IMIS

The experiment was conducted using a full factorial design with seven Asterionella genotypes (S24, S26, S37, S38, S43, S49, and S53) and five different temperatures (1°C, 6°C, 11°C, 16°C or 21 ± 0.5°C) in six replicates per treatment combination, resulting in 35 experimental combinations and 210 experimental units. The temperature levels represent the full range of temperatures experienced by the Asterionella population in Lake Maarsseveen over the course of a year. All cultures were placed in temperature-controlled water baths and light was set to discontinuous illumination at 160 ± 10 µmol quanta m-2 s-1 provided by cool, white fluorescent lamps on a 14:10 h light:dark cycle. All genotype cultures were acclimatized stepwise to their experimental temperature so that each genotype culture spent between 12 and 42 generations in semi-continuous batch culture at their experimental temperature prior to the start of the experiment (i.e., 2 weeks for the 21°C cultures to 13 weeks for the 6°C cultures). All experimental treatments were started on the same day. Six replicates of each clonal subculture were inoculated at ~10,000 cells mL-1 in 60 mL CHU-10 medium. This inoculum size was small enough to ensure that the culture medium could support several generations of population growth before either light or nutrient availability could become limiting (i.e., before the culture would enter stationary growth phase). The cultures were shaken twice per day and their positions randomized within their water bath once each day. Samples were taken on day 0, 1 and 2, after that on every other day until day 12 for temperature treatments 11°C to 21°C and at day 0, 1, 3, and every third day until day 21 for temperature treatments 1°C and 6°C, all at the same moment of the light cycle. The samples were fixed with a glutaraldehyde-formaldehyde fixative and stored cool and in the dark. The number of Asterionella cells mL-1 was calculated from optical density measurements at 720 nm in 5cm cuvettes on a Lambda 800 UV/VIS spectrometer (PerkinElmer Inc., Waltham, MA, USA) whereby the specific light extinction to number of cells conversion factor was determined for each genotype-temperature combination separately. Maximum population growth rates were calculated by fitting a least squares linear regression to the linear part of each time course of natural log transformed algal densities. Cell length and width (in µm) were measured for 30 cells each of all six replicates per genotype-temperature combination using an inverted microscope (DMI 4000B; Leica Microsystems CMS GmbH, Wetzlar, Germany) and the image analysis program Cell-D (Olympus Soft Imaging System GmbH, Munster, Germany). Care was taken to measure no cells in the process of division, as this would inflate the width measurements. SAV and biovolume were calculated from length and width measurements whereby the Asterionella cell was assumed to have the form of an elongated cuboid. The change in cell length, width, biovolume, and SAV per generation was calculated by subtracting the averages of day 0 samples from that of day 9 (for 1°C treatments) and day 10 (for 11 and 21°C treatments) samples and by dividing that difference by the temperature and genotype specific generation time for all six replicates of all genotypes at temperature treatments 1°C, 11°C and 21°C. Hence, negative values mean that the cell trait has decreased over time; positive values mean that the cell trait has increased over time.