Pollen limitation influences the divergences in mating system and floral traits between two sympatric <i>Halenia</i> species

To investigate differences in pollination limitation and mating system between <i>H. elliptica</i> and <i>H. grandiflora</i>, we conducted a two-part experiment:1. <b>Manipulative Pollination Experiment</b><br>During the peak flowering season in 2022, manipulative pollination experiments were carried out at three sites (YS, RS, and BG) at different times corresponding to the flowering peak. For each species at each site, 120 plants were randomly selected and three flower buds per plant were marked at the first or second node. These plants were randomly assigned to one of four treatments (30 plants per treatment):<b>Open pollination:</b> Flowers were exposed to natural pollinators.<b>Emasculation:</b> Anthers were removed before anthesis, and flowers were exposed to pollinators.<b>Supplementary hand-pollination:</b> Flowers were hand-pollinated with pollen from pre-netted flowers 10 m away, while still exposed to pollinators.<b>Net-bagged:</b> Flowers were bagged to exclude pollinators.Fruits were collected five weeks after pollination, and seed set per flower was calculated as the proportion of fully developed seeds relative to total ovules.2. <b>Molecular Paternity Analysis</b><br>To assess the mating system, leaf samples and three mature fruits were collected from 10 randomly selected plants per species at each site. Offspring (10 per plant) were germinated and stored for molecular analysis. DNA was extracted from offspring and parental samples using respective kits, and all samples were genotyped at 14 microsatellite loci.Genotyping was performed via PCR with results analyzed using GENEMAPPER 4.0. Data were organized for input into COLONY software, which estimated selfing rates (s) and inbreeding coefficients (F) for each species using a full-likelihood approach with defined parameters and error rates.