Environmental DNA from avian residual saliva in fruits and its potential uses in population genetics

Buccal cells are a valid source of vertebrate DNA for genetic analysis, typically collected with mouth swabs on captured individuals. Yet, advances in DNA storage and processing now permit recovering genomic material from traces of saliva in food remains. For example, the feeding habits of canopy-dwelling frugivorous birds, e.g. large macaws (Psittaciformes), could present an ideal opportunity to obtain environmental DNA (eDNA) from partially consumed fruits. We tested this by collecting tropical almond (Terminalia catappa) fruits, eaten and discarded by scarlet macaws (Ara macao), and processed them using three different collection/storage methods. We successfully isolated DNA molecules from macaw residual saliva in fruits. This genetic material allowed the amplification of 7 microsatellite markers and of the CHD region of the avian sex chromosomes using two different primer pairs. Macaw eDNA concentration (mean 12 ng/µl) was similar to the reported in other avian studies using buccal swabs but overall microsatellite (60% success) and CHD-gen (20% success) amplification was low. The best results were obtained for samples preserved in ethanol. We conclude that saliva eDNA in partially consumed food items is an underused source of non-invasive bird DNA for genetic analyses. Based on our results, we recommend that a combination of specialized collection swabs or ethanol stored swabs along with commercial DNA extraction kits be used. Protocols should be modified accordingly to reach a consistent level of individual identification and gender determination that closely matches traditional sampling.