Download Deleterious effects of recombination and possible

doi: 10.1111/jeb.12196
Deleterious effects of recombination and possible
nonrecombinatorial advantages of sex in a fungal model
! EZ-VILLAVICENCIO*, A. J. M. DEBETS†, M. SLAKHORST†, T. GIRAUD‡
M . L OP
& S. E. SCHOUSTRA†
*Origine, Structure, Evolution de la Biodiversit!e, UMR 7205 CNRS-MNHN, Mus!eum National d’Histoire Naturelle, Paris, France
†Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands
‡Ecologie, Syst!ematique et Evolution, Universit!e Paris-Sud, Orsay, France
Keywords:
Abstract
experimental evolution;
fungi;
natural selection.
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
Correspondence: Manuela L!
opez-Villavicencio, Origine, Structure,
Evolution de la Biodiversit!e, UMR 7205 CNRS-MNHN, Mus!eum
National d’Histoire Naturelle, CP39, 57 rue Cuvier, 75231 Paris
Cedex 05, France. Tel.: +33 1 40 79 36 74; fax: +33 1 40 79 35 94;
e-mail: [email protected]
1968
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
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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 matingtype 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).
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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.
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(a) Conidiophore
emerging from the
mycelium carrying
asexual spores
(i.e., conidia).
Asexual spore
Asexual
(g) Mycelium with haploid nuclei that
divide through mitosis to allow propagation
Sexual spore
Sexual structure
develops
Sexual
Haploid selfing or
outcrossing
(b) Two nuclei initiating a dikaryotic cell
(fruiting initial); nuclei are either recruited from
the same mycelium (resulting in haploid
selfing), or from different mycelia (resulting in
outcrossing)
(f) Mature fruiting bodies vary in size
and contain up to 105 sexual spores
(c)
Dikaryons within
each fruiting body
proliferate, being
genetically identical
(e) Dikaryons fuse to diploid nuclei, all
identical within a given fruiting body; they
undergo meiosis and a mitosis, resulting in 8
haploid spores in each ascus
(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. Fruiting initials differentially proliferate depending on
resource uptake and/or maternal investment. Eventually dikaryons fuse.
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.
Fig. 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.
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
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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).
(a)
(b)
Fig. 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.
1971
vs.
vs.
vs.
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Materials and methods
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.
inoculation of 100 lL 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 lL 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 lm against ~150 lm 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.
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
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 lL
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Advantages of sex beyond recombination
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 9 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.
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 9 B and
between strains C 9 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
1973
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 9 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 9 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
Fig. 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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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
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 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 variation
d.f.
MS
F Ratio
(a)
Size of cleistothecium
Error
1
14
0.0462387
0.0068
7.4340
(b)
Outcrossing status
Error
1
78
144116.2
403
357.2085
P>F
0.0109
<0.0001
Table 1a). This suggests some positive effects of outcrossing, although the outcrossing rate among large
cleistothecia remained low (Fig. 4a; Table 1a).
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 hypothe-
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1968–1978
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Advantages of sex beyond recombination
1975
(a)
(a)
(b)
(b)
(c)
Fig. 5 (a) Mean and SE of the fitness for large and small haploselfed 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.
Fig. 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.
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 variation
d.f.
MS
(a)
Strain
Size of the cleistothecium
Error
1
1
77
0.00734
2.99022
0.36273
0.0203
8.2437
0.8872
0.0053
(b)
Size of the cleistothecium
Error
1
59
14627.64
1055.74
12.1095
0.0009
F
P
sis: 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).
Discussion
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
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! EZ-VILLAVICENCIO ET AL.
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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 haploselfed 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
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1968–1978
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Advantages of sex beyond recombination
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
1977
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
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
Aanen, D. & Hoekstra, R. 2007. Why sex is good: on fungi and
beyond. In: Sex in Fungi: Molecular Determination and Evolutionary Implications (J. Heitman, J.W. Kronstad, J.W. Taylor & L.A.
Casselton, eds), pp. 527–534. ASM Press, Washington, DC.
Alexopoulos, C.J., Mims, C.W. & Blackwell, M. 1996. Introductory Mycology. John Wiley & Sons, New York, NY. 869p.
Bernstein, H., Byerly, H.C., Hopf, F.A. & Michod, R.E. 1985.
Genetic damage, mutation, and the evolution of sex. Science
229: 1277–1281.
Billiard, S., L!
opez-Villavicencio, M., Devier, B., Hood, M.,
Fairhead, C. & Giraud, T. 2011. Having sex, yes, but with
whom? Inferences from fungi on the evolution of anisogamy
and mating types. Biol. Rev. 86: 1–6.
Billiard, S., L!
opez-Villavicencio, M., Hood, M. & Giraud, T.
2012. Sex, outcrossing and mating types: unsolved questions
in fungi and beyond. J. Evol. Biol. 25: 1020–1038.
Bruggeman, J., Debets, A.J.M., Wijngaarden, P.J., de Visser,
J.A.G.M. & Hoekstra, R.F. 2003a. Sex slows down the accumulation of deleterious mutations in the homothallic fungus
Aspergillus nidulans. Genetics 164: 479–485.
Bruggeman, J., Debets, A., Swart, K. & Hoekstra, R. 2003b.
Male and female roles in crosses of Aspergillus nidulans as
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1968–1978
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1978
! EZ-VILLAVICENCIO ET AL.
M . L OP
revealed by vegetatively incompatible parents. Fungal Genet.
Biol. 39: 136–141.
Bruggeman, J., Debets, A.J.M. & Hoekstra, R.F. 2004. Selection arena in Aspergillus nidulans. Fungal Genet. Biol. 41:
181–188.
Callac, P., Jacobe de Haut, I., Imbernon, M., Guinberteau, J.,
Desmerger, C. & Theochari, I. 2003. A novel homothallic
variety of Agaricus bisporus comprises rare tetrasporic isolates
from Europe. Mycologia 95: 222–231.
Colegrave, N. 2002. Sex releases the speed limit on evolution.
Nature 420: 664–666.
Cove, D.J. 1976. Cholorate toxicity in Aspergillus nidulans: the
selection and characterisation of chlorate resistant mutants.
Heredity 36: 191–203.
van Diepeningen, A.D., Varga, J., Hoekstra, R.F. & Debets,
A.J.M. 2008. Mycoviruses in Aspergilli. In: Aspergillus in the
Genomics Era (R. Samson & J. Varga, eds), pp. 133–176.
Wageningen Academic Publishers, Wageningen, The Netherlands.
Fraser, J.A., Giles, S.S., Wenink, E.C., Geunes-Boyer, S.G.,
Wright, J.R., Diezmann, S. et al. 2005. Same-sex mating and
the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 437: 1360–1364.
Galagan, J. & Selker, E. 2004. RIP: the evolutionary cost of
genome defense. Trends Genet. 20: 417–423.
Gifford, D., Schoustra, S.E. & Kassen, R. 2011. The length of
adaptive walks is insensitive to starting fitness in Aspergillus
nidulans. Evolution 65: 3070–3078.
Giraud, T., Yockteng, R., L!
opez-Villavicencio, M., Refr!egier, G.
& Hood, M.E. 2008. Invited Mini-Review: Mating system of
the anther smut fungus Microbotryum violaceum: selfing under
heterothallism. Eukaryot. Cell 7: 765–775.
Goddard, M.R., Charles, H., Godfray, J. & Burt, A. 2005. Sex
increases the efficacy of natural selection in experimental
yeast populations. Nature 434: 636–640.
Gorelick, R. & Carpinone, J. 2009. Origin and maintenance of
sex: the evolutionary joys of self sex. Biol. J. Linn. Soc. 98:
707–728.
Gouyon, P.-H. 1999. Sex: a pluralist approach includes species
selection. (One step beyond and it’s good). J. Evol. Biol. 12:
1029–1030.
Gouyon, P.H., de Vienne, D.M. & Giraud, T. 2012. Sex and
evolution. In: Handbook of Evolutionary Thinking (P. Huneman, T. Heams, G. Lecointre & M. Silberstein, eds), in
press.
Hadany, L. & Otto, S. 2007. The evolution of condition-dependent sex in the face of high costs. Genetics 176: 1713.
Hiremath, S.S., Chowdhary, A., Kowshik, T., Randhawa,
H.S., Sun, S. & Xu, J. 2008. Long-distance dispersal and
recombination in environmental populations of Cryptococcus
neoformans var. grubii from India. Microbiology 154:
1513–1524.
Johnson, S., Lively, C. & Schrag, S. 1995. Evolution and ecological correlates of uniparental reproduction in freshwater
snails. Cell. Mol. Life Sci. 51: 498–509.
Knop, M. 2006. Evolution of the hemiascomycete yeasts: on
life styles and the importance of inbreeding. BioEssays 28:
696–708.
Kozlowski, J. & Stearns, S.C. 1989. Hypotheses for the production of excess zygotes: models of bet-hedging and selective
abortion. Evolution 43: 1369–1377.
Lehtonen, J., Jennions, M.D. & Kokko, H. 2011. The many
costs of sex. Trends Ecol. Evol. 27: 172–178.
Lindman, H.R. 1975. Analysis of Variance in Complex Experimental Design. W. H. Freeman & Co Ltd., San Francisco, CA.
Murphy, H.A. & Zeyl, C.W. 2010. Yeast sex: surprisingly high
rates of outcrossing between asci. PLoS ONE 5: e10461.
Neiman, M. 2004. Physiological dependence on copulation in
parthenogenetic females can reduce the cost of sex. An. Beh.
67: 811–822.
Nunney, L. 1989. The maintenance of sex by group selection.
Evolution 43: 245–257.
Otto, S.P. 2009. The evolutionary enigma of sex. Am Nat. 174:
S1–S14.
Perrin, N. 2012. What uses are mating types? The ‘developmental switch’ model. Evolution 66: 947–956.
Pontecorvo, G., Roper, J.A., Hemmons, C.M., MacDonald,
K.D. & Bufton, A.W.J. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 5: 141–238.
Pringle, A. & Taylor, J.W. 2002. The fitness of filamentous
fungi. Trends Microbiol. 10: 474–481.
Schoustra, S.E., Debets, A.J., Slakhorst, M. & Hoekstra, R.F.
2007. Mitotic recombination accelerates adaptation in the
fungus Aspergillus nidulans. PLoS Genet. 3: e68.
Schoustra, S., Rundle, H.D., Dali, R. & Kassen, R. 2010. Fitness-associated sexual reproduction in a filamentous fungus.
Curr. Biol. 20: 1350–1355.
Stearns, S.C. 1987. The selection-arena hypothesis. Experientia
Suppl. 55: 337–349.
de Vienne, D.M., Giraud, T. & Gouyon, P.-H. 2013. Lineage
selection and the maintenance of sex. PLoS ONE 8: e66906.
de Visser, J.A. & Elena, S.F. 2007. The evolution of sex: empirical insights into the roles of epistasis and drift. Nat. Rev.
Genet. 8: 139–149.
Received 6 February 2013; revised 3 May 2013; accepted 8 May 2013
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1968–1978
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY