FLAVONOID SYSTEMATICS OF NORTH AMERICAN LUPINUS SPECIES (LEGUMINOSAE) by KEVIN WILLIAM NICHOLLS B.Sc.(Hons .), University College of Wales, Aberystwyth, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF"BRITISH COLUMBIA August 1981 © Kevin William Nicholls, 1981 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date I-»TT> c i i / -in \ 11 ABSTRACT This study was an assessment of the usefulness of flav- onoids as taxonomic markers in the genus Lupinus (Leguminosae). The genus itself is readily recognizable, but, in North America, specific boundaries are poorly defined. This is probably the result of a combination of considerable morphological plasticity and hybridization (particularly amongst the outcrossing peren• nial taxa). At the outset, a detailed analysis of L u p i n u s flavonoids was made. Fifty-six compounds were identified, the majority being flavones based on apigenin, luteolin and less commonly acacetin and chrysoeriol. Glucose was the only sugar attached to flavonoids but was found C-bonded at the 6- or 8- positions and 0-bonded at the 5,7,3' or 41-positions. Some flavonols and isoflavones were also identified. The reliability of flavonoids as specific characters was next tested. Fresh or dried plant material had identical flav- onoid content, and plants of varying ages were chemically iden• tical. Geographically, quantitative variation in flavonoid con• tent was noted in an analysis of Lupi nus seri ceus by high pres• sure liquid chromatography. A sharp discontinuity in orientin levels was seen between populations north and south of Great Salt Lake, Utah, lending support to the subspecific ranking of the southerly population by Fleak (1971). Subsequent analysis of plants grown in uniform conditions showed the flavonoid dif• ferences to have been a phenotypic response. i i i Since flavonoids appeared to be infraspecifically stable characters, an interspecific study of 73 taxa was made. Prin• cipal components analysis exposed 7 chemical groups. Six of these accumulated some novel flavonoids peculiar to each group. The seventh group had profiles containing no unusual compounds. One species (L_. di f f usus) had a very complex profile and was classed as a monospecific group. Using.the literature and personal observations it was seen that in 5 groups the chemical affinities paralleled morphological affinities thereby strengthening the integrity of the groups as natural units. Group C, however, was divided into two on the basis of morphological discontinuities as was the seventh group (F).which contained a collection of lupines with doubtful affin• ities (chemical or morphological). Overall, it is concluded that flavonoids are good specific characters in L u p i n u s . Their distribution, interspecifical1y, lends support to the recognition of some previously described subgeneric groupings that were based on morphology. In other cases, flavonoid data suggests affinities between some taxa that hitherto were considered unrelated. i v Table of Contents Abstract ii List of Tables vii ListofFigures ix I INTRODUCTION 1 Tribal position of Lupi nus 2 Subgeneric taxonomy of Lupi nus 5 Some reasons for the confusing taxonomy of L u p i n u s 7 Previous chemotaxonomic studies on Lupinus . 10 Economic aspects of Lupinus 15 Thesis aims 17 II FLAV0N01D CHEMISTRY OF LUPINUS . 18 Introduction 18 Materials and methods I. Flavonoid extraction andpurif ication . 20 Materials and methods II. Flavonoid iden• tification 24 Results.. 29 III INFRASPECIFIC VARIATION IN LUPINE FLAVONOIDS . 35 Introduction 35 The effect of plant age on the flavonoid content of a Lupinus species 38 The effect of drying and storage on the flavonoid content of a Lupi nus species. 43 Geographical variation in the flavonoid content o f Lupinus species Experiment I. A preliminary study . .46 Experiment II. Variation in Lupinus s e r i c e u s . 53 Materials and methods. 53 High Pressure Liquid Chromatography . .55 HPLC of flavonoids 57 HPLC of-Lupinus sericeus flavonoids . 58 Results and discuss si on 59 Principal components analysis (PCA) . 62 Results and discussion of PCA on Lupi nus _ sericeus flavonoid data, 65 Canonical variates analysis (CVA). 68 Results and discussion of CVA on L u p i n u s sericeus populations . .69 The analysis of orientin accumulation in L u p i n u s sericeus populations . 73 Experiment III. The determination of the pheno- typic or genotypic nature of orientin variation in Lupinus-sericeus 78 IV INTERSPECIFIC FLAVONOID VARIATION IN LUPINUS . 81 Materials and methods 81 Data generation and analysis 81 Discussion. .99 V OVERVIEW 122 Flavonoids as taxonomic characters in Lupi nus 122 Relationships in Lupi nus 126 Convergence 128 VI SUMMARY 130 References 133 APPENDIX I. .Plants and Sources 141 APPENDIX II. Sol vent, systems and media used in TLC of flavonoids from L u p i n u s 151 APPENDIX III. UV spectral absorption maxima of some flavonoids from Lupi nus 152 APPENDIX IV. Raw HPLC data: peak heights of 17 flav• onoids from 181 individuals of Lupi nus sericeus . 154 APPENDIX V. Two-dimensional chromatograms (2-D maps) the 73 lupine taxa that were analysed. v i i List of Tables I. A comparison of subgeneric groupings of European lupines by Nowacki & Prus-Glowacki (1971) with that of Tutin et aj_. ( 1968) using alkaloid and morphological data,respectively . 12 II Subgeneric groupings of North American lupines from alkaloid and serological studies by Nowacki (1960) and Nowacki & Prus-Glowacki (1971) ... 13 III Colour reactions of lupine flavonoids under UV light and resultant structural inferences ... 25 IV The flavonoids isolated from 73 taxa of North American Lupinus 34 V The distribution of flavonoids in various stages of maturity of Lupi nus arboreus 41 VI The distribution of flavonoids in Lupinus arboreus samples following drying and storage 44 VII The distribution of flavonoids among individuals of Lupinus arboreus from ten populations. ... 48 VIII The distribution of flavonoids among individuals of Lupinus bico1or from ten populations .... 51 IX Table of eigenvectors of the 17 flavonoid variables for-'the first three "component axes of PCA . 67 X Analysis of variance for orientin accumulation in Lupinus sericeus populations 74 XI Orientin accumulation means analysed by Duncan's Multiple Range Test for 32 populations of Lupinus seri ceus . 75 XII HPLC peak heights of orientin from 20 Lupinus sericeus individuals grown from seed in uniform conditions 80 XIII Data Set I: the distribution of flavonoids in 73 Lupinus taxa 83 XIV Table of eigenvectors of the 56 flavonoid variables for the first three component axes of PCA on Data Set I . 87 XV Data Set III: The distribution of 11 flavonoid- types in 73 Lupinus taxa 92 v i i i XVI Table of eigenvectors of the 11 f 1 avonoid-type variables for the first three component axes of PCA on Data Set III 96 XVII Summary of Lupinus groupings from principal com• ponents analysis on Data Sets I and III . 98 XVIII Summary table of the groups of Lupinus taxa de• limited by flavonoid data and morphological evidence 119 i x List of Figures 1 The numbering system of flavonoid carbon atoms. 19 2 Wesley-Moser rearrangement of a C-glycof1avone during prolonged treatment with strong acid. 28 3 Two-dimensional TLC map of the flavonoids from Lupinus arboreus at 6 months. , . 40 4 Map to show the locations of the Lupinus arboreus and L_. bi col or•col 1ecti ons'monitored.for infraspec^ ific flavonoid variation .47 5 Map to show the locations of 32 populations of Lupinus sericeus monitored for infraspecific flavonoid variation 54 6 Diagrammatic representation of a high pressure liquid chromatograph system, 56 7 HPLC of a typical Lupinus sericeus individual . .60 8 The relationship between OTU 1 s compared with respect to two attributes (A and B) 63 9 Ordination of the Fig.8 OTU's with 1st and 2nd com• ponent axes 64 10 Ordination of 181 individuals of Lupinus sericeus by PCA on HPLC data 66 11 Ordination of 32 populations if Lupinus sericeus by CVA on HPLC data. 70 12 Ordination of 32 populations of Lupinus sericeus from CVA. Population centroids are drawn with +/- 1 standard deviation to show infrapopu1 ational variabi 1 ity 71 13 Ordination of 73 lupine taxa from PCA on Data Set I. Components I /11. 85 14 Ordination of 73 lupine taxa from PCA on Data Set I. Components 11 /111 86 15 Biosynthetic interralationships of lupine flavonoids 91 16 Ordination of 73 lupine taxa from PCA on Data Set III. Components 1/11 94 17 Ordination of 73 lupine taxa from PCA on Data Set III. Components 11 /111. ......... 95 ACKNOWLEDGEMENTS I would like 'to express my gratitude to my supervisor, Prof. Bruce A. Bohm for his continuing interest, enthusiasm and assis• tance throughout the course of my studies and also for his generous financial support for field work. My other committee members were Profs. Wilfred B. Schofield and Fred R. Ganders. Their advice, encouragement and critical reading of this manu• script is greatly appreciated. I should also like to thank the following people: Profs.