Fastq files related to publication: At Palmyra Atoll, the fish‐community environmental DNA signal changes across habitats but not with tides

In August 2017, water samples were taken from 22 sites (Figure 1; Supporting Information Table SS1) by filling sterile Nalgene jars with surface water while wearing nitrile-free gloves. Depth and tide direction were noted for the intertidal sites (though, regrettably, temperature was not recorded). Five sites on submerged reef (2 on the fore reef, and 3 on the inner reef, 1 sample at each reef site corresponding to 2 pooled 250-ml samples taken within 10 m), 1 site in the deep-water lagoon (which is ignored here) and 16 intertidal sand-flat sites (1 sample of 250-ml per site, because higher turbidity limited the volume that could be passed through a filter) were sampled. Unfortunately, the difference in sample volumes taken from the two habitats means that when comparing reef sites to sand-flat sites, the relative proportion (rather than total counts) of fish species per sample associated with reef habitat or sand-flat habitat had to be analysed. All 16 sand-flat sites on outgoing tides and 7 of these sites again on incoming tides were sampled. The 29 samples put on ice were transported to the Palmyra Atoll lab, where they were filtered within 2 h. Because of the remote location and lack of vacuum pumps, the samples were pushed through a 0.22-um Sterivex™ filter unit, using a sterile 50-ml syringe. All filters were preserved using Longmire's solution (Longmire et al., 1997; Renshaw et al., 2015) and kept refrigerated until extraction in February 2019 (the authors did not collect fish, perform surgical procedures, stress fish in experiments, cause lasting harm to sentient fishes or involve sentient un-anaesthetised animals). DNA extractions and PCR preparations were performed in a pre-PCR room, separate from all areas where post-PCR products are used. For each filter, DNA was extracted separately from both the individual filter capsules and the Longmire's preservation solution within the capsules, resulting in 58 samples (e.g., two extractions for each of the 29 samples). After lysis buffer (720 μl buffer ATL for capsule and 180 μl for the solution extraction; Qiagen Inc., Hilden Germany) and proteinase K (80 μl and 20 μl, respectively) was added, the samples were incubated at 56°C for 24 h on a rotating incubator. A modified DNeasy™ Blood & Tissue protocol (following Spens et al., 2017) was used to extract the eDNA before amplification. Each set of 29 extractions had its corresponding extraction blank: a new Sterivex™ filter capsule with 720 μl buffer ATL and 80 μl proteinase K was used for the filter set, and a sterile microcentrifuge tube with 180 μl of ATL and 20 μl of Proteinase K was used for the Longmire's solution set. DNA was PCR amplified using the following primer sets with an Illumina Nextera™ adaptor modification: MiFish 12S Universal and MiFish 12S Elasmobranch (Miya et al., 2015), 18S (V8-V9; Bradley et al., 2016) and CO1 (mlCOIintF and jgHCO2198; Leray et al., 2013). PCR amplification was carried out in triplicate using a 15- μl reaction volume containing 3 μl of extract, 7.5 μl of QIAGEN Multiplex Taq PCR 2x Master Mix, 4.20 μl of dH2O and 0.15 μl of each primer (10 μmol l–1). Each set of 29 extractions had its corresponding PCR blank. For these, dH2O was used instead of DNA extract. Although CO1, 18S, MiFish 12S Universal and MiFish 12S Elasmobranch were sequenced, only the 12S data were analysed here, because these barcodes had the best coverage for fishes (CO1 detected 48 species, and 18S detected 3); nevertheless, the other barcodes give insight into different taxa at Palmyra Atoll. Thermocycler settings for 12S reactions included an initial denaturation at 95°C for 15 min, followed by 13 touchdown cycles of (a) 94°C for 30 s; (b) annealing beginning 69.5°C for 45 s, decreasing by 1.5°C until 50°C was reached; and (c) extension at 72°C for 45 s. Subsequently, 35 additional cycles were run with a 50°C annealing temperature, followed by a 10-min final extension at 72°C. PCR replicates were pooled after verifying successful amplification. Then the PCR products were cleaned using Serapure magnetic beads (Faircloth & Glenn, 2014) to remove <100 bp fragments. Cleaned samples were quantified using the Qubit dsDNA BR Assay (Thermofisher Scientific, Waltham, MA, USA). Even numbers of molecules were pooled from each primer set for indexing. Dual i7 and i5 Illumina Nextera indices (Illumina, San Diego, CA, USA) were incorporated by amplification using Kapa HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA) in 25-μl reactions of 12.5-μl mix, 0.625 μl of each index and sample volumes amounting to 10 ng of DNA for each sample. The thermocycler setting began with a 95°C incubation for 5 min, followed by 12 cycles of 98°C for 20 s, 56°C for 30 s and 72°C for 3 min, and ended with a 72°C incubation for 5 min. Indexed PCR products were again bead-cleaned and quantified. DNA libraries were pooled evenly and submitted for sequencing to the Technology Center for Genomics & Bioinformatics (TCGB) at the University of California, Los Angeles, where they were run on an Illumina MiSeq™ with Reagent Kit V3 (2 × 300 bp) at a goal depth of 40,000 reads in each direction per marker per sample.2017年8月，研究人员佩戴无丁腈手套，使用无菌纳基因瓶（Nalgene jars）采集了22个采样点的表层水样（图1；补充信息表SS1）。记录了潮间带采样点的水深与潮汐方向，但遗憾的是未记录水温。本次采样共覆盖5个水下礁采样点（2个礁前、3个礁内，每个礁点采集1份样本，对应10米范围内2份混合的250毫升水样）、1个深水泻湖采样点（本次研究未纳入该样本）以及16个沙质潮间带采样点（每个采样点采集1份250毫升水样，因浊度较高，可过滤的水样体积受限）。由于两种生境的采样体积存在差异，因此在比较礁生境与沙质潮间带生境的样本时，需针对每个样本中鱼类物种的相对占比（而非总计数）进行分析。研究人员对退潮时的全部16个沙质潮间带采样点，以及涨潮时其中7个采样点进行了重复采样。共收集29份样本，置于冰上运输至帕尔迈拉环礁实验室，并在2小时内完成过滤。因采样地点偏远且缺乏真空泵，研究人员使用无菌50毫升注射器将水样推入0.22 μm的Sterivex™滤器单元中完成过滤。所有滤膜均使用朗米尔保存液（Longmire's solution）进行保存（Longmire等，1997；Renshaw等，2015），并于4℃冷藏至2019年2月进行提取（本研究未采集鱼类样本、未实施外科手术、未对鱼类施加实验胁迫、未对有感知能力的鱼类造成永久性伤害，也未使用未麻醉的有感知动物）。DNA提取与PCR制备均在PCR前实验室中完成，该区域与所有使用PCR后产物的区域相互隔离。针对每个滤膜，研究人员分别从滤膜胶囊内部以及胶囊内的朗米尔保存液中提取DNA，最终得到58份样本（即29份原始样本各对应2次提取）。加入裂解缓冲液（滤膜胶囊使用720 μL ATL缓冲液，保存液提取使用180 μL ATL缓冲液；凯杰公司（Qiagen Inc.），德国希尔登）与蛋白酶K（分别对应80 μL与20 μL）后，将样本置于旋转孵育箱中56℃孵育24小时。采用改良的DNeasy™血液和组织提取试剂盒操作流程（遵循Spens等，2017）在扩增前提取环境DNA（eDNA）。每29份提取样本对应1份提取空白：滤膜组空白使用新的Sterivex™滤器胶囊，加入720 μL ATL缓冲液与80 μL蛋白酶K；保存液组空白使用无菌微量离心管，加入180 μL ATL缓冲液与20 μL蛋白酶K。使用以下带有Illumina Nextera™接头修饰的引物组进行PCR扩增：MiFish 12S通用引物（MiFish 12S Universal）、MiFish 12S软骨鱼引物（MiFish 12S Elasmobranch，Miya等，2015）、18S（V8-V9区；Bradley等，2016）以及CO1（细胞色素氧化酶亚基I，mlCOIintF与jgHCO2198；Leray等，2013）。PCR扩增设置3次技术重复，反应体系总体积为15 μL，包含3 μL DNA提取液、7.5 μL QIAGEN多重Taq PCR 2×预混液、4.20 μL无菌去离子水，以及0.15 μL的每种引物（浓度为10 μmol·L–1）。每29份提取样本对应1份PCR空白，以无菌去离子水替代DNA提取液作为模板。尽管对CO1、18S、MiFish 12S通用引物与MiFish 12S软骨鱼引物的扩增产物进行了测序，但本研究仅分析12S测序数据，因该条形码对鱼类的覆盖度最优（CO1检测到48个物种，18S仅检测到3个）；不过其余条形码可用于解析帕尔迈拉环礁的其他类群。12S反应的热循环参数为：95℃初始变性15分钟，随后进行13个降落式循环：(a) 94℃变性30秒；(b) 退火温度从69.5℃开始，每循环降低1.5℃直至50℃，退火时长45秒；(c) 72℃延伸45秒。随后进行35个循环，退火温度维持50℃，最终72℃延伸10分钟。验证扩增成功后，将技术重复的PCR产物混合。随后使用Serapure磁珠（Serapure magnetic beads，Faircloth & Glenn, 2014）对PCR产物进行纯化，以去除小于100 bp的片段。使用Qubit双链DNA BR检测试剂盒（Qubit dsDNA BR Assay，赛默飞世尔科技（Thermofisher Scientific），美国马萨诸塞州沃尔瑟姆）对纯化后的样本进行定量。将每个引物组的样本按分子数等量混合以进行索引扩增。采用Kapa HiFi HotStart预混液（Kapa HiFi HotStart ReadyMix，Kapa生物系统公司（Kapa Biosystems），美国马萨诸塞州威尔明顿）进行双索引扩增，反应体系总体积25 μL，包含12.5 μL预混液、0.625 μL的每种索引引物，以及对应10 ng DNA的样本体积。热循环参数为：95℃预孵育5分钟，随后12个循环：98℃变性20秒、56℃退火30秒、72℃延伸3分钟，最终72℃孵育5分钟。完成索引扩增的PCR产物再次进行磁珠纯化与定量。将DNA文库等量混合后，提交至加州大学洛杉矶分校基因组与生物信息学技术中心（Technology Center for Genomics & Bioinformatics, TCGB）进行测序，使用Illumina MiSeq™测序仪与V3试剂试剂盒（2×300 bp读长），目标为每个样本的每个标记物在每个测序方向获得40,000条读数。