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.