Morphological and DNA analysis of pollen grains on butterfly individuals reveal their flower visitation history.

Many butterfly species are conspicuous flower visitors. However, understanding their flower visitation patterns in natural habitats remains challenging due to the difficulty of tracking individual butterflies. Therefore, we aimed at establishing a protocol to solve the problem using the Common five-ring butterfly, Ypthima argus (Nymphalidae: Satyrinae). Focusing on the pollen grains attached the butterfly's body surface, we examined validities of two pollen analyses based on pollen morphology and DNA markers (ITS1 and ITS2), in addition to the classical route census method. We captured thirty-nine butterflies from mid-April to early July and collected pollen grains from each individual. Morphological and DNA analyses of collected pollens identified eighteen and thirty-four taxa of insect pollinated plants respectively, including woody plants such as Castanopsis. The DNA analysis detected as many as thirteen plant taxa from a single butterfly, indicating its high sensitivity for detecting flower visitation. We detected more plant taxa in May when many individuals were flying. This is assumingly related to the post emergence days of the butterflies with more foraging experience. We also found that fluctuations of pollen grain numbers of Leucanthemum vulgare and Erigeron philadelphicus on individual butterflies depend on their flowering periods overlapping partly. Consequently, we conclude that pollen morphology and DNA barcoding analysis, and field observations are mutually complementary techniques, providing an integrated pollen analysis method to study the pollination ecology of butterflies.