Optimisation of a pollen DNA metabarcoding method for diet analysis of flying-foxes (Pteropus spp.)

. Determining the diet of fl ying-foxes can increase understanding of how they function as pollinators and seed dispersers, as well as managing any negative impacts of large roosts. Traditional methods for diet analysis are time consuming, and not feasible to conduct for hundreds of animals. In this study, we optimised a method for diet analysis, based on DNA metabarcoding of environmental DNA (eDNA) from pollen and other plant parts in the faeces. We found that existing eDNA metabarcoding protocols are suitable, with the most useful results being obtained using a commercial food DNA extraction kit, and sequencing 350 – 450 base pairs of a DNA barcode from the internally transcribed spacer region (ITS2), with ~550 base pairs of the chloroplast rubisco large subunit ( rbcL ) as a secondary DNA barcode. A list of forage plants was generated for the little red fl ying-fox ( Pteropus scapulatus ), the black fl ying-fox ( Pteropus alecto ) and the spectacled fl ying-fox ( Pteropus conspicillatus ) from our collection sites across Queensland. The diets were determined to comprise predominantly Myrtaceae species, particularly those in the genera Eucalyptus , Melaleuca and Corymbia. With more plant genomes becoming publicly available in the future, there are likely to be further applications of eDNA methods in understanding the role of fl ying-foxes as pollinators and seed dispersers.