Functional Ecology © 2016 British Ecological Society One of the most dramatic examples of biome shifts in the geological record is the rapid replacement of C 3  vegetation by C 4 grasses in (sub-) tropical regions during the Late Miocene–Pliocene. Climate-driven biome shifts of this magnitude are expected to have a major impact on diversification and ecological speciation, especially in grazing taxa. Mycalesine butterflies are excellent candidates to explore the evolutionary impact of these C 3 /C 4 shifts on insect grazer communities. Mycalesine butterflies feed on grasses as larvae, have radiated spectacularly and occur in almost all extant habitats across the Old World tropics. However, at present, we lack a comprehensive understanding of the larval ecology of these butterflies and this hampers investigations of co-evolutionary patterns among the geographically parallel radiations of mycalesine butterflies and the remarkable evolutionary history of their host plants. By conducting several experiments under defined environmental conditions, we demonstrate that the feeding history of mycalesine larvae on C 3 and C 4 grasses can be traced by analysing δ 13 C in the organic material of the adult exoskeleton, while values of δ 18 O in the adult reflect atmospheric humidity during larval development. To show the power of these isotopic proxies for ecological studies, we analysed the isotopic composition of organic material obtained from adult butterflies sampled in two extensive longitudinal surveys. We observed strong associations among the larval ecology, habitat preferences of the adult butterflies and patterns of seasonality, such that mycalesine species that inhabit open environments are more opportunistic in their host plant choice but utilize C 3 grasses more frequently during the dry season. Crucially, the ability to process the less palatable C 4 grasses appears to be phylogenetically clustered within mycalesine species, suggesting that novel feeding adaptations may have evolved in response to the ecological dominance of C 4 grasses in open savanna habitats. A lay summary is available for this article.