Sexual isolation is a critical form of reproductive isolation in the early stages of animal speciation, yet little is known about the genetic basis of divergent mate preferences and preference cues in young species. Heliconius butterflies, well known for their diversity of wing color patterns, mate assortatively as a result of divergence in male preference for wing patterns. Here we show that the specific cue used by Heliconius cydno and Heliconius pachinus males to recognize conspecific females is the color of patches on the wings. In addition, male mate preference segregates with forewing color in hybrids, indicating a genetic association between the loci responsible for preference and preference cue. Quantitative trait locus mapping places a preference locus coincident with the locus that determines forewing color, which itself is perfectly linked to the wing patterning candidate gene, wingless. Furthermore, yellow-colored males of the polymorphic race H. cydno alithea prefer to court yellow females, indicating that wing color and color preference are controlled by loci that are located in an inversion or are pleiotropic effects of a single locus. Tight genetic associations between preference and preference cue, although rare, make divergence and speciation particularly likely because the effects of natural and sexual selection on one trait are transferred to the other, leading to the coordinated evolution of mate recognition. This effect of linkage on divergence is especially important in Heliconius because differentiation of wing color patterns in the genus has been driven and maintained by natural selection for Mü llerian mimicry.Heliconius ͉ Lepidoptera ͉ sexual isolation ͉ speciation

Abstract. Introgressive hybridization is an important evolutionary process and new analytical methods provide substantial power to detect and quantify it. In this study we use variation in the frequency of 657 AFLP fragments and DNA sequence variation from 15 genes to measure the extent of admixture and the direction of interspecific gene flow among three Heliconius butterfly species that diverged recently as a result of natural selection for Mü llerian mimicry, and which continue to hybridize. Bayesian clustering based on AFLP genotypes correctly delineated the three species and identified four H. cydno, three H. pachinus, and three H. melpomene individuals that were of mixed ancestry. Gene genealogies revealed substantial shared DNA sequence variation among all three species and coalescent simulations based on the Isolation with Migration (IM) model pointed to interspecific gene flow as its cause. The IM simulations further indicated that interspecific gene flow was significantly asymmetrical, with greater gene flow from H. pachinus into H. cydno (2Nm ϭ 4.326) than the reverse (2Nm ϭ 0.502), and unidirectional gene flow from H. cydno and H. pachinus into H. melpomene (2Nm ϭ 0.294 and 0.252, respectively). These asymmetries are in the directions expected based on the genetics of wing patterning and the probability that hybrids of various phenotypes will survive and reproduce in different mimetic environments. This empirical demonstration of extensive interspecific gene flow is in contrast to a previous study which found little evidence of gene flow between another pair of hybridizing Heliconius species, H. himera and H. erato, and it highlights the critical role of natural selection in maintaining species diversity. Furthermore, these results lend support to the hypotheses that phenotypic diversification in the genus Heliconius has been fueled by introgressive hybridization and that reinforcement has driven the evolution of assortative mate preferences.

Introgressive hybridization is an important evolutionary process and new analytical methods provide substantial power to detect and quantify it. In this study we use variation in the frequency of 657 AFLP fragments and DNA sequence variation from 15 genes to measure the extent of admixture and the direction of interspecific gene flow among three Heliconius butterfly species that diverged recently as a result of natural selection for Miillerian mimicry, and which continue to hybridize. Bayesian clustering based on AFLP genotypes correctly delineated the three species and identified four H. cydno, three H. pachinus, and three H. melpomene individuals that were of mixed ancestry. Gene genealogies revealed substantial shared DNA sequence variation among all three species and coalescent simulations based on the Isolation with Migration (IM) model pointed to interspecific gene flow as its cause. The IM simulations further indicated that interspecific gene flow was significantly asymmetrical, with greater gene flow from H. pachinus into H. cydno (2Nm = 4.326) than the reverse (2Nm = 0.502), and unidirectional gene flow from H. cydno and H. pachinus into H. melpomene (2Nm = 0.294 and 0.252, respectively). These asymmetries are in the directions expected based on the genetics of wing patterning and the probability that hybrids of various phenotypes will survive and reproduce in different mimetic environments. This empirical demonstration of extensive interspecific gene flow is in contrast to a previous study which found little evidence of gene flow between another pair of hybridizing Heliconius species, H. himera and H. erato, and it highlights the critical role of natural selection in maintaining species diversity. Furthermore, these results lend support to the hypotheses that phenotypic diversification in the genus Heliconius has been fueled by introgressive hybridization and that reinforcement has driven the evolution of assortative mate preferences.

Recent models of mate preference evolution suggest that direct selection on alleles at preference loci and correlated evolution of preference with locally adapted mating cues are more likely to drive the evolution of assortative mate preference than reinforcement. Mate preference evolution in mimetic Heliconius butterflies has been attributed to all three forms of selection, but here we show that reinforcement has been critical. By examining geographical variation in assortative mating and male mate preference among seven populations of three hybridizing Heliconius species from Costa Rica, we found pronounced character displacement of preference such that sexual isolation was enhanced in areas of interspecific contact. Of the different explanations for the evolution of assortative mate preference, only reinforcement is dependent on interspecific contact in this system. Thus, the observed pattern of reproductive character displacement of mate preference is best explained as a product of indirect selection generated by natural selection against nonmimetic hybrids.

The reliability and, to a lesser extent, the validity of the newly created Number Sense Test was evaluated with a group of 40 3-to 5-year-old children attending Head Start. Six number sense skills (e.g., counting, number identification, addition-subtraction) and children's feelings about school were assessed both before and after instruction either in 2 of these skills (i.e., counting and number identification) or in insects (i.e., an attention control condition). The Number Sense Test was found to have high internal consistency and to be a valid means of assessing pre-to posttest improvements in instructed skills as a function of condition. Except for addition-subtraction, training effects did not generalize to noninstructed number sense skills. In neither condition did instruction have a harmful effect on children's largely positive feelings about school.