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 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-specific sequences could be identified with high taxonomic resolution
for 97% of samples. Out of the seven metabarcodes, just four (one for
species identification, one for sexing and two for diet) 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.