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, mostly concentrated within or near National Parks. This spatial association might be attributed to availability of wild ungulate carcasses free from toxic veterinary drugs. Hence, quantification of vulture diets is critical to test this hypothesis. We describe a validated “field-to-benchtop-to-desktop” metabarcoding workflow for assessing the species- and sex-specific diet of these obligate scavengers from non-invasively collected faecal samples. Seven metabarcodes were designed to simultaneously identify the vulture species, sex and diet species.  The 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...