Pollen limitation experiment
We set up a pollination experiment to quantify pollen limitation and its
magnitude in each species across our sites. Our experimental setup
consisted of 120 plants of each of our two species, grown in a
greenhouse from seed, then transplanted into grow bags at each study
site (20 plants of each species at each of the six sites). We also had 4
pollen donor plants per species at each site in case we could not
collect enough pollen from our experimental plants to cross with all
open flowers. Seeds were planted at the WCH greenhouse for S.
lycopersicum and the CBG greenhouse for C. pepo on 1 April 2021.
Between 29 May and 02 June 2021, seedlings were transplanted into
7-gallon fabric grow bags at each site using organic potting soil
(Cowsmo Compost, Cochrane, WI). Plants were fertilized once per month
and were scheduled to be watered three times per week, however the
growers at each site made decisions about increasing or decreasing
watering based on local weather conditions.
For each species, we applied a pollen addition treatment to ten
individuals at each site, while the other ten served as unmanipulated,
open-pollinated controls (i.e ., ambient pollination). Plants were
placed in two to four rows, depending on the space available at each
site, and arranged in a paired design, with each pair consisting of one
control and one supplemental plant placed side by side, 30 cm apart, to
account for potential fine-scale variation within each site
(e.g ., shade). Treatments were assigned randomly within each
pair. We applied supplemental outcross pollen to the stigmas of all open
flowers on pollen-supplemented plants twice weekly, weather permitting,
as hand-pollination was not possible during rain. We pollinated C.
pepo flowers by collecting pollen from the anthers of open male flowers
with a paintbrush and transferring it directly to the stigma of open
female flowers on a different plant. We pollinated S.
lycopersicum flowers using VegiBee Garden Pollinators (VegiBee, China),
vibrating tools that mimic insect sonication. We sonicated each open
flower on a supplemental plant, collecting the pollen in a spoon and
using it to pollinate all open flowers on another experimental plant by
dipping the stigmas into the pollen. At each visit, we counted all open
flowers on control and supplemental plants. On supplemental plants, if
there were any flowers that had opened and closed since our last visit,
causing us to miss the window of opportunity for pollination, we marked
these flowers using twist-ties and did not include them in our
assessment of pollen limitation.
We harvested fruits twice weekly, counting and harvesting all fruits
that were at the stage that local farmers consider ready to harvest. ForS. lycopersicum , this is when the fruits reach a deep orange
color and readily come off the vine when tugged gently. For C.
pepo , this is when the fruits are between two and four inches in
diameter. Half of the fruits harvested from each individual plant were
transported to and frozen at the CBG for measures of reproductive
success. If an individual produced more than ten fruits in one week, a
subset of five fruits were used. We considered both ecologically and
agriculturally relevant measures of reproductive success: the proportion
of flowers that set fruit, number of seeds produced per fruit, mass of
fruit, and size of fruit. We measured fruit size using diameter in the
longest direction between the stem end and blossom end. We did not
consider size or mass measures for C. pepo because fruits
continue to grow larger the longer they stay on the plant, far beyond
the optimal size for harvesting, so size and mass reflect when the
fruits were harvested. We measured the magnitude of pollen limitation as
the difference in reproductive success between control and
pollen-supplemented plants for each measure of reproductive success.