A PHYLOGENETIC ASSESSMENT OF LYCASTE AMD ANGULOA (ORCHIDACEAE) By ANGELA RYAN A thesis submitted for the degree of Doctor of Philosophy in the University of London DEPARTMENT OF CHEMISTRY UNIVERSITY COLLEGE LONDON 2001 ProQuest Number: U642610 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642610 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Parsimony analysis has been used to examine the phylogenetic relationships of two genera of Neotropical orchids, Lycaste and Angulos. Within these genera, difficulties occur when assigning names to plants using traditional morphological techniques. Many herbarium specimens are in bad condition and some descriptions are incomplete. To date, infrageneric classifications have been based on very few diagnostic characters. Here, three approaches have been evaluated: A systematic analysis of the morphology, an examination of two regions of DMA sequence and an analysis of the chemical composition of the floral fragrances. Morphological analysis showed that Lycaste is not monophyletic. There was a clear division between species currently ascribed to sect. Fimbriatae and the other species of the genus. Selection of characters introduced an element of subjectivity into the analysis and it was shown that exclusion of a single character could significantly affect the topology of relationships. Parsimony analysis of the sequences of both ITS and matK placed Lycaste sect. Fimbriatae closer to Angulos than to the other species of Lycaste. Neomoorea was identified as nearest neighbour to Lycaste and Angulos. A combined analysis of ITS and morphological data gave congruent results. Morphological and sequence analyses also indicated that the single pendent species, L. dyeriana, should remain within sect. Fimbriatae and that the taxonomic status of the subspecies of L. macrophylla should be revised. None of the analyses provided sufficient resolution to address the sectional treatment of the remaining Lycaste species. To answer this question, comparison of sequence data from a faster evolving region of DNA will be required. The floral fragrance composition of 28 species and subspecies of Lycaste and Angulos was determined. In its current form, the data was found to be unsuitable for addressing phylogenetic relationships at species level and above but may prove useful for population studies. CONTENTS Acknowledgements 12 Glossary 14 1 Introduction 16 1.1 General Introduction 16 1.2 The Orchidaceae and their classification 19 1.2.1 Infrageneric Classification of Lycaste 24 1.2.2 Infrageneric Classification of Angulos 28 1.3 Morphology 33 1.3.1 The Morphology of Lycaste 33 1.3.2 The Morphology of Angulos 40 1.4 DNA and Plant Phylogeny 44 1.5 The role of floral fragrance in Orchid Pollination 47 2 Morphological Analysis 53 2.1 Introduction 53 2.1.1 Orchid Seed Morphology 56 2.1.2 Parsimony Analysis 59 2.2 Materials and Methods 62 2.3 Results 63 2.3.1 Character selection and coding 63 2.3.1.1 Vegetative Characters 64 2.3.1.2 Floral Characters 66 2.3.1.3 Seed Characters 74 2.3.2 Data Analysis 89 2.3.3 Seed morphology 92 2.4 Discussion 93 Molecular and Cytogenetic Analysis 101 3.1 Introduction 101 3.2 Materials and Methods 105 3.2.1 Cytology 105 3.2.2 Extraction of DNA 105 3.2.3 PGR 107 3 3.2.4 Sequencing 108 3.2.5 Data Analysis 110 3.3 Results 110 3.3.1 Cytology 110 3.3.2 Analysis of the ITS Region 110 3.3.3 Analysis of matK 111 3.3.4 Combined sequence analysis 115 3.3.5 Combined sequence and morphological analysis 118 3.4 Discussion 119 Osmophores 122 4.1 Introduction 122 4.2 Materials and Methods 126 4.3 Results 127 4.3.1 Staining with neutral red 127 4.3.2 SEM 134 4.4 Discussion 139 Floral Fragrance Analysis 141 5.1 Introduction 141 5.1.1 The Origins of Floral Fragrance 143 5.1.2 Fragrance Composition and Pollination Strategy 153 5.1.3 Floral Fragrance and Phylogeny 157 5.1.4 Sample Preparation Techniques 158 5.2 Materials and Methods 164 5.2.1 Sample Collection 164 5.2.2 Sample Analysis 167 5.2.3 Phylogenetic Analysis 170 5.3 Results and Discussion 170 5.3.1 Variation of Fragrance With Time 172 5.3.2 Infra-species Fragrance Variation 177 5.3.3 Inter-species Fragrance Variation 180 5.3.4 Phylogenetic analysis 188 5.3.5 The Fragrance Composition of Hybrids 190 Conclusions 205 References 208 Appendices Appendix 1 The classification of Lycaste and Anguloa species 232 Appendix 2 Voucher Information 253 Appendix 3 Morphological character states 263 Appendix 4 ITS and matK sequences 267 Appendix 5 The floral fragrance composition of Lycaste and 276 Anguloa species Appendix 6 Chromatograms of the fragrances of Lycaste and 300 Anguloa species List of Figures Figure 1.1 The geographical range of Lycaste and Anguloa. 17 Figure 1.2 Sectional treatment of Lycaste species, after Fowlie 29 (1970) and Oakeley (1993). Figure 1.3 Lycaste aromatica (Graham ex Flooker) Lindl., drawn by 35 Judi Stone. Figure 1.4 Lycaste cinnabarina Lindl., drawn by Judi Stone. 37 Figure 1.5 Anguloa clowesii Lindl., drawn by Judi Stone. 42 Figure 1.6 Schematic representation of the location of matK, from 46 Soltis and Soltis (1998). Figure 1.7 Schematic diagram of the rDNA repeat unit in plants, 47 from Soltis and Soltis (1998). Figure 2.1 Subtribe Lycastinae Schltr., after Dressier (1981,1993). 54 Figure 2.2 One of 226 successively weighted most parsimonious 90 trees showing cladistic relationships within subtribe Lycastinae based on 47 morphological characters. Figure 2.3 One of 4164 successively weighted most parsimonious 91 trees showing cladistic relationships within subtribe Lycastinae based on 46 morphological characters. Figure 3.1 One of more than 6000 successively weighted most 112 parsimonious trees showing cladistic relationships within Lycastinae based on ITS sequence data. Figure 3.2 One of the nine successively weighted most 113 parsimonious trees showing relationships within Lycastinae based on matK sequence data. 5 Figure 3.3 One of the six successively weighted most 114 parsimonious trees showing relationships within Lycastinae based on \TS/matK sequence data. Figure 3.4 One of the three successively weighted most 116 parsimonious trees showing cladistic relationships within subtribe Lycastinae, based on 47 morphological characters and ITS sequence data. Figure 3.5 One of the two successively weighted most 117 parsimonious trees showing cladistic relationships within subtribe Lycastinae, based on 46 morphological characters and ITS sequence data. Figure 5.1 The biosynthesis of fragrance chemicals by plants, after 144 Mann (1994). Figure 5.2 The biosynthesis of terpenoids, from Banthorpe and 145 Charlwood (1980). Figure 5.3 The biosynthesis of monoterpenoid skeletons, after 146 Banthorpe (1994) and Croteau and Karp (1991). Figure 5.4 Biosynthetic routes to the formation of cyclic 147 sesquiterpenoids, after Banthorpe (1994). Figure 5.5 The biosynthesis of sesquiterpenoid skeletons, after 148 Banthorpe (1994). Figure 5.6 The biosynthesis of longibornanes, from Banthorpe and 149 Charlwood (1980). Figure 5.7 The formation of ionones, from Croteau and Karp 149 (1991). Figure 5.8 Biosynthesis of malonyl coenzyme A, from Luckner 150 (1990). Figure 5.9 A mechanism for the biosynthesis of aliphatic 150 compounds, after Luckner (1990). Figure 5.10 The biosynthesis of phenylpropanoids, from Croteau 152 and Karp (1991). Figure 5.11 The biosynthesis of phenylacetic and 4-hydroxyphenyl- 153 acetic acids, from Luckner (1990). Figure 5.12 The Analysis of Floral Fragrances. 165 Figure 5.13 The daytime fragrance cycle of Lycaste aromatica (raw 173 area counts). Figure 5.14 The daytime fragrance cycle of Lycaste aromatica (area 174 counts relative to toluene). Figure 5.15 Lycaste aromatica'. Variation in fragrance composition 176 during the first seven days of anthesis. Figure 5.16 Lycaste aromatica'. Variation in fragrance composition 179 of seven different specimens. Figure 5.17 Lycaste depper. Variation in fragrance composition of 181 seven different specimens. Figure 5.18 Monoterpenoids from the floral fragrances of Lycaste 184 and Anguloa. Figure 5.19 Sesquiterpenoids from the floral fragrances of Lycaste 185 and Anguloa. Figure 5.20 One of the 45 successively weighted most 186 parsimonious trees showing cladistic relationships in Lycaste and Anguloa, based on individual floral fragrance compounds. Figure 5.21 One of the three successively weighted most 187 parsimonious trees showing cladistic relationships within Lycaste, based on individual floral fragrance compounds. List of Tables Table 1.1 Early classification of orchids, after Lindley (1830-1840). 21 Table 1.2 A morphological key to Anguloa species, from Schlechter 32 (1916). Table 3.1 Chromosome counts for Lycastinae and related 103 subtribes. Table 3.2 ITS and matK primer sequences. 107 Table 3.3 PGR cycle sequence conditions for ITS and matK. 108 Table 4.1 Fixation and dehydration protocol for SEM examination 127 of osmophores. Table 5.1 The headspace composition of Stephanotis floribunda 166 (Apocynaceae) when sampled from a glass flask and Rilsan bag. Table 5.2 Analytical conditions for floral fragrance analysis. 169 Table 5.3 Biosynthetic groups used for phylogenetic analysis. 172 Table 5.4 Lycaste aromatica'. Variation in fragrance composition 175 over a seven day period.