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

An understanding of the factors affecting the diet composition of critically endangered Gyps vultures in the Indian subcontinent has important applications to the design of effective conservation strategies. After suffering a massive decline (~99%) in numbers caused by veterinary use of the nephrotoxic drug diclofenac, vultures now persist in very low numbers, mostly concentrated within or near National Parks. This spatial association might be attributed to the availability of wild ungulate carcasses free from toxic veterinary drugs. Hence, quantification of vulture diets and the factors affecting them is critical to test this hypothesis. Here, we describe and validate a robust \"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 targeting mitochondrial and nuclear genes were used to simultaneously determine the vulture species identity, sex and species consumed. The amplicons were sequenced using 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. Using this strategy enabled identification of species and sex for all samples. Diet species-specific sequences could be identified with high taxonomic resolution for 93% of samples. Out of the seven metabarcodes, just four were sufficient to meet the objectives. From this preliminary analysis, domestic livestock seem to be 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 species, paving the way for a better understanding of the role protected areas play in persistence and recovery of the remaining Gyps vulture populations in the wild.