Background/Question/Methods The extent to which plants depend on pollinators for seed production, and their vulnerability to genetic Allee effects caused by scarcity of compatible mates in small populations, are determined primarily by their breeding systems. Gladiolus inandensis is endemic to the highly fragmented Sandstone Plateau Sourveld grasslands of eastern South Africa. To assess its vulnerability to pollinator loss and genetic Allee effects, we investigated its pollination biology and breeding system. Gladiolus is characterized by an unusually wide diversity of pollination systems. G. inandensis has “gullet” flowers conforming to a long-tongued bee pollination syndrome, but it pollinators have not been recorded. We tested the predictions that this species is obligately dependent on pollinators for seed set, and that the most important of these pollinators are long-tongued bees. To determine the breeding system, we performed controlled pollination experiments in two populations using inflorescences from which insects were excluded. On each plant, one flower received each of the following treatments: unmanipulated; self-pollinated; cross-pollinated; emasculated. We scored fruit set and seeds per fruit. Fluorescence microscopy revealed the fates of pollen tubes in self- and cross-pollinated pistils. We characterized floral colour using reflectance spectrometry, and floral scent using dynamic headspace extractions and gas chromatography-mass spectrometry.

Results/Conclusions With insects excluded, no unmanipulated or emasculated flowers set fruit whereas all crossed flowers did. This rules out apomixis and autonomous selfing. The small proportion (0.19) of selfed flowers that set fruit set fewer seeds than crossed flowers. Growth of pollen tubes was blocked more often in selfed than in crossed pistils. Thus, G. inandensis is self-incompatible but a small amount of selfing may occur, potentially resulting in a mixed mating system. Supplemental cross-pollination increased fruit and seed set even in inflorescences open to insects, indicating pollen limitation. Like most other insect-pollinated flowers that appear white to humans, those of G. inandensis absorb UV. The flowers emit caryophyllene, p-methyl-anisole, linalool and other volatiles typical of generalist insect-pollinated flowers. The flowers were visited by long-tongued bees (Amegilla spp., as predicted) but also by honeybees (Apis mellifera, possibly an artifact of nearby managed hives), small bees, and bombylid bee flies. All visitors carried predominantly Gladiolus pollen. G. inandensis' self-incompatibility may reduce its seed production if self-incompatibility alleles become fixed in small populations. Its inability to set seed autonomously makes it dependent on pollinators, though its vulnerability to extirpation of particular insects depends on how effective a pollinator each visitor is.