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.