Experimental manipulation of flowers to determine the functional modes and fitness consequences of self‐fertilization: unexpected outcome reveals key assumptions

Summary Reproductive assurance is often invoked to explain the repeated transition from outcrossing to selfing among plants. The most common experimental test of this hypothesis uses floral emasculation (anther removal) to manipulate the capacity of flowers for autogamous self‐pollination. The coastal dune endemic C amissoniopsis cheiranthifolia exhibits striking variation in floral morphology and the mating system across its geographic range in C alifornia, and reproductive assurance is likely to have played a role in the transition from large flowers and predominant outcrossing to small flowers and predominant selfing. To compare variation in the timing of self‐pollination and the extent to which it provides reproductive assurance in large‐ vs. small‐flowered populations, we emasculated flowers at different times during anthesis and measured the effects on both seed production and the proportion of seeds self‐fertilized vs. outcrossed estimated using marker genes. We also manipulated large‐ vs. small‐flowered phenotypes within two morphologically variable populations. As predicted from floral development, small‐flowered plants self‐pollinated before flowers open, whereas large‐flowered phenotypes did not. Comparing flowers that were emasculated before closing suggested that a seemingly well‐developed mechanism for delayed selfing (anthers contacting the stigma when flowers close) does not increase seed production. Using emasculation to manipulate selfing assumes that removing anthers from flowers does not compromise outcrossing. However, while not affecting the capacity of flowers to produce seed, emasculation shortened floral longevity, thereby reducing opportunities for outcrossing. Genetic estimation of the mating system revealed a negative effect of emasculation on the proportion of seed outcrossed, even in small‐flowered populations, further suggesting that flowers lacking anthers are unattractive to pollinators. These side effects of emasculation prevent a clear interpretation of the reproductive assurance value of selfing. Given that much of what we know about reproductive assurance in natural populations comes from emasculation experiments, our results suggest that the assumptions of this approach, which are rarely verified, require much more serious consideration.