doi: 10.1111/j.1420-9101.2012.02495.x

REVIEW Sex, outcrossing and mating types: unsolved questions in fungi and beyond

S. BILLIARD*, M. LO´ PEZ-VILLAVICENCIO ,M.E.HOODà &T.GIRAUD§– *Laboratoire de Ge´ne´tique et Evolution des Populations Ve´ge´tales, UMR CNRS 8016, Universite´ des Sciences et Technologies de Lille – Lille1, Villeneuve d’Ascq Cedex, France Origine, Structure, Evolution de la Biodiversite´, UMR 7205 CNRS-MNHN, Muse´um National d’Histoire Naturelle, Paris Cedex, France àDepartment of Biology, McGuire Life Sciences Building, Amherst College, Amherst, MA, USA §Ecologie, Syste´matique et Evolution, Universite´ Paris-Sud, Orsay cedex, France –Ecologie, Syste´matique et Evolution, CNRS, Orsay cedex, France

Keywords: Abstract ascomycete; Variability in the way organisms reproduce raises numerous, and still asexual reproduction; unsolved, questions in evolutionary biology. In this study, we emphasize that basidiomycete; fungi deserve a much greater emphasis in efforts to address these questions breeding systems; because of their multiple advantages as model eukaryotes. A tremendous diploid selfing; diversity of reproductive modes and mating systems can be found in fungi, gametophytic selfing; with many evolutionary transitions among closely related species. In addition, haploid selfing; fungi show some peculiarities in their mating systems that have received little mating systems; attention so far, despite the potential for providing insights into important oomycetes; 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 mainte- nance 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.

fields of biotechnology, breeding and artificial selection Introduction (Whitton et al., 2008), conservation biology (e.g. Fre´ville The process of reproduction is one of the most variable et al., 2007), species invasion (Barrett, 2011) and path- traits in the living world, with organisms giving rise to ogen evolution and control (Shea et al., 2000; Zuk, progeny either clonally or sexually, and in the latter case 2009). by selfing or outcrossing (see the Glossary for defini- The prevalence of sexual or asexual reproduction is tions). Understanding what factors shape an organism’s highly variable among eukaryotes, with the vast majority reproductive mode is of fundamental importance because of taxa being able to perform sexual reproduction either patterns of inheritance drastically affect major evolution- exclusively (e.g. mammals) or alternatively with clonality ary and ecological processes (adaptation, e.g. Charles- (e.g. aphids, ciliates, many angiosperms and fungi). worth & Charlesworth, 1995; Otto, 2009; colonization, Sexual reproduction implies the succession of haploid e.g. Busch, 2011; Richards, 2003), as well as the applied and diploid phases, transitions occurring by , where recombination and chromosomal segregation Correspondence: Sylvain Billiard, Laboratoire de Ge´ne´tique et Evolution des occur, and syngamy, where two haploids fuse. The Populations Ve´ge´tales, UMR CNRS 8016, Universite´ des Sciences et long-term persistence of eukaryotes relying exclusively 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; on asexual reproduction is rare and is thought to include e-mail: [email protected] 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 extended haploid life stage such as ascomycete fungi, and some plants (Judson & Normark, 1996). Even in mosses, ferns and some algae, selfing is sometimes these textbook examples of asexuality, however, recent possible through the fusion of two mitotic descendants studies have suggested the occurrence of cryptic sex, in of the same haploid cell, which is called intrahaploid particular, because all the genes required for the meiotic mating, intragametophytic selfing (Hedrick, 1987), same- machinery are maintained in the genomes (Schurko clone mating (Perrin, 2012), gametophytic selfing (Epinat et al., 2009; Halary et al., 2011). & Lenormand, 2009) or haploid selfing (Billiard et al., Mating systems, governing which haploids fuse at 2011). Diploid selfing and haploid selfing can have syngamy, are also highly variable: most species under- very different consequences for genetic structure, and go predominantly selfing or outcrossing, whereas a few care should be taken in the application of different species show a mixed-mating system in both animals terms to describe their occurrence in eukaryotes. and plants (e.g. Jarne & Auld, 2006; Igic & Kohn, The proximate mechanisms controlling mating systems 2006). Outcrossing results from the syngamy between are also highly diverse (sometimes called ‘breeding haploid cells produced by separate diploid individuals, systems’, Neal & Anderson, 2005); obligate outcrossing whereas selfing results from the syngamy between can result, for instance, from the existence of (i) separate haploid cells produced by the same diploid individual sexes (even though outcrossing may not be the main (Figs 1 and 2). Selfing occurs in plants and animals force responsible for the evolution of separate sexes), through the fusion of gametes produced from meioses (ii) sexual morphs (e.g. heterostyly in angiosperms, of a single diploid individual, referred to as diploid where two morphs with different stigma and pistil selfing. In some eukaryotes, in particular those with an lengths coexist in populations, mating being only possible

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Fig. 2 Illustration of the different modes of reproduction and mating systems in fungi. between individuals of different morphs), (iii) molecular Evolutionary biologists attempt to explain the deriva- recognition mechanisms (e.g. self-incompatibility sys- tion of particular reproductive modes in terms of fitness tems in angiosperms), or (iv) asynchrony in the produc- benefits and costs. Sexual reproduction can induce tion of male and female gametes in an individual (see fitness costs through energetic, genetic transmission Barrett, 2010 for a review). The tendency for selfing can and demographic constraints compared to asexual repro- result, for instance, from nonopening hermaphroditic duction (see Otto, 2009; Lehtonen et al., 2012, for flowers (cleistogamy) or developmental proximity be- reviews). When sex requires males that contribute only tween gametes from the same individual (Giraud et al., with their genes and do not directly produce offspring, a 2008). allowing females to reproduce independently Biologists have struggled for more than a century to of males, either asexually or through various forms of identify factors responsible for the evolution of such self-fertilization, is expected to invade because its prog- preference for outcrossing vs. selfing or for sexual vs. eny grows at twice the rate of the ancestral lineage (the asexual reproduction. Sexual reproduction has appar- famous ‘two-fold cost of males’; Maynard Smith, 1978). ently been lost independently in diverse eukaryotes Recombination can furthermore break locally adapted (reviewed in fungi, Billiard et al., 2011; Lobuglio et al., combinations of alleles at multiple loci, which is called 1993; Lo´ pez-Villavicencio et al., 2010; Kuhn et al., 2001; the recombination load. Even in organisms without in microsporidia, Ironside, 2007; in animals, Simon et al., either separate sexes or outcrossing, however, meiosis 2003; and in plants, Whitton et al., 2008). However, itself can be costly because it takes more time and energy there is growing evidence that some species long thought than mitotic cell divisions. Other costs of sex may apply to be asexual undergo cryptic sex (Burt et al., 1996; in specific systems: cost of finding and courting a mate, Schurko et al., 2009; Lee et al., 2010). Nevertheless, risk of predation or contracting sexually transmitted anciently asexual lineages undoubtedly exist in many diseases or parasitic genetic elements. On the other hand, different taxonomic groups, as revealed, for instance, by sex is considered to provide benefits, both proximate (e.g. the Meselson effect, in which the divergence between DNA repair) and ultimate, by limiting the accumulation alleles in the two nuclei of each cell is so great that it can of deleterious and by creating novel and only be explained by a long divergence without recom- advantageous genetic combinations, especially in envi- bination (Kuhn et al., 2001; Enjalbert et al., 2002; Roose- ronments varying in space and time. Lineages that Amsaleg et al., 2002; Schurko et al., 2009). Similarly, undergo only asexual reproduction or obligate selfing transitions between outcrossing and selfing preferences in fact tend to have high extinction rates (Beck et al., have occurred independently in the evolution of many 2011; Goldberg et al., 2010; Igic et al., 2008; Simon et al., plant groups (for instance, the gain or loss of heterostyly, 2003). e.g. Barrett & Shore, 2008; Busch, 2011; and in animals, Biological groups exhibiting a large diversity of repro- Jarne & Auld, 2006). ductive strategies provide unique opportunities to iden-

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tify the factors underlying the evolution of sex and fitness of the progeny resulting from different modes mating systems. The majority of theories and observa- and systems of reproduction. tions have been based on vertebrate, insect and plant models, whereas other groups of eukaryotes, with even 1. The different modes of reproduction more diverse reproductive strategies, have too often been and mating systems in fungi excluded from the debate (Birky, 1999). Groups such as fungi are excellent models to study these topics (Lee Fungi exhibit a huge variety of life cycles, but we tried to et al., 2010; Billiard et al., 2011; Whittle et al., 2011). draw in Figs 3 and 4 some typical sexual cycles of Fungi include species with obligate sexual species (e.g. filamentous ascomycetes and mushrooms (homobasidio- Microbotryum, Giraud et al., 2008), species exhibiting both mycetes), respectively. Most fungi are able to undergo sexual and asexual reproduction and others appearing both asexual reproduction and sexual reproduction strictly asexual (Taylor et al., 1999; Roose-Amsaleg et al., (Figs 1 and 2), where, as in other sexual eukaryotes, 2002; Schurko et al., 2009); there seem to have been tightly regulated mechanisms determine which haploid multiple transitions from sexuality to asexuality (Lobu- cells can fuse at syngamy. However, additional possibil- glio et al., 1993; Lo´pez-Villavicencio et al., 2010; Coelho ities of syngamy exist in fungi as compared to plants and et al., 2011; Schurko et al., 2009). Fungi are also poten- animals. In fungi considered to be ‘heterothallic’, haploid tially informative because they present a huge diversity selfing is prevented because syngamy can only occur in the degree of haploid and diploid selfing rates (Billiard between haploid cells carrying different alleles at the et al., 2011), and they provide significant advantages over mating type locus ⁄ loci (Fig. 1, Billiard et al., 2011). other eukaryotic models for empirical studies (Goddard However, in homothallic fungi, syngamy can occur et al., 2005, Bruggeman et al., 2003, 2004). For instance, between genetically identical haploid cells, that is, clones, many can easily be cultivated under laboratory condi- resulting in haploid selfing (Figs 1 and 2, Billiard et al., tions, can be cloned, have relatively short generation 2011). Several proximal mechanisms may confer homo- times and can be revived after long-term frozen storage, thallism in fungi: most often each haploid carries two which is then useful in studies of experimental evolution. active mating type alleles (Coppin et al., 1997), whereas Here, we thus would like to argue that fungi have in some species haploid individuals can ‘switch’ which much to bring to questions on the evolution of sex and mating type allele is expressed (e.g. in Saccharomyces mating systems, being tractable experimental models for cerevisiae, Haber, 1998). In other species, syngamy can addressing the advantages and costs of the various simply occur between haploid cells carrying and express- reproductive systems, and would benefit from being ing the same single mating type allele (i.e. ‘same-sex studied further within an evolutionary context. Several mating’, Alby et al., 2009; Fraser et al., 2005; Lin et al., concepts referring to the mating system of fungi, how- 2005; Metzenberg & Glass, 1990). In homothallic fungi ever, need clarification because the same terms (e.g. where mating type switching occurs, haploid selfing selfing and outcrossing) are used for different phenom- results from syngamy between cells genetically identical ena with completely different evolutionary consequences except at the expressed mating type locus. (Giraud et al., 2008; Neal & Anderson, 2005). Further- In both heterothallic and homothallic fungi, two other more, knowledge on the advantages and costs of modes types of syngamy, which are also common in plants and of reproduction and mating systems in fungi can have animals, can occur: (i) syngamy between two different direct applications, for instance, to the numerous fungi meiotic products originating from a single diploid indi- used in industry or that threaten health and agricultural vidual, that is, diploid selfing (more commonly referred production. to simply as selfing in plants and animals, but the term We first describe the different modes of reproduction ‘diploid selfing’ here allows the distinction from haploid and mating systems in fungi, highlighting frequent selfing) and (ii) syngamy between meiotic products misconceptions, and we review their respective evolu- originating from two different diploid individuals, which tionary benefits and costs. We will also consider is called outcrossing (Figs 1 and 2). oomycetes, which are protists, but have long been It should be noted that molecular and developmental studied by mycologists and share with fungi many systems of self-incompatibility exist in plants and animals morphological and genetic peculiarities. We argue that preventing diploid selfing (such as dioecy or self-incom- understanding the evolution of modes and systems of patibility alleles systems in angiosperms), but these are reproduction, especially in fungi, requires (i) assessing absent in true fungi where mating compatibility is their frequencies in natural populations, and we will determined by the genotype of haploid nuclei. Fungal review the (scarce) evidence available in the literature, species are always able to undergo both outcrossing and (ii) mapping these, as well as other life-history traits diploid selfing (Giraud et al., 2008); mating types in fungi onto phylogenies, and (iii) measuring experimentally can only prevent haploid selfing in heterothallic species. the advantages gained by the different modes of Nevertheless, some life-history traits may increase the reproduction and mating systems. We will propose probability of outcrossing in fungi. For example, many some possible experimental settings to compare the species of ascomycetes and rust fungi exhibit a long-

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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. trad mating (see Fig. 2; e.g. the anther smut fungi The random encounter of haploid genotypes should Microbotryum, Giraud et al., 2008). The genetic conse- cause outcrossing to be more prevalent than diploid quences of automixis differ from diploid selfing, due to selfing, although this remains to be investigated by the restriction of the effective population of mating analyses in natural populations. partners and the tendency to retain heterozygosity Quite to the opposite extreme, cases exist where haploid (Kirby, 1984; Hood & Antonovics, 2004). dispersal is very limited or nonexistent and mating occurs Several ascomycete and basiomycete fungi have been among the immediate products of meiosis, a mating characterized as being ‘pseudo-homothallic’, due to their system called automixis (Mogie, 1986), that is, intrate- 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 considered the ‘thallus’. This situation is confusing, haploid nuclei of opposite mating types and from a single especially as the terms ‘homothallism’ and ‘heterothal- meiosis in the dispersed spore (e.g. tetrasperma, lism’ are also used interchangeably with ‘selfing’ and Merino et al., 1996; Raju & Perkins, 1994; Agaricus bisporus, ‘outcrossing’ in both oomycetes and fungi. It is essential Callac et al., 2006; Saccharomycodes ludwigii, Zakharov, to distinguish the types of syngamy illustrated in Figs 1 2005). Here, a developmental process allows, and even and 2 with the consistent use of terminology, because seems to promote, the automictic combination of post- there are different consequences in evolutionary terms, meiotic nuclei. However, here again one can wonder as explained in the section 2 below. whether the cosegregation of two haploid nuclei of More generally, the confusion over the different forms opposite mating types has been selected to favour of the term ‘selfing’ as described above stems from the automixis or to allow universal compatibility under fact that ascomycetes have a long-lasting haploid stage, outcrossing. The presence of nuclei of the two mating so that the ‘individual’ is considered as the haploid types within dispersing spores can indeed ensure that the mycelium or cells. Thus, haploid selfing is called selfing spore will be able to mate with any first encountered in ascomycetes, and it remains essential to distinguish haploid. Interestingly, recent studies have suggested that diploid selfing from haploid selfing because of the pseudo-homothallic fungi may exhibit a mechanism different effects on genetic structure, in particular the favouring outcrossing over automixis. In A. bisporus,a genome-wide distribution of heterozygosity. We there- choice experiment in seminatural conditions has shown fore argue that, even in ascomycetes, the syngamy that outcrossing was indeed more frequent than autom- between identical haploids should be consistently ixis in this pseudo-homothallic mushroom (Callac et al., described by the term ‘haploid selfing’. For the same 2006). In this case, the cosegregation of two meiotic reason, outcrossing for ascomycetes often refers to mating products of opposite mating types in a spore may have with a nonidentical haploid (i.e. ‘nonhaploid selfing’), actually evolved for allowing universal mating compati- which can actually be either diploid selfing or true bility that achieves outcrossing rather than to favour outcrossing in the classical sense for plants and animals automixis (Billiard et al., 2011). It should be noted, (i.e. syngamy between products of meiosis of different however, that the choice experiment was performed in diploid individuals). The use of nonambiguous terms is conditions where spore inoculum was maybe higher than important for understanding the underlying concepts and in natural conditions (Callac et al., 2006) and pseudo- evolutionary consequences, and for rendering the litera- homothallism may also allow intertetrad mating when no ture on fungi useful, without misunderstandings, for other mating is available. In contrast, a few basidiomy- evolutionary biologists less familiar with fungal life cycles. cetes do undergo preferentially automixis, even when gametes from other diploid individuals are available, via a 2. Evolutionary benefits and costs developmental specificity promoting automixis (e.g. the of the different modes of reproduction, anther smut fungi Microbotryum, Giraud et al., 2005). breeding systems (homothallism vs. The terms ‘homothallism’ and ‘’ are also heterothallism) and mating systems used to describe the phenomena in other groups as (selfing vs. outcrossing) fungal-like oomycetes, which have been studied for long by mycologists. Nevertheless, the genetic basis and the 2.1. Costs and benefits of sex evolutionary consequences are strikingly different from true fungi (Fig. 1). In oomycetes, which are phylogenet- The costs and benefits of sex have been briefly presented ically closer to brown algae and diatoms, ‘heterothallism’ in introduction and have been extensively reviewed and ‘homothallism’ are used to describe how sexual elsewhere (e.g. Otto, 2009; Lehtonen et al., 2012). reproduction is started: heterothallic oomycetes cannot However, some classically assumed costs of sex cannot undergo gamete production and sexual reproduction be applied directly to fungi because many species are unless an individual of the opposite mating type is isogamous and determine mating compatibility only present, while homothallic oomycetes can (Fig. 1, see during the haploid stage through molecular means of also Judelson, 2007). Once gametes are produced, diploid nonself-recognition. Furthermore, many sexual fungi selfing and outcrossing are possible in both, homothallic undergo multiple rounds of asexual reproduction for and heterothallic oomycetes. Haploid selfing (a kind of each sexual cycle, thus benefiting both from the recom- syngamy possible in homothallic fungi) is prevented in binational advantages of sex and from the demographic oomycetes due to the lack of mitotic multiplication of advantages of clonality. gametes, as in plants and animals. The terms ‘homothal- lism’ and ‘heterothallism’ thus correspond to different 2.2. Costs and benefits of homothallism phenomena in different organisms. This is probably because of the potential to isolate haploid cell lineages Although it has been traditionally accepted in mycology (therefore considered as the ‘thallus’) in fungi, but this is that homothallism (i.e. the possibility for an haploid not possible in oomycetes, where the diploid stage was individual to mate with any other haploid individual,

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even itself, Fig. 1) evolved to favour haploid selfing, it is universally compatible mutants (homothallic mutants) important to recognize that the potential for homothal- from an ancestral heterothallic population. lism as determined by haploid selfing in the laboratory Homothallism, whatever the proximal mechanism, does not imply that this is the prominent mating system could thus have evolved for universal compatibility of of the species in nature. The prevalence of haploid gametes under outcrossing when there is a cost in finding selfing in nature is difficult to assess. Indeed, most a mating partner and little risk of engaging in haploid homothallic species can also reproduce asexually and selfing, for instance, due to gamete dispersal. In case the these two modes of reproduction cannot be distin- gametes remain near their mother for mating, homo- guished by population genetics analyses. In order to thallism may be selected against to avoid haploid selfing measure the actual frequency of haploid selfing in that brings no recombinational advantage but still incurs nature, genetic analyses of progeny in sexual structures some costs of sexual reproduction (section 2.5 below; collected in nature are required. Moreover, it is not Giraud et al., 2008). If this is the case, a prediction would clear why having sex with an identical haploid would then be that homothallic fungi are predominantly out- be favoured. Two benefits are possible: benefits from crossing. This prediction may seem paradoxical at first segregation and those from recombination. Concerning sight because it is generally believed that homothallism the segregation advantage, Epinat & Lenormand (2009) evolved to promote selfing. Studies on the population showed, in a model with a modifier of haploid selfing genetics analyses in homothallic species are rare, but partially linked to a locus under diploid selection, that several in fact have found evidence of outcrossing (see haploid selfing may be favoured when the decrease in section 3.2). In the homothallic nidulans, it has the mean fitness of offspring due to depres- been suggested that mating occurs preferentially between sion is low relative to the advantages of the transmis- genetically different individuals (which has unfortu- sion of selfing and of the increase in the fitness variance nately and confusingly been called ‘relative heterothal- in offspring. Concerning the recombination advantage, lism’, Pontecorvo et al., 1953). there are no models to our knowledge investigating the Nevertheless, some homothallic fungi (e.g. homothallic possible recombination advantage or costs of homothal- species of Neurospora) have been suggested to rely mainly lism but we can expect a priori that haploid selfing on haploid selfing for reproduction because of the lack of should confer no recombinational advantages over conidia (male gametes) production (Glass & Kuldau, asexual reproduction, and it should still incur some of 1992). In some other ascomycetes, such as the sexual the costs of sex, such as the physiological costs involved Talaromyces species (Stolk & Samson, 1972), the male in utilizing the meiotic machinery and costly sexual structures surround the female organs, which may in fact structures. Other costs of sex should, however, be promote haploid selfing. In these homothallic species that minimal under haploid selfing, in particular avoiding seem to reproduce sexually only via haploid selfing, it is the costs of finding a mate and of risking parasite intriguing why sex is retained instead of asexuality, which transmission. would allow bypassing the physiological costs of meiosis Instead of having evolved to favour haploid selfing, and syngamy. If sex via haploid selfing is disadvantageous homothallism could alternatively be viewed as a lack of over the short term compared to asexual reproduction, discrimination at syngamy: in homothallic species, each because it incurs costs without providing recombinational haploid is compatible with all other haploids in the advantages, it should be lost. It has been proposed that population (including incidentally, genetically identical haploid selfing could, nevertheless, be maintained if it haploids). In fact, several models suggest that such a increased the chance to engage in occasional outcrossing universally compatible mutant should easily invade (Lee et al., 2010); this would work only if outcrossing populations when there is a cost for waiting for a occurs frequently enough to counter the expected rapid compatible mate (Iwasa & Sasaki, 1987). If we consider invasion by asexuals, in which case we should detect such a fungal species experiencing random encounters of events of outcrossing in natural populations. Unfortu- haploids because its gametes widely disperse, homothal- nately, too few studies exist that investigate mating lism as a mutant form should be selected for because it systems in natural populations (see section 3). allows compatibility with all other gametes in the Some advantages of sex that are not related to recom- population. An interesting observation is that some bination have been proposed to exist in fungi, and they species have recently been reported to exhibit polymor- could provide advantages to haploid selfing over asexu- phism for homothallism that may reflect the invasion of ality, as explained in the section 2.3 below. universally compatible mutants (homothallic mutants): in the pathogens Candida albicans and in Cryptococcus 2.3. Advantages of haploid selfing vs. asexual neoformans, both heterothallism and homothallism via reproduction ‘same-sex mating’ are possible (Heitman, 2010 and refs therein; Alby et al., 2009). These species are human A possible advantage of haploid selfing over asexual pathogens, especially in immunocompromised patients. reproduction relies on the advantage of segregation. A possible explanation could be the incipient invasion of Indeed, haploid selfing immediately produces diploid

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Fig. 5 Life cycle of the anther smut Microbotryum violaceum (adapted from Lopez- Villavicencio, 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, progeny, which would develop better while getting rid of which can increase the efficacy of selection for deleteri- lower fitness offspring. This selective resource allocation ous alleles, especially for recessive mutations (e.g. Haag & has been shown to be more stringent in the more costly Roze, 2007). However, this mechanism might be of sexual pathway (Bruggeman et al., 2004). Theoretical evolutionary importance only in species with a signifi- work is required to investigate the extent to which cant diploid phase under which strong selection can act. deleterious mutations rates are sufficiently high to create This is likely not the case in most ascomycetes, the group an advantage for haploid selfing that balances the cost of of fungi in which homothallism is most frequent: the sex. The costs of sex are likely much lower in the case of diploid stage of the life cycle is most often transient in this haploid selfing than for outcrossing, being restricted to group, although some exceptions exist, such as in some those related to physiological costs of meiosis and sexual hemiascomycetous yeasts (Knop, 2006). structures. These physiological costs can, however, be Yet, some empirical studies suggest that there indeed significant (Aanen & Hoekstra, 2007). might be advantages of haploid selfing vs. asexual Sexual reproduction by haploid selfing may also be reproduction associated with the elimination of newly advantageous compared to asexual reproduction when it arisen deleterious mutations. Compared to asexual repro- allows for the purging of parasites. In some ascomycete duction, haploid selfing seems to reduce the accumula- species, DNA and RNA viruses are only transmitted via tion of slightly deleterious mutations in the homothallic asexual spores. Sexual spores produced by either out- A. nidulans (Bruggeman et al., 2004). Haploid selfing does crossing or haploid selfing are free of these parasites not allow the classical purging of deleterious alleles by (Coenen et al., 1997; van Diepeningen et al., 2008). outcrossing between individuals carrying deleterious Similarly, repeat-induced point (RIP mutation) is a mutations at different loci (which is needed to prevent mechanism restricting the proliferation of transposable Mu¨ ller’s ratchet). It has, however, been proposed that elements, occurs only in association with the dikaryotic haploid selfing would be more efficient at purging newly stage that follows mating and precedes karyogamy and arisen deleterious mutations from the population than meiosis (Galagan & Selker, 2004). Also, the sexual cycle is asexual reproduction in A. nidulans. In a mycelium, all also associated with cell rejuvenation through the reset- the nuclei can form spores because no special germ line ting epigenetic signalling and the avoidance of senes- exists. Nuclei can be packed to form asexual spores or can cence. In senescent cultures of Podospora anserina, sexual fuse to form sexual spores after meiosis. The concept of a reproduction restores healthy mitochondria, which is selection arena is proposed such that when deleterious called ‘rejuvenation’ (Silliker et al., 1996). These benefits mutations appear, they could be eliminated by a selective that are not associated with recombination have likely maternal diversion of resources to the nonmutant fittest been linked with sex secondarily, but they can now

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contribute to rendering haploid selfing advantageous over this genetic advantage of sex and recombination can asexual reproduction. explain the evolution and the maintenance of a small Haploid selfing may also occur in nature because sex is rate of sexual reproduction (e.g. Roze, 2009). This is associated with a variety of beneficial functions other consistent with the observation that many species alter- than recombination (reviewed in Burt, 2000). For example, nate asexual reproduction and sexual reproduction, there may be ecological or developmental differences especially in fungi. However, it is still not clear why so between offspring produced by sexual or asexual repro- few species are exclusively asexual given the costs of sex. duction, which then contribute to the maintenance of An explanation may be higher extinction rates in asexual sex. In fact, this hypothesis is supported by numerous species, which would be less able to adapt to changing observations in fungi. In most fungi, sexual spores are, environments or are subject to mutational decay of for instance, considered to be more environmentally Muller’s ratchet over the long term (Gouyon, 1999). This resistant than asexual ones (Aanen & Hoekstra, 2007), so hypothesis is supported by the observation that exclu- that haploid selfing may permit to produce resistant sively asexual taxa seemed to have appeared relatively structures even in the absence of a mating partner. Some recently (Beck et al., 2011). sexual structures, such as basidiocarps, also allow effi- cient spore dispersal by wind, water or animals. Another 2.5. Advantages of heterothallism over homothallism example is when sex is required before the colonization of nutrient source, such as a host: even if sex does not An interesting question is why heterothallism is some- provide recombinational advantages, haploid selfing times favoured over homothallism, whereas heterothal- would allow producing infectious spores when no lism restricts the number of potentially compatible genetically different mating partner is available (Byrnes partners for any gamete. Some authors proposed that et al., 2010; Fraser et al., 2005; Heitman, 2010). Third, it gamete classes such as mating types evolved as a means to has been suggested that sexual reproduction and asexual avoid cytoplasmic conflicts between organelles by enforc- reproduction coexist in many species because the cost of ing their uniparental inheritance (Hurst & Hamilton, sex varies in time (Burt, 2000). For instance, individuals 1992). The idea behind this hypothesis is that uniparental undergo sex rather than asexual reproduction when the inheritance should discourage the evolution of selfish population is crowded because there is no difficulty to cytoplasmic elements, which would replicate faster but find a mate. It would be interesting to assess whether this would be less efficient for providing energy to the hypothesis is supported in fungi. However, assessing the organism. Indeed, it seems highly advantageous for relative frequency of haploid selfing and asexual repro- nuclear genes to limit the opportunity to be associated duction remains difficult in nature because they leave with selfish organelles. Uniparental inheritance of organ- little distinguishing impacts upon genetic structure. elles is efficient for this and in fact most often seems Actually, these evolutionary forces preventing the loss controlled by a mechanism associated with gamete of sex beyond the advantages of recombination have classes, such as the size of the gametes or mating types been proposed to be the main general evolutionary force (Hurst & Hamilton, 1992). However, the association maintaining sex over the short term (Gouyon, 1999; between uniparental inheritance of organelles and gam- Nunney, 1989). A long-term species selection level would ete classes is not necessarily evidence for gamete classes favour species not able to lose sex over the short term having evolved for controlling uniparental inheritance. because it is linked to other essential functions; only The evolution of uniparental inheritance should be easier those species retaining sexual reproduction would be if it can use a pre-existing system of gamete classes that able to persist over the long term because the associated may have evolved for other reasons. In fact, we showed in recombination allows more rapid adaptation and purging a previous review (Billiard et al., 2011) that evidence deleterious mutations. One would only be able to exists in fungi suggesting that gamete classes may have observe only the species in which sex has been evolu- evolved independently of the system regulating unipa- tionarily linked to other essential functions. rental inheritance of organelles. Some species do exhibit mating types, but nevertheless show biparental inheri- tance of organelles (e.g. Coprinus cinereus, May & Taylor, 2.4. Advantages of sexual reproduction over 1988; Neurospora crassa, Yang & Griffiths, 1993), or show asexual reproduction, other than those regarding uniparental inheritance but with a system independent of haploid selfing mating types (e.g. A. bisporus, Jin & Horgen, 1994). On Most of the advantages of sex other than haploid selfing the other hand, in some other fungal species without have been already mentioned above. One of the most mating types, organelles are inherited uniparentally, important advantages of sexual reproduction between which means that mechanisms other than mating types two different genomes is to create new adaptive allelic exist that ensure uniparental inheritance (e.g. in yeasts combinations, often called ‘Fisher–Muller hypothesis’, or with mating type switching mitochondria actively segre- to get rid of deleterious alleles, that is, avoiding Muller’s gate during the first few rounds of cell division; Berger & ratchet (Otto, 2009). Most theoretical papers show that Yaffe, 2000). Also in many ciliates, mating types exist

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despite the lack of any cytoplasmic exchange during between diploid selfing and haploid selfing contributes to syngamy, and thus the impossibility for the evolution of understand why mating types exist in fungi. selfish organelles (Phadke & Zufall, 2009). Therefore, the absence of strict association between organelle uniparen- 2.6. Advantages of diploid selfing over haploid tal transmission and gamete classes as would have been selfing predicted by the hypothesis of Hurst & Hamilton (1992) suggests that other evolutionary pressures are involved in As we have seen in the previous sections, we expect little the appearance and maintenance of gamete classes, such or no advantage of sex when it occurs as haploid selfing, as mating types. The most likely evolutionary sequence especially regarding those advantages of sex that rely on thus seems that gamete classes evolved first and that they recombination. In the case of diploid selfing, genetic allowed the evolution towards uniparental transmission differences exist between the two haploid genomes of the organelles (Maynard Smith & Szathma´ry, 1995). undergoing syngamy and recombination, allowing some A number of other hypotheses have been proposed to of the recombinational advantages of sex to be realized. explain the evolution of mating types, defined here as a In this case, the diploid or haploid progeny produced by molecular mechanism of the gametes allowing discrim- diploid selfing should have a higher fitness than that ination for syngamy, independent of size dimorphism. produced by haploid selfing. We summarize below only the main hypotheses and their limits (see Billiard et al., 2011, for more details). 2.7. Advantages and drawbacks of diploid selfing The advantages put forward to explain the evolution of over outcrossing mating types depend on the specificities of mating types in different organisms. Mating types indeed do not A long-standing question is why so many species restrict the possibility of syngamy in similar ways in all undergo outcrossing instead of diploid selfing. Indeed, organisms. In plants, for instance, mating types are called there is an automatic advantage of diploid selfing self-incompatibility systems and they prevent diploid relative to outcrossing: an individual undergoing diploid selfing (i.e. syngamy between gametes produced by the selfing transmits two copies of its haploid genome in same diploid individual; Figs 1 and 2). In such cases selfed progeny and in many species they can in addition where mating types prevent diploid selfing, it is generally sire offspring by fertilizing outcrossers, whereas these assumed that they evolved precisely to avoid diploid outcrossers cannot fertilize selfers (Fisher, 1941). Fur- selfing, and thus promote outcrossing to limit inbreeding thermore, in species with a predominant diploid phase, depression. This probably also applies to oomycetes. the ability to undergo diploid selfing avoids mating In fungi however, mating types are effective at the partner limitation, that is, selfing provides reproductive haploid stage and therefore do not prevent diploid selfing: assurance. Diploid selfing may, however, be disadvan- any diploid stage of a heterothallic species is heterozygous tageous relative to outcrossing because of inbreeding for the mating type and can therefore produce haploids depression, that is, when recessive deleterious muta- that can undergo diploid selfing (Fig. 2). Mating types in tions are present in a population and are exposed to fungi will only prevent haploid selfing. Besides, inbreed- selection in the homozygous condition due to mating ing depression is not expected to act in predominantly with close relatives. However, in fungi with a predom- haploid organisms, such as ascomycetes, because delete- inantly haploid life cycle, such as most ascomycetes, the rious mutations are not sheltered and can be purged from importance of should be the population as they appear. Inbreeding depression restricted to loci with expression limited to the dikary- therefore has likely played much less of a role in the otic or diploid stages. This has been observed, for evolution of fungal mating types as it does in plants. A example, in N. crassa, the only ascomycete species possible explanation for the evolution of mating types in where inbreeding depression has been investigated: fungi would therefore be to prevent haploid selfing, thus crosses between highly related individuals exhibited affording the benefits of sex associated with recombina- reduced fertility; inbred lines produced deficient peri- tion (Czaran & Hoekstra, 2004). Indeed, if recombination thecia with no or few viable or presented has an advantage, whatever it is, individuals gain this maturation defects (Leslie & Raju, 1985). In advantage only when the haploid genomes that are basidiomycetes in contrast, the dikaryotic stage is recombining are not strictly identical. For example, the predominant in the life cycle; inbreeding depression prevention of Mu¨ ller’s ratchet (i.e. the restoration of may therefore have important consequences and be a haplotypes free of deleterious mutations in a population significant evolutionary force. Inbreeding depression has with no individual carrying zero mutations) requires in fact been found in the basidiomycetes A. bisporus mating between nonidentical haploids. This hypothesis where outcrossed populations showed higher fitness remains poorly investigated, but if valid, then the fitness than inbred ones in several fitness components (Xu, of progeny produced by haploid selfing should be lower 1995). Some mechanisms in fungi may have in fact than the fitness of individuals produced by diploid selfing evolved to control the mating system by limiting diploid (see section 2.6 below). We see here that the distinction selfing or promoting outcrossing (see section 3 below).

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diversity of reproductive systems has been revealed 2.8. Conclusions and prospects (Giraud et al., 2008; Taylor et al., 1999), from exclu- We argue that answering the questions raised above sively clonal propagation, at least in some introduced about the evolution of asexual vs. sexual reproduction, ranges, to a high prevalence of sex, with many homothallism vs. heterothallism, haploid selfing vs. surprising results compared to what was expected from diploid selfing and diploid selfing vs. outcrossing requires in vitro observations. In the ascomycete Botrytis cinerea, (i) measuring what types of syngamy occur in nature (see responsible for grey mould, the human pathogen section 3 below), (ii) retracing their evolution using Coccidioides immitis or the Macrotermes natalensis fungus phylogenies and identifying associated life-history traits grown by termites, sexual structures have never been (see section 4) and (iii) experimentally assessing the observed and these species were long thought to be benefits and costs of the different possible modes of strictly clonal. However, molecular markers revealed reproduction, asexual or sexual, and the mating systems pervasive recombination (Burt et al., 1996; Fournier & of haploid selfing, diploid selfing and outcrossing (see Giraud, 2008; Giraud et al., 1997; De Fine Licht et al., section 5). 2006). In contrast, sexual forms have been observed in the wheat leaf rust Puccinia triticina (Wahl et al., 1984), 3. Modes of reproduction and mating yet genetic analyses of French populations revealed a systems in nature completely clonal structure, with no or very little evidence of genetic recombination (Goyeau et al., In order to understand why particular breeding systems 2007). In the fungus responsible for the yellow rust of (homothallism vs. heterothallism), mating systems and wheat, Puccinia striiformis f.sp. tritici, studies measuring modes of reproduction have evolved, we need to assess linkage disequilibrium in European populations simi- what types of reproduction and syngamy are actually larly indicated prominent clonality (Hovmøller et al., performed in natura. Indeed, in vitro observations do not 2002). Moreover, the high degrees of heterozygosity allow for strong inferences of the reproductive mode in observed at microsatellite markers (Enjalbert et al., 2002) natural conditions. For instance, homothallism have and in sequences associated with ribosomal RNA genes been suggested to evolve either for allowing haploid (Roose-Amsaleg et al., 2002) provided evidence for a selfing or, on the contrary, for allowing higher mate Meselson Effect, where ancient asexual lineages exhibit availability under outcrossing (Billiard et al., 2011). high divergence between their homologous chromosomes Disentangling between these hypotheses requires due to the accumulation of independent mutations on assessing how often in nature homothallic fungi undergo different alleles (Halkett et al., 2005). A Meselson effect haploid selfing, diploid selfing or outcrossing. Similarly, has also been detected in Glomales (Kuhn et al., 2001; the frequency of sexual reproduction in nature cannot be Schurko et al., 2009), similarly indicating very ancient inferred from in vitro observations. Below, we briefly clonality, but recent data showing recombination events review the modes of reproduction and mating systems of and that the meiotic machinery is conserved might fungi (sensu lato, i.e. including oomycetes, and the two suggest that these fungi undergo cryptic sex (Halary et al., main groups of true fungi, ascomycetes and basidiomy- 2011). Sex seems to have been lost several times cetes), as revealed in natura using population genetics independently in certain groups, such as in the Penicillium approaches, although such studies remain scarce. genus (Lobuglio et al., 1993; Lo´ pez-Villavicencio et al., 2010). However, exclusively clonal fungi are scarce, molecu- 3.1. Modes of reproduction: sexual vs. asexual lar markers having most often revealed the occurrence of reproduction at least some degree of recombination, even in species As a measure of relative time or growth, most ascomy- where no sexual stages are known (Burt et al., 1996; Lee cetes are haploid during almost their entire life cycle, as et al., 2010). Besides footprints of recombination based mycelium or yeast, and often they are capable of both on population genetics data, another type of evidence asexual and sexual reproduction. Ascomycetes include indicating that sex occurs regularly in most fungi comes yeasts, filamentous species and many species responsible from the apparent functionality of the mating types for crop or tree diseases. Basidiomycetes include mush- genes, even in species without known sexual structures. rooms, smuts, yeasts and rusts, the latter being also Virtually, all the mating type genes analysed so far indeed responsible for plant diseases and producing large present apparent functional sequences (Debuchy et al., numbers of asexual spores, as do ascomycetes. Linkage 2010). Most ascomycetes actually exhibit mixed repro- disequilibrium among genetic markers and the number ductive systems, with indications of both sexual and of repeated haplotypes isolated from natural populations asexual reproduction (e.g. Dutech et al., 2008; Kiss et al., have been used to infer the occurrence and degree of 2011). However, as mentioned above, a problem is that clonality, although this latter phenomenon is not haploid selfing cannot be distinguished from clonality in distinguishable from haploid selfing by these methods homothallic fungi using most common approaches of (Giraud et al., 2008; Gladieux et al., 2010). A great population genetics.

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In contrast with ascomycetes, mushrooms (homobas- developmental patterns of the promycelium where mei- idiomycetes) do not produce asexual spores, so that osis takes place (Hood & Antonovics, 2000). clonal propagation is restricted to dispersal via mycelia. In oomycetes, an example of contrasted mating systems Clonality appears therefore of minor importance in most in two closely related species is given by Plasmopara viticola mushrooms at the population level (Prospero et al., and Plasmopara halstedii, respectively, responsible for the 2008), although clone growth can occur over quite large downy mildews of grapevine and sunflower. Molecular distances (Anderson & Kohn, 1995; Smith et al., 1992). 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 3.2. Mating systems in fungi: Haploid selfing vs. P. halstedii showed considerable heterozygote deficiency diploid selfing vs. outcrossing (FIS = 0.95), probably due to the limited dispersal ability In the following sections, we review briefly the evidence before mating and thus lack of available sexual partners in for the occurrence of the different types of syngamy in the field (Giresse et al., 2007), whereas the populations of natural populations. the heterothallic (sensu oomycetes) species P. viticola only slightly deviated from Hardy–Weinberg proportions Diploid selfing vs. outcrossing (Chen et al., 2007; Delmotte et al., 2006). The relative importance of diploid selfing vs. outcrossing is most easily examined in basidomycetes and oomycetes Haploid selfing vs. clonality vs. outcrossing because, with their main state being respectively dikary- Haploid selfing is difficult to distinguish from clonality otic and diploid, the FIS index estimated using codomi- using molecular markers as both yield progeny not nant genetic markers can give direct indications about segregating for any marker and yield linkage dis- the degree of diploid selfing. Surprisingly however, such equilibrium over the long term at the population level. studies are still quite rare compared to those in plants or Sampling and genotyping the progeny within sexual animals. structures in natural populations should allow determin- Most basidiomycetes disperse primarily as haploid ing whether haploid selfing actually occurs in nature. basidiospores just before mating, which greatly favours The literature on mating systems in fungi is, however, outcrossing. In fact, heterozygote deficiencies are quite unfortunately blurred by the pervasive confusion be- rare in the few studies having measured FIS in nature in tween haploid selfing and diploid selfing. For instance, a sexual basidiomycetes (Barre`s et al., 2008; Bergemann & study on the ascomycete pathogen Cryphonectria parasi- Miller, 2002; Engh et al., 2010; Franzen et al., 2007; tica, responsible for the chestnut blight, claims to have Kauserud & Schumacher, 2002; Kretzer et al., 2004; shown that this ascomycete has a predominantly out- Rosewich et al., 1999; Roy et al., 2008). Some sexual crossing mating system (Marra et al., 2004). In fact, the basidiomycetes showed heterozygote excess (Amend authors looked at whether the progeny within perithecia et al., 2010; Engh et al., 2010; Rosewich et al., 1999), segregated for some highly polymorphic markers and which has been interpreted as due to disassortative could thus only infer the occurrence of haploid selfing vs. mating (preferential mating with individuals genetically either diploid selfing or outcrossing. Their results inter- more different than the average of the population) or estingly showed that sampling progeny in natural pop- linkage of markers with the MAT or vic (vegetative ulations can reveal whether haploid selfing occurs, and it incompatibility) loci (which maintains high heterozy- was in fact rare in this species (although not completely gosity as fusion can only occur between cells carrying absent, which is surprising for a supposedly heterothallic different alleles at these loci, Hood & Antonovics, 2000). species). Such studies estimating the rate of haploid In contrast to most basidiomycetes, the heterothallic selfing are, however, still too rare, and the questions are Microbotryum violaceum, population genetics studies have confused by ambiguous terms like ‘selfing’ and ‘outcrossing’. shown strong heterozygote deficiency (Delmotte et al., Another example is the study by Pe´rez et al. (2010) 1999; Giraud, 2004), indicating high rates of diploid which also called ‘selfing’ the phenomenon of haploid selfing. Diploid selfing may be common in natural selfing, and estimated that ‘outcrossing’ (i.e. in fact either populations because of a lack of available mating partners diploid selfing or outcrossing) was frequent in the or because of mechanisms favouring diploid selfing homothallic plant pathogen Mycosphaerella nubilosa. compared with outcrossing. In M. violaceum, diploid Recent population genetics studies in yeasts have spores are dispersed and only one may arrive at a time managed to estimate the rates of haploid selfing, diploid on a new resource for growth (i.e. a host plant) (Fig. 3), selfing and outcrossing at the same time by using linkage and because further dispersal of the haploid products of disequilibrium with the mating type locus in these spore germination and meiosis is limited, diploid selfing species capable of mating type switching. Tsai et al. may often be the primary option available. Some data, (2008) estimated that a sexual cycle occurred every however, also indicate that a preference for diploid 1000 asexual cycles in . The sex selfing vs. outcrossing exists (Giraud et al., 2005; Hood & events were estimated to be 94% from within the same Antonovics, 2000) and that this is mediated through tetrad (i.e. the automixis for diploid selfing), 5% with a

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clonemate after switching the mating type (i.e. haploid Another interesting possibility offered by phylogenies selfing) and 1% outcrossed. This was consistent with would be to assess whether associations exist between another study of natural populations estimating out- the evolution of certain mating or breeding systems and crossing at 1.1% in this species (Johnson et al., 2004). other traits. For instance, if homothallism evolves for Murphy & Zeyl (2010) have shown, using an exper- universal compatibility at syngamy, whereas heterothal- imental approach, that S. cerevisiae and S. paradoxus lism evolves for preventing haploid selfing, we may undergo even higher rates of outcrossing (ca. 10–25%) expect that homothallism evolved in outcrossing species when mates are available, much higher than what was in which other mechanisms prevent haploid selfing, such long expected based on the existence of a capsule- as rapid gamete dispersal after meiosis or lack of clonal protecting whole ascus. Earlier experimental studies had multiplication of gametes. If selfing ensures reproductive also estimated intratetrad mating rates at about 75–80% success, we expect that it evolves in species where in vitro (Knop, 2006). finding a mate is challenging. These approaches can be Several other studies have showed high rates of applied with great power to diverse organisms (e.g. in outcrossing in homothallic or pseudo-homothallic spe- corals Kerr et al., 2011) but we first need to acquire data cies, in particular in Cryptococcus and Agaricus (Bui et al., on fungal mating systems in nature (haploid selfing vs. 2008; Callac et al., 2006; Heitman, 2010 Hiremath et al., diploid selfing vs. outcrossing) and on multiple life- 2008; Saul et al., 2008), supporting the idea that homo- history traits, in order to analyse them on phylogenies thallism may have evolved for higher mate availability, together with the mode of reproduction and breeding instead of and ⁄ or in addition to promoting haploid selfing. system (heterothallism vs. homothallism).

4. Utility of phylogenies in the study 5. Experimental designs to distinguish of sex, breeding systems and mating between hypotheses systems Most of the models proposed to explain the origin and Phylogenetic studies can enlighten the evolutionary his- maintenance of sexual reproduction and mating types tory of mating systems and modes of reproduction in fungi. assume fitness differences between the progeny depend- Some studies have shown, for example, that sex has been ing on the mode of reproduction and the mating system independently lost several times in fungal groups such as (see section 2). Experimental approaches may therefore Penicillium (Lobuglio et al., 1993; Lo´ pez-Villavicencio allow testing for the existence and the amount of these et al., 2010). Phylogenetic analysis is also a powerful tool differences using fungi. to study the relative ages of clades depending on the type of The experimental designs we propose are based on the reproduction and thereby to assess whether different comparison of the fitness of the diploid or haploid reproductive systems are advantaged over the long term. individuals produced by different modes of reproduction For example, the advantage of sex and outcrossing would and by different types of syngamy. We note WAR,WHS, be supported if asexual clades and selfing clades appeared WDS and WOC the fitnesses of progeny produced by younger than sexual outcrossing clades. asexual reproduction, haploid selfing, diploid selfing and Phylogenies also allow estimating the ancestral and outcrossing, respectively. Different fungal models may be derivate modes of reproduction in given clades. Phyloge- used to test these different hypotheses. It should be noted nies have shown that homothallism has probably appeared that these fitnesses can be measured either in haploid or from a heterothallic ancestor in some groups of fungi in diploid individuals, depending on the biology of the (Fusarium, O’Donnell et al., 2004; Neurospora, Nygren et al., species under investigation. 2011; Penicillium,Lo´ pez-Villavicencio et al., 2010). In other specific groups such as Aspergillus, sequencing and analyses Does haploid selfing afford the advantages of sex not of mating type genes suggest that heterothallism may have related to recombination? W

ª 2012 THE AUTHORS. J. EVOL. BIOL. JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 14 S. BILLIARD ET AL. asexually. Some experimental work has already been Deleterious mutation accumulation experiments carried out on these aspects in A. nidulans (Bruggeman Recombination is considered to be fundamental to et al., 2004): several lineages were started, some trans- prevent the accumulation of deleterious mutations in ferred between plates exclusively by sexual spores result- sexual populations. Haploid selfing does not allow ing from haploid selfing, and others transferred between recombination between different genetic haploid ge- platesexclusively by asexualspores. Aftera certain number nomes; it therefore does not allow the elimination of of replating events (i.e. several generations), fitness com- deleterious mutations, except in the meiosis where they parisons were made based on sizes of the colonies. Results appear (Bruggeman et al., 2004). Experimental protocols indicated that haploid selfing slowed down the accumu- using fungi can allow testing the importance of recom- lation of deleterious mutations. Similar experiments are bination for the fitness of the progeny after several needed on additional species, and other traits can be used to generations of mutation accumulation. Different lineages estimate the fitness depending on the species, such as can be created with individuals grown in a controlled asexual spore production, sexual reproduction, viability of environment for a given number of generations. the progeny or competitive ability. Other features unre- A generation begins with a single individual produced lated to the recombinational advantages of sex could also by haploid or diploid selfing. Lineages can be maintained be compared in the progeny resulting from asexual for enough generations to permit mutations to appear or reproduction vs. haploid selfing, such as the protection mutational load could be artificially increased. To detect from the proliferation of parasites or transposable elements advantages of recombination, the fitness of the progeny in sexual and asexual structures, or senescence. formed by haploid vs. diploid selfing could be compared. If differences exist (WHS

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in fungi. Yet, estimating inbreeding depression in stimulating discussions. We also thank Hanna Kokko, basidiomycetes and oomycetes would be straightfor- Duur Aanen and two anonymous reviewers for helpful ward, as they have a life cycle with a predominant comments. We acknowledge grants FungiSex ANR-09- dikaryotic and diploid stage, respectively. Inbreeding 0064-01 and NSF-DEB 0747222. S.B. acknowledges post- depression indeed results from the homozygosity of doctoral grants from the CNRS. We apologize to all those deleterious rare mutations in inbred offspring, and can colleagues whose work we may have failed to cite in this be estimated by comparing the fitness between out- article. The authors declare no conflict of interest. crossed dikaryotic or diploid progeny and progeny produced by diploid selfing. Traits that can be measured References to estimate fitness in basidiomycetes and oomycetes include heterokaryon growth rate, primordium forma- Aanen, D. & Hoekstra, R. 2007. Why sex is good: on fungi and tion, number of fertile fruiting bodies and average beyond. In: Sex in Fungi: Molecular Determination and Evolution- weight per fruiting body. ary Implications (J. Heitman, J.W. Kronstad, J.W. Taylor & L.A. Casselton, eds), pp. 527–534. ASM Press, Washington, DC. Alby, K., Schaefer, D. & Bennett, R.J. 2009. Homothallic and Conclusion heterothallic mating in the opportunistic pathogen Candida albicans. Nature 460: 890–893. This study had two main goals: first, clarifying the Amend, A., Garbelotto, M., Fang, Z. & Keeley, S. 2010. Isolation concepts and terminology used to describe sexual by landscape in populations of a prized edible mushroom reproduction in fungi by placing them in an evolution- Tricholoma matsutake. Conserv. Genet. 11: 795–802. ary perspective, and explaining the theoretical costs and Anderson, J.B. & Kohn, L.M. 1995. Clonality in soilborne, plant- benefits of each mode of reproduction and mating pathogenic fungi. Annu. Rev. Phytopathol. 33: 369–391. system, and second, proposing some general experi- Barre`s, B., Halkett, F., Dutech, C., Andrieux, A., Pinon, J. & mental considerations and population genetics analyses Frey, P. 2008. Genetic structure of the poplar rust fungus in fungi to help disentangle hypotheses about the Melampsora larici-populina: evidence for isolation by distance in evolution of the mode of reproduction and mating Europe and recent founder effects overseas. Infect. Genet. Evol. 8: 577–587. systems. In the evolutionary biology literature, many Barrett, S.C.H. 2010. Darwin’s legacy: the forms, function and models and experiments have focused on the question sexual diversity of flowers. Philos. Trans. R. Soc. Lond. Ser. B of why many species undergo outcrossing instead of Biol. Sci. 365: 351. diploid selfing. So far however, very few studies have Barrett, S. 2011. Why reproductive systems matter for the used fungi as models, and even worse, the mating invasion biology of plants. 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Whitton, J., Sears, C., Baack, E. & Otto, S. 2008. The dynamic produce gamete and undergo either diploid selfing or nature of apomixis in the angiosperms. Int. J. Plant Sci. 169: outcrossing. 169–182. Idiomorph: sequences present at the same locus but that Xu, J. 1995. Analysis of inbreeding depression in Agaricus are not derived from a common ancestral sequence (i.e. bisporus. Genetics 141: 137–145. in contrast to alleles); idiomorphs at the mating type Yang, X. & Griffiths, A.J.F. 1993. Male transmission of linear locus in ascomycete fungi determine the compatibility for plasmids and mitochondrial DNA in the fungus Neurospora. Genetics 134: 1055–1062. syngamy. Zakharov, I.A. 2005. Intratetrad mating and its genetic and Isogamy: a feature of species where the two fusing cells evolutionary consequences. Genetika 41: 508–519. at syngamy are of the same size and morphology, often Zuk, M. 2009. The sicker sex. PLoS Pathog. 5: e1000267. yielding the same amount of resources to the zygote (e.g. in basidiomycete fungi, syngamy can be between two Glossary mycelia). Mating system: the realized type of syngamy in nature Anisogamy: a feature of species where the two fusing in terms of the relatedness of the cells involved (e.g. cells at syngamy are of different sizes or morphologies inbreeding or outcrossing). and often do not provide the same amount of resources Mating type: molecular recognition systems determin- to the zygote (e.g. large and small gametes, or as in some ing the compatibility between cells for syngamy where fungi where syngamy involves a spore fusing with a haploid cells expressing identical mating types at syn- hypha or mycelium). gamy cannot fuse. Automixis: syngamy between haploid cells or haploid Mating type switching: a system found in some nuclei derived from the same meiosis, also referred to as ascomycete yeasts where the determinants of alternate intratetrad mating when involving syngamy. mating types are present but the determination of which Breeding system: the cellular or developmental mecha- mating type is expressed can change by localized genetic nisms that determine how syngamy can be achieved for rearrangements. an organism (e.g. homothallism vs. heterothallism in Modes of reproduction: a basic distinction between fungi, hermaphroditism vs. dioecy in plants). asexual and sexual reproduction. Diploid selfing: syngamy between haploid cells derived Outcrossing: syngamy between haploid cells derived from meioses of a single diploid individual. from meioses that occurred in different diploid individuals. Haploid selfing (or same-clone mating, intragamet- Pseudo-homothallism: ability of some fungi to com- ophytic mating, gametophytic selfing): syngamy plete the sexual cycle without the apparent need for a between haploid cells that are mitotic descendants of a syngamy, which is achieved by the presence of two common haploid progenitor, often the cells are geneti- haploid nuclei of opposite mating types and from a single cally identical except for mutation and at the mating type meiosis in the dispersed spore. locus in the case of species with mating type switching. Syngamy: fusion of haploid cells (often gametes, but Homothallism: for fungi, the potential to undergo sometimes mitotically replicating haploid cell or hyphae, haploid selfing; for oomycetes, the potential to undergo as in homobasidiomycetes) for fertilization, which leads diploid selfing without the presence of another individual to zygote formation after karyogamy; karyogamy can with another mating type. occur long after syngamy in fungi, for example, in Heterothallism: for fungi, the requirement for genetic basidiomycetes. differences between haploid cells to undergo syngamy; for oomycetes, the requirement of hormonal signals from Received 26 November 2011; revised 21 February 2012; accepted 22 a diploid individual carrying a different mating type to February 2012

ª 2012 THE AUTHORS. J. EVOL. BIOL. JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY