29Environmental DNA (eDNA) has great potential to complement visual surveys, camera 30 trapping, and bioacoustics in measuring biodiversity. We report here a large-scale attempt 31 to use DNA from leech-ingested bloodmeals to estimate vertebrate occupancy at the scale 32 of an entire protected area: the 677 km 2 Ailaoshan national-level nature reserve in Yunnan 33 province, southwest China. We contracted 163 park rangers to collect leeches in 172 pa-34 trol areas, resulting in 30,468 total leeches, divided over 893 replicate samples. Replicate 35 sampling from each patrol area was achieved by providing rangers with preservative-filled 36 tubes in separate, self-sealing bags, inducing them to distribute collected leeches over mul- 37 tiple bags, which we could sequence separately. We show that combining leech-derived 38 DNA data with Bayesian site-occupancy modeling can produce detailed and useful biodi-39 versity inferences for a wide range of vertebrates in a reasonable timeframe. For example, in 40 1 Ailaoshan, sites at lower elevations and closer to the reserve edge have higher occupancy of 41 domestic species (cows, sheep, goats) compared to sites at higher elevations and closer to the 42 reserve interior. Muntjak deer show similar occupancy patterns to the domesticated species, 43 but otherwise, most species of large mammal wildlife (e.g. sambar, black bear, serow, tufted 44 deer) show the opposite pattern: greater occupancy at higher elevations and closer to the 45 reserve interior. We conclude that leech-derived eDNA can be used to efficiently measure 46 the effectiveness of protected areas in terms of vertebrate biodiversity outcomes and to help 47 us optimize the deployment of management resources within reserves, by providing valuable 48 information on the spatial distributions of vertebrate species and on the environmental and 49 anthropogenic correlates of those distributions. 50 环境DNA (eDNA) 在生物多样性评估方面具有极大的潜力, 可以与现有的生物多样性监测 51 方法, 如目视遇测法、红外相机监测法, 生物声学监测法等形成互补. 本研究首次利用蚂 52 蝗eDNA(即从蚂蝗吸食的血液中提取的DNA) 对位于中国西南部云南省的哀牢山国家自然 53 保护区进行了一个全局的脊椎动物多样性的评估. 在本研究中, 677平方公里的保护区被划 54 分成172个巡逻区, 由163位护林员在巡视过程中采集了总共30468只蚂蝗, 这些蚂蝗根据采集 55 样点, 采集时间及具体数量被合并成了893份样本. 我们将蚂蝗eDNA数据和贝叶斯位点占据 56 模型相结合, 分析推断得到在一定时间范围内哀牢山脊椎动物各物种的具体分布图谱. 例如, 57 哀牢山海拔较低及靠近保护区边缘的地区, 相较于海拔较高及靠近保护区中心的地区, 具有 58 更多的牛, 羊等家养动物; 在野生物种中, 赤麂呈现了与家养动物类似的分布趋势, 而其它大 59 型哺乳动物(如水鹿、黑熊、苏门羚) 则相反, 在海拔较高及靠近保护区中心的区域分布更 60 多. 本研究的结果显示基于蚂蝗的eDNA技术可以提供关于脊椎动物物种的空间分布以及环 61 境和人类活动对这些物种分布的影响等有价值的信息, 让我们可以据此评估保护区对脊椎动 62 物多样性的保护效率, 从而可以帮助优化保护区内管理资源的部署. 63 2 Introduction 64 The difficulty of measuring the effectiveness of protected areas. In 2010, the signatories 65 of the Convention on Biological Diversity, including China, agreed to the 2011-2020 Aichi 66Biodiversity Targets [6]. Aichi Target 11 concerns the safeguarding of biodiversity and sets 67 the goals of placing (A) 17% of terrestrial and inland water habitats in a system of nature 68 reserves that are (B) ecologically representative, (C) well-connected, (D) equitable, and 69 (E) effective. The world has nea...