Estimating the extended and hidden species diversity from environmental DNA in hyper-diverse regions

Updated Marine fish checklist We constructed an extensive species checklist of the ' Bird's Head Peninsula ' (BHP) of West Papua Province' region based on historical fishing records and visual surveys (Kulbicki et al. 2013) including the ecoregions of the study area, extended with species occurring within and in the adjacent ecoregions with similar environments (Allen & Erdmann 2012, Froese 2020), and with the specimen collected and observed during the 2017 survey. Species names were checked and updated using the authoritative reference and searchable on-line database Eschmeyer's Catalog of Fishes (http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp, Fricke 2020). This extensive checklist identifies 2,534 marine fish species including 1,761 species with confirmed occurrences belonging to 582 genera and 144 families ; it also includes 736 species that are present in close regions and similar environments (See Appendix S4). This exceptional fish diversity is subject to a range of threats (Mangubhai et al. 2012, Campbell et al. 2020). Underwater visual census We retrieved data from 186 UVC transects performed during Aug.-Sept. 2014, Sept. 2015 and March 2018 from the Reef Life Survey initiative (https://reeflifesurvey.com). Additionally, we used data from 93 UVC transects performed between 2004 and 2013 in the region (Cinner et al. 2016, Fig. 1). All surveys used standardized protocols with two divers recording fish identity, abundance and size in 5x50m, or 2.5x50m for Cinner et al. (2016), blocks either side of the transect line. The two transect blocks include independent counts that are averaged to characterized the transect (Edgar et al. 2020). Environmental DNA filtering and processing We collected 92 water samples along the south coast of the BHP region of West Papua between October and November 2017 across different reef habitats (estuarine and brackish waters excluded) distributed over an area of 500 km from East to West, with a focus (80 of the 92 samples, or 87%) from the easternmost 210 km sector (Fig. 1). We collected the water samples in DNA-free plastic bags from a dinghy, during closed-circuit rebreather diving (depths between 10 - 100m) as close as possible to the habitat or using Niskin water samplers (depths between 100 - 300m) (Hocdé et al. 2020). Every water sampling session were performed before and never at the same time as fish collection to avoid in situ contamination. We coupled a pressure and temperature sensor to the Niskin bottle to control the sampling depth and characterize the water mass via the vertical temperature profile. For each sample, we filtered 2L of seawater with sterile Sterivex filter capsules (Merck© Millipore; pore size 0.22µm) and disposable sterile syringes. Immediately after, we filled the filter units with lysis conservation buffer (CL1 buffer SPYGEN©) and stored them in 50 mL screw-cap tubes at -20°C. The DNA extraction and amplification were performed following a modified protocol of Pont et al. (2018) including 12 separate PCR amplifications per sample. A teleost-specific 12S mitochondrial rDNA primer (teleo, forward primer-ACACCGCCCGTCACTCT, reverse primer -CTTCCGGTACACTTACCATG, Valentini et al. 2016) was used for the amplification of metabarcoding sequences (see Appendix S1 for laboratory analyses and bioinformatic analyses). Among fish eDNA 12S primers, teleo provides a strong performance to detect fish diversity even in highly diverse ecosystems (Collins et al. 2019, Polanco Fernández et al. 2022). Although alternative fish eDNA primers might cover a larger proportion of fishes in the reference database and hence be more informative on species identification, there is currently no primers located outside the 12S with similar performance (Zhang et al. 2020). We followed a contamination control protocol during both field and laboratory stages (Valentini et al. 2016). Water sample processing included the use of disposable gloves and single-use filtration equipment, and the bleaching (50% bleach) of Niskin bottles between samples. Staffs who performed eDNA filtration were not involved in tissue sampling of fish and used a dedicated workspace to avoid both contact and airborne contamination. Genetic reference database completion During the same survey along the south-western coast of the BHP in West Papua, we collected 1,466 individuals from 413 species, 180 genera and 69 families of fishes along the shore. The specimens were mainly collected by hand or with 4 to 8m long bottom gillnets deployed by open-circuit and closed-circuit divers in the 0-100 m depth range (Hocdé et al. 2020). Some brackish and estuarine fishes were also collected with 10m beach purse seines and pelagic fish with line fishing and spearfishing. We used morphological features and 652 bp CO1 (Cytochrome Oxidase 1) targeted genetic sequencing to identify the specimens. Then we amplified and sequenced the individuals on a large fraction of the 12S mitochondrial rDNA region (480 bp) with two distinct pairs of primers respectively designed for teleosts and elasmobranchs to improve sequencing results. Finally, the 12S teleo region defined in Valentini et al. (2016) was extracted from the obtained sequences to complement the EMBL genetic reference database (European Molecular Biology Laboratory, www.ebi.ac.uk, version 141, downloaded on January 2020, Baker et al. 2000) and improve taxonomic assignments (see Appendix S2 and S3 for the reference database and the methodological details of its completion). To evaluate the completeness of the online database for the teleo region of the 12S mitochondrial DNA, we performed an in silico PCR on the EMBL database with ecoPCR (Ficetola et al. 2010) using the teleo primer sequences, allowing up to 3 mismatches. We compared the generated list of sequenced species to the extensive species checklist of the BHP ecoregion. Among the 1,761 species of the Bird’s Head Peninsula checklist for which presence is confirmed, only 496 species (28%) were sequenced in EMBL for the teleo region. The addition of sequences retrieved from our fish sampling increased this list to 762 sequenced species (43.4%). Additionally, 21 species absent from the historical checklist were collected, or observed and clearly identified, during the development of the genetic reference database (see Appendix S4 for the extensive checklist). Taxonomic assignments The metabarcoding workflow was based on the VSEARCH toolkit and the clustering algorithm SWARM that groups multiple sequence variants into MOTUs (Molecular Operational Taxonomic Units, Mahé et al. 2014) to clean PCR and sequencing errors. We performed taxonomic assignments using the ecotag program (lowest common ancestor algorithm) from the OBITOOLS toolkit (Boyer et al. 2016) against our custom reference database and the global public EMBL genetic database (release 141, downloaded on January 2020). For each MOTU, we chose the taxonomic assignment with the highest similarity from either the custom reference database or EMBL. We only retained the assignments with 100% similarity to either reference database so matching perfectly over the full length of the sequence (see Appendix S1). Some sequences could match at 100% but correspond to several species due to limited taxonomic resolution on our marker region, preventing a taxonomical assignment at the species level. For those sequences, we determined, if possible, the most probable species being detected based on the list of species corresponding to the sequence and the known spatial distribution of those species. For other sequences, it was not possible to narrow down the list of possible species if those are all known to occur in the region or in the vicinity of the region, so these sequences were tagged with a list of possible assignations (Appendix S6) and removed from the analyses. Fish traits The extended fish diversity may be characterized by certain traits or behaviors which may limit the detection by classical (fishing or visual records) and eDNA surveys (Thalinger et al. 2021). To investigate this bias, we retrieved available data on habitat (reef or pelagic), diet, circadian activity, maximum body length, and IUCN (International Union for Conservation of Nature) conservation status for all the species detected by eDNA from Fishbase (www.fishbase.org) and compared them among the different sets of species.

更新版海洋鱼类名录

本研究基于历史捕捞记录与目视调查（Kulbicki等，2013），构建了西巴布亚省鸟头半岛（Bird's Head Peninsula, BHP）区域的全面物种名录，涵盖研究区域的所有生态区；补充了分布于研究区内及邻近相似环境生态区中的物种（Allen & Erdmann, 2012; Froese, 2020），并纳入2017年调查中采集与观测到的标本。

物种名称通过权威参考资料与可检索在线数据库《埃施迈尔鱼类目录》（Eschmeyer's Catalog of Fishes, http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp, Fricke, 2020）进行校验与更新。

本全面名录共收录2534种海洋鱼类，其中1761种为已确认分布物种，隶属于582个属、144个科；此外还纳入736种分布于邻近区域及相似环境中的物种（详见附录S4）。

这一极高的鱼类多样性正面临多种威胁（Mangubhai等，2012; Campbell等，2020）。

水下视觉普查（Underwater Visual Census, UVC）

本研究从珊瑚礁生活调查（Reef Life Survey）项目（https://reeflifesurvey.com）中获取了2014年8-9月、2015年9月及2018年3月期间开展的186条UVC样带数据。

此外，本研究还使用了2004-2013年间该区域内开展的93条UVC样带数据（Cinner等，2016，图1）。

所有调查均采用标准化方案：由两名潜水员在样带两侧的5×50米样方（Cinner等，2016的研究中采用2.5×50米样方）内记录鱼类的种类、丰度与体长。

两个样方的调查数据为独立计数结果，通过取平均值以表征整条样带的调查结果（Edgar等，2020）。

环境DNA过滤与处理流程

2017年10-11月，本研究在西巴布亚省鸟头半岛南部海岸的不同珊瑚礁生境（排除河口与咸淡水水域）中采集了92份水样，采样区域东西跨度达500公里，其中87%的样本（92份中的80份）采集自最东侧的210公里区域（图1）。

水样采集工作在小艇上使用无DNA塑料袋完成，采用闭路循环呼吸器潜水（采样深度10-100米）尽可能靠近生境，或使用尼斯金采水器（Niskin water samplers，采样深度100-300米）进行采样（Hocdé等，2020）。

所有水样采集工作均在鱼类采样前后进行，绝不与鱼类采样同步开展，以避免现场污染。

研究人员将压力与温度传感器连接至尼斯金采水器，以控制采样深度，并通过垂直温度剖面表征水团特征。

每份样本均使用无菌Sterivex过滤胶囊（默克密理博（Merck© Millipore），孔径0.22μm）与一次性无菌注射器过滤2升海水。

过滤完成后，立即向过滤装置中加入裂解保存缓冲液（CL1缓冲液，SPYGEN©），并将其置于50毫升螺口管中，于-20℃下保存。

DNA提取与扩增流程参照Pont等（2018）的改良方案，每份样本需进行12次独立的PCR扩增。

本研究使用硬骨鱼特异性12S线粒体rDNA引物（teleo引物，上游引物：ACACCGCCCGTCACTCT，下游引物：CTTCCGGTACACTTACCATG，Valentini等，2016）扩增宏条形码序列（实验室分析与生物信息学分析细节详见附录S1）。

在鱼类环境DNA引物中，teleo引物即使在极高多样性的生态系统中也能高效检测鱼类多样性（Collins等，2019; Polanco Fernández等，2022）。

尽管其他鱼类环境DNA引物或许能在参考数据库中覆盖更多鱼类类群，从而在物种鉴定中提供更多信息，但目前尚无12S区域外的引物能达到与之相当的性能（Zhang等，2020）。

本研究在野外与实验室阶段均遵循污染防控方案（Valentini等，2016）。

水样处理过程中需佩戴一次性手套、使用一次性过滤设备，并在每份样本采集后用50%漂白剂对尼斯金采水器进行消毒。

负责环境DNA过滤的工作人员不得参与鱼类组织采样，且需使用专用工作区域，以避免接触污染与空气传播污染。

遗传参考数据库完善

在西巴布亚省鸟头半岛西南海岸的同期调查中，本研究沿岸边采集了413种、180个属、69个科的鱼类共计1466个个体。

标本主要通过手捕方式采集，或使用由开路、闭路循环呼吸器潜水员布设的4-8米长底刺网在0-100米水深范围内采集（Hocdé等，2020）。

部分咸淡水与河口鱼类通过10米长滩涂围网采集，远洋鱼类则通过延绳钓与鱼叉捕捞获得。

研究人员通过形态特征与652bp的细胞色素氧化酶亚基1（Cytochrome Oxidase 1, CO1）靶向基因测序对标本进行鉴定。

随后，为提升测序质量，研究人员使用分别针对硬骨鱼与软骨鱼设计的两对引物，对12S线粒体rDNA区域的大片段（480bp）进行扩增与测序。

最后，从所得序列中提取Valentini等（2016）定义的12S teleo区域，以补充欧洲分子生物学实验室（European Molecular Biology Laboratory, EMBL）遗传参考数据库（www.ebi.ac.uk，版本141，2020年1月下载，Baker等，2000），并优化分类学赋值结果（参考数据库及其完善方法细节详见附录S2与S3）。

为评估在线数据库中12S线粒体DNA teleo区域的完整性，本研究使用teleo引物序列，通过ecoPCR工具（Ficetola等，2010）在EMBL数据库中进行虚拟PCR，允许最多3个碱基错配。

将生成的测序物种列表与鸟头半岛生态区的全面物种名录进行比对。

在鸟头半岛名录中已确认分布的1761个物种中，仅有496种（28%）在EMBL数据库中完成了teleo区域的测序。

加入本研究鱼类采样获得的序列后，已测序物种数量增至762种，占比达43.4%。

此外，在遗传参考数据库构建过程中，本研究还采集、观测并明确鉴定了21种未见于历史名录的物种（全面名录详见附录S4）。

分类学赋值

宏条形码分析流程基于VSEARCH工具包与SWARM聚类算法：SWARM算法可将多个序列变异归为分子操作分类单元（Molecular Operational Taxonomic Units, MOTUs，Mahé等，2014），以去除PCR与测序错误。

本研究使用OBITOOLS工具包中的ecotag程序（最低共同祖先算法，Boyer等，2016），基于本研究构建的自定义参考数据库与全球公共EMBL遗传数据库（2020年1月下载的141版）进行分类学赋值。

针对每个分子操作分类单元，研究人员从自定义参考数据库或EMBL数据库中选择相似度最高的分类学赋值结果。

本研究仅保留与任一参考数据库序列完全匹配（相似度100%）且覆盖序列全长的赋值结果（详见附录S1）。

部分序列虽可达到100%相似度匹配，但由于标记区域的分类分辨率有限，可对应多个物种，因此无法完成物种水平的分类学赋值。

针对这类序列，研究人员将根据序列对应的物种列表及这些物种的已知空间分布，尽可能确定最可能被检测到的物种。

另有部分序列，若其对应的所有物种均已知分布于该区域或邻近区域，则无法缩小可能物种的范围，因此这类序列将被标记为可能的赋值列表（附录S6）并从分析中剔除。

鱼类性状

新增的鱼类多样性可能具有某些性状或行为特征，这些特征可能会限制传统调查（捕捞或目视记录）与环境DNA调查的检测效果（Thalinger等，2021）。

为探究这一检测偏差，本研究从Fishbase（www.fishbase.org）中获取环境DNA检测到的所有物种的相关数据，包括生境类型（珊瑚礁或远洋）、食性、昼夜活动模式、最大体长以及世界自然保护联盟（International Union for Conservation of Nature, IUCN）保护等级，并在不同物种组间进行比较。