Abstract Context. Diet variability is a significant driver of seabird decline; however, data on seabird diet composition and trends have been affected by changes in precision and resolution owing to the evolution of different sampling methods over time. We investigated the effectiveness of applying a passive molecular diet method using faeces obtained from the endangered yellow-eyed penguin. Aims. To assess the feasibility of applying DNA metabarcoding methods to yellow-eyed penguin faeces to evaluate diet, and to compare the reliability of diet results derived from adults and chicks, and from latrine versus fresh faecal samples. Methods. We collected 313 faecal samples from yellow-eyed penguins resident on the Otago coast of New Zealand from October 2016 to August 2017. We used polymerase chain reaction (PCR) with mitochondrial 16S cephalopod and chordate primers to amplify prey DNA present in the faecal samples, and tested the completeness of our assembled reference databases based on previous diet research. Amplified prey DNA sequences were then assigned to taxa from our reference databases by using QIIME2. Key results. Mitochondrial 16S chordate PCR primers were effective at identifying 29 fish taxa, with 98.3% of amplified sequences being identified to species or genus level in 193 samples (61.7% collected). There was no significant difference in the number, occurrence or proportion of ray-finned fish prey DNA sequences derived from fresh samples or latrines. Mitochondrial 16S cephalopod PCR primers classified 1.98% of amplified DNA sequences as targets, with 96.5% of these target sequences being identified to species or genus level in 48 samples (15.3% collected), and five taxa identified. Conclusions. We recommend the collection of latrine samples to enable long-term monitoring of the diet of yellow-eyed penguins, which will optimise the trade-off between wildlife disturbance and dietary resolution. Further refinement is needed to identify cephalopod dietary components for yellow-eyed penguins, because our cephalopod primers were not as specific as those used for ray-finned fishes, amplifying a large number (>98%) of non-cephalopod species. Implications. DNA metabarcoding offers a robust and comprehensive alternative to other, more intrusive, seabird diet-assessment methods, but still requires parallel studies to provide critical information on prey size, true diet composition and diet quality.

中文翻译：

---

粪便中的物种：DNA 元条形码作为一种确定濒危黄眼企鹅饮食的方法

摘要上下文。饮食变异是海鸟数量减少的一个重要驱动因素；然而，由于不同采样方法随时间的演变，海鸟饮食组成和趋势的数据受到精度和分辨率变化的影响。我们研究了使用从濒临灭绝的黄眼企鹅身上获得的粪便应用被动分子饮食方法的有效性。目标。评估将 DNA 元条形码方法应用于黄眼企鹅粪便以评估饮食的可行性，并比较来自成人和小鸡以及厕所与新鲜粪便样本的饮食结果的可靠性。方法。2016 年 10 月至 2017 年 8 月，我们收集了居住在新西兰奥塔哥海岸的黄眼企鹅的 313 份粪便样本。我们使用聚合酶链反应 (PCR) 和线粒体 16S 头足类动物和脊索动物引物来扩增粪便样本中存在的猎物 DNA，并根据之前的饮食研究测试了我们组装的参考数据库的完整性。然后使用 QIIME2 将扩增的猎物 DNA 序列分配给我们参考数据库中的分类群。关键结果。线粒体 16S 脊索酸 PCR 引物可有效识别 29 个鱼类分类群，其中 98.3% 的扩增序列在 193 个样本（收集 61.7%）中被鉴定到种或属水平。来自新鲜样品或厕所的鳍状鱼类猎物 DNA 序列的数量、发生率或比例没有显着差异。线粒体 16S 头足类 PCR 引物将 1.98% 的扩增 DNA 序列分类为目标，其中 96。在 48 个样本（收集到 15.3%）中，这些目标序列中有 5% 被鉴定到种或属水平，并鉴定了五个分类群。结论。我们建议收集厕所样本，以便对黄眼企鹅的饮食进行长期监测，这将优化野生动物干扰和饮食解决方案之间的权衡。需要进一步改进以识别黄眼企鹅的头足类食物成分，因为我们的头足类引物不像用于射线鳍鱼的引物那样特异性，扩增了大量 (>98%) 的非头足类物种。影响。DNA 元条形码为其他更具侵入性的海鸟饮食评估方法提供了一种强大而全面的替代方法，但仍需要平行研究来提供有关猎物大小、真实饮食组成和饮食质量的关键信息。