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Why sexual reproduction is so prevalent in nature remains a major question in evolutionary biology. Most of the proposed advantages of sex rely on the benefits obtained from recombination. However, it is still unclear whether the conditions under which these recombinatorial benefits would be sufficient to maintain sex in the short term are met in nature. Our study addresses a largely overlooked hypothesis, proposing that sex could be maintained in the short term by advantages due to functions linked with sex, but not related to recombination. These advantages would be so essential that sex could not be lost in the short term. Here, we used the fungus Aspergillus nidulans to experimentally test predictions of this hypothesis. Specifically, we were interested in (i) the short-term deleterious effects of recombination, (ii) possible nonrecombinatorial advantages of sex particularly through the elimination of mutations and (iii) the outcrossing rate under choice conditions in a haploid fungus able to reproduce by both outcrossing and haploid selfing. Our results were consistent with our hypotheses: we found that (i) recombination can be strongly deleterious in the short term, (ii) sexual reproduction between individuals derived from the same clonal lineage provided nonrecombinatorial advantages, likely through a selection arena mechanism, and (iii) under choice conditions, outcrossing occurs in a homothallic species, although at low rates.
Although much effort has been put to understand why sex is so prevalent among eukaryotes despite its high costs, the maintenance of sexual reproduction remains one of the long-standing fundamental questions in evolutionary biology. Sexual reproduction is expected to provide long-term advantages mainly because recombination increases the genetic variance upon which selection can act and thereby allows more rapid adaptation (Otto, 2009). Nevertheless, sexual reproduction must present advantages sufficient also on the short term, which act fast enough to avoid the invasion of demographically advantaged asexuals. Most of the proposed short-term benefits rely on the recombinatorial benefits leading to the generation of the production of new genetic high-fitness combinations in the progeny (de Visser & Elena, 2007; Otto, 2009). However, it has been difficult to assess how general these potential benefits of recombination are, and it is not clear whether the conditions under which these benefits are sufficient to maintain sex, given the costs are met in nature.
The disadvantages of sex seem to be more obvious and better documented. A major disadvantage is that reshuffling of alleles by recombination breaks apart favourable combinations built by past selection (recombination load) (de Visser & Elena, 2007; Otto, 2009; Schoustra et al., 2010). In addition, when males contribute little or no resources to their progeny, a mutation causing females to reproduce asexually is expected to spread because its frequency doubles at each generation (‘two-fold cost of sex’). Various additional costs associated with mating apply to more specific cases, including costs of finding and courting a mate, risks of predation or of contracting sexually transmitted diseases or parasitic genetic elements (for a detailed review of the costs of sex, see Lehtonen et al. 2011).
The costs of sex are also expected to differ depending on the specificities of the mating system, such as when comparing outcrossing and selfing. Outcrossing, for example, results from the fusion of genetically different haploid cells produced by different diploid individuals, whereas selfing results from the syngamy between haploid cells produced by the same diploid individual, reducing the costs of searching for a compatible mate and of the production of males. In some haploid eukaryotes such as homothallic fungi, mosses and some algae, selfing is also possible through the fusion of two genetically identical haploid clone mates, which is called intrahaploid mating, same clone mating (Perrin, 2012) or haploid selfing (Billiard et al., 2011). Homothallism is a lack of restriction at syngamy, in most of the cases because no differences exist at the mating-type locus between the individuals of these species. Historically, homothallism has been thought to have evolved to promote haploid selfing. However, sex with a clone mate does not result in actual recombination, the supposedly main advantage of sexual reproduction, while still incurring some costs (slower reproduction, maintenance of the meiotic machinery; Billiard et al., 2011, 2012). A transition to asexuality would bypass some of the costs of sex while producing genetically the same progeny. Homothallism has been suggested instead to have evolved to increase the number of available mates as it can be seen as a universal compatibility (Giraud et al., 2008). However, the frequency of outcrossing in homothallic species under choice conditions remains unknown.
Nevertheless, haploid selfing has been suggested to present advantages not related to the generation of novel allele combinations (see Billiard et al. 2011, 2012 for reviews). In particular, haploid selfing has been proposed to be more efficient than asexual reproduction in eliminating newly arisen somatic deleterious mutations in progeny (Bruggeman et al., 2003a). In ascomycete fungi, mitotic divisions occur during the growth of the haploid mycelium, producing cells carrying genetically identical nuclei. During growth, spontaneous mutations are expected to regularly appear. Haploid selfing cannot purge deleterious mutations by recombination in the classic way (i.e. by recombination between two different genomes carrying different DNA regions free of deleterious mutations), but it can be associated with a nonrecombinatorial ‘selection arena mechanism’, allowing the elimination of newly arisen deleterious mutations in the mother mycelium (Stearns, 1987; Bruggeman et al., 2003a, 2004).
The selection arena hypothesis (Stearns, 1987) states that overproduction of zygotes, a widespread phenomenon in nature, is not a waist but can be explained as a mechanism of progeny choice by which only a genetically superior subset will fully develop into new progeny by selective parental investment into the most promising progeny. The existence of a selection arena mechanism has been proposed in plants, animals (including humans) and fungi (Stearns, 1987; Bruggeman et al., 2004). The selection arena hypothesis is based on the assumptions that (i) fruiting initials are cheap (in the case of ascomycete fungi, these are dikaryons established by fertilization of an ascogonial cell by an antheridium nucleus), (ii) fruiting initials require parental investment after conception and (iii) fruiting initials vary in fitness and based on fitness differentially develop either through selective resource allocation by the parental support tissue or through differential capability to attract those resources (Stearns, 1987).
Here, we used Aspergillus nidulans to experimentally study the short-term effects of recombination and the possible advantages of sex nonrelated to recombination. Aspergillus nidulans has a predominantly haploid life cycle (Fig. 1). As most ascomycete fungi, A. nidulans can reproduce both asexually and sexually. Asexual spores (conidia) are produced mitotically, whereas sexual spores can result from meiosis by outcrossing (involving nuclei of two distinctly different strains) or haploid selfing (involving two nuclei of the same strain which are genetically identical except potential recently arisen mutations). The initial step in sexual reproduction is the formation of dikaryotic cells (fruiting initials) by fertilization of a cell of the mycelium containing one maternal haploid nucleus by another haploid nucleus taking the role of the father. Within each fruiting initial, nuclei of dikaryotic cells divide synchronously forming an extensive proliferating dikaryotic tissue (Fig. 1). The extent of development of each of the fruiting initials varies and depends on the investment of the supporting maternal mycelium as both the fruiting body wall and the cytoplasm are of maternal origin (Bruggeman et al., 2003b). This allows the potential of a selection arena (Bruggeman et al., 2004), in which the mother feeds preferentially the sexually produced fruiting initials carrying the fittest dikaryons. Eventually, the proliferated dikaryotic cells undergo nuclear fusion (karyogamy) forming as many identical diploid zygotes that do not divide but directly undergo meiosis, followed by a mitotic division resulting in eight haploid meiotic spores (called ascospores) in a sac-like structure (the ascus). Asci contained in a fruiting body, thus result from different meioses from several but genetically identical zygotes (Pontecorvo et al., 1953). In total, a mature fruiting body may contain 10 000s of such asci, the number being proportional to the amount of maternal resources available.
Figure 1. Aspergillus nidulans life cycle, with both asexual and sexual reproduction. (a) Asexual spores (conidia) are produced mitotically from the mycelium. (b) The sexual cycle starts when two nuclei initiate a dikaryotic cell (fruiting initial); nuclei are either recruited from the same mycelium (resulting in haploid selfing) or from different mycelia (resulting in outcrossing); (c) dikaryons within each fruiting body proliferate, being genetically identical although some nuclei containing newly appeared mutations could exist, making some dikaryons heterozygous; (d) numerous fruiting initials are produced by a given mycelium and can be genetically different (due to somatic mutations and/or outcrossing with different fathers), which constitutes a selection arena (Bruggeman et al., 2003a). Fruiting initials differentially proliferate depending on resource uptake and/or maternal investment. Eventually dikaryons fuse; (e) dikaryons fuse to diploid nuclei, all identical within a given fruiting body; they undergo meiosis and a mitosis, resulting in eight haploid spores in each ascus; (f) mature fruiting bodies vary in size and contain up to 105 sexual spores; (g) spores germinate and give rise to a mycelium with haploid nuclei that divide through mitosis to allow propagation.
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With its particular mode of reproduction (being homothallic, that is, able of both haploid selfing and outcrossing), A. nidulans is an ideal model organism to study the recombinatorial and nonrecombinatorial effects of sexual reproduction. Nonrecombinatorial advantages of sex have indeed been proposed for A. nidulans via a selection arena mechanism, where the newly arisen nuclei containing new mutations in important genes that make them nonfunctional and that could be detected at the dikaryotic stage would be eliminated. The mother then would divert more resources to the progeny of higher genetic quality or, alternatively, the better progeny would be more efficient in resource uptake, leading to larger sexual fruiting bodies containing more ascospores, with more of these offspring (Bruggeman et al., 2003a, 2004). There would be overproduction of fertilized dikaryotic fruiting initials (‘dikaryons’), some of which will produce thousands to ten thousands of ascospores. In small cleistothecia (i.e. fruiting bodies, Fig. 1), dikaryons have not proliferated as much as in large cleistothecia and then will contain less ascospores.
The selection arena is thus expected to be only associated with the sexual pathway in fungi, where a maternal structure feeds at the same time genetically different sexual offspring for some time after progeny production. Although mutations are also expected to pop up in newly produced asexual conidia, these spores receive far less maternal investment as there are no maternal structures feeding them while they multiply. Previous experiments (Bruggeman et al., 2003a, 2004) used available laboratory mutant strains to show that some sexual progeny were not able to develop, but did not test whether a maternal selection indeed acted based on fitness differences of the progeny, in particular to eliminate newly arisen deleterious mutations within a progeny.
We therefore set out here to test whether a homothallic species under choice conditions preferentially performs outcrossing or haploid selfing, and what the benefits and drawbacks are of outcrossing vs. haploid selfing. Specifically, our study (1) assessed the frequency of outcrossing under choice conditions and (2) addressed two hypotheses that relate to the selection arena. We detail these two aims in the following: (1) we asked whether homothallic fungi outcross when mating partners are available and at what frequency; if recombination between different genomes is mostly deleterious in the short term and if haploid selfing allows eliminating most newly arisen deleterious mutations, we expect haploid selfing to be frequent. Outcrossing is, however, expected to be beneficial in the long term, and it is important to assess whether homothallics do outcross at all under choice conditions (Billiard et al., 2012); (2a) we predict that, if recombination is mostly deleterious in the short term, by breaking down favourable allelic combinations, the fitness of sexual progeny produced by outcrossing to be lower than that of the progeny produced by intrahaploid selfing (i.e. involving meiosis and syngamy but without allele reshuffling between two different genomes); (2b) we predict that if a selection arena mechanism exists allowing elimination of newly arisen deleterious mutations within the progeny, more maternal resources will be allocated to fitter progeny. We predict that larger fruiting bodies will then carry higher-fitness progeny than smaller fruiting bodies.
We used A. nidulans strains originated from isogenic strains, with similar fitness but differing by detectable colour and auxotrophic markers and having been independently propagated asexually under laboratory conditions for a large number of generations, during which they likely accumulated genetic differences through novel mutations (Fig. 2a).
Figure 2. Experimental protocol followed showing (a) the origin of the different strains (i.e. single genotype, but somatic mutations) and the methodology followed. The strains A and B originated from the same wild-type strain, whereas the strains C and D originated from different wild-type strains and (b) hypotheses to be tested: the selection arena acting as a nonrecombinant mechanism to eliminate somatic mutations and the deleterious effects of recombination between different genotypes.
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