Experimental design and disturbance treatment
Once in the climate-controlled room, algal individuals were trimmed to 17 g wet weight and incubated in individual tanks containing artificial seawater (ASW; salinity: 24 gL-1) that was prepared from deionized water and sea salt and were bubbled with atmospheric air through an aeration stone inserted into the water with a tube. Five days after the last set of individuals arrived (from the population in Kiel) a new set of samples was obtained (t0) using the vortexing method, after which the disturbance treatment was applied to 10 of the 12 algae from each population (see Figure 1 for a schematic overview of the sampling and experimental design). The treatment constituted a mixture of the antibiotics vancomycin (65 mgL-1) and cefotaxime (70 mgL-1) in a reduced water volume of 1 L. Assuming that a substantial part of microbiota were killed and the microbial community would be severely disturbed, the treatment was terminated after 24 hours, and the algae were rinsed with 1 L ASW. All 40 individuals and pseudo-individuals were then split into two apical fragments of approximately 6 g wet weight and transferred into new separate 2 L tanks with 1.5 L ASW. For half of these thalli, the temperature was controlled at 15 °C and the other half at 22 °C, with both thermal levels containing one of the paired thalli originating from the same individual or pseudo-individual. As the temperature in the climate room was 15 °C, the 22 °C treatment aquaria of were incubated in water-filled basins containing heating elements. As a source of microbiota to reconfigure new microbial communities each aquarium received an inoculum of four 2 cm long branches of undisturbed individuals originating from each of the four populations. To prevent mixing with disturbed thalli the inocula were not directly applied into the aquaria, but instead into 50 mL tubes which contained openings on two sides, sealed with fine mesh (~1mm) to prevent the exchange of algal fragments (see Figure 1). To promote the exchange of microbes, aeration stones were placed inside the tubes containing the inoculum.
The conditions in the climate room were kept constant and epibiota were sampled from all algae after 1 week (t1), two weeks (t2), 4 weeks (t4) and 12 weeks (t12) by harvesting 1 g of tissue and executing the same vortexing method. For practical reasons, we conducted the experiment in a stacked order, where algae were divided into four groups, over which populations and treatments were balanced. Each group underwent the treatment and sampling with a one-day time lag. Within each group populations were balanced and contained two or three thalli per population and treatment. All samplings were conducted with sterilized equipment and tanks were washed with bleach before use. Water was exchanged once per week.