1 INTRODUCTION

Biodiversity of freshwater ecosystems is declining at a far greater rate than terrestrial or marine biodiversity (Reid et al., 2019). This is in part due to biodiversity being concentrated in fresh waters, which harbour 10% of global animal biodiversity despite occupying only 2% of the Earth’s surface (Reid et al., 2019). Declines stem from anthropogenic pressure on freshwater ecosystems related to water abstraction and regulation, pollution, land-use change, overexploitation of biological resources, introduction of non-native species and climate change (Dudgeon, 2019). Rates of freshwater species loss are particularly acute in tropical biodiversity hotspots, including those in Southeast Asia, where levels of endemism and anthropogenic pressures are exceptionally high (Dudgeon et al., 2006; Mittermeier et al., 2011). Most species extinctions in tropical fresh waters are likely to be undocumented, involving species that are yet to be formally described or even discovered, or are not prevented because we lack the data to ascertain their conservation status (Dudgeon, 2003). At the same time, conservation measures to effectively protect threatened tropical freshwater species and their ecosystems, including the identification of key biodiversity areas (Holland et al., 2012), are hampered by a lack of data on their distribution, biology, and ecology.

In comparison to fish, amphibians and other vertebrates, tropical freshwater invertebrates are particularly poorly studied (Dudgeon, 2019; Liew et al., 2020). This is despite the fact that these animals support millions of livelihoods as a food source, and provide crucial ecosystem services, including water purification (Chowdhury et al., 2016; Covich et al., 2004; Macadam & Stockan, 2015). For most of these taxa, we lack even the most basic data, such as numbers and identities of species, as well as identification tools (Morse et al., 2007). New freshwater invertebrate species are described from Southeast Asia every year (Jeratthitikul et al., 2021; Mendoza & Yeo, 2014; Zieritz, Jainih, et al., 2021a), but morphological descriptive work is time-intensive and will not be completed before many of these species will have become extinct (Morse et al., 2007). In recent decades, however, molecular tools have emerged as an alternative for detecting and identifying species, particularly for morphologically variable or cryptic, poorly studied, or yet undescribed taxa. DNA barcoding allows for identification of organisms by matching a short DNA sequence from a given specimen against a reference database, such as the Barcode of Life project (BOLD, http://www.boldsystems.org; Ratnasingham & Hebert, 2013) or GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Whilst taxonomic gaps in these reference databases are ubiquitous (Kvist, 2013; Porter & Hajibabaei, 2018), even in the absence of a database match, data from such unidentified or potentially undescribed species can be retained in analyses as molecular operational taxonomic units (OTUs) or barcode index numbers, leading to a more accurate and complete analysis of species diversity and distribution (Wilson et al., 2016). Since the advent of DNA metabarcoding (Taberlet et al., 2012), multiple specimens representing many different species from a single bulk sample can be processed simultaneously, rendering this a promising tool for rapidly gathering data on freshwater invertebrate diversity and distribution. Several studies have shown that DNA metabarcoding can perform equally well as or better than traditional morphology-based surveys of freshwater invertebrates (Beermann et al., 2018; Elbrecht et al., 2017; Emilson et al., 2017; Jackson et al., 2014; Kutty et al., 2018; Lim et al., 2016).

Despite its great potential for rapid biodiversity assessments in poorly studied tropical systems, DNA metabarcoding of freshwater invertebrates has predominantly been applied in well studied, temperate systems (Andújar et al., 2018; Beermann et al., 2018; Compson et al., 2019; Gardham et al., 2014; Theissinger et al., 2018). The few studies that have applied DNA metabarcoding on tropical freshwater invertebrates to date are restricted in spatial and/or taxonomic scale (e.g. focus on general metazoan diversity in two reservoirs in Singapore (Lim et al., 2016), Chironomidae in a swamp forest in Singapore (Baloğlu et al., 2018) and Southeast Asian dytiscid beetles (Balke et al., 2013)). DNA metabarcoding has not yet been used to improve our knowledge of invertebrate diversity and distribution across tropical river basins, which are known to harbour the bulk of global freshwater biodiversity (Dudgeon, 2000; Dudgeon et al., 2006), let alone identify sites of special conservation importance. One particular challenge in conducting DNA-based surveys in remote tropical regions is a potentially more rapid degradation of DNA due to imperfect storage conditions during field campaigns, for example, because access to ice is not always available. It remains to be quantified how effectively available DNA metabarcoding protocols, including different DNA extraction methods, perform in these circumstances and systems across taxonomic groups.

The present study represents the first to apply DNA metabarcoding to comprehensively assess the benthic freshwater invertebrate fauna across a major tropical river basin. Specific objectives were to: (1) assess performance of two variations of a DNA metabarcoding protocol (with regard to DNA extraction from specimens <1 mm) and identify shortcomings, including quantification of false negatives (through comparison of morphological identification), and discuss potential mitigation measures; (2) identify the most significant gaps in reference databases for freshwater invertebrates of this region; (3) gather new information on tropical freshwater invertebrate species diversity and distribution in the study region; and (4) ultimately draw conclusions regarding the potential and limitations of DNA metabarcoding in tropical freshwater conservation.

中文翻译：

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DNA元条形码揭示了热带淡水无脊椎动物的未知多样性和分布模式

1 简介

淡水生态系统的生物多样性正在以远大于陆地或海洋生物多样性的速度下降（Reid 等人，  2019 年）。这部分是由于生物多样性集中在淡水中，尽管淡水仅占地球表面的 2%，但仍拥有全球 10% 的动物生物多样性（Reid 等人，  2019 年）。下降源于与取水和调节、污染、土地利用变化、生物资源过度开发、引进非本地物种和气候变化有关的淡水生态系统的人为压力（Dudgeon，  2019）。热带生物多样性热点地区的淡水物种损失率尤其严重，包括东南亚地区，那里的特有水平和人为压力异常高（Dudgeon 等人，  2006 年；Mittermeier 等人，  2011 年）。热带淡水中的大多数物种灭绝可能没有记录，涉及尚未正式描述甚至发现的物种，或者由于我们缺乏确定其保护状态的数据而没有被阻止（Dudgeon，  2003）。同时，有效保护受威胁的热带淡水物种及其生态系统的保护措施，包括确定关键的生物多样性区域（Holland et al.,  2012)，由于缺乏关于它们的分布、生物学和生态学的数据而受到阻碍。

与鱼类、两栖动物和其他脊椎动物相比，热带淡水无脊椎动物的研究特别少（Dudgeon，  2019 年；Liew 等人，  2020 年）。尽管这些动物作为食物来源支持数以百万计的生计，并提供重要的生态系统服务，包括水净化（Chowdhury 等人，  2016 年；Covich 等人，  2004 年；Macadam 和 Stockan，  2015 年）。对于这些分类群中的大多数，我们甚至缺乏最基本的数据，例如物种的数量和身份，以及识别工具（Morse et al.,  2007）。东南亚每年都有新的淡水无脊椎动物物种被描述（Jeratthitikul 等，  2021; 门多萨和杨，  2014 年；Zieritz, Jainih, et al.,  2021a )，但形态描述工作是时间密集型的，并且在许多这些物种灭绝之前无法完成（Morse et al.,  2007）。然而，近几十年来，分子工具已成为检测和识别物种的替代方法，特别是对于形态可变或神秘、研究不足或尚未描述的分类群。DNA 条形码允许通过将来自给定标本的短 DNA 序列与参考数据库（例如 Barcode of Life 项目）匹配来识别生物体（BOLD，http://www.boldsystems.org；Ratnasingham & Hebert，  2013) 或 GenBank (https://www.ncbi.nlm.nih.gov/genbank/)。虽然这些参考数据库中的分类学差距无处不在（Kvist，  2013 年；Porter 和 Hajibabaei，  2018 年），但即使在没有数据库匹配的情况下，来自这些未识别或可能未描述的物种的数据也可以作为分子操作分类单位 (OTUs) 保留在分析中) 或条形码索引号，从而对物种多样性和分布进行更准确和完整的分析（Wilson 等人，  2016 年）。自 DNA 元条形码出现以来（Taberlet 等，  2012)，可以同时处理来自单个大块样本的代表许多不同物种的多个标本，使其成为快速收集淡水无脊椎动物多样性和分布数据的有前途的工具。多项研究表明，DNA 元条形码可以与传统的基于形态学的淡水无脊椎动物调查一样好或更好（Beermann 等人，  2018 年；Elbrecht 等人，  2017 年；Emilson 等人，  2017 年；Jackson 等人，  2014 年；Kutty 等人，  2018 年；Lim 等人，  2016 年）。

尽管淡水无脊椎动物的 DNA 元条形码在研究较少的热带系统中具有快速生物多样性评估的巨大潜力，但主要应用于研究良好的温带系统（Andújar 等人，  2018 年；Beermann 等人，  2018 年；Compson 等人，  2019 年） ；Gardham 等人，  2014 年；Theissinger 等人，  2018 年）。迄今为止，在热带淡水无脊椎动物上应用 DNA 元条形码的少数研究在空间和/或分类尺度上受到限制（例如，关注新加坡两个水库中的一般后生动物多样性（Lim 等人，  2016 年），在沼泽森林中的摇蚊科新加坡（Baloğlu 等人，  2018) 和东南亚 dytiscid 甲虫 (Balke et al.,  2013 ))。DNA 元条形码尚未用于提高我们对热带河流流域无脊椎动物多样性和分布的了解，众所周知，热带河流域拥有大量的全球淡水生物多样性（Dudgeon，  2000 年；Dudgeon 等人，  2006 年）)，更不用说确定具有特殊保护重要性的地点了。在偏远热带地区进行基于 DNA 的调查的一个特殊挑战是，由于野外活动期间的储存条件不完善，DNA 可能会更快地降解，例如，因为并不总是可以获得冰。仍有待量化可用的 DNA 元条形码协议（包括不同的 DNA 提取方法）在这些情况和系统中跨分类群的效果如何。

本研究首次应用 DNA 元条形码来全面评估主要热带河流流域的底栖淡水无脊椎动物群。具体目标是：(1) 评估 DNA 元条形码方案的两种变体的性能（关于从 <1 mm 的样本中提取 DNA）并识别缺点，包括假阴性的量化（通过形态学鉴定的比较），并讨论潜在的缓解措施; (2) 确定该地区淡水无脊椎动物参考数据库中最重要的空白；(3) 收集研究区热带淡水无脊椎动物物种多样性和分布的新信息；(4) 最终得出关于 DNA 元条形码在热带淡水保护中的潜力和局限性的结论。