Pollen metabarcoding reveals the origin and multigenerational migratory pathway of an intercontinental-scale butterfly outbreak.

Migratory insects may move in large numbers, even surpassing migratory vertebrates in biomass. Long-distance migratory insects complete annual cycles through multiple generations, with each generation's reproductive success linked to the resources available at different breeding grounds. Climatic anomalies in these grounds are presumed to trigger rapid population outbreaks. Here, we infer the origin and track the multigenerational path of a remarkable outbreak of painted lady (Vanessa cardui) butterflies that took place at an intercontinental scale in Europe, the Middle East, and Africa from March 2019 to November 2019. Using metabarcoding, we identified pollen transported by 264 butterflies captured in 10 countries over 7 months and modeled the distribution of the 398 plants detected. The analysis showed that swarms collected in Eastern Europe in early spring originated in Arabia and the Middle East, coinciding with a positive anomaly in vegetation growth in the region from November 2018 to April 2019. From there, the swarms advanced to Northern Europe during late spring, followed by an early reversal toward southwestern Europe in summer. The pollen-based evidence matched spatiotemporal abundance peaks revealed by citizen science, which also suggested an echo effect of the outbreak in West Africa during September-November. Our results show that population outbreaks in a part of species' migratory ranges may disseminate demographic effects across multiple generations in a wide geographic area. This study represents an unprecedented effort to track a continuous multigenerational insect migration on an intercontinental scale.

Protocol to sequence markers from pollen grains carried by long-range migratory butterflies using metabarcoding.

Molecular identification of pollen carried by insects informs about their history of visited plants. For migratory butterflies, it can be used to trace long-range movements enduring days of flight over thousands of kilometers. Here, we present a protocol to (1) isolate pollen grains from butterfly bodies and (2) prepare metabarcoding libraries for their identification using the internal transcribed spacer 2 fragment, a common barcode used to identify plants. This protocol would be applicable to other insect groups and metabarcoding markers. For complete details on the use and execution of this protocol, please refer to Suchan et al.1 and Gorki et al.2.