2.3 Observations of flower pollinator visit behavior
Five inflorescences and about 20 flowers at each study site (FG and AR)
were selected to observe pollinator visit behavior annually for five
days after nectar addition (FG) and natural conditions (AR) in May in
two consecutive years (2020-2021). Observation date and time was from
May 15 to 20, from 08:00 am to 18:00 pm every year, respectively. The
time of flying between two flowers on a plant, total time of stay per
visit, number of flowers visited per visit, and the length of time spent
visiting each flower by pollinators were observed and recorded.
2.4 Collection of fruits and seeds, and measurement of
their morphological characteristics
All mature fruits of the four types of manual control experiments at six
study sites were collected in April 2020 and April 2021. For each
treatment, 60 randomly selected fruits were used to measure their
lengths, widths and weights. For each collected fruit, its seeds were
collected to measure weight, number and proportion of shriveled seeds.
We used vernier calipers to measure fruit length and width, and an
electronic balance with high accuracy (one-millionth) to measure fruit
and seed weights. Thereafter, 0.02 g seeds taken from each collected
fruit were uniformly distributed in the solution of 20 ml 0.1% agarose.
Average numbers of seed and shriveled seed in 5 µl of the above solution
were counted using an optical microscope and 30 experimental replicates
were selected. Seed total number per fruit (N) and proportion of
shriveled seeds (P) was calculated using the following mathematical
formula (N = 2 * M * ñ * 105, P = (2 * M * n’*
105/ N) * 100%, M is seed weight per fruit, ñ and n’
are the average number of seeds and number of seeds with abnormal
development in 5 µl of the above solution, respectively). All surplus
seeds of each treatment were fully mixed, dried with anhydrous calcium
chloride for 5 days at 22℃ and then
stored at -20℃ for long-term preservation. Appropriate amount of mixed
seeds from each treatment were taken to measure morphological
characteristics using a scanning electron microscope.