Rationale: Many insect species undertake multi-generational migrations in the Afro-tropical and Palearctic ranges, and understanding their migratory connectivity remains challenging due to their small size, short life span and large population sizes. Hydrogen isotope ( δ 2H) can be used to reconstruct the movement of dispersing or migrating insects, but applying δ 2H for provenance requires a robust isotope baseline map (i.e., isoscape) for the Afro-Palearctic. Methods: We analysed the δ 2H in the wings ( δ 2H wing) of 142 resident butterflies from 56 sites across the Afro-Palearctic. The δ 2H wing values were compared to the predicted local growing-season precipitation δ 2H values ( δ 2H GSP) using a linear regression model to develop an insect wing δ 2H isoscape. We used multivariate linear mixed models and high-resolution and time-specific remote sensing climate and environmental data to explore the controls of the residual δ 2H wing variability. Results: A strong linear relationship was found between δ 2H wing and δ 2H GSP values (r 2=0.53). The resulting isoscape showed strong patterns across the Palearctic but limited variation and high uncertainty for the Afro-tropics. Positive residuals of this relationship were correlated with dry conditions for the month preceding sampling whereas negative residuals were correlated with more wet days for the month preceding sampling. High intra-site δ 2H wing variance was associated with lower relative humidity for the month preceding sampling and higher elevation. Conclusion: The δ 2H wing isoscape is applicable to trace butterflies, moths and other terrestrial herbivorous insects that migrate across the Afro-Palearctic range but has limited geolocation potential in the Afro-tropics. The spatial analysis of uncertainty using high-resolution climatic data demonstrated that many African regions with highly variable evaporation rates and relative humidity have δ 2H wing values that are less related to δ 2H GSP values. Increasing geolocation precision will require new modeling approaches using more time-specific environmental data and/or independent geolocation tools.

The oogenesis-flight syndrome reflects the temporal allocation of energy resources between dispersal / migration and reproduction and is a key concept in research on migratory behaviour in animals. In migratory butterflies, host plant abundance and quality may act as environmental cues to switch between the two states, but the mechanisms regulating this process are virtually unknown. Here, we used an experimental set-up to assess how variation in host plant abundance affected the activity of regulatory elements in the painted lady butterfly (Vanessa cardui), a model species for insect migratory behaviour studies. Chromatin immunoprecipitation (ChIP-seq) was used to evaluate histone tail modifications of H3K27ac and H3K4me3, as a proxy for regulatory activity. The results indicate that recently eclosed females that had access to host plants invested in reproduction at an earlier stage and that variation in host plant abundance triggered significant differences in regulatory element activity via histone tail acetylation. The functions of genes in the vicinity of differentially activated regions were primarily associated with metabolism, egg shell formation, female receptivity, muscle activity, pheromone binding and chromosome maintenance. Our results provide a first glimpse into the regulatory underpinnings of how females perceive the environment and allocate resources for either migration or reproduction and a starting point for more detailed understanding of the links between environmental variation, gene regulation and behaviour in butterflies.

Endemics co-occur because they evolved in situ and persist regionally or because they evolved ex situ and later dispersed to shared habitats, generating evolutionary or ecological endemicity centres, respectively. We investigate whether different endemicity centres can intertwine in the region ranging from Alps to Sicily, by studying their butterfly fauna. We gathered an extensive occurrence dataset for butterflies of the study area (27,123 records, 269 species, in cells of 0.5x0.5 degrees of latitude-longitude). We applied molecular-based delimitation methods (GMYC model) to 26,557 COI sequences of Western Palearctic butterflies. We identified entities based on molecular delimitations and the most recent checklist of European butterflies and objectively attributed occurrences to their most probable entity. We obtained a zoogeographic regionalisation based on the 69 endemics of the area. Using phylogenetic ANOVA we tested if endemics from different centres differ from each other and from non-endemics for key ecological traits and divergence time. Endemicity showed high incidence in the Alps and Southern Italy. The regionalisation separated the Alps from the Italian Peninsula and Sicily. The endemics of different centres showed a high turnover and differed in phenology and distribution traits. Endemics are on average younger than non-endemics and the Peninsula-Sicily endemics also have lower variance in divergence than those from the Alps. The observed variation identifies Alpine endemics as paleoendemics, now occupying an ecological centre, and the Peninsula-Sicily ones as neoendemics, that diverged in the region since the Pleistocene. The results challenge the common view of the Alpine-Apennine area as a single “Italian refugium”.