The maintenance of genetic variation in traits under natural selection is a long-standing paradox in evolutionary biology [1][2][3] . Of the processes capable of maintaining variation, negative frequency-dependent selection (where rare types are favoured by selection) is the most powerful, at least in theory 1 ; however, few experimental studies have confirmed that this process operates in nature. One of the most extreme, unexplained genetic polymorphisms is seen in the colour patterns of male guppies (Poecilia reticulata) 4,5 . Here we manipulated the frequencies of males with different colour patterns in three natural populations to estimate survival rates, and found that rare phenotypes had a highly significant survival advantage compared to common phenotypes. Evidence from humans 6,7 and other species 8,9 implicates frequency-dependent survival in the maintenance of molecular, morphological and health-related polymorphisms. As a controlled manipulation in nature, this study provides unequivocal support for frequency-dependent survival-an evolutionary process capable of maintaining extreme polymorphism.Colour-pattern polymorphism in guppies is limited to males and consists of irregular spots of several different structural (blue, green and purple) and pigment-based (yellow, orange, red and black) colours that occur on the body, caudal fin and dorsal fin (Fig. 1). The position, number, size and hue of the spots are highly heritable 5,10 , although the colour saturation (chroma) of orange spots can be influenced by diet 11,12 . Male colouration is highly polymorphic despite being subject to sexual and ecological selection. Female mating preferences usually favour males with the greatest area of orange, although the strength of that preference varies among populations 10,12,13 . Predators also exert selection on colour patterns; they preferentially prey upon males with brighter or more conspicuous colours 14,15 . Despite the apparently strong and directional selection within populations, colour patterns are so variable that any two males are easily distinguishable based on colour pattern alone, unless they are closely related 10 .Several mechanisms have been proposed to explain the maintenance of this extreme polymorphism 10,14 . Mate-choice experiments indicate that females preferentially mate with males bearing rare or novel colour patterns 16,17 . A trade-off (antagonistic pleiotropy) between male sexual attractiveness and offspring viability has also been reported 18 . Both processes could contribute to the maintenance of genetic variation in nature. However, experiments demonstrating these processes were conducted in laboratory environments, and it is not clear whether either process occurs in nature. Another process capable of maintaining polymorphism is a rare-morph survival advantage. This process has been implicated in the maintenance of colour polymorphism in some invertebrates 19,20 and vertebrates 21 , but it has not been tested in the highly polymorphic guppy system. We tested the hypothesis that ma...