DOI: 10.5061/dryad.j3tx95xbd
title: Metabarcoding for parallel identification of species, sex and diet of obligate scavengers: an application to globally-threatened Gyps vultures

description: 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. 
description: We collected fecal samples from a captive individual (Himalayan
 Griffon, <i>Gyps himalayensis</i>) held at the vulture
 conservation breeding centre at Pinjore, Haryana, (March 2018) and from
 wild vultures at sites located in Pench, Kanha, Sariska, Ranthambore and
 Mukundra Hills Tiger Reserves in India (June 2018-December 2018). At each
 field site, we located resident <i>Gyps </i>vulture
 (White-rumped and Long-billed) roosting and nesting sites and collected
 faecal droppings from substrates (soil, leaves, rocks) directly under the
 roost/nest. We swabbed the faecal dropping using a plastic applicatory
 rayon swab dipped in Longmire lysis buffer (Longmire et al. 1997). The tip
 of the swab was broken and stored in a 2 ml vial (with 1 ml buffer) at
 room temperature for up to 30 days, after which it was transported to the
 laboratory and stored at -20°C until DNA extraction. Using the extracted
 DNA from these samples, we prepared library using seven metabarcodes (two
 for vulture species identification, two for sexing vultures and three for
 diet identification). We sequenced the library on the Miseq platform
 using 2x150 bp paired-end sequencing.  The resulting sequence data was
 processed using the OBITOOLS bioinformatics pipeline and subsequently
 filterered using low frequency noise filters based on the experimnental
 controls included. 
description: Miseq raw sequences of the seven metabarcodes used in this study  This ZIP file contains the compressed FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal sample in triplicate using the MiSeq platform. The 96 multiplexed PCR products were indexed using both forward and reverse indices, which were trimmed in the FASTQ files provided. We have provided a list of the 28 samples, two positive controls and six negative controls in the excel file titled List of samples.xlsx.  MiSeq_Reads_Vulture_faecal_samples.zip