Reference database

Due to the lack of reference sequences for the V9 region in common public databases (e.g. NCBI, PR2), we generated a dataset consisting of the V9 region of 102 marine protist strains of our Heterotrophic Flagellate Collection Cologne (HFCC), of which several have not been published yet (Supplementary Data 2). Subsamples of a few milliliters of the sediment of the MUC samples (see above) suspension were cultivated in 50 ml tissue-culture flasks (Sarstedt, Nümbrecht, Germany). Isolation was carried out using a micromanipulator or microtiter plates (liquid aliquot method⁵⁹). All cultures were supplied with sterilized quinoa or wheat grains as an organic food source for autochthonous bacteria. After isolation, the strains were cultivated in 50 ml tissue-culture flasks (Sarstedt, Nümbrecht, Germany) filled with 30 ml Schmaltz-Pratt medium⁶⁰ (35 PSU; per liter 28.15 g NaCl, 0.67 g KCl, 5.51 g MgCl₂ × 6 H₂O, 6.92 g MgSO₄ × 7 H₂O, 1.45 g CaCl₂ × 2H₂O, 0.10 g KNO₃, 0.01 g K₂HPO₄ × 3H₂O). The cultures were stored at 10 °C in the dark. Isolates were characterized morphologically using AVEC high-resolution video microscopy and electron microscopy. For molecular studies, protistan cultures were concentrated by centrifugation (4000 × g, 20 min at 4 °C, Megafuge 2.0R, Heraeus Instruments). Genomic DNA of each isolated protist strain was extracted using the Quick-gDNA^TM Mini Prep Kit (Zymo Research, USA). We amplified a long sequence from the 18S rDNA to the 28S rDNA with the primers 18S-For (5′-AAC CTG GTT GAT CCT GCC AGT-3′, ref. ⁶¹) binding at the beginning of the 18S rDNA and either NLR1126/22 (5′-GCT ATC CTG AGG GAA ACT TCG G-3′, ref. ⁶²) or NLR2098/24 (5′-AGC CAA TCC TTW TCC CGA AGT TAC-3′, ref. ⁶²) binding in the 28S rDNA. PCR reactions were performed in 25 µl PCR reaction mixtures containing 5.5 µl ddH₂O, 1.5 units TAQ (Mastermix, VWR Germany), 2 µl DNA and 2.5 µl of each primer (forward and reverse) at a final concentration of 1.6 nM. The PCR conditions for amplifying the SSU–ITS–LSU region were as follows: pre-denaturation at 98 °C for 2 min, 35 cycles of 98 °C for 30 s, 55 °C for 45 s, and 72 °C for 4 min 30 s; final extension at 72 °C for 10 min. For bodonid strains, a different primer combination was used: 18SForBodo (5′-CTG GTT GAT TCT GCC AGT-3′, ref. ⁶³) + NLR1126/22 (5′-GCT ATC CTG AGG GAA ACT TCG G-3′, ref. ⁶²). Internal primers were used for sequencing (Supplementary Table 2). We established a new reference database for the V9 region by combining the Protist Ribosomal Reference database PR2 v4.11.1 (ref. ³⁴) with the 102 sequences of marine protist strains of the Heterotrophic Flagellate Collection Cologne. Using Cutadapt⁶⁴, the final in-house reference database, called V9_DeepSea³³, was trimmed to the V9 region.