Floral Volatile Compounds Of Several Species Of Calanthe And Habenaria (Orchidaceae) From China

Orchidaceae is one of the most species-rich and widely distributed families of angiosperms.The flowers of most orchids can release volatile compounds during opening,and these compounds play an important role in attracting pollinators to visit their flowers.At present,the research on the flower volatile compounds of orchids mainly focused on the composition,biosynthetic pathways,pharmacology and pollination biology of individual species.Studies on the comparison of floral volatile compounds of congeneric species of orchids are rarely reported.Habenaria and Calanthe are the fourth and fifth largest genera of Chinese orchids,respectively.Most species in these two genera specialized a floral spur.Habenraria species usually have green or white flowers,with nectar in the spurs,which can provide rewards to their pollinators.The pollinators are typically nocturnal moths,although butterflies have also been documented in a few cases.Calanthe species are usually have bright yellow,red or purple flowers,without nectar in the spurs,and does not provide any form of reward to their pollinators.The pollinators are mainly diurnal bees and butterflies.Although pollination biology has been reported for 30 and 10 species in the genera Habenaria and Calanthe,respectively,the floral volatile components in these two genera remain largely unknown.In this thesis,we studied the floral volatile components of 11 Calanthe and 7 Habenaria from China.We used two methods,SPME and DHS,to collect the floral volatile components,was subsequently analyzed with GC-MS.We analysis of the diversity of floral volatile components of Calanthe species with those of Habenaria species and the differences between them We also explored the relationship between floral volatile components and different types of pollinating insects.(1)SPME and DHS were used to collect floral volatile components together.The volatile components collected by the SPME have good reproducibility and richness,while the DHS shows good systematic stability.The results of floeal volatile components of Calanthe and Habenaria showed that SPME was sensitive to terpenoids and aromatics,and DHS was more sensitive to alcohols,esters,ketones and aldehydes.From the results of collecting volatile components,SPME has good collection effect on terpenes,alcohols,esters and aromatics,and DHS has good collection effect not only on terpenes,alcohols,esters and aromatics,but also on alkanes,aldehydes and ketones.(2)A total of 133 volatile components were identified in the flowers of 11 Calanthe species,including terpenes,alcohols,aromatics,alkanes,esters,aldehydes,ketones and ethers,and it was characterized by high abundance of terpenes,alcohols and esters.The volatile components with high contents included(-)-α-pinene,β-caryophyllene,α-farnesene and sabinene.In addition,the relative contents of citronellol,benzyl acetate and tridecane is also high in C.tricarinata,C.brevicornu and C.graciliflora,respectively.The volatile components of C.tsoongiana and C.reflexa without spur not only differ significantly from other Calanthe species with spurs,but also between themselves.In the PCA analysis,PC1,PC2 and PC3 explained 19.5%,12.6% and 9.3% of the variance,respectively,accounting for 41.47%of the total variance,and the higher loadings of terpenes suggest that they have sufficient influence on the composition of the floral volatile components of Calanthe.The CA divided the 11 species of Calanthe into three groups,C.sieboldii and C.reflexa clustered as the first group,C.triplicata clustered separately as the second group,and the rest of Calanthe clustered as the third group.(3)A total of 133 volatile components were identified in the flowers of 7Habenaria species during the most abundant time period of volatile components,including terpenes,alcohols,aromatics,alkanes,ketones,esters,aldehydes,ethers and acids,mainly terpenes and aromatics.The volatile components with high content were β-caryophyllene,Germacrene D,linalool,(3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene and nonanal.The flowers of H.davidii,H.ciliolaris,H.dentata,H.aitchisonii and H.delavayi were detected more volatile components at night,while the flowers of H.finetiana and H.rhodocheila were detected more volatile components during the daytime.In the PCA analysis,PC1,PC2 and PC3 explained24.16%、18.47% and 11.9% of the variance,respectively,accounting for 54.53% of the total variance.The CA divided the 7 species of Habenaria into two groups,in which H.rhodocheila were clustered into a separate group and the other six species of the genus Habenaria were clustered into another group,indicating that the floral volatile components of H.rhodocheila exhibited significant differences from other Habenaria.H.delavayi,H.finetiana,H.dentata and H.aitchisonii clustered together at a distance of 1,indicating that the volatile components of the flowers of these four Habenaria species was similar.(4)Esters,aromatics and aldehydes are the differences between the volatile components of the flowers of Calanthe and Habenaria.The characteristic floral volatile components of Calanthe were esters and those of Habenaria were aromatics and aldehydes.Terpenes were the main floral volatile components of both Calanthe and Habenaria and contributed to their floral scents.CA results showed that the floral volatile components of H.finetiana and H.delavayi were similar to that of Calanthe,and the floral volatile components of H.rhodocheila were different from those of Calanthe and other Habenaria species.(5)Relationships between floral volatile components and different insect pollinators.The main floral volatile components of orchids pollinated by bees were terpenes,those of orchids pollinated by butterflies were mainly terpenes and alcohols,and those of orchids pollinated by moths were mainly terpenes,aromatics and alcohols.H.finetiana,which has not yet been studied for pollination biology,fits the typical butterfly pollination syndrome,with nectar in the spur,without obvious flower scents,and pale white flowers.while cis-3-hexenol,sulcatone,nonanal,and decanal detected in H.finetiana during the day have been shown to elicit strong antennal responses in butterflies,and hormone-like substances(nonanal and methyl salicylate)that attract moths have also been detected at night,so we speculating that there may be daytime pollination by buffterflies and nocturnal pollination by moths in H.finetiana.In addition,floral volatile components such as benzyl acetate,linalool,terpinolene and(Z)-β-ocimene were detected in C.brevicornu,C.tricarinata and C.fargesii that can attract bees,so it is presumed that the pollinators of these three Calanthe species may be bees.The flowers of both C.tsoongiana and C.reflexa have no spurs and inconspicuous odor,presumably their pollinating insects are of the same type,both of which are bees.This study is the systematic exploration of the floral volatile components of Calanthe and Habenaria from China.It provides valuable information to further explore the association between the floral volatile components and different insect pollinators,as well as determining the attraction of floral volatile components to insect pollinators by GC-EAD analysis.But the prediction of their pollinating insect types for plants still needs to be confirmed by future studies on the pollination biology of more species.