Heteromorphic self- incompatibility system

From November 2014 to February 2015, hand pollination was performed on five labeled individuals of each morph in a natural population at the Yongxing Island. Forty inflorescences with unopened flowers were enclosed separately in 40-mesh bags. We performed four treatments: (1) self-pollination to test self-compatibility; (2) intra-morph pollination (illegitimate cross); (3) inter-morph pollination (legitimate cross); (4) netted without hand-pollination; and also (5) marked some flowers without treatments as natural control. At the end of the flowering period, the mesh bags were removed to allow fruits to mature naturally. Three months after pollination, fruit set was recorded and statistical analyses were performed by G-test.

Pollen-tube growth was examined in vivo. Newly open virgin flowers in the evening were hand-pollinated by fresh pollen with above (1), (2) and (3) treatments. After pollination, the styles with ovary were collected per time interval (1, 3, 6, 12 and 24 h) and fixed in FAA. In lab, after softening in 10% Na₂SO₃ (100 °C) for 6 h, pistils stained with aniline blue were observed by fluorescence microscope⁷⁵.