Molecular Phylogeny Of Gymnosperms And The Origin Of Ephedra Alkaloids

The gymnosperm is one of the major clades of extant seed plants. The phylogeny of the gymnosperm, especially the systematic position of Gnetales is an attractive issue for investigating seed plant evolution. In the present study, we sampled 48 genera of the gymnosperm, and obtained molecular data sets for nuclear 18S rDNA, chloroplast rbcL, matK and rps4, and mitochondria coxI. Phylogenetic analyses were carried out based on the five genes and their combined data sets, respectively, using the most parsimony method (MP), maximum likelihood method (ML), and neighbor joining method (NJ). The effect of the third genetic code on construction of phylogenetic tree was also explored. The results indicate that the molecular data of each gene support different hypotheses. First, NJ, MP and ML analyses of 18S rDNA give "Gnetifer" trees, which suggests that gymnosperms are sister to angiosperms with a low bootstrap support at 37%, and that both are monophyletic. Conifers is monophyletic and is sister to Gnetales. Second, NJ, MP and ML analyses of rbcL gene support "Gnetales-Sister" trees. In this tree, Gnetales, as a basal lineage of seed plants, is sister to angiosperms and other gymnosperms. Gymnosperms include angiosperms along with cycads, and therefore become paraphyletic. Third, NJ, and MP analyses of matK gene support "Gnetales-Sister" trees, while ML analysis of matK supports "Gnetifer" trees. The two gene trees only have difference in the position of Gnetales. The phylogenetic relationships among other families are the same, but with different bootstrap support. Conifers becomes monophyletic. Fourth, the rpsA gene trees, which have an apparent different topology from other gene trees, nest Gnetales within conifers and unit Gnetales with non-Pineceae conifers, with bootstrap support of 70%. Therefore, conifers is paraphyletic. Finally, ML analysis of the data set combined five genes strongly supports "Gnetifer" trees. The divergence of gymnosperms and angiosperms forms the first internal node of the phylogenetic tree. Cycads is considered as the most primitive gymnosperms, with ginkgos next to it. Gnetales is sister to conifers, which is monophyletic with bootstrap support of 92%, and the bootstrap support is 82%. Whether cutting of the third genetic code or not has no effect on the topology of phylogenetic trees. This study proved that enlarging the number of sampling species could greatly improve the accuracy of analysis and indicated that "Gnetifer" trees might be generated by biased sampling.Ephedra plantslie in the basal systematic position of Gnetales and its phylogeney has been a focus for a long time. Ephedra rhytidosperma has a close relationship with the ancient ephedroid megafossils, and therefore is presumed as the most primitive species among extant Ephedra. The present study explored the systematic position of Ephedra rhytidosperma according to chloroplast matK and rbcL gene, ribosomal 18S gene, and ITS sequences. A coarse estimate of the divergence time of Ephedra rhytidosperma was also performed. The results suggest no evidence for Ephedra rhytidosperma to be the primitive group of Ephedra, but it has a close relationshiop with Ephedra equisetina. According to the molecular clock of rbcL gene, the divergence time for Ephedra rhytidosperma and Ephedra equisetina is 10.85±2.44 Mya.The essential oil of Ephedra is another important secondary metabolite besides ephedrine. This study sampled six Ephedra populations from Inner Mongolia Autonomous Region and determined their chemical composition by GC-MS Method. The results show that there are two chemotypes in Ephedra. One consists abundant sesquiterpene and fatty acid compositions, high-concentration phytol (15.73%), y-eudesmol (7.77%) and eudesm-7(11)-en-4-ol (6.13%). The other chemotype mainly comprises α-terpineol (36.56-52.23%) and p-methoxystyrene (1.55-13.74%), and has a low phytol, eudesmol and eudesm-7(11)-en-4-ol content.Ephedrine is a characteristic component in Ephedraceae plants, distributing in many kinds of plants, such as Sida acuta, Catha edulis and Taxus baccata. However, no ephedrine was found in America Ephedra. This study using phylogenetic trees of gymnosperms, attests to the fact that ephedrine in gymnosperms had an independent origin through the methods of reconstruction of ancestral state and sensitivity analysis, and also approved that America Ephedra lost ephedrine in the process of evolution.