DNA extraction and PCR amplification
All samples were centrifuged at 11,000 xg for 30 minutes to harvest diatom cells. Then, supernatant was discarded and pellet was resuspended in 200 µL of nuclease-free water. DNA was isolated using the Power Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, California, US) following the manufacturer instructions and its concentration was measured with Nanodrop 1000 (NanoDrop Technologies, Wilmington, Delaware, US). A 312 bp fragment of rbc L gene (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) was amplified by PCR using an equimolar mix of degenerate primers with overhang adapters for Illumina sequencing (Table 2) (Rivera et al., 2017). For all samples, we performed six PCR reactions on 50 µL of reaction mixture containing 10-20 ng/µL of extracted DNA, 2 U of Platinum II Taq Hot-Start DNA Polymerase (Invitrogen, Grand Island, New York, US), 10 µL of 5X Platinum II PCR Buffer, 0.5 µM of each primer, 5 µL of dNTP mix (2mM each), 10 µL of Platinum GC Enhancer and 9.6 µL of nuclease-free water. PCR conditions included an initial denaturalization step at 94 °C for 4 min followed by 40 cycles of denaturalization at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 68 °C for 30 s, and a final extension step at 68 °C for 10 min. PCR products were visualized with ultraviolet light in a 1.5% agarose gel stained with ethidium bromide. Bands targeting the rbc L barcode gene were excised off from agarose gel and then DNA extracted with Clean-Easy Agarose Purification Kit (Canvax Biotech, Córdoba, Spain) following the manufacturer instructions, except for elution volume, which was 15 µL.