蝴蝶蘭屬 (Phalaenopsis) 植物是蘭科植物的成員之一，主要分佈於南中國 (South China)、印度中國 (Indochina)、印度 (India)、東南亞 (Southeast Asia) 及澳洲等熱帶亞洲地區。根據最新的分類資料顯示，本屬約有66種原生種。這些植物也都極具有觀賞價值，目前已有成千上萬的雜交品種經由人工的方法被培育出來。雖然蝴蝶蘭屬植物是那樣的漂亮且受歡迎，但是蝴蝶蘭親緣關係方面的研究卻相當缺乏。
Christenson (2001) 最近將朵麗蘭屬 (Doritis) 及金氏蝶蘭屬 (Kingidium) 處理為蝴蝶蘭屬的同物異名 (synonym)，並將所有的成員分成五個亞屬 (subgenus)，分別為Proboscidioides, Aphyllae, Parishianae, Polychilos及Phalaenopsis。本研究擬以分子的證據來進一步闡釋由Christenson (2001) 所修訂的蝴蝶蘭屬植物的系統分類及親緣關係，並進一步探討蝴蝶蘭屬植物的演化趨勢。首先藉由分析核基因組 (nuclear genome) 的核糖體核酸 (ribosomal DNA, rDNA) 內轉錄間隔區 (internal transcribed spacer, ITS) 及葉綠體基因組trnL的intron, trnL-trnF的基因間隔區 (intergenic spacer, IGS) 及 atpB-rbcL的基因間隔區，來探討現今尚存活的大部分蝴蝶蘭屬植物的親緣關係及演化趨勢。在親緣關係的研究方面，結果支持Christenson (2001) 將朵麗蘭屬 (Doritis) 及金氏蝶蘭屬 (Kingidium) 處理為蝴蝶蘭屬的同物異名 (synonym)的觀點；然而在屬內的五個亞屬中，僅有Parishianae亞屬為單支系類群 (monophyletic group)。並且也發現同為具有四個花粉塊 (pollinia) 的分類群具有相近的親緣關係 (phylogenetic relationship)，這些類群包含Proboscidioides, Aphyllae, Parishianae及蝴蝶蘭亞屬內的兩個節，分別為Deliciosae 及 Esmeralda。這一群具有四個花粉塊的蝴蝶蘭植物亦有地理分佈相近的共同點，他們主要分佈於南中國、印度中國及印度等地區；有別於分佈於印尼、菲律賓及馬來西亞等地具有兩個花粉塊的蝴蝶蘭類群。在整個蝴蝶蘭屬植物的演化趨勢方面，藉由核基因組及葉綠體基因組的分子證據，配合花粉塊的演化趨勢推估分佈於南中國喜馬拉雅山山區的Aphyllae亞屬這一群植物為蝴蝶蘭屬植物的起源類群，向印度中國及印度發展出其它具有四個花粉塊的類群。再經由兩個途徑往東南亞分佈及演進，發展出具有二個花粉塊的蝴蝶蘭類群，其一是經由印度中國到達菲律賓的古老島嶼發展出Phalaenopsis亞屬；另一是經由馬來半島向婆羅州分佈及演進，發展出Polychilos亞屬。此外，由分子資料及地質歷史的訊息可以估算DNA序列的取代速率 (substitution rate)，結果顯示蝴蝶蘭ITS及葉綠體DNA序列每年每個位置的取代速率 (substitutions/site/year) 分別為2.4~4.7 x 10-9 及3.9~7.8 x 10-10。依上述DNA取代速率可以估算蝴蝶蘭其它類群的分離時間，初步估算分佈於菲律賓的P. lueddemanniana 複合種群的形成約在Pleistocene期間；而Deliciosae 節與Stauroglottis節的分離時間約在21~10.5 百萬年前 (Mya)。
另外，也利用分子證據來探討蝴蝶蘭屬內三個複合種群之分子親緣、生物地理及演化趨勢。首先針對P. amabilis 複合種群 (P. amabilis complex)進行分析，其成員計有P. amabilis, P. amabilis subsp. moluccana, P. amabilis subsp. rosenstromii, P. aphrodite, P. aphrodite subsp. formosana 及 P. sanderiana，這些成員在分類上界定不易，因此常會因觀點不同而有新的修正。本研究藉由分析核基因組的核糖體核酸內轉錄間隔區 (ITS) 來探討此一相近分類群的分子親緣、生物地理及其演化趨勢。結果顯示，除了P. aphrodite及 P. aphrodite subsp. formosana 無法明顯區分外，其它上述的分類群皆可以加以區分，並且顯示不同地區的P. amabilis 族群已有分化，僅有分佈於菲律賓巴拉望及印尼沙巴地區的族群無法明顯區分。分子證據也顯示，目前被處理為物種階級的P. sanderiana 無法與P. amabilis的不同族群加以區隔，因此並不支持將P. sanderiana處理為獨立的物種。在P. amabilis複合種群的演化趨勢方面，經由蝴蝶蘭屬的親緣關係樹得知，同樣分佈於菲律賓群島的P. schilleriana 及其相近類群與P. amabilis類群具有共同的祖先，因此以P. schilleriana的類群為外群，可以推估出P. amabilis類群的起源類群為P. aphrodite。P. aphrodite 向北有一分支推進至台灣南部，目前處理為P. aphrodite subsp. formosana；有兩個途徑向南推演，其一是經由菲律賓巴拉望 (Palawan)，演化出P. amabilis，再向婆羅州及蘇門達臘推進；另外經由菲律賓名達那峨 (Mindanao)，演化出P. sanderiana，再往新幾內亞或蘇拉威西演化出P. amabilis subsp. rosenstromii或 P. amabilis subsp. moluccana，其中 P. amabilis subsp. rosenstromii 再往澳洲北部推進。
其次探討P. sumatrana的複合種群，這一群包含P. sumatrana及 P. corningiana，及一群已被處理為P. sumatrana的同物異名之物種P. zebrina。藉由分析核基因組的核糖體核酸內轉錄間隔區 (ITS)、及葉綠體基因組trnL的intron, trnL-trnF的基因間隔區及 atpB-rbcL的基因間隔區來探討此類群的親緣關係及演化趨勢。葉綠體的資料顯示，此類群的物種無法明顯的加以區分；在核基因組 (ITS) 的證據顯示，P. sumatrana及P. corningiana 依然無法區分，但P. zebrina 可以與上述兩物種加以區分。因此，由分子證據建議將這一群稱為P. zebrina的植物獨立出來。在P. violacea複合種群的演化趨勢方面，經由親緣關係樹，可以推估出P. sumatrana複合種群的起源類群可能為P. zebrina，此物種在婆羅州演化出來，並演變出P. corningiana及P. sumatrana 二個物種，其中P. sumatrana藉由冰河所形成的陸橋，漸漸擴展至蘇門達臘、馬來半島及巴拉望等。
最後研究P. violacea的複合種群，這一群包含P. violacea及 P. bellina二個物種。其中P. violacea又有分為兩個型(馬來亞型及蘇門達臘型)。本研究針對核基因組的核糖體核酸內轉錄間隔區 (ITS) 及葉綠體基因組 trnL的intron, trnL-trnF的基因間隔區及 atpB-rbcL的基因間隔區來探討此類群的親緣關係及演化趨勢。葉綠體的資料顯示，此類群的物種無法明顯的加以區分；在核基因組 (ITS) 的證據顯示，P. bellina及P. violacea的兩個型亦無法加以區分，僅有分佈於印尼蘇門達臘西南的小島-蒙達威 (Mentawai Is.) 的 P. violacea與其它物種或族群可以加以區分。由分子證據建議，產於蒙達威的P. violacea可以處理為另一物種。以目前的證據並無法推測此複合種群的起始類群為何？但可以初步推測產於蒙達威的P. violacea 植物為較近期演變出來的類群。

Species of Phalaenopsis Blume (Orchidaceae) are found throughout tropical Asia, namely South China, Indochina, India, Southeast Asia, and Australia. This genus is comprised of approximately 66 species according to the latest classification. Most of them possess commercial value. Thousands of Phalaenopsis cultivars have been grown for commercial goals. Although this orchid is very beautiful and popular throughout the world, studies on the molecular systematics and phylogenetic relationships among these orchids are still deficient.
Phylogenetic trees inferred from the internal transcribed spacers 1 and 2 (ITS1+ITS2) region of nuclear ribosomal DNA (nrDNA) and chloroplast DNAs (cpDNAs), including the intron of trnL, the IGS of trnL-trnF, and the IGS of atpB-rbcL, were used to clarify the phylogenetics and evolutionary trends of the genus Phalaenopsis (Orchidaceae). Molecular data are provided to clarify the latest systematics of the genus Phalaenopsis as suggested by Christenson (2001). He treated the genera of Doritis and Kingidium as synonyms of the genus Phalaenopsis and divided it into the five subgenera of Proboscidioides, Aphyllae, Parishianae, Polychilos, and Phalaenopsis. The results concurred that the genera Doritis and Kingidium should be treated as synonyms of the genus Phalaenopsis as suggested by Christenson (2001). The subgenera of Aphyllae and Parishianae were both shown to be monophyletic groups, and to be highly clustered with the subgenus Proboscidioides and two sections (including sections Esmeralda and Deliciosae) of the subgenus Phalaenopsis, which have the same morphological characters of four pollinia as well as similar biogeographies. Furthermore, neither the subgenus Phalaenopsis nor Polychilos was found to be a monophyletic group in this study. In addition, the phylogenetic tree indicates that Phalaenopsis is monophyletic and does not support the existing subgeneric and sectional classification.
The phylogenetic tree of the genus Phalaenopsis is basically congruent with the geographical distributions of this genus. Based on the tree, two major clades were separated within the genus Phalaenopsis. The first clade, having four pollinia, included sections Proboscidiodes, Parishianae, and Esmeralda, of which are distributed in South China, India, and Indochina. The second clade, bearing two pollinia, included the sections Phalaenopsis, Polychilos, and Fuscatae, of which are distributed in Malaysia, Indonesia, and the Philippines. In addition, the biogeography of the genus Phalaenopsis is congruent with the historical geology of the distribution regions of this genus as well. According to molecular evidences, biogeography, historical geology, and the evolutionary trend of pollinia number of orchid, evolutionary trends of the genus Phalaenopsis were deduced. The subgenus Aphyllae was suggested to be the origin of Phalaenopsis and South China was suggested to be the origin center of Phalaenopsis. In addition, there were two dispersal pathways of Phalaenopsis from the origin center to Southeast Asia. In one pathway, Phalaenopsis species dispersed from South China to Southeast Asia, in particular the Philippines, using Indochina, older lands of the Philippines (Mindoro, Palawan, Zamboanga, etc.) as steppingstones, from which the subgenus Phalaenopsis developed. In the other pathway, Phalaenopsis species dispersed from South China to Southeast Asia, in particular Indonesia and Malaysia, using the Malay Peninsula as a steppingstone, from which the subgenus Polychilos developed.
Furthermore, molecular data and geological dating were used to estimate the substitution rates of DNA from the genus Phalaenopsis based on the hypothesis of the molecular clock. The substitution rates of both ITS and cpDNA data from the genus Phalaenopsis were 2.4~4.7 x 10-9 and 3.9~7.8 x 10–10 substitutions/site/year, respectively. The substitution rates of ITS data of the genus Phalaenopsis are approximately six times those of cpDNA. Based on the substitution rates, the divergence time among most of the P. lueddemanniana complex was estimated to have been during the Pleistocene. The section Deliciosae separated from the section Stauroglottis at 21~10.5 Mya.
Furthermore, the phylogenetics of the close species of Phalaenopsis will be evaluated based on molecular data, involving three groups of close Phalaenopsis species, namely the P. amabilis complex, P. sumatrana complex, and P. violacea complex. For the first complex, the internal transcribed spacer 1 and 2 (ITS1+ITS2) regions of nuclear ribosomal DNA (nrDNA) were applied to evaluate the phylogenetics of the P. amabilis complex, namely P. amabilis, P. amabilis subsp. moluccana, P. amabilis subsp. rosenstromii, P. aphrodite, P. aphrodite subsp. formosana, and P. sanderiana. Based on molecular data, each of species/subspecies from the P. amabilis complex with the exception of P. aphrodite and its subspecies could be separated from each other. Phalaenopsis aphrodite from different locations and its subspecies could not be separated from each other, but all of them were separable from different populations/subspecies of P. amabilis. In addition, P. sanderiana was nested within both P. amabilis and its subspecies. These results do not support P. sanderiana being treated as a separate species from P. amabilis. In addition, I suggest that P. aphrodite is the origin of the P. amabilis complex and originated in the Philippines. Phalaenopsis amabilis and P. sanderiana descended from P. aphrodite (or its ancestor). Based on the phylogenetic tree, evolutionary trends of the P. amabilis complex were suggested. Within evolutionary trends of P. amabilis complex, two different lineages with different dispersal pathways were suggested. First, P. aphrodite, dispersed into Palawan and evolved to be P. amabilis, thereafter further dispersing into Borneo and Sumatra. Second, P. aphrodite dispersed into southern Mindanao and evolved into P. sanderiana, thereafter further dispersing into Sulawesi and New Guinea, from which P. amabilis subsp. moluccana and P. amabilis subsp. rosenstromii developed, respectively.
For the second complex, the phylogenetic relationship of the P. sumatrana complex, namely P. sumatrana, P. corningiana, and P. zebrina, was detected based on the ITS1 and ITS2 regions of nrDNA, the intron of trnL, and the IGS of atpB-rbcL of cpDNA. The P. sumatrana complex includes the two species of P. sumatrana and P. corningiana, as well as a problem species, P. zebrina, according to the concepts of Sweet (1980) and Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, accessions of P. sumatrana cannot be separated from those of P. corningiana. Furthermore, accessions of P. zebrina can be separated from those of both P. sumatrana and P. corningiana. In addition, analyses of both sequences of the trnL intron and atpB-rbcL IGS of cpDNA apparently cannot discriminate among these three species of the P. sumatrana complex. Based on the molecular data of this study, plants of P. zebrina might be treated as a separate species from both P. sumatrana and P. corningiana. In the evolutionary trend of the P. sumatrana complex, plants of P. zebrina were deduced to be the relative origin group of the P. sumatrana complex based on the phylogenetic tree and biogeography. In addition, plants of both P. sumatrana and P. corningiana might have descended from plants of P. zebrina.
For the third complex, the phylogenetic trees inferred from the internal transcribed spacer 1 and 2 (ITS1+ITS2) regions of nuclear ribosomal DNA (nrDNA), the intron of trnL, and the intergenic spacer of atpB-rbcL of chloroplast DNA (cpDNA) were used to clarify the phylogenetic relationships of the P. violacea complex. The complex includes the two species of P. violacea and P. bellina, according to the concept of Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, P. bellina could not be separated from most populations from P. violacea with the exception of the population distributed on Mentawai Is., Indonesia. In addition, analyses of both the intron of trnL and the IGS of atpB-rbcL of cpDNA apparently could not discriminate among the three species of the P. sumatrana complex. Based on the morphological characters, P. violacea from Mentawai Is. bears a long floral rachis and was separable from the other groups of the P. violacea complex. Therefore, the results in this study have a trend to treat the population of Mentawai Is. of the P. violacea complex as a separate species from P. violacea. In the evolutionary trend of the P. violacea complex, Mentawai plants of this complex might be descended from those of Sumatra/the Malay Peninsula according to the phylogenetic analysis and biogeography.

Tables of Contents

List of Tables………………………………………………………………………I
List of Figures…………………………………………………………………….VI
Chinese Abstract……………………………………………………………………IX
English Abstract……………………………………………………………………XI

Introduction in General…………………………………………………………..1

Chapter 1
Molecular Phylogeny, Biogeography, and Evolutionary Trends of the Genus Phalaenopsis (Orchidaceae)
Abstract………………………………………………………………………………4
Introduction…………………………………………………………………………5
Materials and Methods………………………………………………………………10
Results………………………………………………………………………………15
Discussion……………………………………………………………………………32
Conclusions……………………………………………………………………………54
Literature Cited………………………………………………………………………56
Tables…………………………………………………………………………………67
Figures…………………………………………………………………………………89
.
Chapter 2
Phylogenetics, Biogeography, and Evolutionary Trends of the Phalaenopsis amabilis Complex Inferred from ITS1 and ITS2 of Nuclear DNA
Abstract………………………………………………………………………………119
Introduction…………………………………………………………………………120
Materials and Methods………………………………………………………………123
Results and Discussion………………………………………………………………125
Conclusions……………………………………………………………………………131
Literature Cited………………………………………………………………………132
Tables…………………………………………………………………………………135
Figures…………………………………………………………………………………140

Chapter 3
Phylogenetics, Biogeography, and Evolutionary Trends of the Phalaenopsis sumatrana Complex Inferred from Nuclear DNA and Chloroplast DNA
Abstract………………………………………………………………………………150
Introduction…………………………………………………………………………150
Materials and Methods………………………………………………………………151
Results and Discussion………………………………………………………………153
Literature Cited………………………………………………………………………157
Tables…………………………………………………………………………………159
Figures…………………………………………………………………………………162

Chapter 4
Phylogenetics, Biogeography, and Evolutionary Trends of the P. violacea Complex Inferred from Nuclear DNA and Chloroplast DNA
Abstract……………………………………………………………………………176
Introduction…………………………………………………………………………176
Materials and Methods………………………………………………………………177
Results and Discussion……………………………………………………………178
Literature Cited………………………………………………………………………182
Tables………………………………………………………………………………184
Figures…………………………………………………………………………………188
List of Tables
Chapter 1
Table 1. Comparison of the systematics of the genus Phalaenopsis between Sweet (1980) and Christenson (2001)…………………………………………………67

Table 2. List of the 52 Phalaenopsis species of this study, their systematic classification, and geographical distributions…………………………………69

Table 3. Lengths of internal transcribed spacer 1 (ITS1) and ITS2 and GenBank accession numbers from 52 Phalaenopsis species plus the five taxa of related genera………………………………………………………………………71

Table 4. Number of characters, variable sizes, and genetic distances of the two-parameter method of Kimura among the 52 Phalaenopsis species based on the analyses of different DNA fragments of this study……………………72

Table 5. Number of informative sizes, and genetic distances of the two-parameter method of Kimura among 52 Phalaenopsis species plus the four outgroups based on the analyses of different DNA fragments of this study……………72

Table 6. Genetic distances of the two-parameter method of Kimura method inferred from the ITS1+ITS2 of nrDNA among the 52 taxa of the genus Phalaenopsis plus the five related species…………………………………………………73

Table 7. The lengths and accession numbers of the trnL-trnF IGS, the trnL intron, and IGS of atpB-rbcL from 52 Phalaenopsis species plus the five taxa of related genus…………………………………………………………………………74

Table 8. Genetic distances of the two-parameter method of Kimura method inferred from the trnL intron among the 52 taxa of the genus Phalaenopsis plus the five related species……………………………………………………………76

Table 9. Genetic distances of the two-parameter method of Kimura method inferred from the trnL – trnF intergenic spacer among the 52 taxa of the genus Phalaenopsis plus the five related species……………………………………77

Table 10. Genetic distances of the two-parameter method of Kimura method inferred from combined the trnL intron with the trnL-trnF IGS among the 52 taxa of the genus Phalaenopsis and the five related species………………………………………………………………78

Table 11. Genetic distances of the two-parameter method of Kimura method inferred from the atpB-rbcL IGS among the 52 taxa of the genus Phalaenopsis and four related species…………………………………………………………79

Table 12. Genetic distances of the two-parameter method of Kimura method inferred from combined data of sequences of the intron trnL, the trnL-trnF IGS, and the atpB-rbcL IGS among the 52 taxa of the genus Phalaenopsis and four related species………………………………………………………………80

Table 13. Genetic distances of the two-parameter method of Kimura method inferred from combined data of sequences of the ITS1 and ITS2 of nrDNA, the trnL intron, the trnL-trnF IGS, and the atpB-rbcL IGS among the 52 taxa of the genus Phalaenopsis and four related species………………………………81

Table 14. Genetic distances of the two-parameter method of Kimura derived from combined data of sequences of the internal transcribed spacer 1 (ITS1), ITS2, the trnL intron, the trnL-trnF intergenic spacer (IGS), and the atpB-rbcL IGS among six subgenera/sections of the four-pollinium Phalaenopsis divided according to the suggestions of the phylogenetic tree of this study…………………………………………………………………………82

Table 15. Tajima’s neutrality tests of data of sequences of both ITS and chloroplast DNA obtained from the genus Phalaenopsis plus the outgroups of this study…………………………………………………………………………82

Table 16. Tajima’s relative rate test for the ITS data set between species of the sections Amboinenses and Zebrinae distributed on the Sunda Shelf and species of the Phalaenopsis lueddemanniana complex, with P. lobbii (subgenus Parishianae) as the reference group………………………………83

Table 17. Tajima’s relative rate test for the chloroplast DNA data set between species of the sections Amboinenses and Zebrinae distributed on the Sunda Shelf and species of the Phalaenopsis lueddemanniana complex, with P. lobbii (subgenus Parishianae) as the reference group………………………………83

Table 18. Comparisons of internal transcribed spacer sequences of sections Amboinenses (with the exception of the P. lueddemannina complex) and Zebrinae distributed on the Sunda Shelf with species of the P. lueddemanniana complex distributed in the Philippines used to deduce the substitution rate of the genus Phalaenopsis between them based on the geological events of the combination of the Philippines and Borneo (5~10 Mya)…………………………………………………………………………84

Table 19. Comparisons of chloroplast DNA data sets of the group of the sections Amboinenses (with exception of the Phalaenopsis lueddemannina complex) and Zebrinae with that of species of the P. lueddemanniana complex to deduce the substitution rate of the genus Phalaenopsis between them based on the geological events of the combination of the Philippines and Borneo (5~10 Mya)…………………………………………………………………84

Table 20. Tajima’s relative rate test for the internal transcribed spacer data set among species of the Phalaenopsis lueddemanniana complex, with P. lobbii (subgenus Parishianae) as the reference group………………………………85

Table 21. Tajima’s relative rate test for the chloroplast DNA data set among species of the Phalaenopsis lueddemanniana complex, P. lobbii (subgenus Parishianae) as the reference group…………………………………………85

Table 22. Number of differences of the internal transcribed spacer data set among species of the Phalaenopsis lueddemanniana complex with the exception of P. hieroglyphica……………………………………………………………86

Table 23. Number of differences of the chloroplast DNA data among species of the Phalaenopsis lueddemanniana complex……………………………………86

Table 24. Putative divergence times among species of the Phalaenopsis lueddemanniana complex with the exception of P. hieroglyphica calculated by the substitution rate of 2.4~4.7 x 10-9 substitutions/site/year of the internal transcribed spacer sequences of the genus Phalaenopsis obtained from this study………………………………………………………………………87

Table 25. Divergence times among species of the Phalaenopsis lueddemanniana complex calculated by the substitution rate of 3.9~7.8 x 10-10 substitutions/site/year of chloroplast DNA of the genus Phalaenopsis obtained from this study……………87

Table 26. Tajima’s relative test between the section Stauroglottis and the Phalaenopsis lueddemanniana complex, with P. lobii (subgenus Parishianae) as the reference group…………………………………………………………88

Table 27. Number of differences and divergence times between species of the section Deliciosae and the section Stauroglottis (Phalaenopsis lindenii was excluded from the chloroplast DNA data set) obtained from substitution rates of both the internal transcribed spacer and cpDNA data sets…………………………88

Chapter 2.
Table 1. A list of the 39 accessions of the Phalaenopsis amabilis complex, namely P. amabilis, P. aphrodite, and P. sanderiana, and their different geographical distributions.………………………………………………………………135

Table 2. Lengths of ITS1 and ITS2 and GenBank accession nos. of the 39 accessions of the Phalaenopsis amabilis complex……………………………………136

Table 3. The genetic distance matrix of the Kimura two-parameter method among the 39 accessions of the Phalaenopsis amabilis complex based on ITS1 and ITS2 of nrDNA…………………………………………………………………137

Table 4. Genetic distances among 13 inter-populations/subspecies/species of the Phalaenopsis amabilis complex based on ITS1 and ITS2 of nrDNA ………138

Chapter 3.
Table 1. A list of 16 accessions from three closely Phalaenopsis species of P. sumatrana, P. corningiana and P. zebrina, and their different geographical distributions………………………………………………………………158

Table 2. Lengths of ITS1 and ITS2 and GenBank accession nos. of the 14 accessions of the Phalaenopsis sumatrana complex……………………………………158

Table 3. Lengths and G+C contents of the trnL intron and IGS of atpB-rbcL among the 14 accessions of the Phalaenopsis sumatrana complex…………………159

Table 4. Genetic distance matrix among the 14 accessions of the Phalaenopsis sumatrana complex based on the ITS1 and ITS2 of nrDNA…………………159

Table 5. Genetic distance matrix of the intron of trnL among the 14 accessions of the Phalaenopsis sumatrana complex……………………………………………160

Table 6. Genetic distance matrix among 14 accessions of the Phalaenopsis sumatrana complex based on analysis of the IGS of atpB-rbcL…………………………160

Chapter 4.
Table 1. A list of 15 accessions from the two closely Phalaenopsis species of P. bellina and P. violacea and their different geographical distributions……182

Table 2. Lengths of ITS1 and ITS2 and GenBank accession numbers of the 14 accessions of the Phalaenopsis violacea complex………………………183

Table 3. Lengths and G+C contents of the trnL intron and the IGS of atpB-rbcL among the 14 accessions of the Phalaenopsis violacea complex……………184

Table 4. Genetic distance matrix of ITS1 and ITS2 of nrDNA among the 14 accessions of the Phalaenopsis violacea complex…………………………185



List of Figures
Chapter 1.
Fig. 1. Correlation between the distribution pattern and pollinia number of different subgenera of Phalaenopsis……………………………………………………89

Fig. 2. The six major biogeographic regions of the world………………………………90

Fig. 3. Putative map of Southeast Asia 30 Mya…………………………………………91

Fig. 4. Comparison of Southeast Asian lands between Pleistocene times and the present time…………………………………………………………………92

Fig. 5. Localities and sequences of primers for amplifying and sequencing the internal transcribed spacer 1 (ITS1) and ITS2 of nrDNA………………………………93

Fig. 6. Localities and sequences of primers for amplifying and sequencing the trnL intron (UAA) and the intergenic spacer (IGS) of trnL (UAA)-trnF (GAA)……93

Fig. 7. Localities and sequences of primers for amplifying and sequencing the intergenic spacer (IGS) of atpB-rbcL…………………………………………93

Fig. 8. Neighbor-joining tree of 52 Phalaenopsis species plus the five outgroups obtained from internal transcribed spacer 1 (ITS1) and ITS2 sequences………94

Fig. 9. Sequence alignment of different lengths of the atpB-rbcL intergenic spacer of chloroplast DNA from an individual of Phalaenopsis gibbosa…………………95

Fig. 10. Sequence alignment of different lengths of the trnL intron of chloroplast DNA from an individual of Phalaenopsis lowii………………………………96

Fig. 11. Sequence alignment of different lengths of the intergenic spacer of atpB-rbcL of chloroplast DNA from an individual of Phalaenopsis lowii…………………97

Fig. 12. Neighbor-joining tree of 52 Phalaenopsis species plus the five outgroups obtained from sequence comparison of the trnL intron………………………98

Fig. 13. Neighbor-joining tree of 52 Phalaenopsis species plus the five outgroups obtained from sequence comparisons of the trnL-trnF intergenic spacer……99

Fig. 14. Neighbor-joining tree of 52 Phalaenopsis species plus the five outgroups obtained from sequence comparisons of combined data of the trnL intron and the trnL-trnF intergenic spacer………………………………………………100

Fig. 15. Neighbor-joining tree of 52 Phalaenopsis species plus the four outgroups obtained from sequence comparisons of the atpB-rbcL intergenic spacer...101

Fig. 16. Neighbor-joining tree of 52 Phalaenopsis species plus the four outgroups obtained from sequence comparisons of combined data of the trnL intron, the trnL-trnF IGS, and the atpB-rbcL intergenic spacer…………………………102

Fig. 17. Neighbor-joining tree of 52 Phalaenopsis species plus the four outgroups obtained from sequence comparison of combined data of internal transcribe spacer 1 (ITS1) and ITS2 of nuclear DNA, the trnL intron, the trnL-trnF intergenic spacer (IGS), and the atpB-rbcL IGS…………………………103

Fig. 18. Matrix of geographical distributions of the genus Phalaenopsis……………104

Fig. 19. Biogeographical tree of the genus Phalaenopsis constructed by the Neighbor-joining method…………………………………………………104

Fig. 20. Comparisons between phylogenetic relationships of the 52 Phalaenopsis species plus the four outgroups obtained from the combined data of nuclear and chloroplast DNA and the geographical distributions of the genus Phalaenopsis………………………………………………………………105

Fig. 21. Evolutionary phylogenetic tree of the genus Phalaenopsis inferred from the combined data of the internal transcribed spacer of nrDNA and chloroplast DNA reconstructed by minimum-evolution method………………………106

Fig. 22. Evolutionary phylogenetic tree of the genus Phalaenopsis inferred from the combined data of the internal transcribed spacer of nuclear DNA and chloroplast DNA reconstructed by the minimum-evolution method and rooted based on the subgenus Aphyllae……………………………………107

Fig. 23. Evolutionary trends of the genus Phalaenopsis obtained from this study plotted on a map of the geographical distribution of this genus…………108

Fig. 24. Sequence alignment of the ITS sequences from the five clones of Phalaenopsis ×intermedia plus the species of the sections Phalaenopsis and Stauroglottis………………………………………………………………109

Fig. 25. The phylogenetic tree of five clones of Phalaenopsis ×intermedia plus species of the sections Phalaenopsis and Stauroglottis of the genus Phalaenopsis inferred from ITS data………………………………………112

Fig. 26. (a) Phylogenetic subtree of the section Phalaenopsis obtained from combined data of the trnL intron, the trnL-trnF intergenic spacer (IGS), and the atpB-rbcL IGS of chloroplast DNA. (b) Phylogenetic subtree of the section Phalaenopsis obtained from internal transcribed spacer 1 (ITS1) and ITS2 of nuclear DNA…………………………………………………………………113

Fig. 27. Evolutionary phylogenetic subtree of both the section Amboinenses and the P. lueddemanniana complex inferred from the combined data of the internal transcribed spacer of nuclear DNA and chloroplast DNA data constructed using the minimum evolution method………………………………………114

Chapter 2.
Fig. 1. Geographical distributions of Phalaenopsis amabilis, P. aphrodite, and P. sanderiana……………………………………………………………………139

Fig. 2. Comparison of Southeast Asia lands between Pleistocene times and the present time………………………………………………………………………140

Fig. 3. Sequence alignment of ITS1 and ITS2 of nrDNA from the 39 accessions of the Phalaenopsis amabilis complex………………………………………………141

Fig. 4. Minimum evolution tree of 39 accessions from three closely Phalaenopsis species and their subspecies, namely Phalaenopsis amabilis, P. aphrodite, P. sanderiana, P. amabilis subsp. moluccana, P. amabilis subsp. rosenstromii, P. aphrodite subsp. formosana, plus three groups, namely P. stuartiana, P. schilleriana, and P. philippinensis, obtained from sequence comparisons of the ITS region of rDNA………………………………………………………146

Fig. 5. Evolutionary phylogenetic tree of minimum evolution rooted based on Phalaenopsis aphrodite, the origin group of the P. amabilis complex suggested by this study…………………………………………………………147

Fig. 6. To map eolutionary trends of the Phalaenopsis amabilis complex obtained from this study on the distribution of this complex…………………………148

Chapter 3.
Fig. 1. Geographical distributions of P. sumatrana, P. corningiana, and P. zebrina………………………………………………………………………161

Fig. 2. The sequence alignment of ITS1 and ITS2 of rDNA from the14 accessions of the P. sumatrana complex……………………………………………………162

Fig. 3. The sequences alignment of the intron of trnL of chloroplast DNA from the 14 accessions of the P. sumatrana complex……………………………………164

Fig. 4. The mutational hot spot of length variations within the intron of trnL of chloroplast DNA from the P. sumatrana complex………………………………167

Fig. 5. The sequences alignment of the IGS of atpB-rbcL of chloroplast DNA from the14 accessions of the P. sumatrana complex……………………………………168

Fig. 6. The Neighbor-joining tree of the 14 accessions from Phalaenopsis sumatrana complex plus outgroups, namely Phalaenopsis cornu-cervi and P. fuscata, obtained from sequence comparisons of the ITS region of rDNA……………171

Fig. 7. The Neighbor-joining tree of the 14 accessions from Phalaenopsis sumatrana complex plus one outgroup, namely P. fuscata, obtained from sequence comparisons of the intron of trnL of chloroplast DNA………………………172

Fig. 8. The Neighbor-joining tree of the 14 accessions from Phalaenopsis sumatrana complex plus one outgroup, namely P. fuscata, obtained from sequence comparisons of the IGS of atpB-rbcL of chloroplast DNA………………173

Fig. 9. Evolutionary trend of P. sumatrana complex based on the phylogenetic tree…………………………………………………………………………174

Chapter 4.
Fig. 1. Geographical distributions of Phalaenopsis bellina and P. violacea………186

Fig. 2. Sequence alignment of ITS1 and ITS2 of rDNA from the14 accessions of the Phalaenopsis violacea complex……………………………………………187

Fig. 3. Sequences alignment of the intron of trnL of chloroplast DNA from the 14 accessions of the Phalaenopsis violacea complex…………………………189

Fig. 4. Mutational hot spot of length variations within the intron of trnL of chloroplast DNA from the Phalaenopsis violacea complex………………………………192

Fig. 5. Sequence alignment of the IGS of atpB-rbcL of chloroplast DNA from the 14 accessions of the Phalaenopsis violacea complex…………………………193

Fig. 6. The neighbor joining tree of the 14 accessions of the Phalaenopsis violacea complex plus the outgroup of P. fuscata obtained from sequence comparisons of the ITS region of rDNA……………………………………………………197

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