Parallel evolution of morphological traits is widely reported, providing evidence for the role of local conditions in driving adaptive divergence. Comparatively, fewer studies have tested for parallelism in behaviour, and it is less clear to what extent heritable behavioural shifts contribute to adaptive divergence. We exploit repeated incipient speciation across altitudinal gradients to explore behaviour and physiology in Heliconius butterflies adapted to high-elevation. We performed common garden experiments with H. chestertonii, a high-altitude specialist from the Colombian Cordillera Occidental, and H. erato venus, a low-elevation proxy for the ancestral population, and compared our results to existing data for an equivalent Ecuadorian taxa-pair. Using broad-scale climatic data, we show that both pairs diverge across similar ecological gradients, confirmed using localised data loggers in the ranges of H. chestertonii and H. e. venus. We further show that H. chestertonii and H. e. venus have divergent activity patterns, attributable to different responses to microclimate, and life histories. Finally, we provide evidence for parallelism in these traits with H. himera and H. e. cyrbia. We propose that this is a result of selection associated with independent colonisations of high-altitude forests, emphasising the importance of heritable behavioural and physiological adaptations during population divergence and speciation.

The excel file contains, in order, a list of supplementary literature (Supplementary Material 1), raw data measurements (S1-S7), PCA outputs (S8-S9), mixed model outputs (S10) and semi-partial R squared measures for mixed models (S11). The .rmd file contains annotated R scripts for performing the analyses presented in the manuscript. This report is accompained by descriptions explaining the use of different packages and functions. The .xlsx file can be opened with Microsoft Excel. The .Rmd file can be opened with R. Supplementary figures can be opened with the majority of image viewers and the captions file with text editor.