台灣蘿藦亞科(夾竹桃科)分子親緣關係之探討

台灣蘿藦亞科植物共10屬21種，可依據生長習性、葉部、花器等形態特徵作良好區分，然而不論藉由較大的外觀型態乃至黏液腺體均無法良好的解釋蘿藦亞科內之親緣關係(Phylogeny)，本研究利用兩個核內基因片段ITS (Internal Transcribed Spacer)、G3DPH (Glyceraldehyde-3-phosphate dehydrogenase)與葉綠體trnL-F (tRNA-UAA to tRNA-GAA)分別以不同方法建構台灣蘿藦亞科之親緣關係，結果顯示尖尾鳳族(Asclepiadeae)、吊燈花族(Ceropegieae)與牛嬭菜族(Marsdenieae)均為單系群，在屬的位階下，牛嬭菜與絨毛芙蓉蘭均為牛嬭菜屬植物，然而無論是基因樹或形態上均不支持牛嬭菜屬為單系群，本屬植物在種以上的位階需要再研究是否具有系統分類上的意義。牛皮消屬內之催吐白前節與屬內其他植物分屬不同譜系且催吐白前節與鷗蔓屬成為並系群，汁液顏色與副花冠型態亦支持此結果，符合前人研究建議催吐白前節與鷗蔓屬合併。疏花鷗蔓與台灣鷗蔓為鷗蔓屬較原始的分類群，而光葉鷗蔓、蘇氏鷗蔓、海島鷗蔓、呂氏鷗蔓與鷗蔓等5個物種僅有G3DPH片段提供足夠的序列差異建構譜系樹，結果顯示呂氏鷗蔓、海島鷗蔓、鷗蔓為鷗蔓屬基部的分類群，本研究建構之分子親緣關係支持現行分類處理，且G3DPH片段可用於解釋鷗蔓屬內物種親緣關係。Asclepiadoideae is comprised by 10 genus and 21 species in Taiwan, morphological characters from leaf, flower to colleters which require microscopic techniques, can used to delimit each species well, but the phylogeny within Asclepiadoideae is still unclear. In this study, two nrDNA ITS(Internal Transcribed Spacer)、G3DPH (Glyceraldehyde-3-phosphate dehydrogenase), and cpDNA trnL-F (tRNA-UAA to tRNA-GAA) were used to reconstruct the phylogeny by different analysis. Results showed that three major groups, Asclepiadeae, Ceropegieae and Marsdenieae, were evident using different gene sequence data. At genus level, Marsdenieae is not monophyletic, M. formosana and M. tintoria are morphological distinct species, the classification of Marsdenieae should be reexamined. The species from sect. Vincetoxicum and other Cynanchum species formed two distinct clades, sect. Vincetoxicum and Tylophora are paraphyletic. This is also supported by latex color and corona characters which indicate that Vincetoxicum and Tylophora should be lumped together. T. oshimae and T. taiwanensis are ancestral taxa in Tylophora, relationships among T. brownii, T. sui, T. insulana, T. lui and T. ovata are not accessible using ITS and trnL-F due to poor sequence variation. However, data from G3DPH has successfully grouped T. ovata, T. lui and T. insulana in the basal position. In conclusion, this study successfully reconstructed phylogeny of Asclepiadoideae using molecular data, in which G3DPH would be especially useful to reconstruct the phylogeny of Tylophora in Taiwan.Contents Page 摘要 …..……..………………………………………………………………………...i Abstracts …………………………..…………………………...…………………….ii Contents ………………………………………………...…………………..……….iii Contents of table …………………………………………...………………………..iv Contents of figure ……………………………………………..……………………..v 1. Introduction ……………………………………………………………………….1 2. Materials and Methods …………………………………………………………...5 Plant materials ……………………………………………………………………..5 Genomic DNA extraction and PCR amplification………………………………....5 Gel electrophoresis analysis ……………………………………………………….7 Subclone and sequencing …………………………………………………………. 7 Phylogenetic analysis ……………………………………………………………...8 3. Results ……………………………………………………………………………12 Sequence characterization ………………………………………………………..12 Phylogenetic analysis …………………………………………………………….13 4. Discussion ………………………………………………………………………..16 5. Conclusion ……………………………………………………………………….21 6. References ………………………………………………………………………..22 7. Appendix ………………………………………………………………………....75 Contents of table Page Table 1. List of Asclepiadoideae species in Taiwan…………………………...…….39 Table 2. Sample location…………………………………………………..…………40 Table 3. Gene region and specific primers used in this study……………………….41 Table 4. Basic information of ITS, trnL-F and G3DPH in Asclepiadoideae………..42 Table 5. Sequence alignment of ITS region………………………………………….43 Table 6. Sequence alignment of trnL-F region……………………………...……….48 Table 7. Sequence alignment of G3DPH……………………………………………54 Contents of figure Page Fig. 1. Structure of different locus and primers………………….…………………..25 Fig. 2. The substitution distribution of ITS region sequence……………….………..26 Fig. 3. The substitution distribution of trnL-F region sequence……………….…….27 Fig. 4. The substitution distribution of G3DPH region sequence……………………28 Fig. 5. Maximum parsimony phylogram based on ITS region………………………29 Fig. 6. Maximum likelihood phylogram based on ITS region……………………….30 Fig. 7. Phylogram obtained from the Bayesian analysis based on ITS region……….31 Fig. 8. Maximum parsimony phylogram based on trnL-F region……………………32 Fig. 9. Maximum likelihood phylogram based on trnL-F region……………………33 Fig. 10. Phylogram obtained from the Bayesian analysis based on trnL-F region…..34 Fig. 11. Maximum parsimony phylogram based on G3DPH region………………...35 Fig. 12. Maximum likelihood phylogram based on G3DPH region…………………36 Fig. 13. Phylogram obtained from the Bayesian analysis, based on G3DPH region...37 Fig. 14. Maximum parsimony analysis of G3DPH with morphological characters...3