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.