Nectar Robbing Behavior By Bees Affects The Reproductive Fitness Distyly Plant Tirpitzia Sinensis(Linaceae)

Due to the mechanical mismatch between the body size of visiting animals and floral characters,some animals will directly pierce the corolla tube and obtain the nectar,rather than pollinating the plant,which is the nectar robbing.Nectar robbing is common in nature and can directly affect plant reproductive success by influencing the female and male fitness,and can indirectly affect plant reproductive success by influencing the pollinator behavior.Nectar robbing has negative,neutral and positive effect on plants.Nectar robbing is an important driving force for plant evolution.Previous studies on nectar robbing mainly focus on homotypic plants.It remains unclear how the nectar robbing affect the reproductive success of distyly plants,whether the effects vary on the different morphs and whether the defense strategies of two morphs are different.Our previous study showed that the flower of distylous Tirpitzia sinensis(Linaceae)consists of five green sepals and five white petals forming a floral tube.The long tongue hawkmoths are the effective pollinator and honeybees and bumblebees are the main nectar robbers.The distylous T.sinensis is selfincomoatible.To explore the direct effect and indirect effect of nectar robbing on distylous plant reproductive success and the defense strategies of two morphs,the T.sinensis is taken as materials.We investigate and compare the nectar robbing rate between L-and S-morphs in the field.The floral traits,the rhythm variation in nectar volume and sugar concentration,nectar secondary metabolites(Ultra Performance Liquid ChromatographyQuadrupole /time-of-flight mass spectrometers)between robbed and unrobbed flowers of the two morphs.The nectar robber behavior was observed.The direct effects of nectar robbing on female and male components of plant fitness and the indirect effects of nectar robbing via pollinators were tested.Here are the main results:(1)There was no significant difference in the nectar robbing rate between long-styled morph(L-morph)and short-styled morph(S-morph).For L-morph,the sepal width,flower width,petal width and tube diameter were significantly larger in unrobbed flower than those of robbed flower.For the S-morph,the sepal width,flower width,petal width and pistil length were significantly larger in unrobbed flower than those of robbed flower.The thickness of the tube of robbed flowers was also smaller than that of unrobbed flowers in the L-morphs.Petal size and sepal size showed a significant positive correlation with flower size.In general,the flower size of unrobbed flowers was larger,with thicker tube and larger sepals compared to robbed flowers.(2)The artificial nectar robbing significantly decreased the nectar volume and sugar concentration in total and from 0800 to 1100,the nectar volume of artificial nectar robbing flowers and unrobbing flowers in both morphs were increased indicating that the flowers secreted nectar continuously.From 1100 to 1700,the artificial nectar robbing flowers in both morphs barely secreted nectar and the unrobbing flowers in two morphs continuously secreted nectar.The content of sucrose in nectar was the highest,and nectar robbing had no significant effect on the sugar content of nectar.The UPLC-Qtof analysis of secondary metabolites of nectar showed that a total of 216 metabolites in 10 categories were detected in the nectar,including 71 terpenoids,28 flavonoids and 26 alkaloids.After nectar robbing,twenty-one chemicals were up-regulated and six substances was down-regulated in the S-morph,and ten substances were up-regulated and six substances were down-regulated in the L-morph.Especially in the S-morph,the composition and accumulation of nectar metabolites were significantly changed after nectar robbing,mainly in terpenoids,flavonoids and alkaloids.(3)The pollen germination rate of nectar unrobbing flowers as pollen receipt was significantly higher than that of nectar robbing flowers as pollen receipt in the L-and S-morph,while there was no significant difference in pollen tube length among different treatments.The seed sets of intermorph pollination treatment when normal flowers as pollen receipt was significantly higher than that of the natural,natural nectar robbing,than that when flowers with artificially cutting a robber hole as pollen receipt and that when flowers with artificially removing the nectar as pollen receipt.The seed sets of normal buds followed open pollination treatment as control were significantly higher than that of buds with obvious nectar robber hole in nature followed open pollination.seed sets of the L-morph and the S-morph as pollen recipient had no significant difference.(4)The visitation frequency of hawkmoths to unrobbed flower was significantly higher than that of robbed flower.it showed no significant preference in the L-morph and showed preference for visiting unrobbed flowers in the S-morph.This study showed that the physical structure and nectar secondary materials of T.sinensis can defence unfavorable nectar robbers,and the nectar chemical composition varied more significantly in the S-morph.Nectar robbing showed a negative effect on female fitness by reducing pollen germination and a neutral effect on male fitness,and nectar robbing also mediated pollinator behaviors,preferring to visit unrobbed flowers in S-morph.In this study,we clarified the reproductive strategy of the T.sinensis by exploring the effects of nectar robbing on plant phenology,chemical traits and reproductive success.This study provides a theoretical basis for effectively promoting successful plant reproduction and maintaining biodiversity from the perspective of nectar robbing behavior.