Vertical gradients in microclimate, resource availability and interspecific interactions are thought to underly stratification patterns in tropical insect communities. However, only a few studies have explored the adaptive significance of vertical space use during the early stages of reproductive isolation. We analysed flight-height variation across speciation events in Heliconius butterflies representing parallel colonisations of high-altitude forest. We measured flight-height in wild H. erato venus and H. chestertonii, parapatric lowland and mountain specialists respectively, and found that H. chestertonii consistently flies at a lower height. By comparing our data to previously published results for the ecologically equivalent H. e. cyrbia (lowland) and H. himera (high-altitude), we found that the species flying closest to the ground are those that recently colonised high-altitude forests. We show that these repeated trends largely result from shared patterns of ecological selection producing parallel trait-shifts in H. himera and H. chestertonii. Although our results imply a signature of local adaptation, we did not find an association between resource distribution and flight-height in H. e. venus and H. chestertonii. We discuss how this pattern may be explained by variation in forest structure and microclimate. Overall, our findings underscore the importance of behavioural adjustments during early divergence mediated by altitude-shifts.

We characterised the local forest structure by measuring the trunk diameter at breast height (m) and the average distance (m) between neighbouring trees of common tree species along 15–20m sections of the trails used for collecting butterflies. For the tallest trees, we measured the angle of elevation to the treetop with a clinometer. The tangent of this angle was then multiplied by the distance to the tree from the point of observation to estimate height. Shorter trees were measured using measuring tapes.

We measured the height (m) of all the Passiflora (Passifloraceae) hostplants that were visually identified along the trails used to collect butterflies. We also measured the height (m) of the flowering plants that butterflies were observed feeding from at least once. 

In our study, flight-height is defined as the length of the line perpendicular to the ground level from the point where individuals were first observed, as estimated with a measuring tape. Each measured individual (H. e. venus n=33 and H. chestertonii n=39) was stored, ensuring no repeated observations.