Pollen metabarcoding as a tool for tracking long-distance insect migrations

Insects account for a large portion of Earth's biodiversity and are key players for ecosystems, notably as pollinators. While insect migration is suspected to represent a natural phenomenon of major importance, remarkably little is known about it, except for a few flagship species. The reason for this situation is mainly due to technical limitations in the study of insect movement. Here, we propose using metabarcoding of pollen carried by insects as a method for tracking their migrations. We developed a flexible and simple protocol allowing efficient multiplexing and not requiring DNA extraction, one of the most time¿consuming part of metabarcoding protocols, and apply this method to the study of the long¿distance migration of the butterfly Vanessa cardui, an emerging model for insect migration. We collected 47 butterfly samples along the Mediterranean coast of Spain in spring and performed metabarcoding of pollen collected from their bodies to test for potential arrivals from the African continent. In total, we detected 157 plant species from 23 orders, most of which (82.8%) were insect¿pollinated. Taxa present in Africa¿Arabia represented 73.2% of our data set, and 19.1% were endemic to this region, strongly supporting the hypothesis that migratory butterflies colonize southern Europe from Africa in spring. Moreover, our data suggest that a northwards trans¿Saharan migration in spring is plausible for early arrivals (February) into Europe, as shown by the presence of Saharan floristic elements. Our results demonstrate the possibility of regular insect¿mediated transcontinental pollination, with potential implications for ecosystem functioning, agriculture and plant phylogeography.GT is supported by the Marie Curie Actions FP7‐People‐2013 IOF (project 622716) and by the MINECO programme Juan de la Cierva Incorporación (IJCI‐2016‐29083). Funding was provided by the statutory funds of W. Szafer Institute of Botany, Polish Academy of Sciences to TS and MR, by the British Ecological Society (grant LRB16/1015) to GT, by project CGL2016‐76322 (AEI/FEDER, UE) and by the Committee for Research and Exploration of the National Geographic Society (grant WW1‐300R‐18) to GT and RV.Peer reviewe