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.