Pollination Ecology Of Two Sympatric Orchids, Changnienia Amoena And Cypripedium Japonicum

Orchids with highly elaborate and unique pollination mechanisms had a prominent role in providing evidence for natural selection and for the advantages of cross-pollination in the early history of evolutionary biology. The study of pollination ecology of orchids could provide insights into understanding of some key questions of plant evolution, such as reproductive isolation of speciation, adaptive evolution and evolution of mating system. The present sudy focuses on plant-pollinator interaction, intraspecific morphological variation and pollen flow of two orchid species, Changnienia amoena and Cypripedium japonicum, in Shennongjia Mountains, with the hope to explore the adaptive evolution and evolutionary trend of pollination system. In addition, microsatellite markers were preliminarily developed in C. amoena for use in the analysis of gene flow and mating system. 1. Anthecology of the endangered deceptive orchid, Changnienia amoena The field observation and experiment in C. amoena were investigated at two sites in two consecutive years. The results showed that the orchid was self-compatible and outcrossing species. It is exclusively pollinated by bumblebees, but the pollinators were different in two sites. In Longmenhe, Bombus (Diversobombus) trifasciatus was the primary effective pollinator, whereas B. (Tricornibombus) imitator was the only pollinator in Guanmenshan. These two bumblebee species could be treated as a functional group because of the same position of carrying pollinarium and similar pollinating behaviors although their abundance at the local sites and foraging behavior were different. At the population level the flowering spectrum of C. amoena was not markedly skewed. The visitation of bumblebees is mainly in the first half of the flowering period. Flower visit of bumblebees was brief due to the absence of food reward. Pollinarium removal and pollinia deposition took place when bumblebees withdraw from the flower. Natural fruit set in this species is only 6%-12%, with obvious variation across years and among populations. The low fruit set resulted mainly from pollinator limitation because 87.5% of individuals set fruits under hand-pollination. In comparison to Calypso bulbosa that belongs the same subtribe Calypsoinae, C. amoena suffered less from glaciation, and its pollination environment was relative stable, and so this species developed a stabilized pollination system. These results imply that it is important to conserve the pollinator community together with the food plants when conservation management for this endangered species is undertaken. 2. Population size, pollinator visitation and pollen flow in C. amoena To test the hypothesis that in non-rewarding plants the pollination intensity should increase with increasing population size, I examined pollinarium removal and fruit set in 10 populations of C. amoena. Pollen flow was estimated by marking the pollinium with microtags and colored stains. The results showed that the percentage of pollen removal was significantly negatively related to population size in one of the two years. In the other year, this negative relationship was not significant. Although the spatial pattern varied in different populations, the visits of pollinator occurred randomly except for one population where bumblebee nests were found. The low density-dependent selection is a mechanism maintaining reproductive success of C. amoena. Pollen flow pattern typically shows a leptokurtic distribution, with most dispersal over short distances and incidental dispersal over much longer distances. Mean distance of pollen flow varied among sites, 7.3 m in Longmenhe and 10.6 m in Guanmenshan, respectively. The pollinium transfer occurred mainly among the related individuals within the population, and few or even no pollinia was imported because of long distance isolation between populations. 3. Morphological variation of C. amoena and its adaptive significance Investigation of intraspecific morphological variation is critical for the understanding of evolutionary and ecological processes, and may provide insights for uncovering the roles of natural selection and gene flow in the distribution and abundance of species diversity. In the present study, I analyzed statistically the variation patterns of 13 morphological traits for 15 populations in three localities of C. amoena. The results showed that there was significant difference (P<0.01) in most morphological traits among three localities. UPGMA analysis showed that the populations of three localities were clustered into three main clades, in which the populations from Xinning and Lushan formed one clade, while the Shennongjia populations formed the other. It is noteworthythat Shennongjia populations were separated into two subclades corresponding to the populations of Longmenhe and Guanmenshan, respectively. The functional morphological characters of pollinators are significant difference in Longmenhe and Guanmenshan. Correlation analysis between the fitness (pollinia removal) and floral morphology indicated that the morphological differentiation resulted from pollinator-mediated selection. The transplant experiment indicated that the local pollinator showed selective visits to the transplanted orchids. These results have important implications for our understanding of the evolutionary processes and mechanism of C. amoena. 4. Anthecology of lady's slipper orchid, Cypripedium japonicum The anthecology of rewardless C. japonicum, with typical Japonicum-lip, was studied in six populations. Hand-pollinations experiments demonstrated that the species is self-compatible, but dependent on insects for pollen transfer. At population level the flowering spectrum is positively skewed, with the typical feature of rapid reaching the peak anthesis. The pollinium of the orchid is exclusively transferred by three bumblebee species. Because of lack of nectar and collectable pollen, bumblebees' visits are infrequent and short, mainly focusing on the early and peak anthesis. Natural fruit set is only 4.3%-8.5%, which mainly resulted from limited pollinators. Despite very low visitation rates, the chance of a removed pollen mass fertilizing another ovary was 15.3% to 23.7%. The hypothesis that bumblebees were attracted by clustered, synchronized blooming rewardless flowers was rejected, because I found a weak negative relationship between the clusters sizes and effective pollinator visitations (i.e. the proportion of plants exporting pollen). The percentage of fruit set in the clustered plants are not significantly different from randomly intermingled plants, indicating that female fertility is dependent of flower density. The evidence from anthecological and morphological studies indicated that C. acaule and C. japonicum are sister species pair with disjunct distribution between eastern Asia and North America, in the transitional position that the species with sticky pollen mass have evolved to the taxon with waxy pollinia. 5. Isolation and characterization of microsatellite loci in C. amoena To study the impact of mating system on genetic population structure and theestimation of gene flow among populations, variable and codominant genetic markers are needed. In present study, microsatellite markers in C. amoena was developed. The genomic DNA was converted into the fragments and hybridized to biotinylated microsatellite oligonucleotides and captured on streptavidin-coated paramagnetic beads (Dynal). Unwanted DNA was washed away and captured′ DNA was cloned and sequenced, and then the largely enriched fragments containing SSR were obtained. Primers were then designed according to the sequences flanking the repeat motifs and used for polymorphism analysis. The result showed that 51.4% (18) sequences containing the repeat motifs was found in 35 clones, indicating that efficiency of SSR enrichment by Dynabeads is high. Four primer pairs were designed. This method would establish a foundation for microsatellites isolation of plants in Cypripedium and analysis of population genetic structure and evolutionary pattern of microsatellites of this genus in the future.