Dataset: T. rotula Common Garden Experiment

Eight of the freshly-isolated T. rotula cells (3 cells each from PopA and PopB, 2 from PopC) were brought into culture and maintained at 18°C, ~100 μmol photons m⁻²s⁻¹, and an 18:6 light to dark cycle. The isolates underwent antibiotic treatment 1-4 months before the start of the experiment. Antibiotic treatment consisted of 1.5 mL of culture and 150 mL of F/2 media with 100 μg L⁻¹ of imipenem (Fisher Scientific) in 250 mL flasks on shaker tables at about 70 RPM for up to 96 hours before being transferred into F/2 media. Successful antibiotic treatment was identified by staining live cultures with 1% SYBR and visually verifying the absence of bacteria on a Nikon inverted Epifluorescent microscope both after the initial antibiotic treatments and before the start of the experiment. Isolates were maintained in exponential growth prior in F/2 media to the beginning of the experiment.

To begin the common garden experiment, 10L of whole seawater was collected from the pier from the URI Graduate School of Oceanography (41.49°, - 71.42°) on September 15, 2018. Whole seawater was filtered twice through 1μm polycarbonate track etch membrane filters (PCTE; Sterlitech) into autoclaved flasks to capture the free-living in situ bacterial community and remove grazers and large phytoplankton. Following filtration, 150 mL aliquots of the 1μm filtered seawater were added to sterile 250 mL Erlenmeyer flasks and amended with sterile F/10 vitamins and nutrients. All treatments were conducted in triplicate and included single strain flasks that were inoculated with a final concentration of 1,000 T. rotula cells mL⁻¹. We also had two control treatments: a bacterial control consisting of 150mL quadruplicates of the in situ bacterial community amended with F/10 vitamins and nutrients and a media control consisting of 150 mL triplicates of sterile seawater amended with F/10 vitamins and nutrients. All treatments were maintained at 18°C, 100 μmol photons m⁻²s⁻¹, an 18:6 light:dark cycle, and 70 RPM on shaker tables.

On day 5, 100 mL of each culture flask was filtered onto a 0.2-μm Express Filter (Millipore), flash frozen in liquid nitrogen and kept at -80°C until DNA extraction using the Gentra Puregene Yeast/Bacteria kit (Qiagen) following the manufacturer’s protocol.

The V4-V5 region of the bacterial 16S rRNA gene was amplified using the 515F-Y and 926R primers. PCR reactions were performed in triplicate 20 μl reactions containing 20 ng template DNA, 1X Taq Buffer, 0.5 μM of each primer, 200 μM of dNTPs, and 0.4 U of Taq DNA polymerase (Lucigen). The thermal cycling conditions were 2 min at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min at 50°C, 30 s at 72°C, a final extension of 72°C for 10 min. Triplicate PCR reactions were pooled and gel purified. Libraries were sequenced at the Duke Center for Genomic and Computational Biology on a MiSeq 2x250bp run (Illumina).