Partial restoration of mutational robustness after addition of genetic polymorphism and in the presence of sexual selection

The interaction between mutational (i.e. genetic) robustness, cryptic genetic variation and epistasis is currently under much debate, as is the question whether mutational robustness evolved under direct selection or as a by-product of environmental robustness. Here we report that mutational robustness was restored in a mutant line of the butterfly Bicyclus anynana after the spontaneous mutation, comet, appeared in a genetically polymorphic wild type population. The comet mutation modified two phenotypic traits known to be under sexual selection in this butterfly: the dorsal forewing eyespot, which is normally round, but became ‘comet’-shaped, and the androconia, the structures producing the male sex pheromone, which were reduced in size. The comet mutant line remained phenotypically stable for ∼7 seven years, but when outcrossed to the genetically polymorphic wild type population, the outcrossed comet line surprisingly recovered the wild type phenotype within 8 generations. This suggests that mutational robustness against the comet mutation was recovered in the comet outcrossed line by epistatic interactions with the genetic polymorphism originating from wild types. The extent of wild type phenotype recovery in the comet outcrossed line was trait- and developmental temperature-dependent, such that mutational robustness was partially recovered at high, but not at low developmental temperatures. We hypothesized that sexual selection through mate choice, which is sex-reversed between developmental temperatures in this butterfly, could produce mutational robustness at a high (but not at a low) temperature. Females are the choosy sex and exert stabilizing or directional selection on male secondary sexual wing traits but only at higher temperatures. Male mating success experiments under semi-natural conditions then revealed that males with the typical comet mutant phenotype suffered from lower mating success compared to wild type males, while mating success of comet males resembling wild types was partially restored. Altogether, we document the roles of cryptic genetic variation and epistasis in restoration of mutational robustness against a spontaneous mutation with known fitness effects, and we provide experimental evidence, for the first time to our knowledge, that sexual selection can produce mutational robustness.


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Phenotypic variation is the raw material for selection that is ubiquitous for most traits in natural 59 populations. The amount of phenotypic variation can, however, differ dramatically within and among 60 populations, i.e. some traits are invariant within species while being highly variable among closely 61 related species (Flatt 2005). There is ample evidence that the amount of phenotypic variation often 62 does not reach its full potential (i.e. there is less variation than could be present), because phenotypes 63 are robust to mutations or to environmental perturbations (Masel and Siegal 2009; Masel and Trotter 64 displayed a stable phenotype in the laboratory for at least seven years, while reared at various 109 developmental temperatures ; Brakefield and French 1999;Brakefield 2001). 110 In this study, we outcrossed the comet inbred line to the wild type population that displays high 111 levels of heterozygosity ( Van't Hof et al. 2005) in order to restore the genetic polymorphism typical of 112 the wild type population around the comet mutation. Surprisingly, in the next few generations we 113 observed that most individuals of the outcrossed comet line that were reared at 27°C degrees and 114 developed in the wet seasonal form, did not express the comet phenotype and could not be distinguished 115 from wild types. Loss of the comet phenotype in the outcrossed comet line suggested that mutational 116 robustness against the comet spontaneous mutation was restored by epistatic interactions with the 117 genetic polymorphism that was cryptic in the wild type population. Yet, when reared at 20°C and 118 developing in the dry seasonal form, the outcrossed comet line again fully expressed the comet 119 phenotype. Hence, mutational robustness against the comet mutation was dependent on the 120 developmental environment of the outcrossed comet individuals. In order to document these qualitative 121 observations, we quantified the effect of the comet mutation in the outcrossed comet line on both 122 morphological (eyespot size and shape, androconia presence and size) and physiological (amounts of 123 male sex pheromone components) secondary sexual phenotypic traits by comparing outcrossed comet 124 with wild types reared at various temperatures typical of the dry (20°C) and wet (27°C) seasonal forms. 125 It remained unclear, however, why mutational robustness against comet would be restored at a high but 126 not at a low rearing temperature. We hypothesized that sexual selection through female preference for 127 round-shaped and small to mid-shaped eyespot pupils and/or for large male sex pheromone quantities 128 may have fuelled the rapid recovery of phenotypic robustness in the outcrossed comet line. We thus 129 expected that sexual selection should act at the high but not at the low rearing temperature given that 130 sexual roles are plastic and females are the choosy sex only at a high temperature (Prudic et al 2011). 131 To test this hypothesis, we performed behavioral experiments to compare the mating success of males 132 from the outcrossed comet (cc) line displaying the comet (reared at 20°C) or the wild type (reared at 6 27°C) phenotype with wild type (++) males competing for wild type (++) wet seasonal females reared 134 at 27°C. 135

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Insects 137 An outbred wild type population of the African butterfly, Bicyclus anynana (Lepidoptera: 138 Nymphalidae), was established in 1988 from over 80 gravid females collected from a single source 139 population in Malawi, Africa. B. anynana larvae were maintained on a maize-based diet (Zea mays), 140 whereas adults were fed mashed banana (Musa acuminata). High levels of heterozygosity were 141 maintained by using laboratory population sizes that ranged between 400 and 600 adults per generation 142 (Brakefield 2001; Van't Hof et al. 2005). The wild type population was reared in climate rooms at a set 143 of different temperature (20-27°C) and humidity regimes (60 to 80% RH) that represent the natural 144 range of environmental variation present in the field. The two extreme temperatures, 20°C (± 1°C) and 145 27°C (± 1°C), represent the developmental temperature typical of the dry and wet seasonal forms under 146 laboratory conditions, respectively. 147 The comet line founded before 1998 is formed by homozygous "cc" individuals displaying 148 pear-shaped ("comet-shaped") instead of round eyespots on the dorsal and ventral sides of foreand 149 hind-wings ( Fig. 1; Brakefield et al. 1998;Brakefield and French 1999;Beldade et al, 2009). Genetic 150 diversity within the comet line is expected to be low, first due to the initial bottleneck as this 151 spontaneous recessive mutation occurs very rarely in the wild type population, and second because the 152 comet line was subsequently kept in the laboratory at a relatively small population size for years. In 153 this study, we thus restored the genetic diversity at loci other than comet was restored. The collected 154 F1 generation (c+) displayed a wild type phenotype and was crossed among itself to produce a F2 155 generation in which ¼ of the individuals displayed the comet phenotype and were "cc", similarly to 156 findings in Beldade et al (2009). These F2 comet "cc" individuals were selected to produce the next 7

Effect of comet mutation on male wing secondary sexual traits 160
To quantify the phenotypic effect of the comet mutation and assess the effect of developmental 161 temperature on its expression, we reared 3 wild type and 8 comet families obtained from eggs collected 162 in the outcrossed comet line about 6 to 8 generations after the F2 generation at 5 temperatures: 19, 21.5, 163 23, 24.5 and 27°C. Eggs were collected from the outcrossed cc line and from the wild type population. 164 We measured the following male traits: (i) pupil length/width ratio of the dorsal forewing posterior 165 eyespot pupil (measured as the maximal length of the pupil parallel to the wing vein and the width as 166 the maximum width perpendicular to the length), (ii) pupil area of the dorsal forewing posterior eyespot 167 pupil (approximated from the area of an ellipse with pupil length as major axis and pupil width as 168 minor axis), (iii) the area of the first androconial patch located on the forewing ventral side, (iv) the 169 area of the second androconial patch located on the hindwing dorsal side, (v) the presence/absence of 170 a well-developed hairpencil (functionally associated with the forewing androconia), and (vi) 171 presence/absence of a well-developed hairpencil (associated with the hindwing androconia). 172 Hairpencils were considered to be well-developed when at least 10 hairs were present. These six 173 morphological traits are either directly or indirectly (i.e. androconia size) involved in sexual selection 174 (Nieberding et al. 2012;Bacquet et al. 2015). We also estimated the area of the forewing and hindwing 175 by measuring the area between 4 landmarks on each wing. For all morphometric measurements, we 176 recorded the x y coordinates of different landmarks by projecting an image of each morphological 177 structure of interest from a stereomicroscope equipped with a camera lucida onto a graphical tablet. 178 The x y coordinates were then converted into areas or lengths taking into account the magnification 179 and the number of pixels between the coordinates. 180 Virgin males were sampled for determining male sex pheromone (MSP) quantities at ages 3, 7, 14 and 185 21 days for individuals kept at 27°C and ages 3, 7, 14 and 28 days for individuals kept at 20°C. MSPs 186 were extracted and quantified as described previously (Nieberding et al. 2008). Briefly, one forewing 187 and hindwing per individual were soaked during 5 minutes in 600µl of hexane, after which 1 ng/µl of 188 internal standard (palmitic acid) was added. Extracts were then analyzed on a Hewlett-Packard 6890 189 series II gas chromatograph (GC) equipped with flame-ionization detector and interfaced with a HP-190 6890 series integrator with nitrogen as carrier gas. The injector temperature was set at 240°C and the 191 detector temperature at 250°C. A HP-1 column was used and temperature increased from the initial 192 temperature of 50°C by 15°C/min up to a final temperature of 295°C, which was maintained for 6 min. 193

Effect of comet phenotype on male mating success 195
To test for behavioral effects of the comet mutation on male mating success, we performed behavioural 196 experiments competing wild type (++), heterozygote (c+) and outcrossed comet (cc) males for mating 197 success. Two behavioral experiments were performed that aimed at comparing mating success of wild 198 type males and comet males that showed both abnormal pupil shapes and lacked androconia 199 (experiment 1), or comet males that had normal pupil shapes but lacked androconia (experiment 2). 200 Specifically, for experiment 1: wild type males were obtained from eggs of the wild type stock 201 population; comet males were obtained from the outcrossed comet line (F3 generation); heterozygote 202 males (c+) by crossing 32 F2 cc virgin females from the outcrossed comet line with 30 wild type males, 203 and 30 F2 cc males from the outcrossed comet line with 28 virgin wild type females, in two separate 204 cages. Eggs of the three treatments (cc, c+ and ++) were collected for 10 days and reared mostly at 205 27°C, although eggs from replicates 2 and 3 of experiment 1 were kept at the beginning of their 206 development at 20°C in order to delay emergence of the adults. 207 We noted that about 10% of cc males (60 out of 600 males) in the outcrossed comet F3 208 generation displayed wild type eyespots, while androconia remained typically "comet-like" with the affected male mating success, we crossed these 60 comet F3 males with 50 comet F3 females that had 211 also more rounded eyespots to produce the F4 generation of the comet outcrossed line, which were used 212 in experiment 2. The F4 generation of the outcrossed comet line produced mostly males with a wild 213 type eyespot shape but comet-like reduced androconia. We compared the mating success of these F4 214 outcrossed comet males with that of male heterozygote (c+) and wild type (++) males obtained as 215 described above for experiment 1. 216 In both behavioral experiments, groups of 3 to 10-day old virgin males were released in a 217 spacious tropical greenhouse that provided a semi-natural environment for B. anynana. Male genitalia 218 were dusted with colored fluorescent powder (Joron and Brakefield 2003; Nieberding et al. 2008). In 219 experiment 1, males (cc, c+ and ++) competed for matings at a 1:1:1 ratio, with group numbers ranging 220 from 60 to 75 males per group. In experiment 2, wild type (++), heterozygote (c+) and comet (cc) 221 males were released in a proportion of 1:1:2 to mimic an environment in which the wild type phenotype 222 (represented by both ++ and c+ males) was as abundant as the comet phenotype, with numbers ranging 223 between 25 to 60 males per group. In both experiments, 3 to 10-day old virgin wild type females (50 224 to 130 per replicate) were released the following morning, to obtain approximately a 2:1 male:female 225 ratio. Males competed for matings during 72 h, after which females were inspected under ultraviolet 226 illumination for fluorescent dust transferred during mating to assess female mate choice. Double 227 matings occurred occasionally (approximately 1 in every 20 matings) and were scored as 1:1. 228 Experiment 1 was repeated three times, and experiment 2 was repeated twice. 229 230

Statistics 231
All statistical analyses were performed with R 2.12.0 (R Development Core Team 2010), using the 232 lme4 package (Bates et al. 2015). To test for effects of comet on wing morphology, we used mixed 233 models with family as a random variable, and type (outcrossed comet or wild type), temperature (as 234 continuous variable) and their interaction as fixed explanatory variables. We used a normal error 235 distribution for the continuous variables androconial patch area and eyespot pupil size, and a binomial 236 distribution for the hairpencils, which were scored as present or absent. Eyespot pupil ratio data was 237 log transformed and pupil surface was square root transformed to improve homoscedasticity and 238 normality of residuals. For model parameter inference, we used Markov Chain Monte-Carlo 239 simulations (i.e. the mcmc function from the lme4 package) for normal models and approximate z tests 240 for binomial models. Temperature values were centered on the maximum value (27°C). The "type 241 effect" parameter, therefore, corresponds to the difference between comet and wild type at 27°C. For 242 pupal and androconial patch size, wing area (centered on the mean) was also added as an explanatory 243 variable to control for wing size. 244 To analyze sex pheromone quantities, individuals reared at 20°C and 27°C were analyzed 245 separately, because selected age classes differed between the two temperatures. We used linear models 246 with MSP titers as dependent variables and age, type (outcrossed comet or wild type) and their 247 interaction as explanatory variables. These explanatory variables were tested with type II F tests (nested 248 models comparison, with main effects tested after removing their interaction from the full model). 249 To analyze effects of the comet mutation on male mating success, replicated G tests of goodness 250 of fit were used as described by Sokal & Rohlf (1995). A single G test of goodness of fit was computed 251 for each replicate independently and three additional G statistics were calculated: a heterogeneity G 252 test to test whether the different replicates show the same trend, a pooled G test based on the pooled 253 dataset for all replicates and a total G test based on the sum of the single G statistics produced for each 254 replicate. 255

Effect of comet mutation on male wing secondary sexual traits 257
Within 6-8 generations following outcrossing, we quantified the phenotypic traits affected by the comet 258 mutation by comparing eyespot shape and size as well as androconia size between families from the 259 wild type population and from the outcrossed comet line. When reared at 27°C, the phenotypes of the 260 outcrossed comet line had almost completely recovered the wild type phenotype: eyespot pupil shape 261 (circular compared to the elongated pupils of the original inbred comet line), size of the second 262 androconial spot, and presence of the first androconial hairpencil were similar between outcrossed 263 comet families and wild type families (Fig 2, Table 1). In contrast, phenotypes of outcrossed cc families 264 displayed increasing differences compared to the wild type when developmental temperature was 265 decreased (Fig 2, Table 1). Thus, several generations after outcrossing, the effect of the comet mutation 266 had become strongly temperature-dependent for all traits except the size of the second androconial 267 patch, and the effect of the mutation was uncoupled across the set of six affected traits (Table 1). 268 269

Effect of comet mutation on male sex pheromone quantities 270
The quantities of male sex pheromone (MSP) components were compared between wild type and comet 271 males randomly chosen from the outcrossed comet line to test if morphological changes induced by the 272 comet mutation affected MSP production. MSP production did not differ between wild type and 273 outcrossed comet males reared at a higher temperature, but differed strongly at the lower temperature. were reared at 20°C. The production of MSP2 was almost completely suppressed in all ages in 279 outcrossed comet males reared at 20°C, due to absence of the androconia (Fig 3; (Fig. 3). MSP1 284 and MSP3 titers at a single age class (14-day old) in outcrossed comet males were similar to MSP titers 285 of the younger age class in wild type males (8-day old). Thus the rate of increase of MSP1 and MSP3 12 288

Effect of comet phenotype on male mating success 289
Mating success of males with comet or wild type phenotypes was compared during two mating 290 competition experiments under semi-natural conditions in a large tropical greenhouse. In the first 291 experiment, we used outcrossed comet males of the F3 generation, most of which (540/600) had reduced 292 androconia and modified eyespot pupils typical for comet mutants (Brakefield, 1998;Brakefield and 293 French 1999). Mating success of outcrossed comet males (cc) was significantly lower than that of 294 heterozygote (c+) or wild type males (++) and was similar for all three replicates: both the total and 295 pooled G-tests were significant, as well as the single G-tests for two out of three replicates (Table 2). 296 During the second experiment only outcrossed comet males with circular-shaped eyespot pupils and 297 reduced androconial hairpencils from the F4 generation were selected to compete for matings. Mating 298 success of outcrossed comet (cc) males was significantly lower than that of heterozygote (c+) or wild 299 type (++) males in the first replicate, but not in the second replicate, with non-significant pooled and 300 global G-tests (Table 2). In both experiments, outcrossed comet (cc), heterozygote (c+) and wild type 301 (++) males were recaptured in similar proportions to those at which they were released (all G-tests 302 were non-significant at the 0.05 level; of developmental temperatures for at least 7 years following isolation of the mutant from the wild type 313 population (Brakefield 1998(Brakefield , 2001Brakefield and French 1999). While a quarter of the F2 generation 314 issued from the crossing between heterozygote (c+) individuals displayed, as expected, the comet 315 phenotype, the latter faded away within the next few generations within the outcrossed comet line when 316 individuals were reared at a high developmental temperature. Our question was why phenotypes 317 returned to wild type values, and why this happened only at the higher developmental rearing 318 temperature. 319 Over the years significant progress has been made in understanding the molecular basis of 320 phenotypic robustness to genetic perturbations. Epistatic interactions, molecular chaperones (such as 321

Role of sexual selection in mutational robustness 358
Importantly, in our comet case study the genetic polymorphism present in the wild type population was 359 not sufficient to maintain mutational robustness -otherwise the comet mutant would not have appeared 360 in the wild type population in the first place. Genetic polymorphism merely allowed partial restoration 361 of mutational robustness after polymorphism was added to the original comet mutant line. This 362 suggests that genetic polymorphism alone was not sufficient to maintain mutational robustness. As the anynana, we suggest that sexual selection against the comet phenotypic led to the restoration of 365 mutational robustness against the mutation. In the wild type B. anynana population the eyespot and 366 androconial traits that are affected by the comet mutation are known targets of sexual selection. Here, 367 we showed that wild type males had higher mating success compared to comet males, and moreover, 368 that outcrossed comet males with 'less extreme' (i.e., closer to the wild type) phenotypes have higher 369 mating success than males with more extreme phenotypes. For outcrossed comet males with less 370 pronounced eyespot and androconial deformations, mating success was higher than the outcrossed 371 males with more pronounced changes in eyespot and androconial traits. Decreased mating success of 372 these outcrossed comet males may be due to their larger (compared to wild type) eyespot pupils 373 (Robertson and Monteiro 2005), and reduced MSP transfer to female antenna during courtship as a 374 consequence of the reduced second hairpencil and androconial spots (Nieberding et al. 2008). MSP 375 amounts presents on male wings are indeed correlated with androconia spot areas (Nieberding et al. 376 2012). Strong sexual selection on phenotypically diverse outcrossed comet males likely led to very 377 rapid allelic changes at loci other than comet, which interacted epistatically with the comet mutation to 378 produce more wild type phenotypes. 379 The importance of sexual selection in driving trait evolution has long been recognized 380 (Andersson et al. 1998), but it has remained elusive whether selection, including sexual selection, could 381 play a role in evolving phenotypic robustness. Sexual section is often assumed to be a directional force 382 triggering the evolution of exaggerated traits (i.e. traits with disproportionate scaling), but sexual 383 selection can also be a stabilizing force that either rapidly increases, or reduces, differentiation in male 384 traits over generations. A first modeling study by Fierst (2013) suggested that female mate preferences 385 increase male phenotypic robustness under three different sexual selection scenarios compared to a 386 randomly mating population. Her theoretical results imply that female choice leads to selection 387 pressures that affect mutational robustness, which thus has the potential to develop in any population 388 experiencing sexual selection (Fierst 2013). Our results suggest that sexual selection restored 389 mutational robustness against the spontaneous comet mutation within a few generations at high rearing 390 temperature, likely by stabilizing selection for comet phenotypic variants that were closer and closer 391 to the wild type trait values. To the best of our knowledge we provide the first experimental evidence 392 suggesting sexual selection may act as a driver for restoring mutational robustness. 393 The comet phenotype was originally temperature-independent and pleiotropically affected 394 several sexually selected traits. Outcrossed comet individuals displayed phenotypic plasticity for trait 395 expression in response to temperature and the expression of several abnormal traits became uncoupled. 396 At 27˚C outcrossed comet males recovered the first androconial hairpencil and formed an eyespot 397 similar in shape to that of wild type males. This observation of trait-and temperature-specific recovery 398 of mutational robustness in outcrossed comet mutants excludes the possibility that the comet mutation 399 was lost as a consequence of introducing the wild type background. A study on D. melanogaster 400 revealed that insertional mutations of 16 genes led to temperature-dependent phenotypic effects on 401 wing size, where no differences were found at 18˚C, but smaller wing sizes were found at 27˚C ( sex at 20°C. Females may, therefore, have induced directional selection for comet males displaying preference for wild type eyespot and hairpencil characters was the basis for strong sexual selection on 417 comet modifier loci that were introduced into the population through outcrossing with wild type 418 individuals, and that this selection brought the comet mutation under the control of a temperature-419 dependent genetic switch. This switch thus suppresses many aspects of the comet phenotype at 27°C, 420 but not at 20°C. 421 It is important to note one weakness of our work, which is that we tested sexual preferences of 422 wild type and not of comet females, where we assumed that both would have similar preferences for 423 male traits. This may not be true, because, for example, learning through imprinting of male phenotypes 424 during sexual maturation is known to affect female sexual preferences in insects, also in B. anynana 425 (e.g. Westerman et al, 2012). Comet females may thus have learned to prefer the male comet phenotype 426 because they grew up together. Learning is biased in B. anynana, however, in that females can learn to 427 prefer supra-natural sexual stimuli, but not reduced wing ornamentation and thus females may not be 428 able to learn to prefer drab comet males (Westerman et al, 2012). Assortative mating with similar 429 phenotypes may also affect sexual preferences of comet females toward comet male phenotypes 430 although we have no evidence for assortative mating in B. anynana. It is important to note, however, 431 that we observed no restoration of phenotypes for the comet mutation during the 7 years the inbred 432 comet line was kept in the laboratory when comet females had no choice to mate with other males than 433 phenotypic comet ones. 434 In conclusion, this study provides, to the best of our knowledge, a first empirical example that 435 suggests that genetic polymorphism and sexual selection can underlie the rapid evolution of increased 436 phenotypic robustness of abnormal phenotypes towards wild types. We documented a fortuitous 437 example where cryptic genetic variation had been decoupled from the arising of a new spontaneous 438 mutation: genetic polymorphism present in the wild type was added to the mutant isogenic line after 439 this mutation was observed to be stably present. This approach, following the fate of spontaneous 440 mutations decreasing mutational robustness after adding different levels of genetic polymorphism 441 around the mutation, could be useful to implement as a novel method to experimentally assess the 442 effect of background genetic polymorphism on the restoration of mutational robustness in more natural 443 settings, as evolution proceeds. 444 Tables 559 Table 1: Model estimates for 6 male morphological traits involved in sexual selection from wild type 560 and comet mutants (type) across 5 breeding temperatures. The four first models are linear mixed models 561 with family as random effect (not shown) and normal error distribution. The inference on model 562 parameters is based on 10000 MCMC simulations. The two last models (presence/absence of well-563 developed hairpencils) are generalized linear mixed models with family as random effect (not shown), 564 binomial error distribution and logit link function. The inference on parameters is based on approximate 565 z tests. The temperature values are centered on 27°C so that type effect estimates the difference between 566 comet and wild type at 27°C. ( + ) The type x temperature interaction was not significant for the first 567 androconial hairpencil (p>0.99), but this model had some estimation problems due to the high 568 proportion of "presence" in wild type individuals; yet the graphs (Fig 2 panels E, F) show that the 569 models provide a good fit of the data and that there is no doubt that the differences observed between 570 comet and wild type for the androconial hairpencils depend on temperature (i.e. significant interaction) 571 too. 572