Pollen Chemical Defense Mechanism Of Two Dipsacus Species

Pollen grains in bee-pollinated plants have dual function not only as a vector of male gametes but also as an essential larval food for bees.Pollen grains groomed by bees and stored in the scopae are normally no longer useful for pollination,accounting for a large proportion of pollen loss during transfer.To explore the adaptive strategy of conflicting interest between plants and bee pollinators,we studied the pollen chemical defense mechanism in two herbs in Dipsacus L.(Dipsacaceae).As one of the major Traditional Chinese Medicine,previous studies on Dipsacus species focused on the chemical composition analysis,pharmacology,quality evaluation,application and development,finding that these Dipsacus species usually have the saponin VI in roots,shoots and leaves.It remains unclear whether this type of secondary metabolites occurs in nectar or pollen,which may potentially affect pollinator behavior.Studies have showed that the toxic secondary materials in nectar influence pollinator behavior,and consequently plant reproductive success.In the field,we observed that some bumblebees visiting capitula of Dipsacus chinensis and D.asper were densely dusted with pollen,but discarded the pollen not trying to pack them into the corbicular of the hind legs.Why do some bumblebees groom the Dipsacus pollen but others not?To explore the ecological function of secondary metabolites in pollen grains,we compared pollinator behaviors and the effects on pollen transfer in field populations of Dipsacus chinensis and D.asper.Our main results are:First,to assess the pollination systems in D.chinensis and D.asper,we observed pollinator visits for five years,and measured both vegetative and reproductive traits,and recorded flowering phenology.We also measured nectar sugar concentration and volume at different time from morning to night and conducted five pollination treatments.Small flowers on the capitula of the two Dipsacus species were white and radial with high sugar concentration(D.chinensisi 53%,D.asper 45%)and volume(D.chinensis 5.0?L,D.aspen 3.5?L)of nectar flowering from July to August on the mountain slopes,suggesting generalized pollination systems.Floral visitors included bumblebee,hoverfly,butterfly,solitarybee,beetle,fly,hawkmoth and moth,but bumblebees were the most important pollinators.Fruit set among open-pollination,hand self-and cross-pollination treatments was not significantly different,indicating that reproductive success in these two species was not limited by pollen.We observed that different bumblebee species had different pollen grooming behaviors on the Dipsacus capitula.Second,to quantify whether the bumblebees collect the Dipsacus pollen and detect secondary materials in plant tissues,we counted Dipsacus pollen grains on the bee body and calculated the pollen percentage in the corbicula.The free amino acid kind and content were analyzed using amino acid analyzer.To examine which tissues contain saponin VI,we collected tissues of root,stem,leaf,corolla as well as nectar and pollen from the two species separately and analyzed using high-performance liquid chromatography(HPLC).We observed that B.festivus effectively groomed the Dipsacus pollen,but B.friseanus,B.trifasciatus and B.remotus did not.The proportion of Dipsacus pollen in the corbicula of B.festivus was significantly higher than that of B.friseanus in Yunnan Province and B.remotus in Hubei Province,China.The proportion of D.asper pollen in the corbicular of B.festivus and B.trifasciatus was not significantly different.Moreover,pollen grains on B.friseanus,B.remotus and B.trifasciatus body were dramatically more than on B.festivus.The kind and content of amino acid in Dipsacus pollen were high and abundant.The content of saponin VI was relatively higher in the pollen and root than in other organs.No saponin was detected in the nectar at trace level.The toxic component of saponin VI in the Dipsacus pollen may deter the grooming behavior in some bumblebee species.Third,to explore whether the saponin VI disturb bumblebees behavior and whether the saponin tolerance ability of pollen-grooming and pollen-ungrooming bumblebees are different,we designed a series of artificial nectar treatments with different concentrations saponin in both real and artificial flowers.We observed that higher saponin VI decreased bumblebee foraging behavior,including visiting rates,visiting time per flower and nectar absorbing of one visit.The saponin tolerance ability of pollen-grooming bumblebees,B.festivus was higher than that of pollen-ungrooming B.friseanus,B.remotus and B.trifasciatus.To assess the benefit of ungrooming behavior and the biological significance of producing toxic pollen,we compared pollen removal and receipt by pollen-grooming bumblebee B.festivus and pollen-ungrooming bumblebees B.friseanus,B.remotus and B.trifasciatus.We observed that pollen removal by B.friseanus and B.remotus was significantly lower than by B.festivus.However,pollen receipt by B.friseanus and B.remotus was significantly higher than by B.festivus.Pollen removal by B.trifasciatus and B.festivus was not significantly different and pollen receipt by B.trifasciatus was significantly higher than by B.festivus.Fruit set of D.chinensis and D.asper(YN)among open-,self-and cross-pollination was not significantly different.While fruit set under open pollination was significantly lower than under slef and cross-pollination in Hubei population of D.asper,indicating that pollen limitation existed.Pollinator visitation rates and pollination efficiency were relatively lower in HuBei.Our investigations of bumblebee grooming behavior and its effects on pollen removal and receipt demonstrated that a chemical protection of pollen resource facilitates pollen transfer through altering pollinator behaviors of pollen collection.