Two different experiments were performed on the same set of populations, using different plants. Samples of 32 and 28 plants were taken from 2 much larger populations and were manipulated by having 0-5 of their stigma lobes removed. One site was at Gothic Townsite located near the Pump House at the junction of Gothic and Old Copper Creek Roads (PH population). In the PH population 18 hermaphroditic and 10 female plants were sampled. The other site was located at the top of the Research Meadow (RM population) of the RMBL and had 23 hermaphroditic and 16 females plants sampled. As hermaphrodites and females were not equally distributed between the populations at different sites, more females were sampled at the Research Meadow. Both plots were fenced using DEER-X^© protective deer fencing in order to prevent herbivory on the plants. Each plant was marked with flagging denoting plant number, and each manipulated flower was marked with a jewelry tag denoting plant number, date and treatment for reference upon return.

Before treatments (stigma removal) could be applied to hermaphroditic plants, they were emasculated while they were in their male phase in order to avoid self-pollination. This was done by removing the stamens with forceps and then removing 0-5 stigma lobes from the closed stigma. Female plants had their stigmas removed prior to their opening and receptivity. Once the stigma lobes open, they bend back and are chemically available to receive pollen and become fertilized. Thus the manipulations were done prior to this state to ensure the stigma lobes weren’t pollinated before they were removed. For both morphotypes, full treatments were preformed, i.e. 0,1,2,3,4 and 5 stigma lobes were removed, when enough flowers were available.

After the unreceptive stigma lobes were removed (treatment phase) and the flowers began to set fruit, the remaining stigma lobes were collected and stained in order to count pollen grains. The stigma lobes were collected using forceps, and then transferred to slides. Slides were prepared in the laboratory before going out into the field by putting two drops of basic Fuchsin gel (Kearns and Inouye, 1993) in two different areas on the slides to allow for 2 flowers’ stigma lobes to be applied per slide. The gel was re-liquefied in the field using a lighter or alcohol burner, at which point the remaining stigma lobes were applied and covered with a cover slip. These were then squashed with the back of the lighter to aid in dying and recognition of the pollen on each stigma lobe (Kearns and Inouye, 1993). After the slides were allowed to dye for 2-4 hours, pollen grains were then counted using 100x magnification on a compound microscope for each individual stigma lobe and for the total fruit.  

Once the fruits began to set- about two weeks after flowering, preliminary seed set was estimated for manipulated flowers by noting how many carpels were beginning to swell. These will be compared to final seed set in case certain carpels began to swell but never set true seed. Final seed set was collected by clipping the entire developing fruit at the stem once it had turned brown, but before seeds were dispersed (approximately 2-3 days before maturity). These were placed in small coin envelopes and allowed to finish maturation and release seeds on their own. Seed set will be compared to pollen loads of individual stigmas and total flowers using Regression and ANOVA statistics.

The observational portion of the experiment was performed by looking at unmanipulated plants. Once fruits had begun to elongate (approximately 1-2 weeks after pollination), full stigma lobe sets were collected with forceps and then transferred to slides. Slides were prepared and pollen grains counted as described above.

These pollen loads were also compared to the preliminary seed set for the individual flowers that they were taken from, again by taking note of how many carpels had begun to swell. Final seed set will be taken in the same way as noted above, in order to compare actual versus estimated seed set.

 

Many plants in nature fail to set the full seed set that they are anatomically capable of producing. This may be due to factors such as pollen limitation. The effects of stigma lobe removal, and natural pollination among stigma lobes, were tested in two populations of the gynodioecious plant Geranium richardsonii, which has the potential to set 5 seeds. The amount of stigma lobes removed limited seed set, and the majority of naturally pollinated plants received pollen on all 5 stigma lobes. It appears that there is a 1:1 anatomical relationship between stigma lobe and ovule, and this consequently affects the seed set- the less stigma lobes that are pollinated, the lower the seed set.