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Keywords:

  • experimental evolution;
  • fungi;
  • natural selection

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

image

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).

image

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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Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Strains, media and culture conditions

For this study, we used the available A. nidulans strains carrying genetic markers allowing differentiating between outcrossed and selfed progeny. The origin of strains is described in Fig. 2a and the hypothesis tested in Fig. 2b. We used four different strains, each representing a single genotype. Two chlorate-resistant strains of A. nidulans, hereafter A and B, were derived from a wild strain originating from the Glasgow collection. These two strains differed initially only at a colour marker and at two different mutations involved in the same metabolic pathway of the nitrate reductase activity: strain A produces green conidia and presents a mutation in the niaD gene, a structural gene for the nitrate reductase apoenzyme, whereas strain B produces yellow conidia and harbours a mutation in a cnx gene, regulating the synthesis of a co-factor for the nitrate reductase (Cove, 1976). The colour and auxotrophic mutations are neutral in terms of fitness when growing with the supplementation of urea. These two strains have been maintained and propagated asexually for more than 350 asexual generations under laboratory conditions, leading to evolved strains that have likely accumulated extra mutations in their genomes (Fig. 2a). Strains A and B are in the collection of the Laboratory of Genetics at Wageningen University under the numbers WG655 for green strain (strain A) and WG656 for the yellow strain (strain B) and are available upon request. Strains C and D were collected from soil at two different locations (strain C is strain 709 collected from forest and strain D is 710 collected from bog) in Wales in 1992. Two markers, nia and cn, respectively, had been introduced in these strains to facilitate selection of progeny in crosses. As the strains were isolated from two different locations, they most likely have accumulated a different suite of mutations due to local adaptation and drift.

Unless mentioned otherwise, strains were grown on minimal medium (MM: consisting of NaNO3 6.0 g L−1; KH2PO4 1.5 g L−1; MgSO4.7H2O 0.5 g L−1; NaCl 0.5 g L−1; 0.1 mL of a saturated trace element solution containing FeSO4, ZnSO4, MnCl2 and CuSO4; set at pH 5.8) as described by Pontecorvo et al. (1953), supplemented with 1% of glucose and 10 mm of urea as nitrogen source. All incubations were carried out at 37 °C in culture chambers.

Crosses

We performed crosses between the strains A and B to assess the frequency of outcrossing vs. haploid selfing under choice conditions and the effects of recombination on the fitness of the progeny. These crosses were performed on 15 Petri dishes with minimal medium, by inoculation of 100 μL of a mixed suspension containing conidia of both parental strains A and B in equal quantities. The number of conidia was assessed using a haemocytometer, for generating spore suspensions containing the same amount of spores from each strain. Finally, each of the two different strains was grown separately to obtain progeny produced by haploid selfing. For each strain, five Petri dishes were inoculated with 100 μL of a spore suspension of only one genotype, either the A or B strain. Crosses of strains C and D were performed in a similar way using three Petri dishes.

Testing for outcrossing

Plates were grown for 15 days, until cleistothecia (sexual fruiting bodies) were formed. Because A. nidulans is homothallic, cleistothecia can be produced by either haploid selfing or outcrossing in the presence of a compatible mate. We distinguished whether a cleistothecium was the product of haploid selfing or outcrossing by growing the progeny on nonsupplemented medium. As both parental strains need urea supplementation in the medium, progeny from haploid selfing cleistothecia would not grow without supplementation. In contrast, a quarter of the ascospores from cleistothecia produced by recombination between the two parental strains will be able to grow on nonsupplemented medium.

For crosses of strains A and B, we sampled 90 cleistothecia (45 among the largest and 45 among the smallest) from each of the 15 plates inoculated with the two parental strains. Large and small cleistothecia were grown in separated plates. The sampled large cleistothecia had a diameter of ~ 250 μm against ~150 μm for the sampled small cleistothecia. Cleistothecia were cleaned by rolling them on 3% water–agar plates to eliminate conidia and hyphae sticking to the wall and were then crushed to release the ascospores. They were then plated on nonsupplemented media to detect their genetic make-up: after 2 days of growing at 37 °C, only outcrossed cleistothecia yielded recombinant ascospores able to germinate and produce conidia of two different parental colours. The frequency of outcrossing for each pair was estimated as the number of germinating cleistothecia/90. The number of nongerminating cleistothecia/90 permitted to assess the frequency of the haplo-selfed progeny.

For crosses of strains C and D, we only isolated large cleistothecia and recovered two haplo-selfed cleistothecia and two outcrossed cleistothecia.

Fitness measurements

Fitness was estimated as the number of asexual spores (conidia) that the progeny in a sexual fruiting body is able to generate when growing on solid medium for 5 days. For each treatment, we placed a droplet of 3 μL of the saline solution containing a minimum of 300 sexually derived progeny (ascospores) in the middle of a Petri dish; previous work has shown that there are no founder effects on final fitness once at least 100 progeny are present to found a new fungal colony (Gifford et al., 2011). Each fitness measure was repeated three times for each individual. Plates were incubated at 37 °C, the optimal temperature for A. nidulans growth, for 5 days, covered with black plastic to avoid desiccation.

The amount of offspring produced (fitness units) was estimated as the diameter of the colony × the percentage of surface covered by spores (conidia). Although the diameter of the colony (MGR–mycelial growth rate) is usually accepted as a good fitness measure for filamentous fungi (Pringle & Taylor, 2002; Schoustra et al., 2007), we decided to incorporate also the percentage of the surface covered by spores as a way to estimate the total production of offspring. This is an important fitness component as some cultures produced by outcrossed cleistothecia, although presenting normal mycelium growth and colony sizes, showed large sectors without conidia (Fig. 3). Using MGR rather than the fitness measure presented above (fitness units) did not lead to other qualitative results.

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Figure 3. Representative examples of resulting progeny of crosses between the two parental strains used in this study. Note that some recombinant colonies exhibit zones without sporulation.

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Impact of recombination

To test the impact of recombination, we compared the fitness of the progeny produced by haploid selfing (for strain A and strain B separately) vs. the fitness of the outcrossed progeny produced by strains A × B and between strains C × D (Fig. 2b). We were interested in comparing the maximal fitness obtained by the two reproductive strategies. We therefore considered only the progeny produced in large cleistothecia, that we showed contained the fittest progeny (see 'Results'). For the combination of C and D, haplo-selfed progeny of each of the parents and outcrossed progeny were collected and their fitness was measured.

Selection arena

The selection arena posits higher access of maternal resources by genetically superior progeny, either by a preferential maternal diversion of resources or by a more efficient resource uptake and outgrowth of the dikaryotic fruiting initials expected to yield the fittest haploid progeny. To study whether there was indeed a relationship between the size of the cleistothecia and the fitness of its content, we compared the fitness of progeny contained in large vs. small cleistothecia. The comparison was performed for haplo-selfed cleistothecia for strains A and B and for outcrossed cleistothecia produced by A × B. For haplo-selfed cleistothecia, we recovered 20 large cleistothecia and 20 small cleistothecia for each strain. Cleistothecia were cleaned as described above and were then crushed and stored in saline solution to be used in the fitness assays. For each of the 15 couples crossed (A × B, see section 'Testing for outcrossing' above), we identified and recovered four large outcrossed and three small outcrossed cleistothecia and their fitness was measured as described above. The differences in the number of large and small cleistothecia used for the comparisons resulted from the lower availability of small outcrossed cleistothecia.

Statistical analyses

All statistical tests were performed using jmp, version 9 statistical package (SAS Institute Inc., Cary, NC, USA). Normality of the residuals and homoscedasticity of data were tested using Shapiro–Wilk's test. The high number of repetitions required for testing fitness differences between treatments impaired addressing all the questions in a single-experiment trial, given the space needed for Petri dishes. We therefore divided the experiments into two parts and analysed the results separately.

A first experimental set-up was performed to test whether fitness differences existed depending on the outcrossing status (i.e. between the progeny resulting from outcrossing vs. haploid selfing). In this experiment, we therefore generated haplo-selfed progeny, as well as progeny resulting from crosses between strains A and B and between strains C and D. Because we wanted to assess differences in maximum fitness depending on the outcrossing status, we compared the fitness of large cleistothecia only. Therefore, only large cleistothecia were collected in haplo-selfed progeny. For the outcrossed progeny, we collected also small recombinant cleistothecia of the combination A and B to test the selection arena for the outcrossed progeny. We used a one-way anova to test the effect of the outcrossing status (outcrossing vs. haploid selfing) on fitness. To investigate the selection arena hypothesis for outcrossed progeny, we then tested the influence of cleistothecium size and the identity of the parents on fitness.

A second experimental set-up was performed to test the predictions from the selection arena hypothesis in the progeny produced by haploid selfing. We therefore compared differences in fitness between small and large cleistothecia produced by haploid selfing of the combination of strains A and B. We used a two-way anova to test the effects of size of cleistothecia and strain identity on fitness. Finally, we tested selfing preference in the presence of a potential mate; we used a one-way anova including cleistothecium size as factor to control for this effect.

In all analyses, the nonsignificant interactions were stepwise eliminated, whereas main factors were kept even when nonsignificant. Some residuals were not normally distributed, but the distributions of residuals did not look highly skewed. The data were left untransformed because no usual transformation improved the normality and anova is known to be robust to deviations from normality (Lindman, 1975).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Outcrossing in the presence of a potential mate

Most (88%) of the analysed sexual fruiting bodies (cleistothecia) resulting from the inoculation of the suspension containing both strains resulted from haploid selfing, but ca. 12% of the cleistothecia were outcrossed. These results support the view that homothallic species do undergo outcrossing when mates are available, however, at a lower frequency than haploid selfing. The frequency of outcrossing was significantly higher among large than small cleistothecia (Fig. 4a; Table 1a). This suggests some positive effects of outcrossing, although the outcrossing rate among large cleistothecia remained low (Fig. 4a; Table 1a).

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Figure 4. (a) Mean and SE of the frequency of outcrossing among large and small cleistothecia produced by the A and B strains growing together; (b) mean and SE of the fitness (sporulating colony surface area) of colonies from large haplo-selfed mating cleistothecia from the A (green bars) and B (yellow bars) strains and from large outcrossed cleistothecia produced by the recombination between the two strains (spotted bars); (c) Fitness of haplo-selfed progeny of each of the strains C and D and of outcrossed progeny between these two strains. The fitness of the outcrossed progeny is lower than the fitness averaged over the haplo-selfed progeny, showing a deleterious effect of recombination. Error bars show the SE.

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Table 1. anova models for testing what parameters affect (a) the outcrossing status in the progeny produced by large and small cleistothecia (R² of the whole model is 0.21); or (b) the fitness (sporulating colony surface area) in the progeny produced by outcrossing vs. same-clone mating (outcrossing status); in this experiment, only large cleistothecia were sampled (see details in the Materials and methods section). R² of the whole model is 0.82
Source of variationd.f.MSF RatioP > F
(a)
Size of cleistothecium10.04623877.43400.0109
Error140.0068  
(b)
Outcrossing status 1144116.2357.2085<0.0001
Error78403  

Fitness of progeny under outcrossing vs. haploid selfing

Although no detectable fitness differences existed between the two parental strains A and B, the progeny produced by haploid selfing had a higher mean fitness and a lower variance than outcrossed progeny (Fig. 4b and c; Table 1b) (mean fitness strain A = 263.50 units, variance = 11.21; mean fitness strain B = 271.83 units, variance 92.31; mean fitness outcrossed strains of combination A/B = 182.78 units, variance = 738.68). Figure 3 shows pictures of the growth of the parental strains and of the progeny produced by outcrossing.

For the combination of strains C and D, the fitness of the progeny resulting from outcrossing was lower than the average fitness of haplo-selfed progeny from strains C and D (mean fitness over all haplo-selfed progeny = 7701 units, mean fitness outcrossed progeny = 7536 units; one-way anova F1,99 = 10.51; P = 0.002, Fig. 4c), suggesting a deleterious effect of outcrossing. As with the combination of strains A and B, the fitness of haplo-selfed progeny had a lower variance than the outcrossed progeny (mean fitness haplo-selfed progeny strain C = 7492 units, variance = 101.4; mean fitness haplo-selfed progeny strain D = 7859 units, variance = 331.4; mean fitness outcrossed progeny = 7536 units, variance = 737).

Selection arena

The progeny contained in large cleistothecia had a significantly higher fitness than those contained in small cleistothecia. This was true for haploid selfing for both A and B strains (Fig. 5a; Table 2a) and also for outcrossed progeny (Fig. 5b; Table 2b). Overall, these results provide support for the selection arena hypothesis: the large fruiting bodies contained fitter progeny than the small ones, suggesting that fitter offspring in fact received more maternal resources for dikaryon development than did offspring with lower fitness offspring progeny (Fig. 5a and b).

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Figure 5. (a) Mean and SE of the fitness for large and small haplo-selfed cleistothecia produced by the A (green bars) and B (yellow bars) strains; (b) mean and SE of the fitness for large (blue bars) and small (red bars) cleistothecia produced by recombination between the A and B strains. In all panels, an asterisk (*) indicates statistical significance at the 0.05 level.

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Table 2. Models for testing what parameters affect fitness (sporulating colony surface area) in the progeny, among (a) strain and size of cleistothecia (large or small), in the experiment with same-clone mating progeny only (R² of the whole model is 0.60), and (b) size of cleistothecia (large or small), in the experiment with outcrossed cleistothecia only. R² of the whole model is 0.15
Source of variationd.f.MS F P
(a)
Strain 10.007340.02030.8872
Size of the cleistothecium12.99022 8.24370.0053
Error770.36273  
(b)
Size of the cleistothecium 114627.6412.10950.0009
Error591055.74  

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Our results show that (i) homothallic fungi such as A. nidulans do outcross when mates are available, albeit at low frequency; (ii) outcrossed progeny on average had lower fitness than offspring resulting from haploid selfing, suggesting that recombination can be highly deleterious in the short term; the higher variance in the fitness observed for recombinants nevertheless suggests that some rare high-fitness recombinants may also appear. In fact, outcrossing rate was higher in large fruiting bodies than in small ones; (iii) haploid selfing may be advantageous over asexual reproduction due to a selection arena mechanism.

Outcrossing vs. haploid selfing

Our results clearly illustrate that homothallism (the potential for haploid selfing) does not prevent outcrossing to occur (Pontecorvo et al., 1953; Giraud et al., 2008; Billiard et al., 2012), in contrast to the current prevailing view that homothallism means haploid selfing. The occurrence of outcrossing in homothallic species has also been shown in recent experimental studies in other fungal species (Saccharomyces cerevisiae and S. paradoxus (Knop, 2006; Murphy & Zeyl, 2010); Cryptococcus neoformans (Hiremath et al., 2008); and Agaricus bisporus (Callac et al., 2003). Nevertheless, our results suggest that haploid selfing seems to be preferred over outcrossing in this species (but see Pontecorvo et al., 1953), although the adaptive evolutionary role of haploid selfing is still unclear.

Quantifying the impact of recombination: advantageous or deleterious in the short term?

We found that, under controlled and homogeneous conditions, recombination between different genotypes on average had deleterious effects in the short term because the mean fitness of the progeny produced by outcrossing was lower than that of the progeny produced by haploid selfing. Nevertheless, we found that the outcrossing rate was significantly higher in large fruiting bodies than in small ones. This suggests that some rare recombinants could have higher fitness and would grow and multiply faster, leading to larger cleistothecia. We measured the mean fitness of pooled offspring contained within a single cleistothecium, resulting from many different meioses (Fig. 1). It is thus possible that rare high-fitness individuals were produced by recombination, but that our measures failed to detect them because we did not screen each genotype individually among the sexually derived progeny.

Besides, recombination can be advantageous in the mid or long term, for example by allowing the elimination of mutations that in combination are particularly deleterious (due to epistasis) and had accumulated in individual colonies during mycelial expansion. It could also lead to a more rapid adaptive response by bringing together beneficial mutations allowing their fixation in the population (Colegrave, 2002; Schoustra et al., 2007). In fact, we found that the variance in fitness was larger in outcrossed progeny than in the haplo-selfed progeny (Otto, 2009). Furthermore, recombination can be more beneficial in some environments than in others: sex has been experimentally found to be more advantageous in harsher environments where selection is stronger than in more benign ones where selection is reduced (Goddard et al., 2005). In general, sexual reproduction will be triggered when environmental conditions are not optimal for vegetative growth (Schoustra et al., 2010), and then facultative sexual organisms like A. nidulans engage in sex only when under poor conditions (Hadany & Otto, 2007; Schoustra et al., 2010).

Selection arena and interspecies selection for sex?

The fitness difference between progeny from large and small fruiting bodies suggests the occurrence of a selection arena acting in the sexual cycle. During the development of a fungal colony, multiple sexual fruiting bodies begin to develop around 3 days after the hyphae are formed. Although some thrive and develop into large mature fruiting bodies with many progeny, others remain small and at maturity contain fewer ascospores than large ones (Fig. 1). Our results suggest that the maternal, supporting mycelium had thus diverted more resources to its fittest progeny, consistent with the selection arena hypothesis. However, it is difficult to disentangle whether there is preferential maternal investment directed to a subset of fruiting initials or that fruiting initials of high quality attract more resources from the maternal mycelium. Nevertheless, both scenarios are consistent with the selection arena hypothesis. The main point in this hypothesis is that the genetic quality of the progeny determines the amount of maternal resources gained. Even if the amount of resources is not in direct control of the mother, fitter and faster dividing embryos will need to be able to catch more resources faster in order to grow and divide, and we hypothesized that this will also depend in better genetic background.

Such selective resource allocation is less likely to occur in the asexual pathway as asexual spores are ‘cheaper’ (e.g. smaller and less persistent) and because asexual conidia do not depend on the maternal tissue to multiply after production. These associations may prevent the evolution of bypassing meiosis within the sexual maternal structures or to develop a selection arena in the asexual pathway.

The selection arena acting in the sexual pathway is one of the several advantages nonrelated to recombination that have been proposed (Billiard et al., 2012; Gouyon et al., 2012). Sex in fungi has indeed been shown to provide several well-documented short-term benefits that are independent of recombination. For instance, repeated induced point mutation (RIP) acts to inactivate parasitic-repeated elements via mutations (Galagan & Selker, 2004) when passing through meiosis in haploid species. The sexual pathway also helps eliminating DNA and RNA viruses by producing virus-free progeny, while viruses are transmitted in asexual spores (van Diepeningen et al., 2008). Either constraints or mid-term benefits of recombination may prevent the evolution of these mechanisms in the asexual pathway.

More generally in eukaryotes, sexual reproduction is often associated with essential functions – other than recombination – such as the production of dispersal and stress-resistance structures, and this has been proposed to be a mechanism allowing maintaining sex in the short term (Nunney, 1989; Gouyon, 1999). In the case of fungi and aphids for instance, sexual spores or eggs are better protected and can resist more extreme environmental conditions than asexual ones (Aanen & Hoekstra, 2007). Sex can have an important role in some pathogenic fungi for the production of dispersal or infectious structures, which are sexual (Fraser et al., 2005). In other organisms, asexuality cannot evolve for developmental reasons, such as genomic imprinting in mammals or developmental instability in Drosophila (Gouyon, 1999). Sex (independent of recombination) can also be a mechanism allowing DNA structural repair (Bernstein et al., 1985) and ‘rejuvenation’ in eukaryotes, via epigenetic reset which will erase developmental errors that will otherwise accumulate in asexuals (Gorelick & Carpinone, 2009). Finally, the sexual pathway (regardless of the occurrence of recombination) can be essential for the production of some progeny, even if parthenogenetic; parthenogenetic dependence on copulatory stimuli has been reported in several species of vertebrate and invertebrate (Neiman, 2004). Species where such ‘side effects’ of sex exist cannot easily lose sexual reproduction, even if recombination is deleterious in the short term, and in the long term, sex will be maintained because it will prevent the accumulation of deleterious mutations and will allow for faster adaptation. In contrast, other species where sex is not linked to essential functions can become asexual due to the short-term costs of sex and then will go extinct (Nunney, 1989; Gouyon, 1999; de Vienne et al., 2013).

A frequent criticism to the interspecies selection for sex is that selection should then favour the evolution of the short-term benefits in the asexual pathway, such as resistant asexual spores or in our case a selection arena in the asexual pathway. However, the interspecies selection for sex takes this possibility into account, stating that (i) some beneficial traits not associated with recombination are linked to the sexual structures; (ii) the species in which those links between beneficial traits and sex are not strongly constrained can see their beneficial traits evolve to be unlinked from sexual structures; (iii) once the link between beneficial traits and sex no longer exists, these species can become asexual due to the two-fold advantage of asexuality because there are no longer short-term advantages associated with sex; (iv) these asexual species will disappear on the long term as they cannot adapt rapidly enough; (v) the species that remain will be only those species that cannot easily break the association between sex and the beneficial traits, with the strongest constraints preventing the loss of sex (see, for examples, de Vienne et al. (2013), Gouyon (1999), Gouyon et al. (2012) and Nunney (1989).

Fungi have so far been little used for investigating the maintenance of sex (but see Aanen & Hoekstra, 2007), despite the multiple advantages they present for experimental protocols. Our study illustrates that fungi are ideal eukaryote models for studying the evolution of sex thanks to their experimental advantages and the diversity in their reproductive strategies. In particular, fungi are organisms allowing studying the evolution of sex from an unprecedented perspective by permitting decoupling sex from recombination, which is a key point in the maintenance of sexual reproduction.

The selection arena should be an important mechanism in fungi in general, principally in all the fungal species where the development of the sexual progeny is maintained by resources from the maternal tissue, as is the case of most ascomycetes (Alexopoulos et al., 1996). Other studies have highlighted the potential of a selection arena during zygote production in other systems, such as marine invertebrates and plants (Johnson et al., 1995; Kozlowski & Stearns, 1989; Lively & Johnson, 1994).

In conclusion, we show here experimentally that sex can be advantageous for other reasons than the recombinatorial benefits that have long been considered the main advantage of sexual reproduction. We showed that recombination can even be deleterious in the short term, and other forces may be acting to maintain sexual reproduction despite this disadvantage.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank S. Billiard, D. Roze, B. Nieuwenhuis, D. Aanen, A. de Visser and two anonymous reviewers for helpful discussions and/or comments in previous versions. MLV acknowledges funding from the ATM Microorganisms-MNHN 2012-2013. TG acknowledges the grant FungiSex ANR-09-0064-01. SES acknowledges funding through a European Union Marie Curie Fellowship.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References