Step-wise evolution of temperature-mediated phenotypic plasticity in eyespot size across nymphalid butterflies

There are two disparate views regarding phenotypic plasticity. One regards plasticity as a derived adaptation to help organisms survive in variable environments while the other views plasticity as the outcome of flexible, non-canalized, developmental processes, ancestrally present in most organisms, that helps them colonize or adapt to novel environments e.g., a pre-adaptation. Both views of plasticity currently lack a rigorous, mechanistic examination of ancestral and derived states and direction of change. Here we show that the origin of phenotypic plasticity in eyespot size in response to environmental temperature observed in Bicyclus anynana butterflies is a derived adaptation of this lineage. Eyespot size is regulated by temperature-mediated changes in levels of a steroid hormone, 20E, that affects proliferation of eyespot central cells expressing the 20E receptor (EcR). By estimating the origin of the known physiological and molecular components of eyespot size plasticity in a comparative framework, we showed that 20E titer plasticity in response to temperature is a pre-adaptation shared by all butterfly species examined, whereas the origin of expression of EcR in eyespot centers, and the origin of eyespot sensitivity to the hormone-receptor complex are both derived traits found only in a subset of species with eyespots. The presence of all three molecular components required to produce a plastic response is only observed in B. anynana. This gradual, step-wise, physiological/molecular response to temperature is a likely adaptation to temperature variation experienced across wet and dry seasons in the habitat of this species. This work supports, thus, the first view of plasticity as a derived adaptation.