18S-metabarcoding data of a mesocosm experiment on the effects of a heatwave today and tomorrow at Sylt Roads, September 2021

To investigate the effects of concurrent environmental change and heatwaves, we performed an incubation experiment in the mesocosm facility of the Alfred Wegener Institute on Sylt with a plankton community sampled from the long-term ecological research station Sylt Roads (https://deims.org/9d5e3aae-d569-4571-8058-96d5bafda2e7) on September 1st, 2021. The surface community was collected with a sampling tub by the RV Mya and incubated in 16 mesocosms for a month. We exposed the community to four conditions in quadruplicate: current conditions (field temperature, 400ppm CO2, N:P of 16), future conditions (+ 3 °C, 1000 ppm, N:P of 25), current conditions plus a heatwave, and future conditions plus a heatwave. Simulation of the heatwaves was conducted by increasing the temperature of the respective mesocosms to + 2 °C in comparison to the controls for six days. This dataset comprises the 18S-metabarcoding data of all big sampling days, which were conducted each week on Mondays, Wednesdays, and Fridays. A total of 500 mL of sample water, pre-filtered over a 150 µm mesh, was carefully vacuum-filtrated (<-200 mbar) onto polycarbonate filters (3 µm nominal pore size, Nucleopore, Whatman, Maidstone, UK). Filters were put into 15 mL polycarbonate tubes and stored in the dark at −80 °C. DNA extraction was performed according to the manufacturer’s protocol using the NucleoSpin Soil extraction kit (Macherey-Nagel GmbH, Düren, Germany). DNA concentration was quantified and normalized to 5 ng µL−1. Amplicons of the variable region 4 (V4) of the 18S rRNA for eukaryotes were generated according to the standard protocol of amplicon library preparation (16S Metagenomic Sequencing Library Preparation, Part #15044223 Rev. B. Illumina, San Diego, CA, USA) using the forward primer CCAGCASCYGCGGTAATTCC and reverse primer ACTTTCGTTCTTGAT including an illumina tail (Bradley et al., 2016). Single samples were indexed using the Nextera XT Index Kit v2 Set A primers (Illumina, San Diego, CA, USA), and the resulting libraries were pooled. Sequencing was performed on a MiSeq sequencer (Illumina, San Diego, CA, USA), producing 300 base pair paired-end gene amplicon reads. Demultiplexing and FASTQ sequence file generation were carried out using the Generate FASTQ workflow of the MiSeq sequencer software. Primers were removed with v2.8 cutadapt (Martin, 2011), and further processing of the sequence data was performed using the v1.18 DADA2 R package (Callahan et al., 2016). In consideration of the read quality, which usually drops towards the 3′-end, the forward reads were trimmed after 240 base pairs, and the reverse reads were trimmed after 210 base pairs. For each pool, error rates were learned independently, and sequences were denoised. Paired-end reads were merged with a minimum overlap of 20 base pairs allowing 0 mismatches, and chimeras were predicted and removed. Taxonomic assignment of the resulting amplicon sequence variants (ASVs) was performed using the reference database PR2 (v4.12.0; Guillou et al., 2013). All scripts can be found on github (https://github.com/AntoniaAhme/HeatwaveNowTomorrowProtists). The accompanying metadata is published on PANGAEA (Meunier 2025; https://doi.pangaea.de/10.1594/PANGAEA.982599).