Growth Assay
The main experiment consisted of a growth assay of all 16 populations (8 genotypes, each with the microbiome either present or absent) in four distinct abiotic environments, a 2 x 2 crossed treatment of light level and nutrient concentration. Each assay was replicated in three flasks. This fully factorial design results in a total of 192 assays (8 genotypes x 2 microbiome treatments x 4 environment treatments x 3 replicates). Ten random individual fronds were used to inoculate each 500mL Erlenmeyer flask filled with 350mL of sterile Hoagland’s E-media, modified to obtain the desired treatment levels, either low nutrients ([N]=500 µg L-1, [P]= 77 µg L-1), or high nutrients ([N]=5000 µg L-1, [P]= 770 µg L-1). The flasks were then placed in four growth chambers in the McGill phytotron (200C, 70% RH, 14/10 light-dark cycle), two of which were set at low light conditions (30 µmols m-2s-1) the other two at high light conditions (300 µmols m-2 s-1). The initial common garden conditions were at intermediate light and nutrient conditions in relation to the low and high treatments in the experimental growth assay. Flasks were plugged with foam stoppers and all transfers were done using sterile techniques. The 48 flasks in each growth chamber were randomly positioned, leaving a 15cm boundary from the chamber wall.
The growth assay lasted for a total duration of four weeks after which population growth rates and plant phenotypes were measured. However, to maintain populations in a state of exponential growth, the growth assay was broken into two two-week assays. After the first two weeks of growth in the treatment environments, before fronds reached complete surface cover (on average ~100 fronds/ flask), 10 randomly sampled fronds from each flask were transferred to an identical treatment flask of fresh media. Furthermore, since inoculating sterile growth media with natural fronds (with intact microbiomes) would likely create conditions with phytoplankton present but important zooplankton grazers absent, prolonged growth could result in plant-algal competition (van Moorsel 2022). By transferring the cultures after only two weeks, phytoplankton remained sparse. The flasks were repositioned randomly in the growth chambers following the mid-assay transfer.
At the end of the experiment the total number of fronds and the number of colonies (groups of attached fronds) were recorded for each flask. From each flask, we randomly sampled 10 individuals (on average ~10 % of the population) for whom we measured frond area and root length by imaging (plants were pressed onto a sheet including a reference ruler and photographed at a standard 20cm distance) and subsequent image analysis using Image J. Only mature individuals (those from which a daughter frond was budding) were included.