Speciation is facilitated when traits under divergent selection also act as mating cues. Fluctuations in sensory conditions can alter signal perception independently of adaptation to the broader sensory environment, but how this fine scale variation may constrain or promote behavioural isolation has received little attention. The warning patterns of Heliconius butterflies are under selection for aposematism and act as mating cues. Using computer vision, we extracted behavioural data from 1481 hours of video footage, for 387 individuals. We show that the putative hybrid species H. heurippa and its close relative H. timareta linaresi differ in their response to divergent warning patterns, but that these differences are strengthened with increased local illuminance. Trials with live individuals reveal low-level assortative mating that is sufficiently explained by differences in visual attraction. Finally, results from hybrid butterflies are consistent with linkage between a major warning pattern gene and the corresponding behaviour, though the differences in behaviour we observe are unlikely to cause rapid reproductive isolation as predicted under a model of hybrid trait speciation. Overall, our results reveal that the contribution of ecological mating cues to reproductive isolation may depend on the immediate sensory conditions during which they are displayed to conspecifics.

Behavioural data was collected in two separate experiments. In our manuscript, it's supplementary R Markdown and our GitHub repository (https://github.com/SpeciationBehaviour/visual_preference_heurippa_linaresi) more detailed explanations can be found.

mounted_female_data_doi--10_dot_1098_slash_rspb_dot_2021-0157.csv: Males of H. heurippa, H. t. linaresi and hybrids between the two species were presented with a dead female of each species, each of which had a camera pointing on it. Chunks with male behaviour were then extracted from video material using motion detection software. We then scored by eye which male individual (identified by a code on the wings) interacted with which female type at what time of the day. For pure males, we additionally scored the broad light environment at the H. heurippa female as 'sunny', 'cloudy' or 'woodland shade'. Finally, we combined this data with measurements from illuminance loggers positioned in the proximity of each female. 

tetrad_data_doi--10_dot_1098_slash_rspb_dot_2021-0157.txt: Two males (one of each species) and two females (one of each species) were put into an insectary cage until the first mating occured (the experiment then ended). The combination of male and female species that mated was noted. For the table, we combined the counts for each specific male-female combination.

mounted_female_data_doi--10_dot_1098_slash_rspb_dot_2021-0157.csv contains several NA values in various columns, specifically columns: "HC_model_ID", "TLC_model_ID", "light_HC", "light_TLC", "grayscale_mean_at_visited", "light_class_HC", "temp", "hum", "HC_type", "exp_line", "pref_other_males". Of major importance for the analyses presented in the paper are the columns 1) "light_HC", 2) "light_TLC" and 3) "light_class_HC". 1) and 2) are illuminance measurements (in lux) taken either at the HC (H. heurippa) or the TLC (H. timareta linaresi) female. Missing values occurred because of technical issues (logger battery empty, etc.). 3) are the broad light environments at the H. heurippa female that we scored by eye from video material. This was only done for interactions of males from pure species, hence all hybrid data has NA (and a few data points for pure males, for which corresponding video frames could not be extracted).