Artificial pollination experiment
We conducted the hand-pollinated experiment on 10 individuals (growing evenly) in June to July 2019 to evaluate mating system. Since the single flower of V. negundo var. heterophylla was small, the inflorescence was used as a unit for treatment. On each plant, 18 inflorescences (6 treatments × 3 replicates) were randomly marked, each single flower in each inflorescence was treated the same way. Note that in the selection of experimental subjects, the selected inflorescence was basically the same in terms of length, height and other dimensions, thus reducing the possibility caused by this problem.
Pollination treatments including: (1) OP (Fig.2a): open-pollinated by pollinators; (2) WP (Fig.2b): emasculated and bagged with mesh bags to detect wind pollination; (3) SSP (Fig.2c): bagged with paper bags and left unmanipulated to detect spontaneous self-pollination; (4) SP (Fig.2d): hand-pollinated with pollen from the same flower using a small brush and bagged to detect manipulated self-pollination; (5) G (Fig.2e): emasculated, hand-pollinated with pollen from another flower of the same tree and bagged to detect geitonogamy; (6) X (Fig.2f): emasculated, hand-pollinated with pollen from another tree and bagged to detect xenogamy. The bagging was carried out at bud phase and all hand-pollinated flowers were bagged with paper pollination bags to exclude pollinators. (4) and (5) were to detect the degree of self-compatibility, and (6) was to detect the degree of hybridization. The number of treated flowers per labeled inflorescence was recorded every day. 9074 flowers of V. negundo var. heterophyllawere treated in total.
Before the six treatments, stigmatic receptivity was checked to ensure that the selected branches were healthy and normally developed. Bagging was carried out at the bud stage. According to Sun et al. (2020), the best pollination time was determined to be from 10:00 am to 12:00 am in view of the pollen viability and stigmatic receptivity at different stages after flowering, and with the help of tweezers, the anther directly contacted the stigma for pollination. The phenological developmental stage of the flower at pollination was during the anthesis. Then we followed up the post pollination development and subsequently collected the progeny of the treated inflorescences for subsequent fruit weight determination and germination experiments.