Polymorphism at a mimicry supergene maintained by opposing frequency-dependent selection pressures Mathieu Chouteaua,1, Violaine Llaurensb, Florence Piron-Prunierb, and Mathieu Jorona aCentre d’Ecologie Fonctionnelle et Evolutive, UMR 5175 CNRS-Université de Montpellier, École Pratique des Hautes Études, Université Paul Valéry, 34293 Montpellier 5, France; and bInstitut de Systématique, Evolution, Biodiversité, UMR 7205 CNRS-École Pratique des Hautes Études, Muséum National d’Histoire Naturelle, Université Pierre-et-Marie-Curie, 75005 Paris, France Edited by Brian Charlesworth, University of Edinburgh, Edinburgh, United Kingdom, and approved June 7, 2017 (received for review February 13, 2017) Explaining the maintenance of adaptive diversity within popula- the evolutionary convergence of signals among defended species ex- tions is a long-standing goal in evolutionary biology, with impor- posed to the same predator communities [i.e., Müllerian mimicry (6)]. tant implications for conservation, medicine, and agriculture. However, polymorphism is sometimes found within populations Adaptation often leads to the fixation of beneficial alleles, and of warningly colored species (10–13). One spectacular example is therefore it erodes local diversity so that understanding the the mimetic polymorphism found in the Amazonian butterfly coexistence of multiple adaptive phenotypes requires decipher- Heliconius numata. This unpalatable species, whose wing-pattern ing the ecological mechanisms that determine their respective variation is controlled by a single supergene locus [called P (14– benefits. Here, we show how antagonistic frequency-dependent 16)], always displays multiple discrete mimetic color patterns selection (FDS), generated by natural and sexual selection acting within populations (6, 17, 18). This coexistence of distinct on the same trait, maintains mimicry polymorphism in the toxic phenotypes within a locality involves the maintenance of one well- butterfly Heliconius numata. Positive FDS imposed by predators protected form and a diversity of rarer forms suffering up to sev- on mimetic signals favors the fixation of the most abundant and enfold more predation (6) (Fig. 1). The ubiquity of polymorphism best-protected wing-pattern morph, thereby limiting polymor- throughout the range of this species suggests that powerful bal- phism. However, by using mate-choice experiments, we reveal ancing mechanisms are countering the strong positive FDS acting disassortative mate preferences of the different wing-pattern on local warning signals. EVOLUTION morphs. The resulting negative FDS on wing-pattern alleles is Warning color patterns may also be used as mate recognition consistent with the excess of heterozygote genotypes at the cues (3, 19–23). Mate preference generally causes positive supergene locus controlling wing-pattern variation in natural assortative mating between individuals with the same pheno- populations of H. numata. The combined effect of positive and type and may reinforce local monomorphism in the warning negative FDS on visual signals is sufficient to maintain a diversity signal. However, sexual preferences may maintain color poly- of morphs displaying accurate mimicry with other local prey, morphism through a preference for rare phenotypes (24) or although some of the forms only provide moderate protection from disassortative mating [i.e., between individuals of different against predators. Our findings help understand how alternative – adaptive phenotypes can be maintained within populations and phenotypes (25 27)], generating negative FDS (3). Indeed, emphasize the need to investigate interactions between selec- under disassortative mating, individuals with a rarer phenotype tive pressures in other cases of puzzling adaptive polymorphism. enjoy increased reproductive success because a larger propor- tion of the population will prefer them. Negative FDS is therefore disassortative mating | selection conflicts | Müllerian mimicry | a consequence of disassortative mating behavior and has been aposematism | warning signals well described for bisexual reproduction, leading to a 1:1 sex ratio (28), and for the self-incompatibility system of plants (29), resulting in the maintenance of a high level of allelic ocal adaptation generally leads to the fixation of locally Lbeneficial alleles and is often associated with directional or polymorphism at the self-incompatibility locus (29, 30). Here, stabilizing selection that maintains populations at distinct phe- notypic optima in different localities (1, 2). Similarly, sexual se- Significance lection can lead to the fixation and reinforcement of divergent characters between populations (3). Natural and sexual selec- Although biological diversity is being lost at an alarming rate, a tion, when operating in the same direction on the same trait, may mimetic butterfly from the Amazon reveals a selective mech- accelerate trait fixation within populations and divergence be- anism that can lead to rich adaptive diversity. In these toxic and tween populations (3–5). When traits are subject to antagonistic vividly colored butterflies, several forms are maintained within selection regimes, however, it is hard to predict the trajectory and populations, accurately mimicking distinct local butterflies, and equilibria of population differentiation and adaptation. Here, we controlled by differentiated alleles of a supergene. Such di- investigate how the interplay of opposing natural and sexual se- versity is not expected because selection by predators pro- lection may account for spectacular adaptive polymorphism in motes the fixation of a single morph. We show that the mating butterfly wing patterns. behavior favors pairings of dissimilar forms and boosts allelic Prey warning signals and defensive mimicry are well-known diversity at the mimicry supergene. Therefore, sexual selection maintains a phenotypic diversity that predators channel and examples of traits where the best protected phenotypes are well refine into a diversity of warning phenotypes, which corre- described and are determined by the most abundant local phe- spond to the signals used by other defended species of but- notype (6, 7). In such cases, defended prey display warning signals terflies in the local community. that are recognized by predators through avoidance learning, such that the protection associated with a warning signal increases with Author contributions: M.C. and M.J. designed research; M.C., V.L., and F.P.-P. performed its frequency of encounter by predators (6). Predator education research; M.C. contributed new reagents/analytic tools; M.C. and V.L. analyzed data; and therefore generates positive frequency-dependent selection (FDS) M.C., V.L., and M.J. wrote the paper. favoring common warning signals, and whose slope depends on The authors declare no conflict of interest. total prey numbers (6, 8, 9). This selection regime restricts the This article is a PNAS Direct Submission. conditions allowing the emergence of polymorphisms and promotes 1To whom correspondence should be addressed. Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1702482114 PNAS Early Edition | 1of5 Downloaded by guest on September 30, 2021 preferences therefore override male preferences and lead to strong disassortative mating. To test for the effects of disassortative mating in nature, we assessed deviations from Hardy–Weinberg genotype frequencies in natural populations of H. numata. The wing pattern supergene P displayed an excess of heterozygote genotypes (He = 0.699, Ho = 0.720, P = 0.040; Fig. 3). In contrast, 20 unlinked micro- satellite markers distributed across the genome showed allele frequencies fitting Hardy–Weinberg expectations or even show- ing some heterozygote deficits (34). This discrepancy between heterozygote excess at P relative to the rest of the genome is expected if disassortative mating acts specifically on P genotypes and generates negative FDS. The overall heterozygote excess is largely driven by a strong deficit of homozygotes for the domi- Fig. 1. Stability of mimicry polymorphism and associated protection within Pbic P < the natural population of H. numata from Tarapoto (Peru). The most common nant allele ( 0.001) underlying the most abundant and best protected phenotype (bicoloratus). Bicoloratus individuals morph (bicoloratus) is well protected from predator attacks, followed by bic tarapotensis and then the rarest, silvana, which suffers high attack rates (6). constitute 49% of this population, of which only 4% are P homozygotes (Fig. 3). Under the sole effect of positive FDS generated by predators, this allele is expected to be quickly for the case of mating preferences associated with wing pattern driven to fixation (6). However, because this allele is dominant bic in H. numata, we examine whether sexual selection may gen- over all other mimetic alleles, P homozygotes can only be erate negative FDS on mimetic morphs opposing the positive formed through the mating of two individuals displaying the FDS due to mimicry and explain the maintenance of stable same bicoloratus phenotype, which rarely happens when mating polymorphism in this species. occurs preferentially between dissimilar forms. Disassortative mating is therefore expected to be especially efficient in ham- Results pering the formation of homozygotes for the dominant allele, bic To assess the role of sexual selection in stabilizing the wing color which may account for their scarcity and explain why the P pattern