside that of the
parental species. This would facilitate the maintenance of
hybrids in a new niche and thereby their persistence as a
new species.
9. Chromosomal speciation
Another mechanism allowing instant speciation is chromosomal speciation. The first model of chromosomal speciation (speciation due to chromosomal rearrangements)
considered that if two isolated populations had fixed
karyotypic differences, and that recombination between
rearranged chromosomes were generating unbalanced
gametes that lowered fitness, between-population gene flow
could be prevented upon secondary contact (White, 1978).
This model was then dismissed on the rationale that rearrangements that cause a sufficient reduction in fitness in
heterozygotes could not be fixed in a population precisely
because of this reduction in fitness. When at low frequency,
the rearrangement will indeed always be in a heterozygous
state, and should not be able to increase in frequency.
Fungi may however be some of the rare organisms where
this speciation scenario could occur because of asexual
reproduction and selfing that allow mutants with karyotypic rearrangements to reproduce without loss of fitness.
New models of chromosomal speciation consider that
the effects of chromosomal rearrangements on recombination rates are more important than those on fitness to
explain speciation: a chromosomal rearrangements creates
a large region of suppressed recombination where one or
more specialization genes can accumulate and lead to the
localized restriction of gene flow, which could eventually
drive the populations to speciation (Noor et al., 2001;
Rieseberg, 2001). Chromosomal rearrangements seem frequently involved in plant speciation, as indicated by the
findings that sterile plant hybrids often recover fertility
after chromosomal doubling, which furnishes an exact
homolog for each chromosome, and by mapping of genetic
incompatibilities, which frequently fall into chromosomal
rearrangements (Rieseberg and Willis, 2007).
In fungi, the small size of chromosomes has long been a
barrier to the study of chromosomal rearrangements and
chromosomal speciation. The invention of the pulsedfield-gradient-gel-electrophoresis allowed the separation
of intact fungal chromosomes and revealed that an extremely high proportion of fungal species exhibited chromosome-length-polymorphism
(CLP).
Chromosomal
rearrangements leading to CLP reported in fungi include
deletions, reciprocal and insertional translocations, chromosome breakage and fusion or complete chromosome
loss, which may in large part be due to transposable elements and other dispersed repetitive sequences (Burnett,
2003; Zolan, 1995).
In the ascomycete Sordaria macrospora and in the basidiomycete Coprinus cinereus, intraspecific sexual crossing of
strains harboring different karyotypes resulted in low fertil-
797
ity in the progeny, concordant with the idea that chromosomal rearrangement can play a role in the speciation
process (Poggeler et al., 2000; Zolan et al., 1994). However,
these karyotypically differentiated strains may also have
differed in their genic content. In order to isolate the effect
of karyotypic rearrangement, Delneri et al. (2003) elegantly
constructed strains of Saccharomyces cerevisiae differing
uniquely by the presence of reciprocal translocations but
otherwise completely isogenic. They showed that crosses
between such strains had lowered spore fertility and proposed that chromosomal rearrangements, for yeasts at
least, are able to provide partial isolation. Chromosomal
rearrangements can thus theoretically have a role in speciation in fungi, but showing that the rearrangements were a
cause of the divergence, and not only its consequence,
remains a challenging task.
10. Role of epigenetic mechanisms in speciation
Major factors responsible for postmating reproductive
isolation are inherited directly as part of the parental
DNA, such as rapidly evolving genes that may interact
incompatibly in hybrids (the Dobzhansky–Müller model)
or chromosomal rearrangements that produce segmental
deletions in meiotic products. However, the recent appreciation that genetic systems function largely under epigenetic
control mechanisms should let us consider that these may
also be involved in reproductive isolation.
Epigenetic mechanisms have important roles in preserving genomic integrity and in defending the genome against
selfish DNA elements, such as transposons. The types of
sequence and structural irregularities in hybrid genomes
may interact with epigenetic controls in ways that limit survival, and in several cases the disabling of epigenetic mechanisms increases the viability of hybrid crosses. For
example, in the Saccharomyces sensu stricto yeasts, a mismatch repair system prevents chiasma formation when
DNA sequences show marked dissimilarity. Overall divergence can so strongly prevent recombination that chromosomes fail to segregate properly during meiosis. Mutation
to MSH2 in the mismatch repair system increased the relative fertility of crosses, even for intraspecific combinations
between distantly allopatric populations, suggesting that
the epigenetic mechanism may play a role in the early
stages of speciation (Greig et al., 2003).
Another hallmark of hybrid genomes is the presence of
repeated and non-syntenic DNA sequences that result from
allopolyploidy or smaller-scale chromosomal and segmental duplications. Failure of proper meiotic divisions is the
cause of unbalanced gametes, and haploids possessing
duplications are often more likely to survive than those suffering major deletions. Epigenetic defenses in fungi against
autonomously replicating DNA elements, such as retrotransposons, are able to detect and silence repetitive
sequences. In Neurospora crassa, sequences in regions of
the genome that fail to pair normally during meiosis are
silenced by a process involving bi-directional transcription
798
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
that presumably feeds into an RNA-interference pathway
(Matzke and Birchler, 2005). This ‘‘Meiotic Silencing of
Unpaired DNA » depends partly upon an RNA-dependent
RNA polymerase (Sad1), which when disabled restores fertility to sterile hybrids between Neurospora species (Shiu
et al., 2001). In a similar way, the genome defense against
transposable elements known as Repeat-Induced Point
Mutation (Galagan and Selher, 2004) may be expected to
act upon the segmental duplications and aneuploidy in
hybrids. This system is found in diverse fungi and causes
hypermutation of sequence repeats that lead to their nonfunctionalization. Thus a variety of epigenetic mechanisms
may contribute to postmating isolation in fungi, but the
extent to which they plays an important role in the speciation process in nature remains largely unknown.
11. Cospeciation and host shifts
In host–parasite and host–symbiont associations, speciation in one of the interacting organisms, particularly the
host, can lead to speciation in the other. As a consequence
of such cospeciations, a strong congruence (topological
similarity) is usually expected between the host and parasite/symbiont phylogenies. High congruence has in fact
been observed in some host–parasite associations involving
animals, such as gophers and lice (Hafner et al., 1994), but
such patterns are not the rule. Host shifts, sorting events
(e.g. extinctions), and duplications of parasite lineages have
been invoked to account for reduced similarity between the
phylogenetic trees of hosts and their parasites or symbionts
(for a review, see Page, 2003). In order to elucidate whether
cospeciations or host shifts have been prevalent, one has
therefore to test if the level of congruence is significantly
higher than that expected by chance (de Vienne et al.,
2007a; Legendre et al., 2002), and then to reconstruct the
most likely evolutionary history of the association. Methods have been developed to do so, implemented in software
such as Treefitter or Treemap (reviewed in Page, 2003,
Chapter 1). These methods however rely on a priori
assumptions about the relative likelihoods of host shift
and cospeciation events, which can be challenging to
estimate.
Many fungi interact with other species, as symbionts or
parasites, but also sometimes as hosts. Most fungi develop
in association with a restricted number of host species and
are therefore good candidates for cophylogenetic studies.
Because fungal spores are most often widely disseminated
in the environment, a high number of spores are likely to
land on non-suitable hosts where for obligate pathogens
and symbionts they must either cause infection or die. Both
opportunities and selection for the utilization of a new host
should therefore be frequent. As a consequence we expect
cophylogenetic analyses dealing with fungi to reveal high
levels of incongruence between host and parasite trees. This
is indeed the case in many plant–fungal associations (Jackson, 2004), such as in the Puccinia-Crucifer (Roy, 2001) or
the Microbotryum-Caryophyllaceae (Refrégier et al., in
press) systems. Host shifts are therefore frequent in many
fungal pathogens, showing that apparently strict host specificity is not sufficient to impede host shifts over the long
term. This idea has also been supported by the observation
of incipient host shifts (Antonovics et al., 2002; Lopez-Villavicencio et al., 2005).
In other fungi however, high levels of congruence are
observed, as in the Golovinomyces-Asteraceae (Jackson,
2004) or the Epichloe-grass associations (Schardl et al.,
1997, in press). Another interesting case of highly congruent phylogenetic histories is that of fungi belonging to the
Agaricaceae and Tricholomataceae families, being simultaneously cultivated by fungus-growing ants and hosts of the
parasitic Escovopsis species of fungi. Research over the last
decade has revealed a high degree of congruence between
both ant and cultivated fungi and between the cultivated
fungi and Escovopsis (Currie et al., 2003 and references
therein), suggesting that cocladogenesis occurred in this tripartite interaction. Interestingly, the fungal symbionts do
not appear to have free dispersal stages, which could
restrain the possibility of host shifts. However, congruence
between host and parasite trees is not always evidence for
rampant cospeciations because host shifts can give rise to
congruent phylogenies if they occur preferentially towards
closely related hosts (de Vienne et al., 2007b). Temporal
concordance therefore needs to be assessed with appropriate tests in order to elucidate whether cospeciation has
occurred (de Vienne et al., 2007b; Hirose et al., 2005;
Schardl et al., in press).
In any case, cospeciations and host shifts are not strictly
speaking modes of speciation. They are rather related to
the evolutionary forces of ecological divergence leading
to speciation. To occur, cospeciation and host shifts
require allopatry or the evolution of reproductive isolation,
following the mechanisms explained above.
12. Asexual fungi
In asexual fungi, the theoretical issues of species formation are completely different from those in sexual organisms.
There is no recombination to break down combinations of
multiple alleles adapted to a given habitat, and the selective
pressure on one gene has an effect on the whole genome. Any
new allele allowing adaptation on a new niche can thus give
rise to a new ‘‘species”. The difficulty in asexual organisms is
rather to understand if, and why, discrete entities exist that
we can recognize as species, instead of continuous distributions of phenotypes/genotypes. Asexual organisms in fact
seem to form discrete species (Fontaneto et al., 2007), and
the hypotheses invoked to explain their existence despite lack
of homogeneizing gene flow are the existence of discrete ecological niches, random processes of extinctions of intermediate genotypes/phenotypes (Coyne and Orr, 2004, pp. 17–22),
or the recurrent apparition of asexual species from sexual
ones (LoBuglio et al., 1993).
The clonal fungus Penicillium marneffei, the causal agent
of disease in immuno-compromised humans, exhibits geo-
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
graphic endemicity despite long-distance migration via
aerially dispersed spores. DNA multilocus typing showed
that different clones of the fungus are associated with different environments (Fisher et al., 2005), which suggested that
adaptation to these environments is constraining the
organism’s ability to successfully disperse in nature. The
population structure in asexual parasites may thus directly
reflect host or habitat adaptation, at all loci, because selection at one locus results in hitchhiking of the whole
genome.
Another example is the Magnaporthe grisea complex,
many species of which are strictly asexual and host-specific.
One of the species of this complex, M. oryzae, an important
fungal pathogen of rice, has been shown to have arisen
recently, possibly in association with rice domestication
(Couch et al., 2005). Isolates from rice, millet, cutgrass,
and torpedo grass appeared also strictly asexual, and to
constitute recent host-specific lineages. These patterns in
the M. grisea complex appear consistent with the idea that
acquisition of abilities to infect new hosts in asexual parasitic fungi can readily form new species because recombination will not prevent the differentiation from the ancestral
populations.
Fungi, with their enormous diversity of modes of reproduction, seem ideal subjects to test the different hypotheses
on the nature of species in non-recombining organisms
(Coyne and Orr, 2004, pp. 17–22). In some asexual fungi
however, recombination can still occur between individuals
via somatic recombination (Bos, 1996), which can be considered as equivalent to sex as regards the speciation issue.
Hyphal fusions between genetically different individuals is
controlled by elaborate vegetative compatibility systems
(Bos, 1996), resulting in a condition of heterokaryosis.
The exchange of nuclei and organelles can lead to parasexuality via highly transient nuclear fusion and subsequent
chromosomal segregation and/or ameiotic recombination
(Fincham et al., 1979). In fungi undergoing such somatic
recombinations, vegetative compatibility groups (VCG)
could be considered as reproductively isolated from each
other and therefore as distinct species. This as been suggested in Aspergillus flavus, where the different VCGs
indeed formed genetically distinct lineages (Ehrlich et al.,
2007).
799
strongly influenced by positive selection, with functions
not directly involved in reproductive isolation (Wu and
Ting, 2004). This is consistent with the Dobzhansky–Müller model (Orr and Turelli, 2001), with the reunion of rapidly evolving genes (for reasons other than reproductive
isolation) with interacting gene products causing incompatibilities in hybrids and thus postmating isolation.
In fungi, little investigations have been undertaken to
understand the genetics of speciation. Fives genes have
been shown to be involved in the intersterility among Heterobasidion species (Chase and Ullrich, 1990), but they
have not been characterized yet. They do not include the
mating type genes, although we could have expected that
the mating type locus could play a role in sexual isolation
(Natvig and May, 1996). Mating types in fact appear not
to be involved in sexual isolation in general among fungal
species, where mating type alleles indeed exhibit extensive
trans-specific polymorphism (James et al., 2006) and can
be functional when transferred in other species (Arnaise
et al., 1993). Mating types may play a role in sexual isolation in other species. It has for instance been shown that
the pheromones and pheromone receptor of Ustilago hordei MAT-1 were necessary and sufficient to make U. maydis
compatible with U. hordei MAT-2 (Bakkeren and Kronstad, 1996). The pheromone signal system may also play a
role in reproductive isolation among heterothallic Neurospora species (Karlsson et al., 2008).
In the cases of reproductive isolation by mere specialization, the genes allowing specialization should also be the
speciation genes (Giraud, 2006a; Giraud et al., 2006). In
asexual species too, genes responsible for host shift could
also be those present at the origin of new species. More
generally, the genes involved in ecological divergence
between incipient species may also be fascinating to discover, as we have seen that they play prime roles in speciation. Also, as discussed above, genes of the mismatch
repair or silencing machineries could be considered as speciation genes in some cases. Because postmating isolation
seems to arise gradually with time (Le Gac and Giraud,
in press; Le Gac et al., 2007b), it is likely that many genes
are involved, possibly by incompatibility between loci (Orr
and Turelli, 2001). Several fungal species complexes should
be amenable to genetic dissection of the postmating incompatibility, which should provide highly illuminating results.
13. Genetics of speciation
14. Conclusion
Knowing which genes are involved in reproductive isolation may help get a better understanding of speciation processes. The genetics of speciation has just begun to shed
some light on the evolution of reproductive isolation, but
the focus so far has been mostly on insects (Wu and Ting,
2004). Genes reported to cause premating isolation are
often involved in sexual pheromones (Dallerac et al.,
2000; Thomas et al., 2003), in host choice for parasites mating on their host (Dambroski et al., 2005), and in gametic
compatibility (Galindo et al., 2003; Lee et al., 1995). Genes
involved in postmating isolation have been found to be
In conclusion, important advances have been made
recently on the speciation in fungi, and they have proved
tractable biological models for the general study of speciation. Fungi also exhibit some specific and interesting modes
of speciation, and many open questions remain which will
be fascinating to explore. Recently developed analytical
methods for studying past gene flow and differentiation
should be useful to determine in which cases fungal speciation by specialization onto novel hosts has occurred in
sympatry (Hey and Nielsen, 2004; Hey et al., 2004). Deci-
800
T. Giraud et al. / Fungal Genetics and Biology 45 (2008) 791–802
phering the genetics of speciation should also prove to be
fascinating, for instance by finding markers segregating
with inviability or sterility in interspecific progeny. Another
promising approach to understand speciation may be
experimental evolution, which has already been used in a
few excellent works in yeast. These experimental evolution
studies showed that assortative mating could evolve under
selection against hybrids as expected by reinforcement theory (Leu and Murray, 2006), and that adaptation to
divergent environments promotes the evolution of postmating isolation (Dettman et al., 2007), as expected in
the Dobzhansky–Müller model. Fungi should bring
many other unique results to the field of speciation in the
coming years.
Acknowledgment
We thank Pierre Gladieux for comments on a previous
version of the manuscript and Jim Kronstad for initiating
this review. We acknowledge the grant ANR-06-BLAN0201.
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