Supplementary material from "Round-trip across the Sahara: Afrotropical Painted Lady butterflies recolonize the Mediterranean in early spring"

The Palearctic-African migratory circuit has been typically associated with birds. Very few insects are known to endure annual trans-Saharan circuits, but the Painted Lady butterfly (Vanessa cardui) is an exception. While it was demonstrated that this species massively migrates from Europe to the Afrotropics during the autumn, the existence of a reverse migration from the Afrotropics to Europe in the early spring remains hypothetical. Here, we analysed wing stable hydrogen isotope values (δ²H) of V. cardui migrants collected from February to April across the circum-Mediterranean region. We assessed their region of natal origin by comparing their wing isotope signature predicted δ²H values (isoscape). The results unambiguously demonstrate a sub-Saharan origin for many individuals, especially those collected in February, representing the first tangible evidence for a reverse northwards trans-Saharan migration in spring. This work supports the view that the Afrotropics (mostly exploited from September to February) is key in the V. cardui Palearctic-African population dynamics. This species relies on both temperate and tropical habitats to complete their multigenerational cycle, an unprecedented adaptation for butterflies and for most migratory insects. Such migratory circuit has strong parallelisms with those of migratory birds.