In this study, we present a comprehensive analysis of how concentrations of soil nutrients nitrogen and phosphorus affect the lifecycle of annual monoecious (separate male and female flowers on the same individual plant) cucumbers through growth, pollinator attraction, and reproduction. We hypothesized that increased nitrogen and phosphorus will increase floral traits attractive to pollinators, but, outside of a certain range, nutritional stress through limitation or excess would negatively affect floral traits and pollinator attraction [8]. Using regression and structural equation modeling, we demonstrate that increasing soil nutrients generally increase vegetative traits and floral display, including flower number and size, as well as the floral rewards produced by male flowers (nectar concentration and volume, and amount of pollen), leading to increased attractiveness to pollinators and resulting in increased fruit production. However, very high soil nitrogen levels can have negative effects on several traits, and increasing phosphorus levels can mitigate some of these impacts. Intriguingly, some floral traits, mainly pollen nutritional quality, nectar sugar composition, and female flower nectar volume and concentration, remain consistent across soil treatments. Thus, these findings suggest that important floral rewards for pollinator attractiveness can be relatively constant to ensure pollination success, even in stressful conditions.
Importantly, while we show that soil nutrients can lead to particular positive outcomes of annual plants, increasing nitrogen alone can lead to toxicity of plants and increasing phosphorus may mitigate this problem, revealing ideal soil nutrient balances or ratios [1, 3, 5, 6, 8, 9, 12, 24, 29]. Phosphorus tended to increase certain traits linearly from 0 to 50ppm (Figs. 1,2; Supplementary Table S2), while increasing N appears to asymptote or even reduce traits above 100ppm (Figs. 1,2; Supplementary Table S2). Therefore, the ideal soil nutrient nitrogen:phosphorus ratio tested was 4:1, which resulted in best overall growth, pollinator attraction, and reproduction (Figs. 1,2; Supplementary Table S3).
We found that soil nutrients had differential effects on floral display and rewards between male and female flowers, which has interesting implications for plant reward systems and pollination ecology. Similar to previous studies on soil nutrients and floral traits, we found that increasing soil nutrients increased the number and size of cucumber male flowers per plant [6–9], nectar concentration of male flowers [13, 14], and pollen number per flower [6, 10–12]. Thus, under ideal nutrient conditions, plants may increase male fitness by producing more gametes (i.e. pollen), making it more attractive (e.g., larger floral display) and rewarding (nectar volume and pollen quantity) to bees, resulting in higher visitation (and possibly pollen dispersal) as we observed.
Pollen quality (protein and lipid concentrations, and protein:lipid ratios) remained the same under all soil conditions, revealing its importance as a physiological and ecological trait of male flowers. There could be two complementary reasons for this. First, pollen quality must remain at a particular level for the pollen’s own nutrient needs for reproduction, including keeping pollen cells alive, preventing desiccation, binding to receptive stigma, and germinating the pollen tube [37, 38]. Thus, pollen quality may be an evolved trait non-negotiable for plants to ensure successful pollination and fertilization. But pollen also serves as a reward for bee visitation. Pollen protein:lipid ratio has been demonstrated to be important for bumble bee (one of the most common cucumber pollinators) attraction and health [21, 39–41] and specific protein:lipid ratios may be important for attraction and the health of preferred pollinators [22]. Thus, maintaining pollen quality is necessary as an honest reward signal to bees.
Similar to male flowers, soil nutrients also influenced female flower size and number with more female flowers increasing fruit number [8]. Yet these traits did not statistically influence bee attraction and visitation, possibly because floral display was dominated by the sheer number of male flowers. All other aspects of female rewards did not change under any soil conditions tested, i.e. nectar volume, concentration, and sugar composition, which are important aspects of pollinator attraction for bumble bees who prefer higher concentrations and volumes [20] and pollinators that may prefer certain sugar composition [19, 42]. Nectar concentration and composition subsequently had no effect on bee visitation rate. Yet we observed that plants with higher female nectar volumes were more frequently visited by bumble bees and resulted in higher fruit set. Female flowers produce substantially more nectar than male flowers suggesting high volume is a primary mechanism for attracting pollinators. Additionally, higher nectar volumes could also increase bee handling time and likelihood of pollen deposition [43]. Therefore, maintaining nectar composition, concentration, and volume is a key ecological trait for pollinator attraction and pollination success that seemed to be buffered in the face of stressful conditions. We observed that all plants produced fruit, and therefore maintaining reward qualities of male and female flowers under a variety of soil conditions may perhaps insure pollinator attraction in stressful environmental conditions.
There are some limitations to our study which could be explored in future research. First, nitrogen and phosphorus treatments were coupled in our study. The highest level of nitrogen was paired with our middle phosphorus treatment at an 8:1 nitrogen:phosphorus ratio; perhaps the negative effect observed at high nitrogen concentrations could have been mitigated by higher phosphorus levels. Further research on the effects of one nutrient independent of one another and across a wider range of nutrient concentrations and ratios would help understand the asymptotic or potential detrimental effect of higher nutrient concentrations of both nitrogen and phosphorus. Second, there may be other detrimental effects on reproductive quality that we did not measure. These include reduced seed number per fruit by reduced pollinator visitation [44–48] and reduced seed viability from soil nutrients either in seed development or for seed germination [49, 50]. Third, we studied cucumbers which have undergone extensive artificial selection for particular traits [51]. Perennial plants, which are less reliant on a single season for reproduction, or non-cultivated plants may exemplify higher plasticity than annual plants [52]. Therefore similar studies should be conducted with wild populations of plants and with different pollination systems.
In this study we present the effects of multiple soil nutrient titers on the entire plant life cycle, with particular attention drawn to the effects of nutrients on floral display and rewards and pollinator attraction and visitation, and finally reproductive success. Nutrients clearly can affect pollination success of annual plants, but these plants may be somewhat buffered through particular consistent or fixed floral traits. However, because this study focused on a single plant species, future assessments of the responses on a diversity of wild and agricultural populations of plants, and their associated pollinator communities, to nutritional stress caused by anthropogenic activity are critical. Additionally, the impacts of soil nutrition-induced changes in plant nutritional rewards on pollinator health should be considered. With the effects of anthropogenic activities likely to increase in scope and scale, evaluating the full extent of the impacts on natural and agricultural ecosystems is essential.