We used a drip-line system to irrigate plants every morning; the irrigation treatment included two levels: 2.2 L water/plant/day and 0.35 L water/plant/day . Mean volumetric water content for plants in the low irrigation treatment was 10% lower than that of plants in the high irrigation treatment . The volumetric water content for plants in the high irrigation treatment ranged from 38 – 51%, whereas that of plants in the low irrigation treatment ranged from 24 – 48%. All analyses are restricted to data collected from July 21 – August 21 during the height of flowering and pollinator visitation.To test for differences in seed set due to pollen deposition from high and low moisture plants, we used florescent powdered pigments to track bee transfer of pollen from male flowers of plants in high and low irrigation treatments to female flowers of plants in both treatment groups . Before allowing bees access to flowers in the morning, we applied powdered DayGlo© fluorescent pigments to male flowers as in Irrigation experiment I. Male flowers on plants in the high-irrigation treatment received one pigment color, male flowers on plants in the low-irrigation treatment received the other pigment color. Each day we switched the color assignments to mitigate for any color preferences exhibited by bees. As in Irrigation experiment I, the number of pigment particles acted as a proxy for the number of pollen grains transported by bees to the stigmas of the female flowers . We measured pollen and pigment deposition as in Irrigation experiment I and seed set as in the Temperature x irrigation experiment. Mean total pigment particle count on bee pollinated stigmas was highly correlated with mean total pollen grain deposition .
To determine the effect of soil moisture from plants donating pollen on seed set, we used a general linear model with one fixed factor, blueberry in container proportion of deposited pigment from plants grown under low irrigation. For all analyses in Irrigation experiment III, plant is the experimental unit of analysis. All but five plants were given at least one chance to set fruit from a pollinated female flower. For plants that set more than one fruit , we used mean values for total seed mass.Reproductive allocation in bee-pollinated squash plants was affected by the manipulation of temperature and soil moisture, but these factors generally had independent effects on floral traits . When effects on allocation were evident, low soil moisture levels were always inhibitory. For both flower sexes, flower size increased with increasing soil moisture and decreased under warmed conditions . Nectar volume increased with increasing soil moisture and increased temperature for male flowers, but this effect was not significant in female flowers . In addition, nectar concentration increased with soil moisture in ambient temperature male flowers but not in female flowers . There were no effects of temperature or soil moisture on the proportion of viable pollen grains in male flowers . Lastly, per plant flower production was unaffected by the manipulation of temperature and soil moisture, except for the potential availability of non-self flowers . The main pollinators observed visiting squash flowers in this experiment were honey bees and squash bees pruinosa. Together these two species accounted for 97% of all floral visits. In general, pollinator visitation rates did not differ with respect to variation in temperature and soil moisture experienced by squash plants , but this experiment was not designed to examine pollinator choice.
The one exception with respect to visitation was that we observed that the cumulative pollen collecting per min for Apis mellifera was higher in warmed plants . There was also a significant interaction between soil moisture and temperature for visits per female flower per minute for Apis and Eucera ; however, these results were not significant when broken down between warmed and ambient treatments. Fruit set and seed set of squash plants increased with increasing soil moisture . Aside from soil moisture, no other measured variable, including pollination type, affected the likelihood of fruit set . In contrast, seed set increased with increasing soil moisture but only for bee-pollinated plants . The seed set of fruits that resulted from hand-pollination did not depend on soil moisture and was almost 27% higher compared to that from fruits that resulted from bee pollination . Based on seed set measurements, the pollen limitation index increased with decreasing soil moisture . There was no effect of temperature on pollen limitation .We assessed the degree to which pollinators influence how experimental warming and drought affect the reproduction of cultivated squash . For those floral traits that responded to the experimental manipulations, low soil moisture levels always resulted in inhibitory effects: smaller flower size, reduced nectar volume and nectar concentration , and a reduced availability of non-self, male flowers. By comparing responses of squash that were either pollinated by bees or by hand, we observed that be pollinated plants experienced increasing levels of pollen limitation as a function of decreasing soil moisture. This result illustrated that pollinators mediate how soil moisture variation affects the outcome of plant reproduction. Additional soil moisture manipulations indicated that drought-induced pollen limitation results, at least in part, from decreased pollen competition resulting from low-levels of stigmatic pollen deposition, reduced viability of pollen produced by plants grown under low soil moisture conditions, and a reduced capacity of self pollinated fruits to respond to increasing soil moisture.
In the present study levels of warming were realistic based on current climate projections , but mean summer temperatures at our coastal study site were mild and even experimentally warmed conditions likely did not result in prolonged physiological stress for squash plants, which are often grown in areas with higher mean summer temperatures compared to those at our study site . In contrast to previous studies that have documented reduced reproductive allocation to warming , two of the three significant responses to temperature in the Temperature x irrigation experiment represented increases in performance . Our study is not unique in this respect; Hoover et al. also report elevated nectar production by pumpkins experiencing experimental warming. Aside from flower size, which decreased under warmed conditions, all of the measured aspects of reproductive allocation in this study either did not respond to the elevated temperature treatment or apparently benefitted from it. The lack of interactive effects observed in Temperature x irrigation experiment may have thus been partly due to a temperature treatment that did not physiologically challenge plants, but other studies also report that the effects of temperature and soil moisture can act independently of one another . In contrast to the effects of temperature, we found that soil moisture variation affected more reproductive traits and that these responses were all negative . Observed effects mirror those reported in previous studies. Known effects of drought, for example, include reductions in flower size and nectar volume . Changes in floral traits caused by drought stress can also reduce visitation by pollinators , but this trend seems to exhibit context specificity and is not universal. Although the responses observed in our system might be expected to reduce visitation by pollinators, our experiment was not designed to measure pollinator choice as open flowers on a given day were all from the same treatment group. Pollinator visitation is the focus of Chapter 2, in which we explicitly test the consequences of floral choice made by specialist and generalist pollinators in an experimental setting where bees can simultaneously visit squash flowers produced by plants grown under different levels of soil moisture. An additional reproductive trait that varied with soil moisture was the availability of non-self, male flowers. Previous studies have also reported changes in flower production in response to drought stress. Al-Ghzawi et al. , Philips et al. , Descamps et al. , for example, all report examples of drought-induced reductions in flower production, plastic planters bulk but this finding is not universal and likely depends on the timing of floral development relative to the onset of drought stress . To examine the role of pollinators in mediating how soil moisture variation affects plant reproductive success, we compared seed set for plants pollinated by bees versus those pollinated by hand and found that bee-pollinated plants grown under low soil moisture levels were pollen limited. To our knowledge, drought-induced pollen limitation has not previously been reported in the literature but could be common given well-known effects of non-lethal, drought stress on various aspects of plant reproductive allocation . Although pollen limitation commonly results from inadequate visitation by pollinators, this mechanism seems unlikely to explain our result given that bee visitation did not increase with increasing soil moisture level. Differential pollen production can also be ruled out as an underlying mechanism for drought-induced pollen limitation given that the amount of pollen produced was independent of soil moisture.
Given that we observed an apparent reduction in the availability of non-self, male flowers as a function of low soil moisture levels, the transfer of self pollen might have been higher for plants grown under low soil moisture conditions. However, we found no evidence for this type of effect through the use of pigments to estimate patterns of pollinator movement. Our results suggest that drought-induced pollen limitation in part stemmed from reduced pollen competition on stigmas that received pollen from bees. Winsor , for example, found that seed set and offspring vigor in C. pepo increased with increasing pollen load and concluded that more intense pollen competition was the underlying cause of this effect. Like Winsor , we observed that seed set increased with increasing pollen deposition . In the Temperature x irrigation experiment, the greater than two-fold higher pollen loads on hand pollinated stigmas, compared to bee-pollinated stigmas, would have heightened pollen competition and allowed only the fastest-growing pollen tubes to achieve fertilization. Consistent with this hypothesis is the fact that the seed set of hand-pollinated plants was independent of the soil moisture levels that these plants experienced while growing. In contrast, the smaller pollen loads deposited by bees would have led to relatively lower levels of pollen competition and a greater opportunity for self pollen or pollen of reduced viability to achieve fertilization. Relaxed pollen competition in bee-pollinated plants could help to explain why we observed drought-induced pollen limitation, but for this mechanism to reduce seed set pollen deposited on the stigmas of must have been of inferior quality. Two lines of evidence support this hypothesis. First, we observed a negative relationship between seed set and the amount of pigment from the male flowers of plants grown under low soil moisture conditions . Moreover, we found that seed set increased with increasing levels of soil moisture experienced by plants contributing non-self pollen . These results taken together suggest that the pollen viability assays used in the Temperature x irrigation experiment may not have been sensitive enough to measure more subtle aspects of pollen viability as measured here. Previous studies that did find significant effects of drought on pollen viability used different assays than the one performed here. Second, we observed that the seed set produced by flowers pollinated with non-self pollen increased with soil moisture, whereas the seed set resulting from flowers pollinated with self pollen did not respond to increasing soil moisture. Although the reduced availability of non-self, male flowers for plants grown under low soil moisture conditions was on some level an artifact of our experimental design , it would seem common for plant species to grow in discrete enough patches for the availability of self pollen versus non-self pollen might influence the outcome of pollination. For example, buffalo gourd, which is the local, native congener of C. pepo, grows in patches of up to five individuals with hundreds of meters separating patches . In conclusion, our results reveal that pollinators can mediate effects of warming and drought on plant reproduction by increasing pollen limitation as a result of decreased pollen competition caused by low-levels of stigmatic pollen deposition, reduced viability of pollen produced by plants grown under low soil moisture conditions, and a reduced capacity of self-pollinated fruits to increase seed set in response to increasing soil moisture. Although this study focuses on only a single plant species, drought-induced pollen limitation could be a common phenomenon based on the frequency with which drought stress affects reproductive allocation in other plant species. Water stress, for example, commonly lowers pollen viability .