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    Evaluating sediment and water sampling methods for the estimation of deep-sea biodiversity using environmental DNA

    High-throughput sequencing resultsA total of 26 million COI reads, 19 million raw 18S V1-V2 reads,, 14 million 18S V4 reads, and 17 million 16S V4–V5 reads were obtained from three Illumina HiSeq runs of amplicon libraries built from pooled triplicate PCRs of 22 environmental samples, 2 extraction blanks, and 4–6 PCR blanks (Supplementary Table S4 online). The in situ pump yielded less raw reads for COI and 16S (Supplementary Fig. S1 online, F = 4.02–14.4, p = 0.0003–0.03), while more raw reads were recovered from both water sampling methods with 18S V4 (F = 6.5, p = 0.007). Water samples generally yielded fewer raw clusters (F = 5.1–35.1, p = 3.2 × 10−6–0.02), except for 18S V4 where numbers were comparable across sample types (Supplementary Fig. S1 online).Bioinformatic processing (quality filtering, error correction, chimera removal, and clustering for metazoans) reduced read numbers to 20 million for COI, 12 million for 18S V1–V2, 11 million for 18S V4, and 10 million for 16S V4–V5, resulting in 10,351 and 17,608 raw OTUs for COI and 18S V1–V2 respectively; 35,538 raw 18S V4 ASVs, and 62,646 raw 16S ASVs (Supplementary Table S4 online). For eukaryote markers, 17–55% of the raw reads remained in PCR blanks after bioinformatic processing, while 50–75% remained in extraction blanks and 52–87% in true samples. In contrast, with 16S, these values were at 87% for PCR blanks, 67% for extraction blanks, and 29–73% for true samples. Thus, negative control samples accounted for 7–13% of bioinformatically processed reads with eukaryotes, compared to 27% with prokaryotes. The vast majority of 16S reads generated by negative controls belonged to a common contaminant of Phusion polymerase kits, which is well amplified in low concentration samples such as negative controls. These reads however accounted for  20 µm size class, and the sampling box targeting both the 2–20 µm and the 0.2–2 µm size classes, detected different community assemblages. For protists, the in situ pump detected higher proportions of ASVs for Bacillariophyta, Ciliophora, Labyrinthulea, or Phaeodarea, while the sampling box detected more cryptophytes, haptophytes, MAST, and telonemians (Fig. 3 18S V4). For prokaryotes, the sampling box detected more diversity in the Alphaproteobacteria, Chloroflexi, or Marinimicrobia (Fig. 3 16S V4–V5). More

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    Angiosperm pollinivory in a Cretaceous beetle

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