Genetic Diversity And Population Structure In Elephantopus L. From South China, And Case Studies Of Phylogenetic And Evolutionary Methods

The genus Elephantopus L. (family Asteraceae, Vernonieae) comprises approximately 30 species distributed in the Neotropics and the Old World. In China, there are two species: E. scaber L., the type species of this genus, and E. mollis Kunth, both occurring in southern and southwestern regions as well as Hong Kong and Taiwan. In particular, E. scaber is a Chinese traditional medicine widely used in the treatment of nephritis, edema, dampness, chest pain, fever and cough of pneumonia, scabies, and arthralgia due to wounding.Inter-simple sequence repeat (ISSR) markers were used to investigate genetic variation in eight natural populations of E. scaber and seven populations of E. mollis from South China, including Guangdong, Hainan and Hong Kong. Eleven primers produced 247 bands across all 184 individuals, of which 243 (98.4%) were polymorphic. Fourteen primers produced 277 bands across all 148 individuals, of which 246 (88.8%) were polymorphic. The average genetic diversity at the species and population levels was estimated to be 0.283 and 0.103, respectively, using mean expected heterozygosity. For E. mollis, they were 0.188 and 0.089. Comparing with other plants with the similar life history, both E. scaber and E. mollis show relatively high intra-population genetic diversity.AMOVA analysis showed the genetic diversity of E. scaber was mainly among populations, so did E. mollis. They had relatively high genetic differentiation value among populations and a relatively low level of gene flow. The effective number of migrants (N_m) among populations based on the F_ST were 0.095 and 0.138 respectively. Cluster analyses based on neighbor-joining method showed the genetic relationships among the populations of E. scaber and E. mollis. The appearance of the population from Hainan was distinct, showing the longest distance to the other populations at genetic level. This is consistent with population genetic theory that small and isolated populations should experience increased random genetic drift and reduced interpopulation gene flow. However, each two populations from the nearest location did not tend to cluster together, indicating the relatively low geographic proximity. Mantel tests indicated that there was no significant correlation between population genetic and geographic distances, but between population genetic patternsof E. scaber and E. mollis. The results obtained from the AMOVA analyses, the cluster analyses, and the Mantel tests all suggested that fragmented local environments and human disturbance, which could obstruct the continuous flow of pollen and seeds, as well as decrease the adaptability to environmental changes and jeopardize the evolutionary potential of this species in the long term, might play important roles in shaping the population structure of E. scaber and E. mollis.The chemical composition of essential oils from E. scaber and E. mollis were steam distilled or extracted by CH₂Cl₂, then analyzed by GC-MS qualitatively and quantitatively. The steam distillation of the whole plant of E. scaber from Yangjiang County yielded a clear and yellowish essential oil. A total of 21 compounds (about 93.3% of the oil) were identified. The major constituents were hexadecanoic acid (42.3%), isopropyl dimethyl tetrahydronaphthalenol (14.1%),β-sesquiphellandrene (8.3%), octadecadienoic acid (5.5%), and phytol (5.2%). As an important sesquiterpene compound, β-sesquiphellandrene has been isolated from a number of medicinal plants, and was found be the most active of several sesquiterpenes with antirhinoviral activity. The common compounds from 12 E. scaber populations and 9 E. mollis populations were β-Caryophyllene, Isocaryophyllene, and Caryophyllene oxide, which were another kind of important sesquiterpene compounds isolated from a number of medicinal plants. Hierarchical cluster analysis classified the leaf oils of E. scaber from 12 locations into two groups. Two main chemotypes of leaf oils in E. scaber were thus identified, one rich in hexadecanoic acid and octadecadienoic acid, and the other rich in the five alkane homologues. Nine E. mollis populations also could be classified into two groups using cluster analysis: one rich in Tetramerhylhexadecenol, and the other one poor in Tetramerhylhexadecenol. Mantel test indicated that there was significant correlation neither between chemotypical variabilities and geographic distances, nor between chemotypical variabilities and genetic distances.With the development of phylogenetic analysis, it becomes more and more important in the molecular biology. In this thesis, I also discussed some phylogenetic methods and their applications in several projects which have been undertaken in our laboratory: 1) for the genetic diversity analyses of E. scaber and E. mollis, a program was compiled for binary data, such as ISSR markers; 2) the evolutionaryrelationships between the protein modifiers and sulfur carrier proteins were inferred after solving the solution NMR structure of ubiquitin-related modifier-1 (Urml) protein in Saccharomyces cerevisa. Both structural comparison and phylogenetic analysis of the ubiquition superfamily, with emphasis on the Urml family, indicated that Urml is the unique "molecular fossil" which has the most conserved structural and sequence features of the common ancestor of the entire superfamily; 3) independent and combined phylogenetic analyses for the DNA sequences from 16 taxa representing 15 species of the genus Ephedra were performed using the maximum parsimony (MP), neighbor-joining (NJ), minimum evolution (ME) and maximum likelihood (ML) methods, indicating that E. rhytidosperma is closely related to E. equisetina. The divergence time between them is then estimated to be 10.85±2.44 Mya based on the results of the relative-rate tests and a known evolutionary rate of rbcL gene.