Fitness Effects of Thermal Stress Differ Between Outcrossing and Selfing Populations in Caenorhabditis elegans

Abstract

The maintenance of males and outcrossing is widespread, despite considerable costs of males. By enabling recombination between distinct genotypes, outcrossing may be advantageous during adaptation to novel environments and if so, it should be selected for under environmental challenge. However, a given environmental change may influence fitness of male, female, and hermaphrodite or asexual individuals differently, and hence the relationship between reproductive system and dynamics of adaptation to novel conditions may not be driven solely by the level of outcrossing and recombination. This has important implications for studies investigating the evolution of reproductive modes in the context of environmental changes, and for the extent to which their findings can be generalized. Here, we use Caenorhabditis elegans—a free-living nematode species in which hermaphrodites (capable of selfing but not cross-fertilizing each other) coexist with males (capable of fertilizing hermaphrodites)—to investigate the response of wild type as well as obligatorily outcrossing and obligatorily selfing lines to stressfully increased ambient temperature. We found that thermal stress affects fitness of outcrossers much more drastically than that of selfers. This shows that apart from the potential for recombination, the selective pressures imposed by the same environmental change can differ between populations expressing different reproductive systems and affect their adaptive potential.

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Vol:.(1234567890)
Evol Biol (2017) 44:356–364
DOI 10.1007/s11692-017-9413-z
1 3
RESEARCH ARTICLE
Fitness Effects ofThermal Stress Differ Between Outcrossing
andSelfing Populations inCaenorhabditis elegans
AgataPlesnar‑Bielak1 · MartaK.Labocha1· PaulinaKosztyła1·
KatarzynaR.Woch1· WeronikaM.Banot1· KarolinaSychta1·
MagdalenaSkarboń1· MonikaA.Prus1· ZofiaM.Prokop1
Received: 11 August 2016 / Accepted: 23 February 2017 / Published online: 3 March 2017
© The Author(s) 2017. This article is an open access publication
expressing different reproductive systems and affect their
adaptive potential.
Keywords Mating systems· Outcrossing· Temperature·
Adaptation· Fitness· Stress
Introduction
Outcrossing, i.e. reproduction by fusing gametes of dis-
tinct individuals, remains one of evolution’s mysteries.
Compared to uniparental reproduction (asexuality or self-
fertilization), it incurs considerable costs, particularly when
associated with the production of males which facilitate
outcrossing but do not themselves bear offspring, while
requiring energy resources that could have been used oth-
erwise (Maynard Smith 1971, 1978; Lloyd 1980; Bell
1982; Uyenoyama 1984; Lively and Lloyd 1990; Anderson
et al. 2010). Nevertheless, a vast majority of animal spe-
cies produce males, suggesting that this mode of reproduc-
tion does bring some significant selective advantages. Most
theoretical explanations proposed to date relate to the role
of recombination. Outcrossing shuffles genes among indi-
viduals, creating new combinations of alleles. Therefore,
it can break apart selection interference between beneficial
and deleterious mutations (Hill-Robertson effect), facilitat-
ing the spread of the former and the purging of the latter
(reviewed by Otto 2009). Importantly, this may also lead
to (some of) the offspring of outcrossing individuals hav-
ing increased fitness in a changing environment (Stebbins
1957). Both these factors can accelerate the rate of adapta-
tion to novel environmental conditions. Thus, the benefits
of outcrossing should be particularly pronounced under
environmental change (e.g. Colegrave 2002; Goddard etal.
2005; Morran etal. 2009a, b; but see; Zeyl and Bell 1997).
Abstract The maintenance of males and outcrossing
is widespread, despite considerable costs of males. By
enabling recombination between distinct genotypes, out-
crossing may be advantageous during adaptation to novel
environments and if so, it should be selected for under
environmental challenge. However, a given environmen-
tal change may influence fitness of male, female, and her-
maphrodite or asexual individuals differently, and hence
the relationship between reproductive system and dynamics
of adaptation to novel conditions may not be driven solely
by the level of outcrossing and recombination. This has
important implications for studies investigating the evolu-
tion of reproductive modes in the context of environmental
changes, and for the extent to which their findings can be
generalized. Here, we use Caenorhabditis elegans—a free-
living nematode species in which hermaphrodites (capa-
ble of selfing but not cross-fertilizing each other) coexist
with males (capable of fertilizing hermaphrodites)—to
investigate the response of wild type as well as obligato-
rily outcrossing and obligatorily selfing lines to stressfully
increased ambient temperature. We found that thermal
stress affects fitness of outcrossers much more drastically
than that of selfers. This shows that apart from the potential
for recombination, the selective pressures imposed by the
same environmental change can differ between populations
The original version of this article was revised. The presentation
of Table1 was incorrect. The values in the column (df.) has been
corrected in this version.
* Agata Plesnar-Bielak
agata.plesnar@uj.edu.pl
1 Institute ofEnvironmental Sciences, Jagiellonian University,
Gronostajowa 7, 30-387Kraków, Poland
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357Evol Biol (2017) 44:356–364
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However, the same change in the external environ-
ment may impose different levels of stress on individuals
and populations differing in breeding system. If this is the
case, then aside the recombination rate, also the strength
of selection may differ between such populations (Parsons
1987; Kondrashov and Houle 1994; Jasnos et al. 2008,
but see; Agrawal and Whitlock 2010), contributing to dif-
ferences in adaptation process. Furthermore, these effects
may be specific to the type of environmental change experi-
enced by the populations.
This has important implications for investigating the role
of recombination in adapting to environmental change. For
example, if a novel environment applied in a study imposes
stronger selection on outcrossing populations (compared
with selfing or asexual ones), leading to faster evolution-
ary response, and this difference in selective pressures is
then neglected when interpreting the results, the higher
adaptation rate may be attributed primarily to the effects of
genetic shuffling. In consequence, generalizing the effects
of such studies may lead to overestimating the recombina-
tion’s impact on adaptation. The reverse scenario may be
true if the novel selective pressure is stronger on selfers or
asexuals. Thus, when investigating the role of outcrossing
in adaptation to novel conditions, it is important to under-
stand how these conditions influence fitness of individuals
expressing particular reproductive strategies.
Many of the studies investigating the problem of male
maintenance associated with the existence of outcrossing
have used Caenorhabditis elegans, a common model spe-
cies in evolutionary and genetic research (Gray and Cutter
2014). C. elegans is androdioecious, with hermaphrodites
coexisting with males. Hermaphrodites are capable of both
selfing and outcrossing with males, but they are not able
to mate with other hermaphrodites (outcrossing occurs only
through mating with males). Sex is determined by the ratio
of X chromosomes to autosomes (Hodgkin 1987) with her-
maphrodites having AA:XX genotype and males AA:X0.
Hence, a male forms when a gamete carrying one X fuses
with a gamete carrying no sex chromosome—which can
happen either through self-fertilization following X-chro-
mosomes nondisjunction during meiosis, or via outcrossing
(since half of the gametes produced by males lack the X
chromosome).
Selfing is a predominant reproductive mode in C. ele-
gans. Male frequencies vary between strains (reviewed
in Anderson et al. 2010). However, in most populations
(including the well-studied laboratory strain N2) they are
very low, often similar to those of nondisjunction events
(Hodgkin 1983; Chasnov and Chow 2002; Teotònio etal.
2006, but see Wegewitz etal. 2008). Adult males and her-
maphrodites show no difference in viability (Hodgkin
1987; Gems and Ridddle 1996, 2000). Although males
survive dauer (an alternative developmental stage induced
by stressful conditions) slightly better than hermaphrodites
(Morran etal. 2009a, b), this difference is very small. Male
fertilization success depends on their frequency in a popu-
lation, being the highest when the proportion of males is
0.2 (Stewart and Phillips 2002). In the N2 laboratory strain
males may sire 70% of the offspring produced in such
populations (Stewart and Phillips 2002). However, these
rates are still too low to prevent a gradual loss of males
from populations. Moreover, as inbreeding depression has
not been recorded in the species (Johnson and Wood 1982;
Johnson and Hutchinson 1993; Chasnov and Chow 2002;
Dolgin et al. 2007), suggesting that prolonged inbreeding
has purged mutation load, offspring resulting from out-
crossing is not predicted to be fitter than offspring of self-
ing hermaphrodites (cf. Anderson etal. 2010). Altogether,
this suggests that males should be easily lost from popu-
lations—which is supported by the results of experiments
performed under standard laboratory conditions (Stewart
and Phillips 2002; Chasnov and Chow 2002; Cutter etal.
2003; Cutter 2005)—and that they do not play important
role in C. elegans evolution.
However, the fact that a large fraction of the genome is
devoted to male functions (Jiang etal. 2001) and that genes
expressed only in males are among the most conserved
in this species (Cutter 2005) questions such reasoning.
Unless C. elegans has become predominantly selfing only
recently, male-specific genes must have been maintained
and conserved by selection acting on males (Loewe and
Cutter 2008). This suggests that outcrossing or/and males
as such have fitness advantage in at least some conditions
and circumstances. Indeed, the hypothesis that outcrossing
becomes favorable in populations adapting to environmen-
tal challenge has been gaining support over the last several
years (Morran etal. 2009a, b, 2013; Teotònio etal. 2012;
Lopes et al. 2008; Carvalho etal. 2014; but see; Theolo-
gidis etal. 2014).
Extensive knowledge about the genetics of C. elegans
allows manipulating its mating system, providing a use-
ful tool for experimental tests of the role of outcrossing
in adaptation. Scientists have identified several mutations
altering dynamics of mating systems in this species (see
Anderson etal. 2010 for review), with mutations in fog-2
and xol-1 genes being among the most frequently used in
evolutionary studies (Stewart and Phillips 2002; Katju etal.
2008; Morran etal. 2009a, b). The first of those genes, fog-
2, produces a protein inhibiting production of sperm in her-
maphrodites homozygous for this locus (Schedl and Kim-
ble 1988; Clifford et al. 2000; Nayak et al. 2005). Thus,
this mutation effectively turns hermaphrodites into females,
enforcing obligate outcrossing in a mutant population.
Mutation in xol-1 gene causes obligate selfing, as it disturbs
dosage compensation rendering males inviable (Miller
etal. 1988; Rhind etal. 1995). The possibility to utilize the
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358 Evol Biol (2017) 44:356–364
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above mutations enables establishment of populations dif-
fering in mating systems. This makes C. elegans a species
in which the hypotheses considering male maintenance and
the role of outcrossing can be precisely tested.
However, as mentioned above, the same change in exter-
nal environment may affect fitness of males, females, and
hermaphrodites differently, hence imposing disparate selec-
tive pressures on different mutants and reproductive sys-
tems. Here, we use replicated lines derived from the N2
strain to investigate the effects of a stressful novel environ-
ment (increased ambient temperature) on fitness of fog-2
(obligatorily outcrossing) and xol-1 (obligatorily selfing)
mutants, as well as wild type, of C. elegans (Fig.1).
Materials andMethods
Animal Culture
We followed standard procedures for culturing and manipu-
lation of C. elegans (Brenner 1974). Animals were grown
on 6 cm Petri dishes with standard Nematode growth
medium (NGM) seeded with 200 µl of OP50 strain of
Escherichia coli (Stiernagle 2006).
We used a wild type N2 (Bristol) strain of C. elegans,
obtained from the Caenorhabditis Genetics Center (CGC,
University of Minnesota, USA). In this strain, the fre-
quency of males is approximately 0.002 (Hodkin et al.
1983; Chasnov and Chow 2002; Teotònio et al. 2006),
which does not exceed the rate at which they are produced
by spontaneous non-disjunction events (Hodgkin et al.
1979; Rose and Baillie 1979; Teotònio et al. 2006). Two
independent isolines (henceforth referred to as source lines
A and B) were established by 20 generations of single her-
maphrodite transfer.
Mating System Manipulations
Two reproductive system-altering mutations were inde-
pendently introgressed into each of the two source lines in
order to obtain two sets of obligatorily selfing, obligatorily
outcrossing, and wild type (facultatively outcrossing) popu-
lations with otherwise similar genetic backgrounds (Fig.1).
To obtain obligatorily selfing lines, xol-1 mutation
tm3055 was introgressed into each of the source lines (car-
rying the wild type xol-1 allele, henceforth: wt), according
to a modified protocol described by Theologidis and col-
leagues (2014). (1) Hermaphrodites from strain TY1807
homozygous for the tm3055 mutation were placed on
Petri dishes with an excess of source line (wt/wt) males
(P generation). (2) The F1 hermaphrodite offspring were
individually isolated and allowed to reproduce by self-
fertilization for 1day, after which they were genotyped to
confirm tm3055/wt heterozygosity (this step was necessary
since C. elegans hermaphrodites can reproduce by self-fer-
tilization regardless of the presence of males, which in this
case would have resulted in tm3055/ tm3055 offspring). (3)
Hermaphrodite offspring (F2) of the verified heterozygotes
were individually placed on Petri dishes and an excess of
source line (wt/wt) males was added to each dish. (4) Their
offspring (F3) were screened for the presence of males once
they reached the L4 stage; the dishes containing males were
discarded. The absence of males indicated that the maternal
(F2) hermaphrodite was homozygous for the tm3055 allele,
making all male offspring inviable (5) From the dishes
containing no males, F3 hermaphrodites were individually
isolated to Petri dishes and allowed to reproduce by self-
fertilization for 1day, after which they were genotyped to
confirm tm3055/wt heterozygosity (see step (2)). Steps 2–5
were repeated eight times in total.
To obtain obligatorily outcrossing lines, fog-2 mutation
q71 was introduced into each of the wt source lines (carry-
ing the wild type fog-2 allele), using a protocol described
by Teotònio etal. (2012). Parental hermaphrodites from a
given source line (wt/wt) were mated with males from the
JK574 strain homozygous for the q71 mutation, and their
hermaphrodite offspring (q71/wt originating from mating
with males and wt/wt originating from self-fertilization
of hermaphrodites) were separately selfed to generate F2.
Twenty F2 hermaphrodites from each of the lines were
picked onto individual plates and let to self. F3 progeny
was checked for phenotype “piano” (accumulation of unfer-
tilized oocytes in the gonads) and absence of F4 progeny,
which indicated homozygosity for fog-2 mutation q71 in
parental hermaphrodite. There were 8 other such cycles of
Genec
Background
Line A
Genotype
Fog-mutated fog-2(q71)
50% males
Wild type wt
< 0.02% males
Xol-mutated xol-1(tm3055)
0% males
Treatment
20°C
25°C
20°C
25°C
20°C
25°C
Line B
Fog-mutated fog-2(q71)
50% males
Wild type wt
< 0.02% males
Xol-mutated xol-1(tm3055)
0% males
20°C
25°C
20°C
25°C
20°C
25°C
Fig. 1 Schematic representation of our experimental design
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359Evol Biol (2017) 44:356–364
1 3
introgression, starting with the F3 fog-females being mated
with an excess of males from the source lines.
Wild-type source lines were subjected to single her-
maphrodite transfer for the time necessary to complete
eight cycles of introgression in the mutant lines, before
being used in the fitness assays, so the inbreeding in all
lines was similar. After completing the introgression (or
single transfers in wild type lines) all the lines were frozen
at −80 °C.
Fitness Assay
All the lines were thawed and placed on Petri dishes at
20 °C. After 4 days (one generation) of acclimatization at
20 °C, the lines were synchronized and cleared of any con-
taminations using bleaching (Stiernagle 2006; briefly: the
procedure involves treating the animals with hypochlorite
solution which dissolves adults and larvae but leaves the
eggs, which are protected by shells, intact). After bleach-
ing, for each source line × breeding system combination,
approximately 2000 eggs were transferred on two 14cm
diameter Petri dishes (1000 eggs per dish), one of which
was subsequently placed at 20 °C, and the other at 25 °C.
The temperature error range of the incubators was 0.5 °C.
The worms were left at the experimental temperatures for
two generations before the fitness assay was performed.
High population densities were prevented by chunking pro-
cedure, i.e., each generation, a small piece (approx. 1cm2)
of agar containing worms was carved and transferred onto
a new 14 cm diameter Petri dish seeded with bacteria
medium. While the numbers of individuals transferred in
this manner are likely to differ between populations, they
were consistently small enough, compared to dish size, as
to ensure ad libitum space and food access (as confirmed
by the excess of bacteria present on old plates when the
chunks were carved), thus minimizing the risk of any den-
sity-dependent effects.
After the two generations of acclimation, for each
source line × breeding system × temperature combina-
tion, 15 hermaphrodites (for wild type and xol-mutated
populations) or 15 pairs (for fog-mutated populations)
in the last larval stage (L4) were individually transferred
to 6cm Petri dishes. Each dish was then returned to its
respective temperature. After 24 h, we transferred the
animals onto new plates, while the dishes with eggs were
left for 2days, until the offspring reached L3/L4 larval
stage. We repeated this procedure (transferring adult
individuals onto new plates while leaving the eggs they
had laid since the previous transfer for further develop-
ment) for 7days. At the L3/L4 stage, the offspring were
counted, and each scored worm was aspired out with a
vacuum pump to prevent counting the same individual
multiple times. Each dish was re-inspected the next day
in order to score the offspring which were overlooked
during the first counting (e.g. because they crawled under
the agar or on the side of the dish). The total number of
offspring (i.e., the lifetime reproductive success) of each
experimental hermaphrodite/pair was then summed up
over all days it had reproduced. We eliminated from the
analysis data obtained from individuals which could not
be found on the plates and therefore we could not be cer-
tain when they finished reproducing.
Statistical Analyses
Data were analyzed using R.3.2.0 (R Core Team 2015).
Proportions of infertile individuals/pairs were ana-
lyzed using Fisher exact test for each of the source lines
separately at each temperature. Hence, four analyses were
performed, comparing infertility rates across breeding
systems (analysis for the source line A at 20 °C, analy-
sis for the source line B at 20 °C, analysis for the source
line A at 25 °C, analysis for the source line A at 25 °C).
Additionally, we compared infertility rates across tem-
peratures applying Fisher exact tests within each mating
system and source line combination.
Data on lifetime reproductive success showed high
heterogeneity of variance. Thus, they were analyzed
using the gls function, implemented in the nlme pack-
age (Pinheiro etal. 2014), which allows to build models
with differing variance structures in the data (Davidian
and Giltinan 1995). Temperature, breeding system and
source line (and all interactions) were included as fixed
effects and the number of offspring was a response varia-
ble. First, we fitted a standard ANOVA with homogenous
variance structure, and visually inspected model residu-
als plotted against factor levels. Based on these plots, in
order to choose the optimal variance structure, we fitted
four further models: (i) a model allowing for differences
in variances among breeding systems, (ii) a model allow-
ing for differences in variances between temperatures,
(iii) a model allowing for different variances among all
combinations of breeding system × temperature, and (iv)
a model allowing for different variances among all com-
binations of breeding system × temperature × source line.
VarIdent variance structure was used in these models,
allowing for differences in variance among levels of nom-
inal variables (Zuur etal. 2009). Each of the models i–iv
was then compared to the standard ANOVA model using
log-likelihood ratio test, which showed that they were all
significantly better than the standard model. Thus, the
four models were ranked using AIC criterion (we could
not perform log-likelihood ratio test as these models are
not nested). Model iv had the lowest AIC score and hence
it is reported in the Results section.
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360 Evol Biol (2017) 44:356–364
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Results
Infertility Rates
At 20 °C, infertility rates were low and did not differ
between the breeding systems (Fisher’s exact tests, source
line A: p = 0.096, source line B: p = 0.343). Two out of 14
assayed pairs were infertile in the fog-mutated line A and
three out of fifteen assayed pairs were infertile in the fog-
mutated line B. There were no infertile individuals in the
xol-mutated lines. In the wild type lines, 0 and 2 individu-
als failed to lay eggs in lines A and B, respectively, out of
15 assayed in each line. In contrast, at increased tempera-
ture, the rates of infertility were strikingly high in the fog-
mutated lines; 10 and 7 out of 15 tested pairs were infer-
tile in the fog-mutated lines compared to 2 and 0 out of
15 in the xol-mutated lines and 1 and 0 out of 15 in wild
type lines (lines A and B, respectively; Fisher’s exact test,
p < 0.001 for both line A and line B).
Fertility rates decreased significantly with temperature
in the fog-mutated line A (Fisher’s exact test, p = 0.008).
The fog-mutated line B also tended to show reduction
in fertility rate, although the trend was not significant
(p = 0.109). Fertility did not vary between temperatures in
the xol-mutated lines (Fisher’s exact test, line A: p = 0.483,
line B: p = 1) or the wild type lines (Fisher’s exact test, line
A: p = 1, line B: p = 0.483).
Lifetime Reproductive Success
Lifetime reproductive success was affected by interac-
tions between breeding system and temperature and,
less strongly, between line and temperature, but not
by the breeding system × line or the three-way interac-
tion (Table 1; Fig. 2), which were hence removed from
the final model. In 20 °C, fog-mutated lines had higher
reproductive success than wild type (p = 0.005) and xol-
mutated (p = 0.002) lines, whereas in 25 °C the situation
was reversed (both p < 0.001) (Fig. 2), which was largely
due to the high levels of infertility in fog-mutated lines (see
above).
Discussion
Our study reveals the reduction of reproductive success at
high temperature in all three breeding systems. This is in
line with previous studies demonstrating that C. elegans
fecundity is highest at 20 °C and declines with increasing
temperature (e.g. Byerly etal. 1976; McMullen etal. 2012;
Petrella 2014, but see Zhang et al. 2015). The decrease of
reproductive function at high temperatures seems to be
associated with functioning of both spermathogenic and
oogenic germ lines (Aprison and Ruvinsky 2014; Petrella
2014), although the relative contribution of these two fac-
tors varies between strains (Petrella 2014).
At 20 °C, the outcrossing (fog-mutated) pairs produced
more offspring than selfing hermaphrodites from both
wild type and xol-mutated lines. It is likely that inhibi-
tion of sperm production resulted in redirecting more
resources into egg production so that a fraction of germ
cells that could not differentiate as sperm developed as
oocytes (Schedl and Kimble 1988). A similar pattern has
been observed by Theologidis and colleagues (2014), who
have found fecundity of fog-mutated females to be higher
than hermaphrodite fecundity at high salinity (although this
effect has not translated into population level).
More interestingly, the influence of temperature on
reproductive success was much more dramatic in the fog-
mutated lines compared to both xol-mutated and wild type
selfing lines (Fig.2). In particular, the proportion of pairs
which did not produce any eggs rose from 0.13 to 0.14 to
0.67 and 0.47 in the fog-mutated lines. This decrease of fer-
tility was only significant in one of the lines; however, in
the other one the clear trend in the same direction was cou-
pled with extremely low numbers of offspring produced by
fertile pairs (only one or two offspring produced by the fer-
tile pairs). In contrast, in selfing lines, while the offspring
number declined with increased temperature, the infertility
rates remained low (xol-mutated: 0.13 and 0.07, wild type:
0.07 and 0).
Such drastic fitness decline in outcrossers under thermal
stress could not be due to elevated mortality in fog-mutated
lines as most individuals either remained alive for at least
1 day (usually longer) after they finished reproduction or
had never started laying eggs. Only two females in each
line were found dead the next day after eggs were recorded.
Similarly, Theologidis and colleagues (2014) found that
Table 1 Effects of temperature, breeding and source line together
with and all the interactions on lifetime reproductive success (num-
ber of offspring produced) of C. elegans hermaphrodites (xol-mutated
and wild type lines) and pairs of males and females (fog-mutated
lines) analyzed using linear model with differences in variances
among breeding systems, temperatures and source lines
Effect df. F p
Temperature 1;167 1392.749 <0.001
Breeding system 2;167 160.915 <0.001
Source line 1;167 14.151 <0.001
Temperature x breeding system 2;167 16.352 <0.001
Temperature x source line 1;167 8.634 0.004
Breeding system x source line 2;167 1.119 0.329
Temperature x breeding sys-
tem x source line
2;167 0.394 0.675
Error 167 – –
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361Evol Biol (2017) 44:356–364
1 3
survivorship differences did not explain decreased out-
crossing rates in high salinity environment. Conceivably,
the pattern observed in our study could have resulted from
male and/or female gamete production failure. Indeed, it
has been shown that increased temperature affects sperm
and oocyte production, ovulation and spermatid activation
in C. elegans (Aprison and Ruvinsky 2014; Petrella 2014).
We might also expect thermal stress to result in elevated
gamete death (McMullen et al. 2012) as increased tem-
perature during ovulation has been shown to reduce gam-
ete viability in some fish species (Pankhurst and Van Der
Kraak 1997). However, low incidence of sterility in selfing
lines proves that both types of gametes successfully func-
tion in hermaphrodites under the same thermal conditions.
Hence, we hypothesize that the higher thermal sensitivity
of obligatorily outcrossing lines may be associated with
mating failure. Temperature is well-known to affect behav-
ior of ectotherms (reviewed by Angiletta 2009) including
reproductive behavior in many species (Wilkes 1963; Linn
and Campbell 1988; Katsuki and Miyatake 2009). Whereas
self-fertilization is a purely physiological process, outcross-
ing requires a complex set of behaviors in C. elegans. First,
males have to respond to chemosensory cues from poten-
tial partners (Simon and Sternberg 2002). Then, they need
to locate the vulva, to which they insert their spicules and
ejaculate (Barr and Garcia 2006). Such a complex process
is likely to be sensitive to environmental conditions, as dis-
turbance at any of its components will result in reduced
mating ability of an animal. We are not aware of any stud-
ies specifically addressing thermal effects on C. elegans
mating behavior, however, it has been shown to be sensi-
tive to intrinsic stress caused by senescence. Chatterjee
etal. (2013) demonstrated that reproductive senescence in
C. elegans males is associated with decreased mating effi-
ciency rather than deterioration of sperm quality or sperm
number.
Elucidating the mechanisms behind the pattern observed
in our study requires further work. Whatever the mecha-
nism, however, our results highlight the fact that the level
of stress created by the same change in external environ-
ment (5 °C increase in ambient temperature) can differ
dramatically between individuals differing in reproduc-
tive mode. Importantly, the sharp decline in mean fitness
in outcrossing lines was also associated with an interesting
pattern of variation: while 57% of pairs were unfertile and
30% only produced 1–6 larvae, four pairs (13%) bred 23,
30, 109 and 282 offspring, respectively. In hermaphrodites,
such a heterogeneous response has been observed only
line A line B
20°C20°C25°
C2
5°C
fog
xol
wt
fog
xol
wt
fog
xol
wt
fog
xol
wt
number of offspring
Fig. 2 Least square means and confidence intervals for the number
of offspring produced by the obligatorily selfing (fog-mutated—
‘fog’), facultatively outcrossing (wild type—‘wt’) and obligatorily
outcrossing (xol-mutated—‘xol’) lines for each of the source lines
and thermal treatments. The estimates were calculated from the linear
model allowing for different variances among breeding systems, tem-
peratures and source lines
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362 Evol Biol (2017) 44:356–364
1 3
under much more severe thermal stress (≥28 °C; Mc Mul-
len etal. 2012).
Such a pattern of response to a stressful environmen-
tal factor can have complex effects on adaptation process
in outcrossing populations. On one hand, very low (or
zero) fitness of most individuals translates to low effec-
tive population size, which increases the impact of genetic
drift and may also lead to inbreeding depression, hamper-
ing adaptive potential and increasing the risk of extinction.
On the other hand, large variation in fitness will generate
strong selection on any traits associated with it, which can
increase the rate of adaptation, as long as there is heritable
variation in these traits (Lynch and Walsh 1998). For exam-
ple, if mating efficiency does indeed strongly contribute to
the reproductive performance of outcrossers, as we hypoth-
esize, high temperature will impose strong selection on
traits associated with mating success. This would further
lead to intense sexual selection over mating, making sexual
selection an important contributor to adaptation process in
populations of outcrossers (Candolin and Heuschele 2008;
Lorch etal. 2003; Plesnar-Bielak etal. 2012).
Importantly, we measured reproductive success of indi-
vidual hermaphrodites or male–female pairs. Extrapolating
these results to population level would make the estimated
disadvantage of outcrossing even more severe: since in
dioecious population only 50% of individuals can bear off-
spring (assuming 1:1 sex ratio). Thus, reproductive output
of females should be at least twice that of hermaphrodites
to offset the cost of males, whereas we showed it to be, on
average, only about 1.5 times larger in 20 °C and 9.4–14.6
times smaller in 25 °C. However, in the population con-
text, a small fraction of males may fertilize all or nearly all
females. Thus, if the observed pattern of outcrossing pairs
fitness at 25 °C was indeed caused by high incidence of
male mating or fertilization failure, reproductive output of
outcrossing populations in thermal stress conditions could
be considerably higher than predicted from our pairs/indi-
vidual based fecundity assays (but see Theologidis et al.
2014). Thus, determining population dynamics of different
reproductive modes under stressful conditions needs exper-
imental verification.
Summarizing, our results have important implications
for investigating the evolution of reproductive modes in
the context of environmental changes. They indicate that
in addition to the level of genetic shuffling, reproductive
modes may differ in the level of selective pressure experi-
enced under the same external environment. Importantly,
the difference in selective pressures, and its relative con-
tribution to the adaptation process, may be specific to the
nature of environmental change and the genetic make-up of
evolving populations, among other putative factors. Thus,
we suggest that future studies should test how hermaph-
rodite, male and female fitness is influenced by a variety
of different stressors, using populations of various genetic
backgrounds, including other C. elegans strains (although
as shown by a comprehensive recent study, genetic diver-
sity in C. elegans is exceptionally low in a global scale;
Andersen et al. 2012) and other species, and also—other
mechanisms determining reproductive mode (here, we
applied two mutations most commonly used for this pur-
pose in our model species, cf. Anderson etal. 2010). Most
importantly, however, the potential for difference in selec-
tive pressures should be taken into account when assessing
the role of outcrossing in adaptation process. Any differ-
ences in adaptation dynamics observed between reproduc-
tive modes should not be attributed solely, or primarily, to
the effects of recombination, without checking for differ-
ences in selection.
Acknowledgements We appreciate the comments of three anony-
mous reviewers, whose suggestions significantly improved our man-
uscript. The study was supported by National Science Centre, Grant
UMO-2013/09/B/NZ8/03317for Z.M.P.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict
of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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