Mating systems, insect pollination and chemical ecology of grassland Protea species (Proteaceae)
Major transitions between vertebrate and insect pollination systems have occurred many times during the angiosperm radiation and are associated with evolutionary modifications in floral traits. In the large ancestrally bird-pollinated African genus Protea (Proteaceae), an evolutionary shift from bird to insect pollination in the genus is suggested by the fruity diurnal scent of flowers in a recently evolved clade of grassland species. In this study, I confirm that four of these grassland Protea species have mixed mating systems and are indeed insect pollinated, and furthermore demonstrate the functional significance of their floral presentation and scent chemistry for attraction of pollinators, specifically cetoniine beetles. The study species, Protea caffra, Protea dracomontana, Protea simplex and Protea welwitschii, have colourful bowl-shaped inflorescences that produce copious amounts of pollen and dilute, xylose-rich nectar. Cetoniine beetles were found to be the most suitable pollinators due to their abundance, size, relatively pure Protea pollen loads, and their preference for the fruity scent and low growth form of these scented Protea species, as demonstrated by choice experiments in which inflorescences were offered at either end of a y-maze or at various heights above the ground, respectively. Bagging and hand pollinations revealed that these Protea species are self-compatible and capable of autonomous selfing. Self progeny of P. caffra were as vigorous as cross progeny in terms of germinability and survivorship to two months. Vertebrate-excluded and open-pollinated inflorescences yielded similar seed numbers for all species. Supplemental hand-pollinations, however, failed to increase seed set substantially, an indication of resource limitation. Outcrossing rates estimated using polymorphisms at eight allozyme loci in progeny from vertebrate-excluded and open-pollinated treatments of P. caffra were no different (t=0.59), indicating outcrossing by insects and an equal or insubstantial contribution from bird pollinators. The fruity-sweet scents of these species were more complex, with higher whole flower and mass-specific emission rates, than those in eight bird-pollinated congenerics. The overall floral scent is shown to be a blend of emissions from various plant parts, especially nectar. Electroantennography (EAG) revealed that the generalist pollinator Atrichelaphinis tigrina responds to a variety of volatile compounds found in fruity Protea scents. Field trapping confirmed that this cetoniine beetle is strongly attracted to ß-linalool (up to 60% of scent profile) and methyl benzoate. In conclusion, this study demonstrates the evolution of beetle pollination and mixed mating systems in a grassland clade of Protea. Volatile compounds that make up the unique (within Protea) fruity scent of the study species are shown to attract beetles, and the emission of large amounts of these compounds was probably a key step in the transition from bird to insect pollination in Protea.