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.

以含有肾毒性药物双氯芬酸的牲畜尸体为食导致数量急剧下降（〜99％）后，吉普斯受到严重威胁现在，印度次大陆上的秃鹰数量很少。大部分都集中在国家公园附近，这可能是由于可获得无毒兽药的野生car体。秃鹰饮食的定量对验证这一假设至关重要。我们描述了一种经过验证的“现场到台式到台式”元条形码工作流程，用于评估从非侵入式收集的粪便样本中这些专性清除剂的物种和性别特异性饮食。设计了七个元条形码以同时识别秃鹰的种类，性别和饮食。在Illumina Miseq平台上对从多重PCR产生的扩增子进行索引和测序。我们包括了对照和每个样品三个重复，以建立一系列非任意阈值来过滤序列数据并消除交叉污染，PCR /测序错误和假阳性。这种策略可以识别所有样品的物种和性别，以及针对特定饮食的序列，从而对97％的样品具有高分类学分辨率。仅四个（用于饮食的两个，用于秃species物种和性别的一个）的元条形码足以满足目标。初步分析表明，在受保护的栖息地内外的样本中，家畜是最常消耗的饮食。我们的方法提供了一种快速可靠的工具，用于描述这些清道夫的饮食中家庭物种和野生物种的消费量的大规模变化，从而为更好地了解保护区在剩余生物的持久性和恢复中的作用铺平了道路。对于秃v种类和性别，每只一个）元条形码足以满足目标。初步分析表明，在受保护的栖息地内外的样本中，家畜是最常消耗的饮食。我们的方法提供了一种快速可靠的工具，用于描述这些清道夫的饮食中家庭物种和野生物种的消费量的大规模变化，从而为更好地了解保护区在剩余生物的持久性和恢复中的作用铺平了道路。对于秃v种类和性别，每只一个）元条形码足以满足目标。初步分析表明，在受保护的栖息地内外的样本中，家畜是最常消耗的饮食。我们的方法提供了一种快速可靠的工具，用于描述这些清道夫的饮食中家庭物种和野生物种的消费量的大规模变化，从而为更好地了解保护区在剩余生物的持久性和恢复中的作用铺平了道路。在野外的秃鹰秃ul。