The evolution of plastic responses to external cues allows species to maintain fitness in response to the environmental variation they regularly experience. However, it remains unclear how plasticity evolves during adaptation. To test whether distinct patterns of plasticity are associated with adaptive divergence, we quantified plasticity for two closely related but ecologically divergent Sicilian daisy species (Senecio, Asteraceae). We sampled c.40 representative genotypes of each species from their native range on Mt Etna and then reciprocally transplanted multiple clones of each genotype into four field sites along an elevational gradient that included the native elevational range of each species, and two intermediate elevations. At each elevation we quantified survival and measured leaf traits that included investment (specific leaf area), morphology, chlorophyll fluorescence, pigment content and gene expression. Traits and differentially expressed genes that changed with elevation in one species often showed little change in the other species, or changed in the opposite direction. As evidence of adaptive divergence, both species performed better at their native site and better than the species from the other habitat. Adaptive divergence is therefore associated with the evolution of distinct plastic responses to environmental variation, despite these two species sharing a recent common ancestor.

To determine how two ecologically divergent Senecio species respond to an elevational gradient, we undertook field experiments and transplanted multiple clones of 20-40 genotypes of each species across an elevational gradient representing the native range of each species and two intermediate eleavations. We transplanted cuttings that allowed us to quantify the response of each genotype by measuring survival and leaf characteristics (pigment content and leaf morphology). We used the software Lamina to quantify leaf shape, and we used a Dualex instrument to quantify leaf pigments (chlorophyll and flavonols).

In as separate laboratory experiment, we grew seeds of each species and tested whether they differed in physiological parameters that included intrinsic water use efficiency, pigment content of their leaves and their ability to regulate heat.

Missing values in the transplant data represent plants that died and therefore have survival data but no morphology data.