Metabarcoding for parallel identification of species, sex and diet of obligate scavengers: an application to globally-threatened Gyps vultures

After suffering a massive decline (~ 99%) in numbers caused by feeding on livestock carcasses containing the nephrotoxic drug diclofenac, critically endangered Gyps vultures now persist in low numbers in the Indian subcontinent. Most are concentrated near National Parks, likely due to availability of wild carcasses free from toxic veterinary drugs. Quantification of vulture diets is critical to test this hypothesis. We describe a validated “field-to-benchtop-to-desktop” metabarcoding workflow for assessing species- and sex-specific diet of these obligate scavengers from non-invasively collected faecal samples. Seven metabarcodes were designed to simultaneouslsy identify the vulture species, sex and diet. Amplicons generated from multiplex PCRs were indexed and sequenced on an Illumina Miseq platform. We included controls and three replicates per sample to establish a series of non-arbitrary thresholds to filter the sequence data and eliminate cross-contamination, PCR/sequencing errors and false positives. This strategy enabled identification of species and sex for all samples and diet-specific sequences with high taxonomic resolution for 97% of samples. Just four (two for diet, one each for vulture species and sexing) metabarcodes were sufficient to meet the objectives. This preliminary analysis suggests that domestic livestock is the most frequently consumed diet item across samples from inside and outside protected habitats. Our method provides a rapid and reliable tool for describing large-scale variation in consumption of domestic versus wild species in the diet of these scavengers, paving the way for a better understanding of the role protected areas play in persistence and recovery of the remaining Gyps vultures in the wild.