Quantifying Pollen Deposition with Macro Photography and ‘stigmagraphs’

The pollen deposited during a single visit by a flower visitor (“single-visit deposition”; SVD) is often measured by removing the stigma from the flower and counting the pollen grains deposited under a microscope. This process precludes study of any subsequent interactions between the flower and later visitors (such as pollen removal from the stigma). Furthermore, if the stigma is excised too soon after the pollinator visit, the flower may be rendered infertile, such that any analyses of fruit or seed yield in relation to pollen deposition must be done indirectly. Here, a method of pollen deposition measurement was developed using macro photography and the open-source image-analysis software program ImageJ/Fiji. Using colour segmentation options within the program, the pollen grains can be distinguished from the background stigmatic surface, and the percentage of stigma coverage can be calculated. This pollen deposition measurement method leaves the sampled flower in the field to develop into fruit, allowing any subsequent yield or quality analyses to be conducted directly.