An Ecological Study Of The Microbial Function In Insect Pollination Of Seed Plants

In nature,the invisible but ubiquitous microorganisms play important ecological roles in different habitats and in diverse biological interaction processes.For instance,floral nectar of angiosperms and pollintion drops of gymnosperms function primarily as pollinator reward and pollen landing site,respectively,yet they may also act as natural mediums for microbes because of their high concentrations of sugar and amino acids.In these highly nutritious habitats,complex microbial activities of propagation,proliferation,extinction and metabolism may occur,which can potentially influence nectar chemistry,pollinator behaviour,pollination process and the subsequent plant fitness.In this research,three of the most species-rich taxa inhabited in our planet,namely,plants,animals and microorganisms,are involved to reveal the ecological functions of microbes in seed plant–pollinator interactions.By focusing on a natural angiosperm community in the Hengduan Mountains of China and a typically entomophilous gymnosperm(Gnetum luofuense)located in the tropical rainforest,we carried out the following three studies:First,we conducted a community-level survey on the abundance and diversity of culturable yeasts and bacteria in the nectar of 13 alpine plants in the Hengduan Mountains,southwest China.The results show that microorganisms were found in82.6%of the 298 natural flowers,of which a high percentage(68.8%)of flowers contained nectar-inhabiting yeasts.However,phylogenetic diversity of microbes is low,only a few species like Metschnikowia reukaufii can flourish in the nectar.Community composition of nectar-inhabiting microbes varied among plant species,as indicated by the principal component analysis,which also demonstrated that the incidences of Metschnikowia,Cryptococcus and Starmerella are the main factors that cause these differences.In addition,the diversity and abundance of microorganisms in nectar samples of flower at late anthesis were significantly higher than that of virgin flowers.Second,we present results on the incidence and density of M.reukaufii in floral nectar of an endemic alpine plant Clematis akebioides in the Hengduan Mountains,using a combination of yeast cultivation and microscopic cell-counting method.The results show that 79.3%of the 58 C.akebioides flowers harboured M.reukaufii,and the mean cell density is high to 7.4×10⁴±1.8×10⁴ cells mm^-3.Furthermore,in the experiments of using real flowers filled with artificial nectar with or without yeast cells,we found that the presence of this cosmopolitan nectar yeast species increased pollinator visitation rate and seed set of C.akebioides in the field.Besides,in the laboratory,we reveal the volatile metabolites produced in the yeast-inoculated nectar which can potentially act as effective floral signals for pollinator attraction,and thus may be responsible for above beneficial interactions.Last,we used Gas Chromatography–Mass Spectrometry(GC-MS)to analyze qualitatively and quantitatively the volatile compounds released by female strobilus of G.luofuense.The results show that the rancid odor was composed of four volatile short chain fatty acids,namely,acetic acid,propionic acid,isobutyric acid and isovaleric acid.These simple volatiles are effective in attracting two of the more important pollinator groups(moths and cockroaches),as we showed in the behavioral bioassays.Furthermore,we used high-throughput sequencing methods to thoroughly explore the composition of bacteria in pollination drops.The results indicate that the two dominant genera of Prevotella and Bacteroides,are capable of producing above fatty acids by metabolizing sugar and amino acids,and thus may act as functional strains for promoting the formation of volatile signals to attract the effective pollinators of G.luofuense.