Common Garden Assay
To determine the sources of phenotypic variation in the field, we
brought samples back to McGill University to use in a common garden
growth assay. Whereas phenotypic variation in the field is due to a
mixture of environmental and genetic sources, growth in a common garden
removes environmental variation, isolating genetic variation. A single
colony of L. minor was collected from each of 3 microsites for
each site and preserved in tubes filled with natural sample water and
stored in the dark during transport.
Back in the lab, all tubes were placed under artificial grow lights (100
µmols/m2/s) until the plants had doubled in number,
consisting of at least two detached colonies which would be used to
found two clonal replicates for each microsite. The common garden assay
was done using 500mL Erlenmeyer flasks. There was a total of 204 flasks
(34 sites x 3 microsites x 2 replicate flasks). Flasks were filled with
350mL of growth media, diluted Hoagland’s E-media ([N]=5000
µgL-1, [P]= 780 µgL-1, pH=7.0
+/-0.05) (recipe in Supplementary information, Table S2), plugged with a
foam stopper, and then autoclaved. A single colony (3-4 attached fronds)
was used to inoculate each flask. These initial fronds were first marked
on their ventral surface with a small dot with a permanent marker to
later track generations. This was to ensure that phenotypes were only
measured on fronds at least two generations younger than those sampled
from the field.
All flasks were placed in one of two identical controlled growth
chambers of the McGill phytotron (200 µmols/m2/s
light, 200C, 70% relative humidity, with a 14/10
light-dark cycle). The two replicates were blocked, with one replicate
of each microsite in each chamber. The 102 flasks in each growth chamber
were randomly positioned, leaving a 15cm boundary from the chamber wall
on all sides. The common garden assay was broken into three 10-day
phases, separated by two transfers. Transferring the plants to fresh
media every 10 days prevented nutrient depletion, all-the-while limiting
the growth of phytoplankton whose differential abundance among flasks
could influence nutrient availability and plant growth. To remove any
maternal or carry over effects, we tracked generations to ensure that we
only measured the phenotypes of plants at least two generations younger
than the initial plants brought back from the field. The first, 10-day
preliminary acclimation phase served to ensure an equal physiological
starting point of all plants before we began to track population growth
rates, and to ensure the removal of all fronds initially present in the
assay. After these 10 days of growth, all fronds marked with a black dot
were discarded, and a single younger colony was randomly selected and
used to inoculate identical flasks with fresh growth media, after the
oldest frond in this colony was again marked. After a second 10 days of
growth, flasks were again removed, and all plants were transferred to
fresh media before being returned to the chambers for a final 10 days of
growth. After each of the two transfer dates, all flasks were returned
to the same growth chambers, but their positions within each were
independently randomized. At the end of the experiment, the total number
of fronds was counted in each flask and used to calculate rates of
exponential population growth (over the final 20 days). From each flask,
we randomly sampled 10 individuals (on average ~15% 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 (Abràmoff et al. 2004). Second generation fronds
(marked with a black dot) were excluded, as were immature fronds (that
didn’t yet have two daughter fronds budding from them).