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Flavonoid systematics of North American Lupinus species (Leguminosae) Nicholls, Kevin William 1981

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FLAVONOID SYSTEMATICS OF NORTH AMERICAN LUPINUS SPECIES (LEGUMINOSAE) by KEVIN WILLIAM NICHOLLS B.Sc. (Hons . ) , U n i v e r s i t y C o l l e g e of Wales, Aberys twy th , 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 t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF"BRITISH COLUMBIA August 1981 © Kevin W i l l i am N i c h o l l s , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 Dat e I-»TT> c i i / -in \ 11 ABSTRACT Th is study was an assessment of the use fu lness of f l a v -onoids as taxonomic markers in the genus Lupinus (Leguminosae) . The genus i t s e l f i s r e a d i l y r e c o g n i z a b l e , bu t , in North Amer i ca , s p e c i f i c boundar ies are poo r l y d e f i n e d . Th i s i s probably the r e s u l t of a combinat ion of c o n s i d e r a b l e morpho log ica l p l a s t i c i t y and h y b r i d i z a t i o n ( p a r t i c u l a r l y amongst the o u t c r o s s i n g pe ren -n i a l t a x a ) . At the o u t s e t , a d e t a i l e d a n a l y s i s of L u p i n u s f l a v o n o i d s was made. F i f t y - s i x compounds were i d e n t i f i e d , the ma jo r i t y being f l a vones based on a p i g e n i n , l u t e o l i n and l e s s commonly a c a c e t i n and c h r y s o e r i o l . Glucose was the on ly sugar a t tached to f l a v o n o i d s but was found C-bonded at the 6- or 8- p o s i t i o n s and 0-bonded at the 5 , 7 , 3 ' or 4 1 - p o s i t i o n s . Some f l a v o n o l s and i s o f l a v o n e s were a l so i d e n t i f i e d . The r e l i a b i l i t y of f l a v o n o i d s as s p e c i f i c cha r a c t e r s was next t e s t e d . Fresh or d r i e d p l an t ma te r i a l had i d e n t i c a l f l a v -onoid c o n t e n t , and p l an t s of va ry ing ages were c h e m i c a l l y i d e n -t i c a l . G e o g r a p h i c a l l y , q u a n t i t a t i v e v a r i a t i o n in f l a v o n o i d con -tent was noted in an a n a l y s i s of Lupi nus s e r i ceus by high p r e s -sure l i q u i d chromatography. A sharp d i s c o n t i n u i t y in o r i e n t i n l e v e l s was seen between popu l a t i ons north and south of Great S a l t Lake, Utah , l end ing support to the s u b s p e c i f i c rank ing of the s o u t h e r l y popu l a t i on by F leak (1971) . Subsequent a n a l y s i s of p l an t s grown in uni form c o n d i t i o n s showed the f l a v o n o i d d i f -f e rences to have been a phenotyp ic r esponse . i i i S ince f l a v o n o i d s appeared to be i n f r a s p e c i f i c a l l y s t a b l e c h a r a c t e r s , an i n t e r s p e c i f i c study of 73 taxa was made. P r i n -c i p a l components a n a l y s i s exposed 7 chemical groups . S ix of these accumulated some novel f l a v o n o i d s p e c u l i a r to each group. The seventh group had p r o f i l e s c o n t a i n i n g no unusual compounds. One spec i es (L_. di f f usus) had a very complex p r o f i l e and was c l a s s e d as a monospec i f i c group. Us ing . the l i t e r a t u r e and personal obse r va t i ons i t was seen that in 5 groups the chemical a f f i n i t i e s p a r a l l e l e d morpho log ica l a f f i n i t i e s thereby s t r eng then ing the i n t e g r i t y of the groups as na tu ra l u n i t s . Group C, however, was d i v i d e d in to two on the bas i s of morpho log i ca l d i s c o n t i n u i t i e s as was the seventh group (F ) .wh ich con ta ined a c o l l e c t i o n of l up ines with doubt fu l a f f i n -i t i e s (chemical or m o r p h o l o g i c a l ) . O v e r a l l , i t i s conc luded that f l a v o n o i d s are good s p e c i f i c cha r a c t e r s in L u p i n u s . T h e i r d i s t r i b u t i o n , i n t e r s p e c i f i c a l 1 y , lends support to the r e c o g n i t i o n of some p r e v i o u s l y des c r i bed subgener i c groupings that were based on morphology. In other c a s e s , f l a v o n o i d data suggests a f f i n i t i e s between some taxa that h i t h e r t o were cons ide red u n r e l a t e d . i v Tab le of Contents A b s t r a c t i i L i s t of Tab les v i i L i s t o f F i g u r e s ix I INTRODUCTION 1 T r i b a l p o s i t i o n of Lupi nus 2 Subgener ic taxonomy of Lupi nus 5 Some reasons f o r the con fus i ng taxonomy of L u p i n u s 7 Prev ious chemotaxonomic s t ud i e s on Lupinus . 10 Economic aspects of Lupinus 15 Thes i s aims 17 II FLAV0N01D CHEMISTRY OF LUPINUS . 18 I n t r oduc t i on 18 M a t e r i a l s and methods I. F l a vono id e x t r a c t i o n a n d p u r i f i c a t i o n . . . 20 M a t e r i a l s and methods II. F l a vono id i d e n -t i f i c a t i o n 24 R e s u l t s . . 29 III INFRASPECIFIC VARIATION IN LUPINE FLAVONOIDS . . 35 I n t r o d u c t i o n 35 The e f f e c t of p l an t age on the f l a v o n o i d content of a Lupinus spec i es 38 The e f f e c t of d r y ing and s torage on the f l a v o n o i d content of a Lupi nus s p e c i e s . . . 43 Geograph ica l v a r i a t i o n in the f l a v o n o i d content o f Lupinus spec i e s Experiment I. A p r e l i m i n a r y study . . . . 4 6 Experiment II. V a r i a t i o n in Lupinus s e r i c e u s . 53 M a t e r i a l s and methods. 53 High Pressure L i q u i d Chromatography . . 5 5 HPLC of f l a v o n o i d s 57 HPLC of-Lup inus s e r i c e u s f l a v o n o i d s . . 58 Resu l t s and d i s c u s s s i on 59 P r i n c i p a l components a n a l y s i s (PCA) . . 62 Resu l t s and d i s c u s s i o n of PCA on Lupi nus _ s e r i c e u s f l a v o n o i d da t a , 65 Canonica l v a r i a t e s a n a l y s i s (CVA). . . 68 Resu l t s and d i s c u s s i o n of CVA on L u p i n u s s e r i c e u s popu l a t i ons . .69 The a n a l y s i s of o r i e n t i n accumula t ion in L u p i n u s s e r i c e u s popu l a t i ons . . . . 73 Experiment I I I . The de te rm ina t ion of the pheno-t y p i c or genotyp ic nature of o r i e n t i n v a r i a t i o n in Lup inus-se r i c eus 78 IV INTERSPECIFIC FLAVONOID VARIATION IN LUPINUS . . 81 M a t e r i a l s and methods 81 Data genera t ion and a n a l y s i s 81 D i s c u s s i o n . . .99 V OVERVIEW 122 F l avono ids as taxonomic cha ra c t e r s in Lupi nus 122 R e l a t i o n s h i p s in Lupi nus 126 Convergence 128 VI SUMMARY 130 References 133 APPENDIX I. . P l an t s and Sources 141 APPENDIX II. Sol vent, systems and media used in TLC of f l a v o n o i d s from L u p i n u s 151 APPENDIX II I . UV s p e c t r a l a b s o r p t i o n maxima of some f l a v o n o i d s from Lupi nus 152 APPENDIX IV. Raw HPLC d a t a : peak he igh ts of 17 f l a v -onoids from 181 i n d i v i d u a l s of Lupi nus s e r i c e u s . 154 APPENDIX V. Two-dimensional chromatograms (2-D maps) the 73 l u p i n e taxa that were a n a l y s e d . v i i L i s t of Tab les I. A comparison of subgener i c groupings of European l up ines by Nowacki & Prus-Glowacki (1971) with that of T u t i n et aj_. ( 1968) us ing a l k a l o i d and morpho log i ca l d a t a , r e s p e c t i v e l y . 12 II Subgener ic groupings of North American l up ines from a l k a l o i d and s e r o l o g i c a l s t ud i e s by Nowacki (1960) and Nowacki & Prus-Glowacki (1971) . . . 13 III Co lour r e a c t i o n s of l up ine f l a v o n o i d s under UV l i g h t and r e s u l t a n t s t r u c t u r a l i n f e r e n c e s . . . 25 IV The f l a v o n o i d s i s o l a t e d from 73 taxa of North American Lupinus 34 V The d i s t r i b u t i o n of f l a v o n o i d s in va r ious stages of matu r i t y of Lupi nus arboreus 41 VI The d i s t r i b u t i o n of f l a v o n o i d s in Lupinus arboreus samples f o l l o w i n g d ry ing and s torage 44 VII The d i s t r i b u t i o n of f l a v o n o i d s among i n d i v i d u a l s of Lupinus arboreus from ten p o p u l a t i o n s . . . . 48 VIII The d i s t r i b u t i o n of f l a v o n o i d s among i n d i v i d u a l s of Lupinus b i co1or from ten popu la t i ons . . . . 51 IX Tab le of e i genvec to r s of the 17 f l a v o n o i d v a r i a b l e s f o r - ' t he f i r s t three "component axes of PCA . . 67 X A n a l y s i s of va r i ance f o r o r i e n t i n accumula t ion in Lupinus s e r i c e u s popu l a t i ons 74 XI O r i e n t i n accumulat ion means ana lysed by Duncan's M u l t i p l e Range Tes t f o r 32 popu l a t i ons of Lupinus s e r i ceus . 75 XII HPLC peak he ights of o r i e n t i n from 20 Lupinus  s e r i c e u s i n d i v i d u a l s grown from seed in uni form c o n d i t i o n s 80 XIII Data Set I: the d i s t r i b u t i o n of f l a v o n o i d s in 73 Lupinus taxa 83 XIV Tab le o f e i genvec to r s of the 56 f l a v o n o i d v a r i a b l e s f o r the f i r s t three component axes of PCA on Data Set I . 87 XV Data Set I I I : The d i s t r i b u t i o n of 11 f l a v o n o i d -types in 73 Lupinus taxa 92 v i i i XVI Tab le of e i genvec to r s of the 11 f 1 avonoid-type v a r i a b l e s f o r the f i r s t three component axes of PCA on Data Set III 96 XVII Summary of Lupinus groupings from p r i n c i p a l com-ponents a n a l y s i s on Data Sets I and III . . . . 98 XVIII Summary t a b l e of the groups of Lupinus taxa de-l i m i t e d by f l a v o n o i d data and morpho log i ca l ev idence 119 i x L i s t of F igures 1 The numbering system of f l a v o n o i d carbon atoms. . 19 2 Wesley-Moser rearrangement of a C-glycof1avone dur ing pro longed t reatment with s t rong a c i d . . . 28 3 Two-dimensional TLC map of the f l a v o n o i d s from Lupinus arboreus at 6 months. , . 40 4 Map to show the l o c a t i o n s of the Lupinus arboreus and L_. bi co l o r•co l 1ec t i o n s ' m o n i t o r e d . f o r i n f r a s p e c ^ i f i c f l a v o n o i d v a r i a t i o n .47 5 Map to show the l o c a t i o n s of 32 popu l a t i ons o f Lupinus s e r i c e u s moni tored f o r i n f r a s p e c i f i c f l a v o n o i d v a r i a t i o n 54 6 Diagrammatic r e p r e s e n t a t i o n of a high p ressure l i q u i d chromatograph system, 56 7 HPLC of a t y p i c a l Lupinus s e r i c e u s i n d i v i d u a l . . 6 0 8 The r e l a t i o n s h i p between OTU 1 s compared with respec t to two a t t r i b u t e s (A and B) 63 9 O r d i n a t i o n of the F i g . 8 OTU's with 1st and 2nd com-ponent axes 64 10 O r d i n a t i o n of 181 i n d i v i d u a l s of Lupinus s e r i c e u s by PCA on HPLC data 66 11 O r d i n a t i o n of 32 popu l a t i ons i f Lupinus s e r i c e u s by CVA on HPLC da t a . 70 12 O r d i n a t i o n of 32 popu l a t i ons of Lupinus s e r i c e u s from CVA. Popu la t i on c e n t r o i d s are drawn with +/- 1 s tandard d e v i a t i o n to show infrapopu1 a t i o n a l v a r i a b i 1 i t y 71 13 O r d i n a t i o n of 73 l u p i n e taxa from PCA on Data Set I. Components I /11 . . 85 14 O r d i n a t i o n of 73 l up ine taxa from PCA on Data Set I. Components 11 /111 86 15 B i o s y n t h e t i c i n t e r r a l a t i o n s h i p s of l up ine f l a v o n o i d s 91 16 O r d i n a t i o n of 73 l up ine taxa from PCA on Data Set I I I . Components 1/11 94 17 O r d i n a t i o n of 73 l u p i n e taxa from PCA on Data Set I I I . Components 11 /111. . . . . . . . . . 95 ACKNOWLEDGEMENTS I would l i k e 'to express my g r a t i t u d e to my s u p e r v i s o r , P ro f . Bruce A. Bohm fo r h is c o n t i n u i n g i n t e r e s t , enthusiasm and a s s i s -tance throughout the course of my s t ud i e s and a l so f o r h is generous f i n a n c i a l support f o r f i e l d work. My other committee members were P r o f s . W i l f r e d B. S c h o f i e l d and Fred R. Ganders. The i r a d v i c e , encouragement and c r i t i c a l read ing of t h i s manu-s c r i p t i s g r e a t l y a p p r e c i a t e d . I should a l so l i k e to thank the f o l l o w i n g peop le : P r o f s . Jack Maze and Gary E. B r a d f i e l d f o r help in running and i n t e r -p r e t i n g computat iona l par ts of t h i s s tudy ; P ro f . Tony Glass f o r help with s t a t i s t i c s ; Mr. Dennis Moore f o r supp l y ing me with p l an t m a t e r i a l ; P ro f . Mary E. Barkworth (Utah S ta te ) f o r a l l ow ing me access to herbar ium m a t e r i a l ; P ro f . David B. Dunn f o r a s s i s -tance in the i d e n t i f i c a t i o n of spec imens. It i s imposs ib l e to name a l l the graduate s t u d e n t s , past and p r e sen t , who have helped me over the l a s t few y e a r s . T h e i r a s s i s t a n c e in the l a b o r a t o r y and dur ing the w r i t i n g and f i n a l t yp ing of t h i s t h e s i s was i n v a l u a b l e as too was t h e i r f r i e n d s h i p . L a s t l y , I thank Les l ey Joy ; her constant companionship and encouragement at home (amidst her own s t u d i e s ) p lus her hard work on f i e l d t r i p s c o n t r i b u t e d immesurably to the comple t ion of t h i s work. F i n a l l y , f i n a n c i a l support in the form of t each ing a s s i s -t a n t s h i p s , graduate s c h o l a r s h i p s and a t r a ve l award to the U.K. i s g r a t e f u l l y acknowledged. For these I thank the UBC Botany Department and the Fa cu l t y of Graduate S t u d i e s . 1 I. INTRODUCTION Lupinus L. i s a l a rge genus na t i ve to western North America and South America with a second cent re o f d i s t r i b u t i o n in Medi ter ranean Europe. The genus can be found in a wide v a r i e t y of h a b i t a t s , i n c l u d i n g sandy beaches and dunes at ,sea 1 evel ( e . g . I. 1i t t o r a l i s) , the suba lp ine zone ( e . g . I. 1ya l1 i i ) , in the deser t s of southwestern USA ( e . g . I. odoratus ) and the a r c t i c reg ions of the Yukon and A laska ( e . g . I. kusche i ) . As i s t y p i c a l o f the Leguminosae, Lupinus spec i es have s ymb io t i c n i t r o g e n - f i x i n g b a c t e r i a in root nodules and are thus ab le to grow in n u t r i e n t d e f i c i e n t s o i l s . Consequent l y , l up ines are important i n v a s i v e herbs on exposed t e r r a i n f o l l o w i n g g l a c i a l r e t r e a t or the a c t i v i t i e s of man. They are p a r t i c u l a r l y abundant on roads ides a long which they q u i c k l y sp r ead . In t h i s way, fo rmer l y i s o l a t e d taxa are o f t en brought i n to sympatry and h y b r i d i z a t i o n is commonly the r e s u l t . This i s e s p e c i a l l y so in pe renn ia l l up i nes and c o n t r i b u t e s in no small way to the taxonomic problems w i th in the genus. At the gener i c l e v e l , Lupinus is r e a d i l y r e c o g n i z a b l e . With few except ions a l l taxa have racemes o f papi11ionaceous f lowers and pa lmate ly compound l e a v e s . Both annual and pe renn ia l l up ines e x i s t . It i s , however, very d i f f i c u l t to determine p r e c i s e l y the number of spec i es in the genus owing to the ex i s t ence of the l a rge number of pub l i shed names and the l ack of modern comprehensive monographic t r e a tmen t s . It i s es t imated that there are about 10 s p e c i e s , a l l annua l s , na t i ve to the Old Wor ld , whereas in North America there are "p robab l y 100 or more" (H i t chcock et a j k , 1961) and perhaps 200 (Tu rne r , 2 1959). For the Americas as a whole , P o l h i l l (1976) es t imates there to be 200 s p e c i e s , Dunn (persona l communication in P lanchero de Rave lo , 1978) suggests 600 taxa with 50% at the i n f r a s p e c i f i c l e v e l and Smith (1938-1952) documented over 600 separate spec i e s . It i s obvious that there are many taxonomic problems below the genus l e v e l in the New World l u p i n e s . H i t chcock et al . (1961) go so f a r as to say that Lupinus i s " p robab l y in a more chao t i c s t a t e than any o ther (genus) to be found in our a rea " (the P a c i f i c Northwest po r t i on of the Uni ted S t a t e s ) . T r i b a l p o s i t i o n o f Lupinus Al though the number of des c r i bed spec ies has i n c reased s t e a d i l y over the l a s t 150 y e a r s , the i n t e g r i t y o f Lupinus as a group of c l o s e l y r e l a t e d p lants has never been in doubt . Indeed, i t i s t h e i r . d i s t i n c t i v e morphology that has r e s u l t e d in the c o n t r o v e r s i a l t r i b a l placements of Lupinus in the '. Leguminosae. O r i g i n a l l y desc r i bed by Tou rne fo r t (1719) , Lupinus L. was p laced in the Genisteae by Adanson (1763) with many o ther genera i n c l u d i n g Geni s ta , Cyt i s us (the brooms) , Laburnum , Me l i1o t i s and T r i f o l i um (the c l o v e r s ) . It i s c l e a r that the s i z e and the v a r i a b i l i t y o f the group t r o u b l e d him: " J ' a u r a i s d e s i r e d i v i s e r c e t t e s e c t i o n en 2, sqavo i r c e l l e des Genets et c e l l e des T r i f l e s ; mais je n ' a i pu t rouve r dans aucune de l eu r s p a r t i e s un c a r a t e r e que marquat des bournes a c e t t e d i v i s i o n . " (I would l i k e to d i v i d e t h i s s e c t i o n in to 2, the brooms and the t r e f o i l s , but I have been unable to f i n d cha rac t e r s in each on which to base such a d i v i s i o n . ) 3 In 1862 Bentham & Hooker r e s t r i c t e d the Genis teae to those taxa with a two- l ipped c a l y x , c l o sed s tamina ! tube and d imorphic a n t h e r s . Later r esea rch has r evea l ed that a l l such taxa have a chromosome number based on 12 (G ibbs , 1966) compared to 7 in the c l o v e r s (H i t chcock et^  a]_. , 1961) and 9 in the t r i b e Poda l y r i eae ( P o l h i l l , 1976). Lupinus was p laced in the s u b t r i b e Spa r t i e ae with Geni s t a , A r g y r o l o b i um, Adenocarpus , La burnum , Ca1i cotome, P e t t e r i a , Spa r t i um and E r i n a c e a ; c h a r a c t e r i z e d by n o n - a r i l l a t e ( e s t r o p h i o l a t e ) seeds . Bentham & Hooker (1962) p laced the s t r o p h i o l a t e seed-bear ing genera , Cyt i s us , Ulex , Hypoca lyptus and Loddi g e s i a , in the s u b t r i b e C y t i s e a e . Th is c l a s s i f i c a t i o n remained s t a b l e fo r almost a century u n t i l Rothmaler (1944) and Hutchinson (1964) r e i n t e r p r e t e d the group in r a the r d i f f e r e n t ways. Rothmaler un i ted Bentham & Hooker 's s u b t r i b e s i n to the C y t i s i n e a e (a l though the name " G e n i s t i n e a e " would have been nomenc l a tu r a l l y c o r r e c t ) . Excluded were Lupinus (which he cons ide red as a monogeneric s u b t r i b e ) , Hypocal ypti is , A rgy ro l obi um and Loddi ges ia . I n c o n t r a s t , Hutch inson r a i s e d Bentham & Hooker ' s s u b t r i b e s , Cy t i seae and S p a r t i e a e , to t r i b a l rank , the Cy t i seae and Gen i s t eae , r e s p e c t i v e l y . F ive genera were removed from the Genis teae to form the Laburneae and a f u r t h e r two, Lupinus and A r g y r o l o b i um, were p laced in a new t r i b e , Lup ineae . In d e l i m i t i n g the Lup ineae , Hutchinson (1964) r ecogn ized ca lyx s i m i l a r i t i e s between Lupi nus and A r g y r o l o b i um . They o therwise appear to be r a the r d i s s i m i l a r genera , p a r t n e r e d , perhaps , more fo r . conven i ence than fo r phy logene t i c c o n s i d e r a t i o n s . Of the c l a s s i f i c a t i o n s a v a i l a b l e , Rothmaler ' s system has been adopted in most cases (G ibbs , 1966; P o l h i l l , 1976; C h r i s t o f o l i n i & C h i a p e l l a , 1977; B isby & N i c h o l l s , 1977) a l though the taxonomic placement of Lupinus i s s t i l l c o n t r o v e r s i a l . The genus has many anomo l i e s , i n c l u d i n g i t s common annual h a b i t , s t i p u l e s , predominance o f b r a c t s , b r a c t eo l e s and m u l t i f o l i a t e l e a v e s , i t s tendency toward v e r t i c i l l a t e f lowers and the f a c t that the ma jo r i t y o f spec i es are na t i v e to the New Wor ld . P o l h i l l (1976) suggests that s i m i l a r i t i e s shared by Lup inus , B a p t i s i a and Thermo ps i s ( the l a t t e r two members of the north temperate t r i b e Poda l y r i eae ) are p o s s i b l y the r e s u l t o f p a r a l l e l e v o l u t i o n as i n v a s i v e herbs before the d ive rgence o f the Genis teae in the Medi ter ranean r e g i o n . P o l h i l l (1976) views A r g y r o ! o b i um as having more a f f i n i t i e s with the A f r i c a n genus Mel obi um of the C r o t a l a r i e a e than with Lup inus . F i n a l l y , s e r o l o g i c a l s t ud i e s by C h r i s t o f o l i n i & C h i a p e l l a (1977) showed Lupinus (2 spp . ) to be qu i te d i f f e r e n t from the Rothmaler ian Genis teae and supported i t s e x c l u s i o n from the t r i b e . Fur thermore , Harborne ( 1969 ), in h is survey of the f l a v o n o i d s of the G e n i s t e a e , determined tha t Lupinus (26 u n s p e c i f i e d spec i e s ) were devoid of i s o f l a v o n e s , compounds common to the r e s t o f the t r i b e . However, t h i s c o n c l u s i o n i s now d i s c r e d i t e d f o l l o w i n g the i s o l a t i o n o f i s o f l a v o n e s from the geniis by Horhammer & Wagner ( 1962 ), Laman ejt aj_. (.1978), Harborne e_t aj_. (1976) and the present author ( N i c h o l l s & Bohm, 1978) . In summary, Lupinus may be regarded as r a the r a " l o s t sheep " , having obvious a f f i n i t i e s to the Genisteae and s i m i l a r i t i e s Cperhaps by p a r a l l e l i s m ) to the Poda l y r i eae (or North American Thermops ideae ) . There a r e , however*, too many 5 d i s s i m i l a r i t i e s to i n c l u d e i t c o n f i d e n t l y in e i t h e r . Subgener ic taxonomy of Lupinus Lupinus was o f f i c i a l l y p laced in to modern taxonomy by Linnaeus (1753) a l though Tou rne fo r t (1719) must be c r e d i t e d with i t s i n t r o d u c t i o n i n to bo t an i c a l l i t e r a t u r e . F ive European annuals and one North American pe renn ia l appeared in Spec ies  PI antarum with L^ . al bus as the type fo r the genus. During the next e igh t y years new spec i es were desc r i bed from North America ( Pu rsh , 1814; N u t t a l l , 1 8 1 8 ; Hooker & A r n o t t , 1841; Bentham & Hooker, 1862 ; Douglas , 1914) bu t , no attempt was. made to * ' c l a s s i f y them in to subgener i c groupings apar t from that o f De Cando l le (1825) who s imply r ecogn ized the annuals and pe renn i a l s as d i s t i n c t g roups . F i n a l l y , in 1835, Agardh pub l i shed the -f i r s t , and to date only;,' world monograph of Lupinus in which he d i v i d e d the genus in to twelve t r i b e s plus a t h i r t e e n t h , the Spec ies Inqu i rendae , c o n t a i n i n g taxa of unknown or doubt fu l r e l a t i o n s h i p s . A second c l a s s i f i c a t i o n was proposed by Watson (1873) fo r the e x t r a t r o p i c a l l up ines of North Amer i ca . In h is r e v i s i o n he d i v i d e d the genus in to three sect i o n s : P Iatycarpus , c o n t a i n i n g annuals with connate c o t y l e d o n s ; L u p i n e11u s , c o n t a i n i n g a s i n g l e annual with s o l i t a r y , a x i l l a r y f l o w e r s ; and Lupinus p r o p e r , with 50 annuals and pe renn i a l s having p e t i o l a t e co ty l edons and mul t i f1owered i n f l o r e s c e n c e s . P iper (1906) , in h is f l o r a of Washington S t a t e , r a i s e d Watson's s e c t i o n s to subgenera and d i v i d e d Lupinus proper in to s i x complexes f o l l o w i n g the format of Agardh. Subsequent l y , the genus has s u f f e r e d the consequences of ove r-zea lous ' s p l i t t e r s ' 6 and s p e c i a l i s t s . Rydberg (1917) desc r i bed 80 spec i es from the Rocky Mountains and ad jacent p l a i n s , p l a c i n g them in 19 s e c t i o n s (one spec i es appeared in two s e c t i o n s ! ) . Then, between 1938 and 1952, Smith produced a s t agge r i ng number of s p e c i f i c d e s c r i p t i o n s . His p reoccupa t ion with documenting minor v a r i a n t s in f lower c o l o u r , indumentum and hab i t r e s u l t e d in h is p u b l i s h i n g Spec ies Lupinorum, a three volume work c o n t a i n i n g over 600 asso r t ed taxonomic e n t i t i e s from North and South Amer i ca . His t reatment of the North American l up ines r ecogn ized the subgenera P I a t y ca rpus , c o n t a i n i n g 4 groups of annuals (one of which was Watson's s e c t i o n L u p i n e l l u s ) , and Lupinus proper with 6 annual and 13 perenn ia l g roups . For Abrams 1 (1944) F l o r a of the P a c i f i c S t a t e s , Smith subd i v i ded the genus i n to 18 groups , perhaps , more as an a id to key ing than as a statement of e v o l u t i o n a r y a f f i n i t i e s . Once a g a i n , as rseen in Rydberg (1917) , some spec i es occu r red t w i c e , in d i f f e r e n t groups . P h i l l i p s (1955) , on the o ther hand, r e cogn ized on ly 16 pe renn ia l spec ies and severa l subspec ies in North America e x c l u s i v e of C a l i f o r n i a but was c r i t i c i z e d by Fleak (1971) fo r not a ccoun t ing adequate ly f o r the v a r i a t i o n inheren t in Lup inus . More r e c e n t l y , Dunn (1957, 1959, 1965) and h is co-workers ( C h r i s t i a n & Dunn, 1970; Hess & Dunn, 1970; Cox, 1974a, b; Fleak & Dunn, 1971; Vaugn & Dunn, 1977; Dunn & Harmon, 1977) have done much to c l a r i f y the taxonomy of Lup inus . Through the use o f a v a r i e t y of techn iques they have c a r r i e d out d e t a i l e d sys temat i c -t reatments of small numbers of t a x a , termed complexes by them, reduc ing many e p i t h e t s to synonymy as a r e s u l t . In a d d i t i o n , at the spec i es and subspec ies l e v e l they have suggested e v o l u t i o n a r y t rends and a f f i n i t i e s w i th in and between complexes , 7 a l l o f which r ep resen t s c o n s i d e r a b l e improvement over prev ious work. In s p i t e o f t h i s , the genus remains l a rge and unwie ldy . Est imates of the number of d i s t i n c t i v e taxa vary enormously from the 1500-1700 names p u b l i s h e d , a l though approx imate l y 150 spec i es is now the accepted f i g u r e fo r North America (Kar tesz & K a r t e s z , 1980). I n t e r s p e c i f i c a f f i n i t i e s remain most ly unc lea r and subgener i c c l a s s i f i c a t i o n s are absent from modern f l o r a s and monographs ( P h i l l i p s , 1955 ; Dunn, 1956 ; H i t chcock et al . , 1961; Dunn & G i l l e t t , 1966). Those that have been proposed over the years are o b v i o u s l y unnatura l (with the p o s s i b l e excep t ion of Watson, 1873). For example, annuals have always been i n i t i a l l y segregated from the p e r e n n i a l s . The annual hab i t is supposed ly a de r i v ed c o n d i t i o n (Hu t ch inson , 1959; Davies & Heywood, 1963) , and i t i s more probable that annuals have • evolved on numerous separate occas ions from d i f f e r e n t pe renn ia l a n c e s t o r s . Some reasons fo r the confus ing . taxonomy of Lupinus : The taxonomy of Lupinus is r i f e with prob lems, p a r t i c u l a r l y in North Amer i ca . S p e c i f i c boundar ies based on morphology are o b s c u r e , e s p e c i a l l y among the pe renn ia l s p e c i e s . The 1500 or so a v a i l a b l e e p i t h e t s can be a t t r i b u t e d to a) a number of b i o l o g i c a l phenomena that are des c r i bed l a t e r , and b) taxonomists who have s imply c a t e g o r i z e d and desc r i bed taxa and i n d i v i d u a l s with no c o n s i d e r a t i o n fo r i n t e r r e l a t i o n s h i p s .and e v o l u t i o n a r y r a m i f i c a t i o n s ( P h i l l i p s , 1955) . Many taxa show a bew i l de r i ng degree of phenotyp ic p l a s t i c i t y . Morpho log i ca l c h a r a c t e r s , i n c l u d i n g indumentum ' ; 8 t y p e , f lower s i z e and c o l o u r , b r a n c h i n g , and hab i t are extremely v a r i a b l e . N e v e r t h e l e s s , some workers , no tab ly Smith (1938-52) , used' such cha rac t e r s fo r r e c o g n i t i o n of spec i es and pub l i shed hundreds of new spec i es d e s c r i p t i o n s . As H i tchcock ejt a]_. (1961) s t a t e , these cha rac t e r s "vary so g r e a t l y in Lupinus that i t i s almost c e r t a i n that they cannot , except in comb ina t i on , be used fo r the r e c o g n i t i o n of t a x a . " V a r i a t i o n i s a l so the r e s u l t of c o n s i d e r a b l e h y b r i d i z a t i o n between t a x a . S t e r i l i t y b a r r i e r s are low in most pe renn ia l l u p i n e s . Bragdtf (1957) found that every c ross tha t she attemp-ted produced good seed set and y i e l d e d p e r f e c t l y f e r t i l e o f f -sp r i ng with normal m e i o s i s . P h i l l i p s (19 57) a l so demonstrated l u p i n e i n t e r f e r t i l i t y but d id d i s c o v e r a number of s t e r i l i t y b a r r i e r s . Fur thermore , K a z i m i e r s k i (.1963 ) found groups of i n t e r -f e r t i l e l up ines that were i n t e r s t e r i l e with members of other groups Csee next s e c t i o n ) . In g e n e r a l , i n t r o g r e s s i o n is l i k e l y to occur whenever two perenn ia l l up ine spec ies are brought i n to contac t (Phi 11ips , 1957) . P r e v i o u s l y i s o l a t e d popu l a t i ons are o f t en brought in to sympatry by the a c t i v i t i e s of man Ce.g. a long r oads i des ) and through na tura l causes . Dunn & G i l l e t t (1966) a t t r i b u t e much of the taxonomic d i f f i c u l t y to be the r e s u l t of s u c c e s s i v e l up ine m ig ra t i ons f o l l o w i n g the r e t r e a t of the P Ie fs tocene g l a c i e r s . They suggest that the northward spread of the a r c t i c and a l p i n e spec i e s l e f t behind popu l a t i ons which then h y b r i d i z e d with more temperate spec i es as they in turn moved northwards in the moderat ing c l i m a t e . Wtth such wide i n t e r f e r t i l i t y , i t i s not s u r p r i s i n g tha t the chromosome compl e'ments of l up i nes are found to be very un i f o rm . 9 Except fo r two c l e a r l y r e l a t e d annual s p e c i e s , JL. t e x e n s i s and L_. subcarnosus , which have a h a p l o i d number of 18 (Tu rne r , 1957) a l l North American taxa have n = 24 ( P h i l l i p s , , 1957 ; Dunn & G i l l e t t , 1966) . A few popu la t i ons have been found with _n =48 but they are m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from t y p i c a l i n d i v i d u a l s . Less taxonomica l1y t roublesome are the smal1 -f lowered annual spec ies wh ich , f o r the most p a r t , are s e l f - p o l l i n a t e d . Dunn ( 1956) found that I. bi col or r e a d i l y set seed in the absence of p o l l i n a t o r s ; i ndeed , the f lowers a t t r a c t few i n s e c t v i s i t o r s . Lupinus nanus, with s l i g h t l y l a r g e r f l o w e r s , r equ i r e s i n s e c t v i s i t a t i o n or s imple mechanica l s i m u l a t i o n o f an i n s e c t l and ing on the keel before s e l f - p o l l i n a t i o n is • e f f e c t e d . The need fo r i n s e c t v i s i t s does present the o p p o r t u n i t y fo r some c r o s s - p o l l i n a t i o n to o c c u r , and, Harding et a l . (1974) found that l a rge- f l owe red v a r i e t i e s o f I. nanus have very high o u t - c r o s s i n g ra tes (approach ing 100%). Consequent l y , Dunn (1956) found I. b i c o1o r to e x h i b i t s l i g h t morpho log i ca l d i s c o n t i n u i t i e s between popu la t i ons which were made up of very uni form i n d i v i d u a l s . On the o ther hand, L.. nanus showed more v a r i a b i l i t y o f i n d i v i d u a l s w i th i n : p o p u l a t i o n s , but owing to g rea te r gene f l ow , popu la t i ons were much more cont inuous in t h e i r v a r i a t i o n . The gene t i c i s o l a t i o n of the s e l f - p o l l i n a t i n g annuals has r e s u l t e d in the fo rmat ion of high s t e r i l i t y b a r r i e r s ; t h u s , a r t i f i c i a l c rosses between I. s u c c u l e n t u s , I. s pa rs i f 1 or us and j . . a r i z o n i c u s f a i l e d (Dunn, 1956) as d id L. texens i s X I. sub.carnos us ( E rbe , 1957) . The h e a v i l y s c e n t e d , l a rge- f l owe red annuals and pe renn i a l s are c l e a r l y adapted to c r o s s - p o l 1 i n a t i o n and show low s e l f -compatabi 1 i t y even when a r t i f i c i a l l y s e l f - p o l l i n a t e d (Bragd/6', 1955) . Th is breed ing sys tem, as wel l as the common ex i s t ence of sympatry and the r e s u l t a n t h y b r i d i z a t i o n have i n t e r a c t e d with the inhe ren t v a r i a b i l i t y o f Lupinus taxa to produce the ' c on fus i ng s i t u a t i o n ev ident in l up ine taxonomy today . Prev ious chemotaxonomic s tud i e s on Lupinus Al though Lupinus is taxonomica l1y c o n f u s i n g , chemical s t ud i e s to help conf i rm or r e f u t e subgener i c c l a s s i f i c a t i o n s have been few. The s tud i e s a v a i l a b l e i n vo l v e the use of a l k a l o i d d i s t r i but ion pat te rns ' (JNowacki , 1960; Ki nghorn et al . , 1980) , p ro t e i n e l e c t r o p h o r e s i s ( S c o g i n , 1973) and s e r o l o g i c a l techn iques ([Nowacki & P rus-G lowack i , 1971). It is not s u r p r i s i n g that most of the e a r l y chemotaxonomic work emanates from Europe where d e t a i l e d s tud i e s were undertaken on the e conomica l l y important Old World t a x a . Only r e c e n t l y has any emphasis been p laced on the more numerous but e conomica l l y neg lec ted New World s p e c i e s . In some cases both Old and New World taxa appear in the same r e s e a r c h , bu t , f o r conven i ence , each group w i l l be ^d i s cussed s e p a r a t e l y . Old World l up ines The c u l t i v a t i o n of Medi ter ranean l up ines in areas away from t h e i r na tura l ranges has r e s u l t e d in the s e l e c t i o n of many c u l t i v a r s and consequent l y numerous s p e c i f i c d e s c r i p t i o n s . However, F l o ra Europea (Tu t in ejt^  al_. , 1968) l i s t s on ly nine qu i te d i s t i n c t i v e spec i es and s u b s p e c i e s , with many e p i t h e t s reduced to synonymy. ^NDwacki ( 1960) ana lysed .'12 taxa to assess the value of a l k a l o i d s in d i s t i n g u i s h i n g groups of c l o s e l y r e l a t e d s p e c i e s . Seven a l k a l o i d s were i s o l a t e d and f i v e i d e n t i f i e d . D i s t r i b u t i o n of the compounds led him to d e l i m i t 4 s e c t i o n s : A l b u s , P i l o s u s , A n g u s t i f o l i u s and Lu teus . The r e a l i t y of these groups as na tura l un i t s was given f u r -ther support by Kaz im i e r sk i (1963) who found high l e v e l s of i n t e r f e r t i 1 i t y w i th in the groups and i n t e r s t e r i 1 i t y between mem-bers of d i f f e r e n t g roups . Nowacki & Prus-Glowacki (1971) r e -examined these taxa and f i v e a d d i t i o n a l ones , us ing s e r o l o g i c a l t e c h n i q u e s . They conc luded that the genus in Europe c o n s t i t u t e s an immuno log i ca l l y d i s con t i nuous u n i t . Combining a l k a l o i d and s e r o l o g i c a l d a t a , they were able to p lace 4 of the a d d i t i o n a l taxa in the e a r l i e r groups and l e f t one t a x o n , L_ h i r s u t u s , in i t s own s e c t i o n . The c l o se agreement between the c l a s s i f i c a t i o n s based on chemis t r y and morphology i s shown in Table I. New Worl d_l up_i nes Whereas the European spec i es o f Lupi nus are m o r p h o l o g i c a l l y d i s t i n c t , s p e c i f i c boundar ies between North American spec i es are obscure and p r o b l e m a t i c . Nowacki (1960) and Nowacki & Prus-Gl owacki (1971) ana lysed 20 North American spec i e s fo r a l k a l o i d a l and s e r o l o g i c a l a f f i n i t i e s . While f i n d i n g them much more complex than t h e i r European r e l a t i v e s , New World l up ines showed much l e s s i n t e r s p e c i f c v a r i a b i l i t y . S e r o l o g i c a l l y , they were a l so very un i f o rm . T h i r t e e n taxa were ass igned to the s e c t i o n M u t a b i l i s (shown fn Tab le II) but the presence of i n t e r n a l s t e r i l i t y ba r -r i e r s l ed K a z i m i e r s k i (1963) to subd i v ide the s e c t i o n in to s e c -t i o n s M u t a b i l i s and Pe r enn i s . C ross ing experiments with s e c t i o n s M u t a b i l i s and Perennis r evea led high i n f r a-g roup i n t e r f e r t i 1 i t y and i n t e r-g roup s t e r i l i t y . TABLE I. A comparison of subgener i c groupings of European lup ines by Nowacki & Prus-Glowacki (1971) with that of Tu t i n e_t aj_. ( 1968) us ing a l k a l o i d and morpholog-i c a l data r e s p e c t i v e l y A l k a l o i d Lupinus taxa Major Taxa a c co rd ing to group a l k a l o i d s Tu t i n et a 1 . 1uteus 1uteus LUTEUS ro thma le r i hi spani cus 1upi n i ne hi s pani cus h i span i cus al bus a lbus ssp . a lbus p e l e s t i n u s 1upani ne a 1 bus s s p . a lbus ALBUS graecus hydroxy-1upani ne a lbus s s p . graecus megalospermus LA4 a lbus s s p . megalos permus j u g o s l a v i a n u s a n g u s t i f o l i u s s s p . r e t i c u l a t u s p i 1 o s u s va r ius s s p . o r i e n t a l i s p a l e s t i nus epi1upani ne n . t . PILOSUS di gi ta tus LA4 va r i us s s p . o r i e n t a l i s v a r i u s v a r i u s s s p . va r ius p r i n c e i n . t . angust i f o l i us 1upanine a n g u s t i f o l i u s s s p . angust i f o l i us ANGUSTIFOLIUS 1 i ni f o l i us c ryp tanthus hydroxy-1 upani n.e angust i f o l i ne angust i f o l i us s s p . r e t i c u l a t u s a n g u s t i f o l i u s s s p . a n g u s t i f o l i u s HIRSUTUS h i r s u t u s mi cranthus n . t . denotes taxa not t r e a t ed in Tu t i n et a l - (1968) TABLE II. Subgener ic groupings of North American l up ines from a l k a l o i d and s e r o l o g i c a l s t ud i e s by Nowacki (1960) and Nowacki & Prus-Glowacki (1971) . A l k a l o i d group Lupi nus taxa andi col a pubescens m u t a b i 1 i s ornatus douglas i i polyphyl1 us p e r e n n i s arboreus noo tka tens i s h a r t w e g i e lega ns a l b i f rons 1 a t i f o 1 i u s Major a l k a l o i d s MUTABILIS* PERENNIS* s pa r t e i ne 1 upani ne 1upi n i ne BARKERI ba rke r i t runca tus 1upani ne hydroxy lupan i ne SUCARNOSUS subcarnosus t exens i s # s p a r t e i ne 1upani ne hydro xylu pa n i n e DENS IFLORUS d e n s i f l o r u s MICRANTHUS nanus po lycarpus s p a r t e i n e 1u pan i ne hydroxy lupan ine N 4/5 * Both groups c h e m i c a l l y s i m i l a r . S u b d i v i s i o n based on cross-a b i l i t y s t ud i e s by Kaz im ie r sk i (1963) . # A l k a l o i d s not de te rmined ; grouping is based on chromosome coun t s : n= 18. Some members of the Perennis group were i n c l uded in another a l k a l o i d study by Nowacki & Dunn (1965) who examined the shrubby pe renn ia l l up ines of coas ta l C a l i f o r n i a . A l though they conc luded that I. arboreus , JL. noo tka tens i s , I. al bi f rons , J_. excub i tus and J_. chamissonis showed c l o se chemical a f f i n i t i e s and i n t e r f e r t i 1 i t y , the l a t t e r pa i r o f spec i es w§s~ omit ted from the c l a s s i f i c a t i o n of Nowacki & Prus-Glowacki (1971) . Recen t l y , another a l k a l o i d survey of 21 spec ies has been pub l i shed by Kinghorn et_ al_. ( 1980) who found very poor agreement between the subgener i c c l a s s i f i c a t i o n o f Smith (1944) and-the a l k a l o i d d i s t r i b u t i o n pa t te rns o b t a i n e d . In a d d i t i o n , they saw c o n s i d e r a b l e seasonal v a r i a t i o n in the p r o f i l e s of some t a x a . As found by Mankinen e_t aj_. ( 1975) and Dunn (personal communica t ion ) , Kinghorn ejt aj_. ( 1980) d i s cove red the a l k a l o i d content to vary i n f r a s p e c i f i c a 11 y at d i f f e r e n t c o l l e c t i o n s i t e s . On the o ther hand, Vaugn & Dunn (.1977 ) found a l k a l o i d s to be very s t a b l e cha rac t e r s at a given geograph ica l l o c a t i o n . L e a f l e t s c o l l e c t e d over 100 years ago and r e c e n t l y from the same l o c a l i t y had i d e n t i c a l compounds. In summary, as Wall er'&..Nowacki ( 1978) c o n c l u d e , the l up ines o f North America are much more d i f f i c u l t to c l a s s i f y by t h e i r a l k a l o i d chemis t ry than are the l up ines of Europe. Cox (.1973 ) employed p ro t e i n e l e c t r o p h o r e s i s in an attempt to revea l r e l a t i o n s h i p s among taxa be long ing to the pe renn ia l c a e s p i t o s e complex in which over 80 taxa have been d e s c r i b e d . He found high i n f r a - and i n t e r - s p e c i f i c v a r i a t i o n and conc luded that the J.. 1 epi dus - caesp i tosus complex is composed o f : c h e m i c a l l y d i s t i n c t popu la t ions r a the r than r e c o g n i z a b l e s p e c i f i c e n t i t i e s . F i n a l l y , S c o g i n ( 1973) a n a l y s e d f o u r t a x a (J_. c a u d a t u s , L. n a n u s , L_. 1epi dus and I. l y a l l i i ) e l e c t r o p h o r e t i c a l l y f o r l e u c i n e a m i n o p e p t i d a s e . The d i s t r i b u t i o n o f t h r e e isoenzyme bands l e d him to s p e c u l a t e on a p o s s i b l e phylogeny between the t a x a . However, i n s o f a r as o n l y f o u r out of perhaps 150 s p e c i e s were a n a l y s e d f o r a s i n g l e enzyme, such s p e c u l a t i o n s are r a t h e r premature. Economic a s p e c t s of Lupinus The a b i l i t y of l u p i n e s to grow on n u t r i e n t d e f i c i e n t s o i l s where o t h e r crops tend to f a i l l e d e a r l y farmers to conclude t h a t l u p i n e s d e p l e t e the s o i l o f i t s f e r t i l i t y . They were thus g i v e n the i n a p p r o p r i a t e name, Wolf Bean, and hence the L a t i n name Lu p i n u s . Nowadays, we are aware o f n i t r o g e n f i x a t i o n , nodules and Rhi z o b i um; f a r from r o b b i n g the s o i l , l u p i n e s e n r i c h i t . L upines have been c u l t i v a t e d i n both the M e d i t e r r a n e a n Regions and the New World f o r over 2000 y e a r s . W i l k i n s o n (1854) r e p o r t e d the presence o f l u p i n e s seeds i n E g y p t i a n tombs and the d o m e s t i c a t i o n o f the Tarwi Bean (L_. mutabi l ; i s ) i s thought to go back to pr e - I n c a n time i n South and C e n t r a l America (Anony- • mous, 1979). The main drawback o f Lupinus spp. as crop p l a n t s i s the predominance of b i t t e r - t a s t i n g and, moreover, poisonous a l k a l o i d s i n the l e a v e s and more i m p o r t a n t l y , i n the p r o t e i n -c o n t a i n i n g seeds. T h i s has not h i n d e r e d t h e i r use as green f e r t i l i z e r s , however. M e d i t e r r a n e a n a n n u a l s , p a r t i c u l a r l y L. a l b u s , L_. 1 uteus and IL. a n g u s t i f o l i us, are now grown e x t e n -s i v e l y not o n l y i n t h e i r n a t i v e a r e a s , but a l s o i n n o r t h -eas te rn Europe, USSR, A u s t r a l i a , New Zealand and l a t e l y , the United States . Removal o f a l k a l o i d s by soak ing tarwi beans in running water has been p r a c t i s e d for c e n t u r i e s by the Andean I nd i ans . Brucher (1968) determined the p ro t e i n content to be 46% in the seeds , a va lue which compares f a vourab l y with soy beans as a food sou r ce . Modern s e l e c t i v e b reed ing o f ' sweet ' s t r a i n s of a l k a l o i d - f r e e l up ines has i n c reased the importance of the Old World annuals as fodder crops and the New World L. mutab i1 is as human and animal f e e d . U n f o r t u n a t e l y , the c l i m a t i c requi rements of the l a t t e r spec i es r e s t r i c t s i t s use to the h igh lands o f South and Cent ra l Amer i ca . It does seem obvious though, that in Nor th :Amer ica where there are perhaps 150 . . / s p e c i e s , there should e x i s t many other taxa with great p o t e n t i a l as p ro t e i n sources a l r eady adapted to the a r i d and semi-a r id c o n d i t i o n s that p r e v a i l in the southwest . Other uses of l up ines i n c l u d e na tura l i n s e c t i c i d e p roduc t ion by L_. m u t a b i 1 i s (He re ra , 1942) and the use of ground, roas ted s eed l i ngs as a c o f f e e s u b s t i t u t e ( Bu rka r t , 1952) . Pardodi (1938) c i t e s I. mill t i f 1 or us as a sand s t a b i l i z e r as are J_. hi 1 ar i anus ( Bu rka r t , 1952 ) and J_. arboreus (H i t chcock et al . , 1961; Marchant , personal communicat ion ) . F i n a l l y , l up ines are grown e x t e n s i v e l y as o rnamenta l s . The ease with which they can be h y b r i d i z e d has r e s u l t e d in many co l ou r v a r i a n t s and c u l t i v a r s among L_. a rbo reus , L_. pol yphyl 1 us , L_. hartwegi and j . . mutabi l i s being grown throughout the world as popular garden f l o w e r s . Thes i s Aims This study is an assessment of the use fu lness of f l a v o n o i d s as taxonomic markers in the genus Lup inus . Lupines are p a r t i c u l a r l y a p p r o p r i a t e fo r such a study fo r seve ra l r easons . It i s a l a rge and e a s i l y r e c o g n i z a b l e genus that forms a conspicuous part of the f l o r a of western North Amer i ca . C o l l e c t i o n o f p lant ma te r i a l i s consequent l y easy . Below the genus l e v e l , as we have seen , Lupinus is taxonomica l1y con fus i ng through i t s phenotyp ic p l a s t i c i t y , sympatry and h y b r i d i z a t i o n . No w ide ly a ccep tab le subgener i c c l a s s i f i c a t i o n e x i s t s at the moment (a l though D.B.Dunn and his co-workers are making progress toward such a t r e a t m e n t ) . F i n a l l y , e v o l u t i o n a r y a f f i n i t i e s between annuals and pe renn i a l s are poor l y known. The u n c e r t a i n t y of spec i e s i n t e r r e l a t i o n s h i p s makes the d i s c o v e r y and a p p l i c a t i o n of new taxonomic ev idence h i g h l y d e s i r a b l e . The aims o f t h i s study a r e : 1 ) a d e t a i l e d a n a l y s i s o f the f l a v o n o i d s o c c u r r i n g in Lup inus ; 2 ) an assessment of the r e l i a b i l i t y o f f l a v o n o i d s as i n f r a s p e c i f i c c h a r a c t e r s ; 3 ) p o s s i b l e c o n s t r u c t i o n of a t e n t a t i v e subgener i c c l a s s i f i c a t i o n based on f l a v o n o i d d a t a ; 4 ) comparison o f t h i s c l a s s i f i c a t i o n with those of other workers whose systems have been based on morphology and to a l e s s e r extent on breed ing i n fo rma t i on and a l k a l o i d da t a . II. FLAVONOID CHEMISTRY OF LUPINUS I n t r oduc t i on Very few p u b l i c a t i o n s have appeared conce rn ing the f l a v o n o i d s of Lup inus , and on ly one study i s chemotaxonomic. Harborne (1969) ana lysed 20 spec i es as part o f a f l a v o n o i d i n v e s t i g a t i o n of the t r i b e Gen i s t eae . His work concen t ra ted on i n t e r g e n e r i c d i f f e r e n c e s and t h e r e f o r e i n t e r s p e c i f i c chemical d i f f e r e n c e s are l a c k i n g , as too are the names o f the l up ine spec i es s t u d i e d . As i s the case in the r e s t o f the Gen i s t eae , Harborne found tha t Lupinus conta ined the f l avones ap igen in and l u t e o l i n , t h e i r co r respond ing C-glycof1 avones and the f l a v o n o l s kaempferol and q u e r c e t i n . Horhammer & Wagner (1962) r r epor ted the presence of the i s o f l a v o n e g e n i s t e i n in L_. po1yphyl1 us and Laman et_ a_l_. (1978) , i n v e s t i g a t i n g J_. a l b u s , L_. 1 uteus , I. angust i fo l i us and L_. po lyphy l 1 us , found the i s o f l a v o n e s g e n i s t e i n , orobol , 5-methy lgen is te in and methylene-d i o x y o r o b o l . The l a s t group of spec i es a l so had the f l a vones a p i g e n i n , l u t e o l i n , a c a c e t i n , d iosmet in and c h r y s o e r i o l toge ther with t h e i r 7-0-glucos ides ; and the f l a v o n o l s k a e m p f e r o l , , . que r ce t i n and i s o r h a m n e t i n , a l l g l u c o s y l a t e d at the 3-pos i t i on (see F igure 1 ) . F i n a l l y , Harborne ejt a_l_. (1978) i s o l a t e d and c h a r a c t e r i z e d l u t e o n e , an i s o p e n t y l i s o f l a v o n e , from the s e e d l i n g s of 12 spec i es of Lup inus . Th is compound i s thought to be a p r e - i n f e c t i o n a l a n t i - f u n g a l agent . 20 M a t e r i a l s and methods 1. F l avono id e x t r a c t i o n and p u r i f i c a t i o n E x t a c t i on_p_rocedure The e x t r a c t i o n procedure desc r i bed here was s tandard fo r a l l t a x a , but so l v en t q u a n t i t i e s v a r i ed with the amount o f p lant ma te r i a l used . (See P lants and Sou rces , Appendix I.) Leaves and p e t i o l e s o f Lupinus were removed at a n t h e s i s , weighed and blended with 200-400 ml of methanol (100% methanol fo r f r e sh or 80% fo r d r i ed p lant m a t e r i a l ) . The s l u r r y was heated to gent le b o i l i n g . A f t e r 30 minutes the e x t r a c t was f i l t e r e d through g lass wool . The p lant ma te r i a l was then re-heated with a second 200-400 ml of'~'fresh methano l , r e - f i l t e r e d and d i s c a r d e d . The two e x t r a c t s were combined and evaporated to dryness i_n vacuo at 4 0 ° C . The green t a r was s l u r r i e d with 5-10 g of C e l i t e A n a l y t i c a l F i l t e r A id and 10-25 ml of hot ( 9 0 ° C ) water and f i l t e r e d through a Buchner f u n n e l . The brown aqueous f i l t r a t e was ex t r a c t ed 3 or 4 t imes with 100 ml a l i q u o t s of wa te r-sa tu ra ted n-butanol in a s epa r a to r y funnel and the o rgan i c l a ye r s removed, combined and evaporated to dryness i_n vacuo. A brown g lass o f pheno l i c ma te r i a l was l e f t . The mate r i a l cou ld be s to red in t h i s form, or d i s s o l v e d in methanol and s t o r e d , or taken d i r e c t l y to the f i r s t column p rocedure . Th in_Layer_CJ2ro m a t o Sr^9!2-^ (TLC) Most TLC was performed on polyamide p l a t e s . These were prepared in the l a b o r a t o r y by b l end ing 15 g of Polyamid DC 6.6 (Macherey-Nagel) with 100 ml of 50% methanol and sp read ing i t on 10 g lass p l a t es (.20 X 20 cm) to a depth of 0.33 mm. Two main so l ven t systems were used r o u t i n e l y in t h i s study 21 al though on numerous occas ions o ther systems were employed. A f u l l l i s t of so l ven t s i s d e t a i l e d in Appendix II. Most commonly used were the ' aqueous ' system (water , n-butano l , a ce tone , d ioxane ; 70 :15 :10 :5 ) and the ' o r g a n i c ' system (benzene, methyl-ethy l ketone , methano l , water ; 5 5 : 2 2 : 2 0 : 3 ) . Less commonly, TLC was performed on c e l l u l o s e p l a tes us ing e i t h e r 10% or 15% aqueous a c e t i c a c i d as s o l v e n t . On these o c c a s i o n s , pre-coated Polygram 0.1 mm MN300 (Macherey-Nagel) c e l l u l o s e sheets were used. F l avono id d e t e c t i o n and v i s u a l i z a t i o n F lavono ids were detec ted on TLC p l a tes under UV l i g h t (366 nm) in which most compounds appear as dark absorb ing s p o t s . A f t e r examinat ion under UV l i g h t , p l a t es were sprayed with a 0.1% w/v s o l u t i o n (.1:1, methano l : water) of di phenyl bor i c a c i d ethanolamine complex ( A l d r i c h Chemica l ) . Under UV l i g h t compounds then appeared v a r i o u s l y co loured but most ly green or y e l l o w . Fur ther co l ou r r e a c t i o n s were observed when the p la tes were sub jec ted to ammonia fumes. S t r u c t u r a l i n f e r ences based on these co lou r r e a c t i o n s are given in Table I I I . I n i t i a l f l a v o n o i d s epa r a t i on Separa t ion o f the c o n s t i t u e n t f l a v o n o i d s from the pheno l i c e x t r a c t commenced with chromatography through a column of Sephadex LH20 (Pharmac ia ) . A 30 x 4 cm column was f i l l e d with an aqueous s l u r r y of water-swol len LH20. Care was taken to remove a i r from a l l the so l ven t s by p l a c i n g them in a f i l t e r 1 f l a s k a t tached to an a s p i r a t o r . The removal of a i r avoids the genera t ion of a i r bubbles w i th in the column dur ing development. The crude pheno l i c e x t r a c t was d i s s o l v e d in .1-2 ml of water and gen t l y p i p e t t e d onto the top of the column. Development of the column i n v o l v e d use o f water , fo l lowed by water-methano! mixtures in increments of 10%. It was found that 1 1 each of water , 10% and 20% methanol removed the ma jo r i t y o f the f l a v o n o i d s . S ince 30%-70% methanol had l i t t l e e f f e c t , on l y 250 ml o f each was used. Development cont inued with 500 ml each of 80% and 100% methanol before the column was f i n a l l y washed with ace tone . The e l u t i o n o f f l a v o n o i d s was monitored by UV l i g h t , under which they appear as dark absorb ing bands. F r a c t i ons of 250 ml were r o u t i n e l y c o l l e c t e d except when t h i s procedure would mix mate r i a l from two bands; in such cases sma l l e r f r a c t i o n s were t aken . A l l f r a c t i o n s were evaporated to dryness in vacuo and the r e s idue taken up in 0.5 ml of methano l . Each was spo t ted oh a pa i r o f polyamide p l a t e s ; one being developed in the o rgan i c so l ven t sys tem, the o ther in the aqueous sys tem. A f t e r s p r a y i n g , f r a c t i o n s having s i m i l a r components were combined. Fo l l ow ing LH20 f r a c t i o n a t i o n , i n d i v i d u a l compounds were p u r i f i e d us ing a v a r i e t y of methods a c co rd ing to the complex i t y and c h a r a c t e r i s t i c s of each f r a c t i o n m ix tu r e . P repa ra t i ve_TLC F r a c t i ons having s imple mixtures of compounds that cou ld be well separated on polyamide in ' a p a r t i c u l a r so l ven t system were banded on p l a tes and developed in that system. I nd i v i dua l f l a v o n o i d bands were detec ted us ing UV l i g h t , marked, the polyamide scraped o f f and the compounds e l u t ed with 80% methanol. ' The f l a v o n o i d s o l u t i o n s were then evaporated to d r y n e s s , d i s s o l v e d in a l i t t l e methanol and chromatographed in a v a r i e t y of so l v en t s to t e s t for p u r i t y . P a r t i t i o n chromatography LH20 f r a c t i o n s c o n t a i n i n g complex mixtures of compounds e x h i b i t i n g s i m i l a r m o b i l i t y were separa ted by p a r t i t i o n chromatography on Av i ce l m i c r o c r y s t a l 1 i n e c e l l u l o s e columns. A wate r-sa tura ted o rgan i c l a ye r formed the mobi le phase whi le water , adsorbed onto A v i c e l , formed the s t a t i o n a r y phase. Av i c e l (20-50 g, or approx imate ly 100 times the weight of e x t r a c t ) was blended with the i n i t i a l o rgan i c phase ; a 60/40 mixture of wa te r-sa tu ra ted e thy l ace ta te and petroleum e the r . The water phase was a p p l i e d to the Av i c e l by the a d d i t i o n to the the b lender o f water in the p r o p o r t i o n o f 0.5 v/w o f Av ice l .-For example, i f 20 g of A v i c e l were being used then 10 ml o f water and perhaps 100-150 ml of wa te r-sa tu ra ted o rgan i c so l ven t would be b l ended . Columns were packed under s l i g h t p ressure of compressed a i r and the f1avono id-mixture , d r i e d onto 1 g of A v i c e l , was c a r e f u l l y loaded onto the column. The columns were then developed us ing so l ven t mixtures with i n c r e a s i n g amounts of e thy l ace ta te and dec reas ing amounts of petroleum ether in the r a t i o s of 6 :4 , 7 :3 , 8 : 2 , 10 :0 . The sequence was then cont inued with methy le thy l ketone : e thy l ace ta te mixtures in the r a t i o s of 2:8, 5 :5 , 7:3 and f i n a l l y 100% methy le thy l ketone. The i n c r e a s i n g p o l a r i t y o f the so l ven t mixtures caused aglycones to be e lu ted f i r s t fo l lowed by monoglycos ides (at approx imate l y 100% ethy l a c e t a t e ) . D i g l y c o s i d e s were not mobi le u n t i l 50% methy le thy l ketone had been reached . As in other co lumns, compounds were monitored by UV l i g h t and f r a c t i o n s taken a c c o r d i n g l y . Polyamide column chromatography A column o f Polyamid SC6 (Macherey-Nagel) e lu ted with any app rop r i a t e so l ven t system cou ld be used i n s t ead of p r e p a r a t i v e TLC. The major advantage over the use of TLC is speed , e s p e c i a l l y when aqueous so l ven t systems are needed. Fur thermore , l a r g e r amounts of mixtures can be handled us ing column p rocedures . A major d isadvantage i s that r e s o l u t i o n is i n f e r i o r to TLC. The SC6 column was p a r t i c u l a r l y usefu l in the s epa r a t i on of a c y l a t ed from non-acy la ted f l a v o n o i d s . Acy l a t ed compounds appeared as spots with low Rf in both aqueous and o rgan i c systems. A c i d i f i c a t i o n of the so l v en t r e s u l t e d in s i g n i f i c a n t l y i nc reased m o b i l i t y . When such a mixture was encoun te red , i t was p laced on an SC6 column and i n i t i a l l y e lu ted with an app rop r i a t e neut ra l s o l v e n t . On the removal of the non-acy la ted compounds the so l ven t was a c i d i f i e d to 5% a c e t i c a c i d whereupon the a c y l a t ed compound.was r e a d i l y e lu t ed in a pure s t a t e . M a t e r i a l s and methods 2. F l avono id i d e n t i f i c a t i o n Thin l a y e r chromatography I n i t i a l l y , s imple obse r va t i ons of Rf and co lou r r e a c t i o n s y i e l d e d c o n s i d e r a b l e i n f o rma t i on r ega rd ing the i d e n t i t y of l u p i n e f l a v o n o i d s . Mabry et^  aj_. ( 1970) des c r i bed s t r u c t u r a l g e n e r a l i z a t i o n s based on the appearance of f l a v o n o i d s on paper under UV l i g h t before and a f t e r fuming with ammonia. Table III shows the s t r u c t u r a l i n f e r ences that can be drawn from the co lou r r e a c t i o n s of compounds on polyamide with boronate spray and ammonia vapours . 25 TABLE I I I . Co lour r e a c t i o n s of l u p i n e f l a v o n o i d s under UV l i g h t and r e s u l t a n t s t r u c t u r a l i n f e r e n c e s . Colour on pi ate before sp ray i ng Colour a f t e r Colour a f t e r S t r u c t u r a l i n f e r e n c e sp ray ing with boronate spray boronate and ammonia fumes purp le d u l l ye l low blue purp le purp le purpl e purp le purp le purp le v a n ous va r ious blue green va r ious va r ious blue green lemon ye l low lemon ye l low orange/ye l low orange/ye l low orange green green orange brown yel1ow/brown most f l a v o n o l s with s u b s t i t u t e d 3-posi-t i o n , most f l avones f l a v o n o l s with f ree 3-pos i t i on f l a vones with sub -s t i t u t e d 5-pos i t i on f l a vones and f l a v -onols with f ree 4 '-OH but no f r ee 3'-0H f l avones with f ree 5 ,7 ,3 ' , 4 ' - ( 0 H ) 4 f l avones with f ree 5,3 ' , 4 ' - ( 0 H ) 3 7-pos i t i on s u b s t i -uted f l a v o n o l s with f ree 5 , 7 , 3 ' , 4 ' - ( 0 H ) 4 f l avone with 4'-0H s u b s t i t u t i o n f l a vone with 4'-0H s u b s t i t u t i o n but f r ee 3'-0H As the number of i d e n t i f i e d compounds i n c r e a s e d , TLC became i n c r e a s i n g l y dominant as an i d e n t i f i c a t i o n too l by means of c o -chromotography of unknown compounds with the known f l a v o n o i d s . I d e n t i c a l R f ' s and co lou r r e a c t i o n s in a minimum of four so l ven t systems was r equ i r ed before two compounds were cons ide red to be i d e n t i c a l UV spectophotometry UV spec t r a were obta ined fo r a l l p u r i f i e d f l a v o n o i d s . Compounds were d i s s o l v e d in methanol and s p e c t r a l s h i f t s r e c -orded a f t e r the a d d i t i o n of sodium methox ide , aluminum c h l o r i d e / h y d r o c h l o r i c a c i d and sodium a c e t a t e / b o r i c a c i d a f t e r the method of Mabry et_ aj_. ( 1970). A Pye Unicam SP1800 ins t rument was used. Peak maxima are l i s t e d in Appendix I I I . A c i d _ h ^ d r , o l y s i s Ac id h y d r o l y s i s was performed on a l l g l y c o s i d e s . A small amount of pure compound was put i n to a t e s t tube with 1 ml of water and 5 or 6 drops of t r i f 1 u r o a c e t i c a c i d (TFA) from a Pasteur p i p e t t e . The tube was heated in a water bath ( 9 0 ° C ) fo r two hours . A f t e r the f i r s t hour a f u r t h e r 5 drops of TFA was added to r ep l a ce that l o s t by e v a p o r a t i o n . (TFA has a b o i l i n g po in t of 7 2 ° C . ) When h y d r o l y s i s was completed the contents of the tube were evaporated to dryness and then taken up in a 1:1 mixture of e thy l ace ta te and water . A f t e r s e p a r a t i o n , the aqueous phase was evaporated to dryness and the sugar r e s i due r e d i s s o l v e d in 1 drop of water . Th i s was spot ted onto an MN300 c e l l u l o s e p l a t e ; a second spot of a s tandard mixture of g a l a c -t o s e , g l u c o s e , a r a b i n o s e , xy lose and rhamnose was a l so a p p l i e d and the p l a te was developed twice in the ' s u g a r ' so l ven t system (e thy l a c e t a t e , p y r i d i n e , water ; 1 0 : 3 . 2 : 2 ) . The p l a t e was then sprayed with a 1:1 mixture of p-an i s i d i ne and p h t h a l i c a c i d in wate r-sa tura ted n-butanol and p laced in an oven ( 1 1 0 ° C ) fo r f i v e minutes for co lou r development. C o n c u r r e n t l y , the ethy l a ce ta te phase was evaporated to d r yness , r e d i s s o l v e d in methanol and chromatographed aga ins t known aglycones us ing the o rgan i c so l v en t system. On numerous o c c a s i o n s , a c i d h y d r o l y s i s f a i l e d to y i e l d any ag lycones or suga r s , even a f t e r ove rn igh t a c i d t r ea tment . Thes r e s i s t a n t compounds were, as UV spec t r a and NMR s tud i e s a l so i n d i c a t e d , C - g l y c o f l a v o n e s . Prolonged t reatment with s t rong ac id sometimes y i e l d e d a second g l y c o s i d e by the Wesley-Moser rearrangement (Chopin & B o u i l l a n t , 1975) c h a r a c t e r i s t i c o f C-g lyco f l avones (see F igure 2 ) . A l k a l i n e h y d r o l y s i s Severa l compounds were de tec ted that gave s i m i l a r co lou r r e a c t i o n s on sp ray ing to wel l known f l a vono id s such as o r i e n t i n v i t e x i n and l u t e o l i n 7-0-g lucos ide . The i r UV spec t r a and a c i d h y d r o l y s i s products were a l so i d e n t i c a l to the co r r espond ing f l a v o n o i d that each resembled . However, in a l l cases t h e i r R f were s t r i k i n g l y d i f f e r e n t from the common compounds. A c y l a t i o n o f the g l y c o s y l a t i n g moiety o f o r i e n t i n , v i t e x i n and l u t e o l i n 7-0-glucos ide was i n f e r r e d and t h i s was subsequent l y c o n f i r m e d , when, on the a d d i t i o n of 1 or 2 drops of ammonia water , the f a m i l i a r parent g l y c o s i d e was produced. The i d e n t i t y o f the a c y l a t i n g moiety remains unknown. A c y l a t i o n by aromat ic ac ids i s d i s coun ted s i n ce there i s no a d d i t i o n a l absorbance peak on the UV spectrum (Harborne & W i l l i a m s , 1975) . It i s t h e r e f o r e 29 probable that the f l a v o n o i d s ca r r y a s imple a l i p h a t i c a c i d . Nuclear Magnetic Resonance spec t roscopy (NMR) Where q u a n t i t i e s permi t ted (5-10 mg), NMR s p e c t r a were obta ined us ing a Var ian EM 390 ins t rument . T r i m e t h y l s i l y l d e r i v a t i v e s were s yn thes i z ed by the a d d i t i o n o f I ' T r i - S i l ' ( P i e r ce Chemical ) to the dry f l a v o n o i d and l e a v i n g i t ( s toppered) f o r an hour or two. The r e a c t i o n mixture was then evaporated to d r ynes s , f i r s t at the water pump and then at a vacuum pump. The r es idue was then d i s s o l v e d in deute ra ted c h l o r o f o r m , the mixture f i l t e r e d in to an NMR tube and t e t r a m e t h y l s i 1ane added as an i n t e r n a l s t a n d a r d . Spec t ra were i n t e r p r e t e d a f t e r Mabry ejt aj_. ( 1970). Resu l ts F i f t y - s i x f l a v o n o i d s were i d e n t i f i e d from 73 North American l u p i n e t a x a . There was a great v a r i e t y in p o s i t i o n and type of g l y c o s y l a t i o n p r e s e n t ; me thy l a t i on o f the B-ring was a l so common. However, the genus shows remarkable conserva t i sm in the g l y c o s y l a t i n g mo ie t y ; g lucose was the on ly sugar that was found a t tached to f l a v o n o i d s throughout the s tudy . A l i s t o f the compounds i s o l a t e d is shown in Table IV and a f u l l d i s c u s s i o n of t h e i r d i s t r i b u t i o n amongst the taxa i s g iven in Chapter 4; the f o l l o w i n g is a summary of the types of f l a v o n o i d s that were found in the genus as a whole. 30 1) F lavones OR ap igen in a c a c e t i n l u t e o l i n chr , ysoer io l di osmeti n R=Rj=H R=CH 3 , Rj=H R = H V R^OH R = H , R ^  = C H 3 R = CH. R 1 = 0H The ma jo r i t y o f compounds i s o l a t e d from Lupinus were based on four f l a v o n e s : a p i g e n i n , a c a c e t i n , l u t e o l i n and c h r y s o e r i o l . A f i f t h f l a v o n e , d i o s m e t i n , was present in small amounts and as one g l y c o s i d e o n l y . i ) C-G lyco f l avones C-Glyco f l avones were the most common f l a v o n o i d s encountered Two of t h e s e , o r i e n t i n and v i t e x i n ( 8 - C - g l u c o s y l 1 u t e o l i n and 8-C - g l u c o s y l a p i g e n i n , r e s p e c t i v e l y ) , were present in 63 of the 73 taxa s t u d i e d . Not on ly were they most f r e q u e n t l y found , they were a l so very dominant in the p r o f i l e s of most l up ines and on numerous occas ions were the on ly type of f l a v o n o i d present (see Table X I ) . Less f requent were the 6-C-glucosy l isomers o f the above compounds, i s o o r i e n t i n and i s o v i t e x i n , r e s p e c t i v e l y . In a d d i t i o n to these s imple C - g l y c o f l a v o n e s , 0-g lucos ides of them were sometimes p r e sen t . One of these gave a green c o l o u r a t i o n on sp ray ing with boronate and had a high Rf in aqueous s o l v e n t s . Fo l l ow ing a c i d h y d r o l y s i s the co lou r r e a c t i o n had changed to lemon ye l low denot ing the l u t e o l i n h y d r o x y l a t i o n p a t t e r n . Th is h y d r o l y s i s product was o r i e n t i n and the co lou r change a f t e r h y d r o l y s i s i n d i c a t e d that the g lucose moiety had been a t tached to the 3 '-0H. The other 0-g lucos ides of C-g lyco f l avones were 31 l e s s complex, having the second sugar a t tached to the C-bonded g l u c o s e . In consequence, they had i d e n t i c a l UV spec t r a to the parent C - g l y c o s i d e . The p o s i t i o n of the sugar-sugar bond in these compounds was not de te rmined . i i ) Methy la ted C-glycof1 avones Me thy l a t i on at the 4 ' - p o s i t i o n of v i t e x i n , and l e ss f r e q u e n t l y o r i e n t i n , was seen in some t a x a . The former f l a v o n o i d i s known as c y t i s o s i d e (8-C-g lucosy l a ca ce t i n ) and was seen to dominate the p r o f i l e s of e igh t t a x a . The l a t t e r (8-C-g lucosy l d i o sme t i n ) was seen on ly in t r a ce amounts in four of those e igh t s p e c i e s . i i i ) F lavone O-g lucos ides Th is c l a s s of compounds was second to C-glycof1avones in dominat ion of l up ine p r o f i l e s . Ap igen in and l u t e o l i n 7-0-g l u c o s i d e s were the most common; sma l l e r q u a n t i t i e s of 7-0-d i g l u c o s i d e s were a l so found . Some taxa were seen to accumulate ap igen in and l u t e o l i n 4 ' - 0 - g l u c o s i d e s which c h a r a c t e r i s t i c a l l y turned brown when sub jec ted to ammonia vapours . Fur thermore , 5-0-glucos ides were i s o l a t e d from f i v e taxa and appeared as b r i gh t blue f l u o r e s c i n g spots under UV l i g h t be fo re and a f t e r s p r a y i n g . H y d r o l y s i s with a c i d y i e l d e d the parent ag lycone with a concommitant co lou r change. It was found that 7-0-glucos ides took up to 2 hours to hydro lyse f u l l y , 4 1 - 0 - g l u c o s i d e s were c leaved w i th in 30 minutes and 5-0-glucos ides hydro lysed almost immediate ly (.within 5 m inu tes ) . 32 i v ) Methy la ted f l avone 0-g lucos ides Acace t i n ( 4 1 -methy l ap igen in ) and c h r y s o e r i o l (3 ' -methy l-l u t e o l i n ) occur red as 7-0-glucos ides and 7-0-d ig lucos ides in severa l t a x a . v) A c y l a t i o n One of the ou t s t and ing f ea tu res of l u p i n e f l a v o n o i d p r o f i l e s was the presence of a c y l a t e d f l a v o n e s . Most compounds that dominated a two-dimensional map (see Appendix V) were a l so present as two d i f f e r e n t acy,lated d e r i v a t i v e s . The f i r s t o f these ( c a l l e d acy l I) has R f ' s in o rgan i c so l ven t s approx imate l y twice that of the parent compound. Acyl II was always r a the r immobi le , s t a y i ng c l o se to the o r i g i n in a l l neut ra l so l v en t sys tems. On a l l o ccas ions the UV spec t r a were i d e n t i c a l to the co r respond ing parent compound which were always p r e sen t . The possess ion of i d e n t i c a l UV spec t r a i n d i c a t e s that the a c y l a t i n g moiety i s a t tached to the sugar . A l k a l i n e h y d r o l y s i s y i e l d e d the parent g l u c o s i d e but no other i d e n t i f i a b l e component. It is t h e r e f o r e suggested that the acy l I compounds are perhaps ace ta te d e r i v a t i v e s of g l y c o s i d e s and the acy l II compounds are charged g l u c o s i d e s . A more complete de te rm ina t ion of s t r u c t u r e s was not undertaken because of i n s u f f i c i e n t q u a n t i t i e s of unknowns and a l s o , the d i f f i c u l t y in a n a l y s i n g the hyd ro l y s a t e fo r small q u a n t i t i e s of the. . f reed a c y l a t i n g mo ie ty . 2) Other f l a v o n o i d s One i s o f l a v o n e was i s o l a t e d from severa l t a x a . This compound was g e n i s t e i n (an isomer of apigenin)-. It was present as an aglycone and a l so g l y c o s y l a t e d at the 7 - p o s i t i o n . It 33 occur red most ly as a minor c o n s t i t u e n t but d id dominate the p r o f i l e s of four t a x a . The f l a v o n o l s kaempferol and que r ce t i n were present in a few Lupinus spec i es but never in major amounts. Both 3-0-g lucos ides and 7-0-glucos ides of the f l a v o n o l s were i s o l a t e d . One unknown f l a v o n o i d type was encountered that was not v i s i b l e on sp ray ing but s low ly appeared as a red c o l o u r a t i o n a f t e r a few hours . The compound was i s o l a t e d and ana lysed by UV spec t ropho tomet ry . A f lavanone type of spectrum was produced and t h i s was f u r t h e r s u b s t a n t i a t e d by a co lou r r e a c t i o n with h y d r o c h l o r i c acid/magnesium (Venkaturaman, 1964). Ac id h y d r o l y s i s y i e l d e d g lucose and an aglycone tha t cont inued to show the same co l ou r r e a c t i o n . A f l avanone g l u cos i de was i n f e r r e d but the s t r u c t u r e of the ag lycone was not de te rmined . TABLE IV. The f l a v o n o i d s i s o l a t e d from seventy-three of North American Lupinus a pi gen in ap. 7-0-glucos ide ap . 7-0-glu. acy l 11 ap. 7-0-d ig lucos ide ap . 5-0-glu. ap . 4 1 - 0 - g l u . ap. 4 ' - 0 - g l u . acy l I ap . 4 ' - 0 - g l u . acy l 11 v i t e x i n v i t e x i n acy l I v i t e x i n acy l II v i t e x i n X ' - O - g l u . i sov i t e x i n i s o v i t e x i n acyl I a c a c e t i n ac . 7-0-glu . a c . 7-0-glu . acy l I a c . 7-0-d ig l u . c y t i sos ide c y t i s o s i d e acy l I c y t i sos i de acy l 11 g e n i s t e i n gen. 7-0-glu . gen. 7-0-glu . acyl I gen . 7-0-glu . acy l 11 f l avanone Hi Rf f l avanone Mid Rf f l avanone Lo Rf 1 u t e o1 in l u . 7-0-glucos ide l u . 7-0-glu . acyl I 1u . 7-0-glu . acyl 11 l u . 7-0-d ig lucos ide 1 u. 5-0-glu . 1 u. 4 ' -0-g lu . 1 u . 4 ' -0-g lu . acy l 11 o r i e n t i n o r i e n t i n acy l I o r i e n t i n acyl II o r i e n t i n X ' - O - g l u . o r i e n t i n 3 1 -0-g1u. i s o o r i e n t i n i s o o r i e n t i n acyl I c h r y s o e r i ol ch r y . 7-0-glu . ch r y . 7-0-glu . acy l I ch r y . 7-0-glu. acy l II c h r y . 7-0-d ig lu . d iosmet in 8-C-g lu . kaempferol kaem. 3-0-glu . kaem. 7-0-glu . q u e r c e t i n quer . 3-0-glu. quer . 7-0-glu . unknown 'green ' 35 I I I . INFRA-SPECIFIC VARIATION IN LUPINE FLAVONOIDS I n t r oduc t i on In the e a r l y days of b iochemica l s y s t e m a t i c s , i n f r a -s p e c i f i c v a r i a t i o n was r a r e l y c o n s i d e r e d ; o f ten on ly one sample of a p lant spec i es would be ana lysed (Harborne , 1975). Chem-i c a l c h a r a c t e r i s t i c s were cons ide red to have advantages over morpho log i ca l cha r a c t e r s s i n ce they cou ld be desc r i bed very ex-a c t l y in terms of s t r u c t u r a l and c o n f i g u r a t i o n a l formulae (Erdtman, 1963) . Fur thermore , a f i n i t e number of enzymat i ca l1y c o n t r o l l e d b i o s y n t h e t i c s teps cou ld be e l u c i d a t e d thereby making phytochemica ls c o n c e p t u a l l y c l o s e r to genes than such general f ea tu res as l e a f shape, ha i r t y p e , e t c . This view led some workers to regard chemica ls as fundamental cha r a c t e r s that were l e s s a f f e c t e d by e x t r i n s i c f a c t o r s than are gross morph-o l o g i c a l f e a tu res ( H a r r i s o n , 1964). C u r r e n t l y , t h i s reverence to phytochemica ls is gone; they are cha ra c t e r s l i k e any o t h e r , and , i f used as a bas is f o r taxonomic judgements, must be proven to be as r e l i a b l e i n f r a s p e c i f i c a l 1 y as any type of morph-o l o g i c a l or c y t o l o g i c a l cha rac t e r that i s s i m i l a r l y used (Hey-wood, 1963). Harborne (1975) notes that the amount of f l a v o n o i d v a r i a -t i o n amongst p lant popu l a t i ons i s not c o r r e l a t e d with the amount of morpho log i ca l v a r i a t i o n w i th in a s p e c i e s . In a d d i -t i o n to no t ing gene t i c d i f f e r e n c e s , F luck (1963) c i t e s i n -t r i n s i c f a c t o r s such as d i u r n a l i t y , ontogeny and s e a s o n a l i t y p lus e x t r i n s i c f a c t o r s such as s o i l and c l ima te as r e s p o n s i b l e fo r i n f r a s p e c i f i c chemical v a r i a t i o n . In consequence, there i s a cont inuous range of v a r i a t i o n between spec i es that are constant in t h e i r f l a v o n o i d s to those in which d i s t i n c t chemical races e x i s t . Most commonly, p lant spec ies are constant in t h e i r p roduc t ion of major compounds and v a r i a b l e in t h e i r p roduc t ion of minor c o n s t i t u e n t s (Harborne , 1975). T i s s u t & Egger (1972) found d i f f e r e n c e s in f l a v o n o l g l y c o s i d e s of young versus o ld leaves of Quercus , T i l i a and Cory !us whi le spec i es of three o ther t r ee genera , A e s c u l u s , Be tu la and Fra xi n us , showed no d i f f e r e n c e s . Increase in anthocyan in p igmentat ion has long been known in p lants grown in co ld temperatures (Ove r ton , 1899; A l s t o n , 1959). F ranc i s & Dev i t t (1969) showed that wa te r logg ing the roots of T r i f o l j um s ubterraneum caused decreased l e v e l s of i s o f l a v o n e s in the leaves and stems. Moore ejt aj_. ( 1972 ) found popu la t ions of PI a n't a go mari t i ma to be c h e m i c a l l y i d e n t i c a l wor ldwide , except fo r 6-hyd roxy l u t eo l i n , which was s t r a n g e l y absent from 4 of 27 European p o p u l a t i o n s , 1 of 6 North American popu la t i ons and from a l l 10 s i t e s in South Amer i ca . F i n a l l y , Aye (1969) d i s cove r ed that Car ex cu r t a and ' . _C. magel1anlca popu la t i ons were i n v a r i a b l e in t h e i r f l a v o n o i d s whereas in C. m i c r o g l o c h i n and C. mac!ovi ana l u t e o l i n 7-0-g lu cos i de is con f i ned to nor thern popu l a t i ons and is absent or r ep l aced by t r i e in 7-0-glucos ide in the sou th . The use fu lness o f f l a v o n o i d s as taxonomic markers in Lupinus can be assessed on ly a f t e r s i m i l a r i n f r a s p e c i f i c s tud i e s have been e v a l u a t e d . To t h i s end, t h i s chapter des c r i be s the f l a v o n o i d s of a number of l up ines t e s t ed fo r developmental changes , geographic and phenotypic v a r i a t i o n and 37 the e f f e c t s of d r y ing and s t o r i n g of p lant mate r i a l p r i o r to chemical a n a l y s i s . 38 The e f f e c t of p lant age on the f l a v o n o i d content of a  Lupinus spec i es Lupinus arboreus Sims, was s e l e c t e d fo r t h i s and s i m i l a r s tud i e s because of i t s occur rence on the B r i t i s h Columbia coast and consequent ease of c u l t i v a t i o n . As a necessary p r e l i m i n a r y s t e p , a d e t a i l e d study of the f l a v o n o i d s o f I. arboreus was undertaken ( N i c h o l l s & Bohm, 1978) to y i e l d pure compounds fo r use in comparat ive TLC. Mater ia l s_and_methods Seeds were c o l l e c t e d in September 1976 from one mature shrub growing w i ld on the campus of the U n i v e r s i t y o f B r i t i s h Co lumbia . A voucher specimen i s depos i t ed in the UBC herbarium.' Germinat ion was s t a r t e d in e a r l y A p r i l . It was found that r a p i d germinat ion cou ld be e f f e c t e d by p r i c k i n g 2 or ch i pp ing away a smal l area of t e s t a (perhaps 0.2 mm ). The seeds were then p laced on wet f i l t e r paper in a p e t r i d i sh were they absorbed water and swe l led w i th in a few hours . The d ishes were kept at room temperature and in the dark . Emergence of the r a d i c l e occur red in 3-4 days and the s eed l i ngs were ready fo r p o t t i n g w i th in a week. The s e e d l i n g s were p lanted in a 50/50 mixture of f i n e sand and s t e r i l i z e d p o t t i n g s o i l in 4" p l a s t i c pots and kept in a greenhouse fo r two weeks. They were watered d a i l y f o r three days and t h e n , fo r two days , i r r i g a t e d with the aqueous s o l u t i o n obta ined from b lend ing nodules that had been cut from the roots of mature I. arboreus p lants c o l l e c t e d l o c a l l y . Th is b r o u g h t a b o u t the i n f e c t i o n of the s e e d l i n g roots with Rhi zo bi um and the r e s u l t a n t development of root n o d u l e s . , 39 A f t e r two-weeks a l l the p lants were t r a n s f e r r e d outdoor i n to co ld-frames where they were watered and re-pot ted as r e q u i r e d . Two p lan ts were harves ted at the end o f each o f 1, 2, 3, 6, 12 and 24 months. Each p lant was sub jec ted to a methano l i c e x t r a c t i o n . The crude green ex t r a c t was spo t ted at the corner of a polyamide p l a te and developed in two d i r e c t i o n s ; f i r s t in the aqueous so l v en t system, and, a f t e r d r y i n g , in the o rgan i c s o l v e n t . F l avono ids were i d e n t i f i e d by comparing t h e i r chromatographic behav iour and co lou r r e a c t i o n ^ with those of known Compounds'.;i so l ated from a bulk sample of L. arboreu»s des c r i bed in N i c h o l l s & Bohm ( 1978). Resu l t s and D i s cuss i on F igure 3 shows a t y p i c a l two-dimensional map of the f l a v o n o i d s of a Lupinus arboreus i n d i v i d u a l . A t o t a l of s i x -teen compounds were i d e n t i f i e d and t h e i r d i s t r i b u t i o n in p lants of va r ious ages i s shown in Table V. V i t e x i n , o r i e n t i n and c y t i s o s i d e ( 4 ' - 0 - m e t h y l v i t e x i n ) dominated the p r o f i l e s , wh i le minor c o n s t i t u e n t s found were a p i g e n i n , l u t e o l i n , t h e i r 7-0-g lucos ides , 4 1 - 0 - m e t h y l o r i e n t i n and a mobi le a c y l a t e d o r i e n t i n compound ( o r i e n t i n acy l I ) . Traces of q u e r c e t i n 3-0-monoglucoside and 3-0-d ig lucos ide , v i t e x i n acy l I, c y t i s o s i d e acy l I and a pa i r o f immobile (probably charged) a c y l a t ed o r i e n t i n and v i t e x i n compounds was a l so no ted . Twelve comp-ounds were present in a l l i n d i v i d u a l s a n a l y s e d ; the on ly v a r i a b l e f l a v o n o i d s were some that were present in t r a c e amounts. Each member of the pa i r s of p lants harves ted at each time was i d e n t i c a l in i t s f l a v o n o i d c o n t e n t . FIGURE 3. Two-dimensional TLC map of the f l a v o n o i d s from Lupinus arboreus at 6 months. aqueous sol vent di rec t i on <f a = ap igen in b = ap igen in 7-0-glucos ide c = v i t e x i n d = v i t e x i n acyl I e = v i t e x i n acy l II f = c y t i s o s i d e g = c y t i s o s i d e acyl I h = l u t e o l i n i = l u t e o l i n 7-0-glucos ide j = o r i e n t i n k = o r i e n t i n acyl I 1 = o r i ent i n acyl 11 m = 4 1 - 0 - m e t h y l o r i e n t i n organi c so l ven t » d i r e c t i o n 41 TABLE V. The d i s t r i b u t i o n of f l a v o n o i d s i n v a r i o u s stages of m a t u r i t y of Lupinus a r b o r e u s . P l a n t age 1 month 2 months 3 months 6 months 1 year 2 years OJ T 3 OJ •i— O J OJ •o to -o -o 'i—• o •r- to o (S> c o 3 o l—i o 't— o i — o 1—i 1—l o 4-> 3 ' C D 3 i—i C i— •r-I — 1—1 i — , — 1— 1—1 • — i OJ C D T 3 cn »—1 >> >> C D •1— 1 1 i o O i 1— r— J- o O o r— , — rei ca o >> >> o 1 1 i >> >> 1 o O 1— o o C O o o OJ O J 0} (0 ca >1 ro ro T 3 T J -a c c •t— •I— • r - c sz £ c +-> •r— •r— r— •r— £= c IS) w </) OJ 4-> +-> c • C •j—• • r- o o o 1— 1— + J +-> + J E OJ OJ O J O J X X X </) to to o o c C c I O C J C D CJ1 . OJ OJ OJ •r— •r- •i— <u OJ CU O J 0) o s- S-•r- •,- + J 4-> + J 4-> +-> + J +-> "r- •1— 1 OJ OJ CL CL •r- •i— >. >> 3 t- s- S- - 3 3 ca ca > > > O o i — • o o •. o cr 0 0 + + • + 0 0 0 + 0 0 0 • + + • + 0 0 • 0 + 0 + 0 0 • + + • + 0 0 • 0 + + 0 0 • + + • + 0 0 • 0 + 0 0 0 • + + • + + 0 0 • 0 + 0 + 0 0 + + 0 0 • 0 + 0 + + • denotes a major c o n s t i t u e n t o denotes a minor c o n s t i t u e n t + denotes a t r a c e c o n s t i t u e n t 42 I t i s obvious t h a t t h e r e i s l i t t l e change i n the major f l a v o n o i d c o n t e n t o f I. arboreus d u r i n g development. Those t r a c e compounds t h a t are v a r i a b l e show no apparent o n t o g e n e t i c t r e n d s , t h a t i s , t h e i r presence i s i n t e r m i t t e n t and not c o r r e l a t e d to i n c r e a s i n g m a t u r i t y . T h e r e f o r e , i t i s p o s s i b l e to o b t a i n a r e a s o n a b l y a c c u r a t e q u a l i t a t i v e assessment of the f l a v o n o i d s of t h i s s p e c i e s at any time d u r i n g i t s development. I t s h o u l d be noted a l s o t h a t every h a r v e s t time was d u r i n g s p r i n g , summer or f a l l . No w i n t e r c o l l e c t i o n s took p l a c e and c o n s e q u e n t l y , the p o s s i b i l i t y of seasonal f l u c t u a t -i o n s o c c u r r i n g d u r i n g the w i n t e r months cannot be r u l e d o u t . Indeed, one would expect chemical changes i n l e a v e s f o l l o w i n g w i n t e r f r o s t damage or the onset of senescence t h a t was seen on the e x p e r i m e n t a l p l a n t s i n the w i n t e r . For chemotaxonomic s t u d y , the a d v i s a b i l i t y o f a v o i d i n g use of damaged p l a n t m a t e r i a l i s o b v i o u s . In summary, i t can be concluded t h a t J_. a r b o r e u s , grown under s t a n d a r d i z e d c o n d i t i o n s , shows no major o n t o g e n e t i c v a r i a t i o n i n f l a v o n o i d c o n t e n t . 43 The e f f e c t of d ry ing and s torage on the f l a v o n o i d content of  Lupinus M a t e r i a l s and methods Lupinus arboreus was a l so used f o r t h i s s tudy . Leaves and p e t i o l e s (600 g f r e sh weight) were p icked from a s i n g l e mature shrub growing on the campus of UBC. The p lant mater -i a l was tho rough l y mixed and then d i v i d e d i n t o three equal p o r t i o n s . The mix ing avo ided the p o s s i b i l i t y that any p a r t i c u l a r sample came from a s p e c i f i c part of the p lant ( h i g h , low, sun-s ide e t c . ) . The f i r s t p o r t i o n was e x t r a c t e d immediate ly us ing the procedure desc r i bed in Chapter II. The f l a v o n o i d s were i s o l a t e d , p u r i f i e d and i d e n t i f i e d . The second p o r t i o n was a i r - d r i e d fo r one week before be ing sub-j e c t e d to the same e x t r a c t i o n p rocedure . The t h i r d po r t i on was a i r - d r i e d fo r a week and then s to red in a paper bag at room temperature f o r two years p r i o r to e x t r a c t i o n and f l a v o n o i d i d e n t i f i c a t i o n . Resu l ts and d i s c u s s i o n As shown in Table VI there are no s i g n i f i c a n t d i f f e r e n c e s between the three po r t i ons of p lant m a t e r i a l . The on ly v a r i a t i o n . t h a t does appear is a s l i g h t i n c r ease in the amount of ag lycones in the d r i ed l e a v e s . Th i s probably r e s u l t s from a minor r e l e a se of g l y c o s i d a s e s dur ing the d r y i ng pe r iod caus ing some sugar c l eavage . Th is was not s u f f i c i e n t l y great to a l t e r s i g n i f i c a n t l y the l e v e l o f g l y c o s i d e s in the d r i ed p l a n t s . T h e r e f o r e , the use of d r i ed or f r e sh p lant ma te r i a l f o r f l a v o n o i d work i n Lupinus is e q u a l l y v a l i d s i n ce the compounds are p e r f e c t l y s t a b l e . TABLE VI. The d i s t r i b u t i o n of f l a v o n o i d s in Lupi nus arboreus samples f o l l o w i n g d ry ing and s t o r a g e . Treatment of p lant ma te r i a l cu - a - o •1— • r -CO CO c o 1—1 O •1— o i—i 1—1 O 4-> 3 3 1—1 £ , — 1—I i — 1— i — I—1 1—1 cu CO 1 l—l 1—1 >> o >> o CO 1 (  , u o >> ra ra o >> CJ >> o o r~ ac ac de de de ra ra ">> *i— • 1 — c £ c: +-> ' 1— c to co </> cu c e • 1 — • 1 — •1— o O o i — i — +J +-> £ <L> cu X X X to CO CO o o c 1 CO c o cu cu CU •r— • r - cu cu cu cu cu o •r— • r - +J +J -t-> 4-> 4-> •f-> •p- • I — 1 O- Q . 'r— • r - • I — >> >> >> =5 3 s- 5- s- -fO ra > > > O o o i — i — o o o f r e sh + o « o o « o o + o » o o + d r i e d fo r 1 week o o • o o • o o + o • o o + d r i e d and s to red o o « o o « o o + o » o o + fo r two years • denotes a major c o n s t i t u e n t o denotes a minor c o n s t i t u e n t + denotes a t r a ce c o n s t i t u e n t 45 Indeed, Bohm (unpubl.) found t h a t l e a v e s of Con i mi t e l l a  w i 1 1 i a m s i i ( S a x i f r a g a c e a e ) c o l l e c t e d and d r i e d over 80 y e a r s ago c o n t a i n e d mono-, d i - and t r i g l y c o s i d e s i n normal amounts and no excess of aglycones to suggest major g l y c o s i d i c break-down to have o c c u r r e d . Moreover, N i k l a s & G i a n a s s i (1977) found t h a t p r e s e r v e d l e a v e s of the ext.i net ..jgenus '-ZeT.ko va (Ulmaceae), 30 m i l l i o n y e ars o l d , from v o l c a n i c ash se d i m e n t s , s t i l l c o n t a i n e d g l y c o s i d e s . The r e l e v a n c e o f t h i s p r e s e n t s t u d y , spanning merely two y e a r s , l i e s i n the f a c t t h a t a l l t h r e e p o r t i o n s o f the l e a v e s of I. arboreus were known to be c h e m i c a l l y i d e n t i c a l at the o u t s e t . C o n s e q u e n t l y , the f l a v o n o i d p r o f i l e o f the two year o l d l e a f l e t s can be com-pared d i r e c t l y to i t s o r i g i n a l c o n d i t i o n ; something t h a t c o u l d not be done i n the case of the Coni mi t e l l a and Zelkova s t u d i e s . 46 G e o g r a p h i c a l v a r i a t i o n i n the f l a v o n o i d c o n t e n t o f  Lupinus t a x a . Experiment I : A p r e l i m i n a r y study Two s p e c i e s were t e s t e d f o r g e o g r a p h i c a l i n f r a s p e c i f i c v a r i a t i o n . Both t a x a , I. arboreus and I. bi c o1o r , are p r e d o m i n a n t l y c o a s t a l and are found from southern B r i t i s h Columbia to C a l i f o r n i a . The p e r e n n i a l shrub J.. arboreus i s r e s t r i c t e d to the immediate c o a s t and i s o f t e n a dominant member of the v e g e t a t i o n o f mature dunes. Lupinus bi c o l o r , an a n n u a l , i s more widespread and may be found at low a l t i t u d e s w e l l i n l a n d on sandy, open a r e a s . M a t e r i a l s _ a n d methods Dr i e d l e a f m a t e r i a l ( a p p r o x i m a t e l y 0.05 g) was removed from herbarium specimens t h a t r e p r e s e n t e d c o l l e c t i o n s t hroughout the ranges of the two s p e c i e s (see F i g u r e 4 and Appendix I ) . Ten samples of each s p e c i e s were t a k e n . The l e a v e s were ground up i n 80% methanol, f i l t e r e d and the e x t r a c t evaporated to dryness i_n vacuo. The e x t r a c t s were r e d i s s o l v e d i n 0.5 ml methanol and s p o t t e d at the c o r n e r s of 10 X 10 cm polyamide TLC p l a t e s . Two-dimensional d e v e l o p - , ment u s i n g aqueous and o r g a n i c s o l v e n t s was c a r r i e d out and the p r o f i l e s compared. R e s u l t s and d i s c u s s i o n 1) Lupinus arboreus Table VII shows the d i s t r i b u t i o n o f f l a v o n o i d s among the ten p o p u l a t i o n s sampled. A s u b j e c t i v e assessment of t h e i r r e l a t i v e c o n c e n t r a t i o n s was made by o b s e r v a t i o n s o f spot s i z e and c o l o u r i n t e n s i t y f o l l o w i n g s p r a y i n g . I t i s 47 FIGURE 4 . Map to show the l o c a t i o n s o f the Lupinus arboreus (•) J_. b i c o 1 o r (x) c o l l e c t i o n s monitored f o r i n f r a -s p e c i f i c f l a v o n o i d v a r i a t i o n . TABLE VI I . The d i s t r i b u t i o n of f l a vono id s among i n d i v i d u a l s of Lupinus arboreus from ten p o p u l a t i o n s . 1—1 c r— •r— • • 1— >> +-> 3 >1 o i—i c i — • i—i o ro i—i i—i cu CD 3 i—i as 'r~ • 1 . i — • . • i — • — 4- 3 o CD , — 1—1 • • 3 3 >> >> o i — 1 ••— =3 1 >> >> 1—1 1—* 3 3 i — i — o o 1— r— CD CO - o 1—• cn o o cu r— i— CD CD ra ro o 1 . 1 CD i ro CO 1— 1— CD CD 1 1 • p - o c o c 1 o .,— >> >> c I 1 o o c tz c s - 1 • r - 1 'r— o i c iSi CJ o •i— o o 1 1 •i— •I— •f— OJ +J CO SZ 1 _ •r— •r- •r- o rc to , — 1 1 - - +-> £ o CU CU X X X tsi o c c 1 IS) • O • cn CU CU OJ •r- . . CU cu cu cu o >> >> S- s -+J +J +-> +-> +J -t-> +J 1 S- S- cu cu Popu la t i on ap ap ap • l — > > •1— > >> O >> CJ >> o 3 3 3 3 or i~ o or ch o 3 cr 3 cr UBC, Vancouver , BC 0 0 0 0 • 0 0 + 0 0 0 + + South Pender Is. , BC + 0 • + 0 • 0 + + 0 • 0 0 0 V i c t o r i a , B C + 0 • + 0 0 + + 0 + • 0 0 + Point Rober t s , WA + • • + 0 0 0 0 + • 0 0 Monroe, WA • • + 0 0 0 + + • 0 0 + Newport, OR + 0 • + 0 0 0 + • 0 0 + P i s t o l R i v e r , OR 0 0 • + 0 0 + 0 • 0 0 Crescent C i t y , CA 0 0 • + 0 • 0 0 • • • 0 Westpor t , CA + + • + 0 . 0 0 0 • • • L i t t l e Sur R i ve r , CA 0 0 0 • • 0 • 0 0 0 + 4^ OO c l e a r tha t one spec imen, that from L i t t l e Sur R i v e r , C a l i f o r n i a , i s s t r i k i n g l y d i f f e r e n t from the o ther n i n e . A d i s c u s s i o n of t h i s r e s u l t f o l l ows s e p a r a t e l y . Nine of the ten popu la t i ons were q u a l i t a t i v e l y very s i m i l a r . Nine compounds were common to a l l p l an ts and two other f l a v o n o i d s were present in a l l but one p l a n t . Q u a n t i t a t i v e l y , however, some d i f f e r e n c e s were apparent . V i t e x i n and o r i e n t i n were the major f l a v o n o i d s th roughou t ; three compounds, c y t i s o s i d e and two a c y l a t ed o r i e n t i n d e r i v a t i v e s v a r i ed from being major components in some p lan ts to be ing ;minor components in . o t h e r s . Ap igen in 7-0-glucos ide ranged from major q u a n t i t i e s in two p lants to t r a c e q u a n t i t i e s in one i n d i v i d u a l . S ix compounds showed q u a l i t a t i v e d i f f e r e n c e s rang ing from minor amounts to complete absence , but i t i s . p o s s i b l e that the compounds are present in c o n c e n t r a t i o n s too low to be det-< ected by TLC methods. Th is i s e s p e c i a l l y p o s s i b l e in the case of ap igen in and l u t e o l i n 7-0-glucos ide which were absent from j u s t one p l a n t . On the o ther hand, 4 ' - m e t h y l o r i e n t i n does seem to be more i n t e r m i t t e n t and may i n d i c a t e rea l q u a l i t a t i v e d i f f e r e n c e s . The 'In-dj. vd dual from L i t t l e Sur' R iver i s consp i cuous l y d i f f e r e n t from the other c o l l e c t i o n s . The f l a v o n o i d p r o f i l e i s dominated by l u t e o l i n , i t s 7-0-glucos ide and 4 ' -0-g luco-s i d e . Th is p lant possesses qu i t e a d i f f e r e n t set of b i o s y n -t h e t i c c a p a b i l i t i e s not seen in other L. arboreus p l a n t s . Hence, we see 4 1 -0-gl ucosy l a t f on , 3 1 - O ^ J i i e t h y l at ion and no s yn thes i s of C-glycof1 avones. G e o g r a p h i c a l l y t h i s i n d i v -idua l i s in a t y p i c a l t r e e - l u p i n e area and macroscop ic exam-i n a t i o n of the p lant r e vea l s noth ing unusua l . However, 50 mic ros cop i c study r evea l s the presence of c o n s i d e r a b l e indumentum on the upper su r f a ce of the l e a f l e t s . Dunn & Gi l 1e t t ( 1966 ) d e s c r i b e I. arboreus as possess ing a d a x i a l l y g labrous l e a f s u r -f a c e s ; a l l o ther specimens in the study f i t t e d t h i s d e s c r i p t i o n . Fur thermore , they s t a t e that the presence of such pubescence on the upper su r f a ce may i n d i c a t e some i n t r o g r e s s i o n with another t axon . The a t y p i c a l f l a v o n o i d p r o f i l e obta ined from t h i s p lant would c e r t a i n l y support t h i s s p e c u l a t i o n . 2 ) Lupinus bi col or The d i s t r i b u t i o n o f f l a v o n o i d s and an approximate assessment of t h e i r c o n c e n t r a t i o n s i s shown in Table V I I I . E ighteen com-pounds were i d e n t i f i e d of which twelve were present in every p lant i n v e s t i g a t e d . O r i e n t i n and v i t e x i n dominated the p r o f i l e s o f a l l the p lan ts except fo r one in which o r i e n t i n and l u t e o l i n 7-0-g l u c o s i d e were the major f l a v o n o i d s . The compounds that were present as major -components showed no q u a l i t a t i v e d i f f e r e n c e s in L. b i c o1o r , but some minor and t r ace c o n s t i t u e n t s were i n t e r m i t t e n t in t h e i r d i s t r i b u t i o n . These d i s c o n t i n u i t t e s > h o w e v e r , showed no v a r i a t i o n in the b i o -s y n t h e t i c r e p e r t o i r e s of the d i f f e r e n t p l a n t s . For example, the presence or absence of ap igen in and l u t e o l i n aglycones i s of l i t t l e consequence s ince both compounds are present as g l y c o -s ides in j . . b i c o 1 o r . S i m i l a r l y , the absence of ap igen in 7-0-g lucos i de from p lants that con ta in the 7-0-d ig lucos ide i s equa l l y undramat i c . In c o n c l u s i o n , there i s no s i g n i f i c a n t q u a l i t a t i v e i n terpopul a t i o n a l d i f f e r e n c e s in the f l a vono id s of L.. arboreus and L. b i c o l o r . oo r— o oo oo 3 ; ' < 00 -o —1 OJ o PJ —1 3" 3" O OJ o D> 3 fD 0J CU to o 3 < -o CO O fD 3 to to _Ja <-+ o OJ c 1— O 3 3 c+ ri- fD o o — ' m i . CU O CU CU -s -s CO << OJ fD t« O O 1. fD r+ < ca • O O O CU 1—1 — i . i — i o o • O —'. o » to O i—i • r+ —•• • 3 i — i c~> o << CO — i • o CA CA , CA o i i« 03 BC te —. 3" o O 3 fD 3> o o + o + + + o o + + o o o o o o + + o o o o o o o o o o o o o o o o o o + + + 0 + + + + + + + + + + + + + + o o o o + o + + ap igen in ap . 7-0-glu . ap. 7-0-d ig lu . v i t e x i n v i t e x i n acyl I v i t e x i n acy l II v i t e x i n X '-O-glu i s o v i t e x i n i s o v i t . acyl I 1 u t e o l i n l u . 7-0-glu. 1 u or 7-0-diglu . en t in en t i n acy l I o r i e n t i n acy l II o r i e n t i n X '-O-glu i s o o r i e n t i n i s o o r . acy l I - a a . 0 -•• •a to c c+ —• -i 01 ->. c+ cr sz O r+ 3 -"• to O O -b Cu < O 3 O i , CL to ro 3 to 3 CL CL £ CU 52 The l a c k o f chemical d i s t i n c t n e s s o f l u p i n e p o p u l a t i o n s i s r e f l e c t e d i n the morphology of L. arboreus but not i n L^ . b i c o l o r . The former taxon has l a r g e , h e a v i l y s c e n t e d f l o w e r s t h a t are o b v i o u s l y adapted to o u t c r o s s i n g ; the s p e c i e s has low seed s e t when grown i n the absence o f p o l l i n a t o r s ( B r a g d 0 , 1955). In consequence, g e n e t i c d i f f e r e n c e s , whether they c o n t r o l morph-o l o g y or c h e m i s t r y , are masked by high l e v e l s of h e t e r o z y g o s i t y ( G r a n t , 1971). Lupi nus b i c o l or on the o t h e r hand, has s m a l l unscented f l o w e r s and i s p r e d o m i n a t e l y s e l f - p o l l i n a t e d . T h i s l a c k o f o u t c r o s s i n g l e a d s to u n i f o r m i t y w i t h i n p o p u l a t i o n s and d i s c o n t i n u i t i e s between them, as Dunn (1955) noted. Such g e n e t i c i s o l a t i o n has l e d to the development of d i s t i n c t m o r p h o l o g i c a l v a r i a n t s . T h i s v a r i a t i o n l e d Smith (1944) to r e c o g n i z e f o u r v a r i e t i e s and Munz (1959) l i s t e d s i x s u b s p e c i e s i n C a l i f o r n i a . I t s h o u l d be n o t e d , however, t h a t no chemical b a s i s f o r such d i v i s i o n s was apparent from the f l a v o n o i d s u r v e y . I n f r a s p e c i f i c q u a n t i t a t i v e v a r i a t i o n i n f l a v o n o i d s i s shown to o c c u r i n L u p i n u s . The questionsv.that a r i s e from E x p e r i -ment I a r e : 1) does the v a r i a t i o n have any g e o g r a p h i c a l or eco-~ l o g i c a l p a t t e r n , or are the v a r i a b l e i n d i v i d u a l s d i s t r i b u t e d at random throughout the range o f the s p e c i e s ? 2) Is t h i s v a r i a t i o n an e n v i r o n m e n t a l l y induced phenomenon or i s i t an e x p r e s s i o n of g e n o t y p i c d i f f e r e n t i a t i o n ? To answer these q u e s t i o n s , Experiments II and I I I were designed to m o n i t o r , more f u l l y and a c c u r a t e l y , the f l a v o n o i d v a r i a t i o n i n a Lupinus s p e c i e s . 53 Experiment I I : V a r i a t i o n i n Lupinus' s e r i c e u s Pursh„ L up i n u s s e r i ceus was used f o r t h i s more d e t a i l e d study be-cause of i t s wide d i s t r i b u t i o n and c o n s i d e r a b l e m o r p h o l o g i c a l v a r i a b i l i t y . I t grows i n the d r y , y e l l o w p i n e / s a g e b r u s h areas from s o u t h e r n B r i t i s h Columbia and A l b e r t a to n o r t h e r n A r i z o n a . H i t c h c o c k e t a_l_.. (1961) d e s c r i b e t h i s taxon as v a r y i n g "even .' more than the m a j o r i t y of our o t h e r l u p i n e s , i n both f l o r a l and v e g e t a t i v e c h a r a c t e r s . " I t s pubescence may be s p a r s e to abundant, s h o r t appressed to l o n g and s p r e a d i n g . Flowers vary i n s i z e , c o l o u r and the amount of a d a x i a l pubescence on the ••'* e banner p e t a l . Hence, 23 synonyms are l i s t e d f o r the s p e c i e s by H i t c h c o c k ejt aj_. (1961) w h i l e Fleak (1971) regarded 61 t a x a as b e l o g i n g to the L^ . s e r i ceus complex. I t i s thus an i d e a l taxon i n which t o s t u d y whether m o r p h o l o g i c a l and g e o g r a p h i c p a t t e r n s are p a r a l l e l e d by d i s t i n c t i v e f l a v o n o i d p a t t e r n s . M a t f l C i ^ l s . a n d ^ M ^ t h ^ d s A t o t a l of 181 i n d i v i d u a l s o f L^ . s e r i c e u s were c o l l e c t e d from 32 p o p u l a t i o n s i n the western U n i t e d S t a t e s and Canada d u r i n g the summers of 1978/79. F i g u r e 5 maps the l o c a l i t i e s of the p o p u l a t i o n s . From 2 to 10 p l a n t s were o b t a i n e d from each p o p u l a t i o n as shown i n Appendix IV. Each p l a n t ( o r p a r t thereofY was p l a c e d i n a paper bag and a i r - d r e i d . A voucher specimen was pr e p a r e d from each p o p u l a t i o n . From each i n d i v i d u a l one gram of d r i e d l e a f l e t s was e x t r a c t e d i n the usual way (see Chapter I I ) i n r e a d i n e s s f o r h i g h p r e s s u r e l i q u i d chromatography. 54 FIGURE 5. Map showing the l o c a t i o n s of 32 p o p u l a t i o n s of Lupinus  s e r i c e u s monitored f o r i n f r a s p e c i f i c f l a v o n o i d v a r i a t i o n . 55 High Pressure_ (Per formance ) L i q u i d Chromatography (HPLC) The use of HPLC to i n v e s t i g a t e the f l a v o n o i d s of i n d i v i d u a l specimens of Lupinus s e r i c e u s was p r e f e r r e d fo r a number of r e a -sons . Not on ly i s .it a f a s t method fo r the q u a l i t a t i v e a n a l y s i s o f complex mixtures of compounds, but a l s o , by s t a n d a r d i z i n g e x t r a c t i o n , i n j e c t i o n and so l ven t g rad i en t p rocedu res , a compara-t i v e measure of f l a v o n o i d q u a n t i t i e s i s o b t a i n e d . The techn ique has become widespread on ly in the l a s t s i x to e igh t years ( K i ng -s t o n , 197 9 ) . Th is r e s u l t s from the i n c r e a s i n g a v a i l a b i l i t y o f i n s t r umen ta t i on and advances in column packing m a t e r i a l s . High pressure 1 i q u i d chromatography i s s imply a r e f i n e d method of column or l i q u i d chromatography. It i n vo l ves the use of a pump (see F igure 6) to fo r ce a pre-set flow of so l ven t through a column of very f i n e , uni form packing m a t e r i a l . The so l ven t may be a u t o m a t i c a l l y mixed in a so l v en t programmer set to d e l i v e r a p r e c i s e g r a d i e n t . Compounds are i n j e c t e d in to the so l ven t p r i o r to en t e r i ng the column. A f t e r s e p a r a t i o n , c o n s t i t -uents are monitored by a UV absorbance d e t e c t i o n system as they leave the column. Markham (1975) l i s t s f i v e advantages of HPLC over other chromatographic methods: i ) shor t a n a l y s i s t ime ; i i ) high r e s o l u t i o n ; i i i ) easy q u a n t i f i c a t i o n ; i v ) no requirement f o r d e r i v a t i z a t i o n o f compounds; v) no r i s k o f thermal decompo-s i t i o n of compounds. The l a t t e r two advantages are in d i r e c t comparison to gas chromatography of f l a v o n o i d s . A l l types of LC ( l i q u i d - s o l i d , p a r t i t i o n , ion exchange and e x c l u s i o n chromatography) can be mod i f i ed to be performed on HPLC equipment. But , in p r a c t i c a l te rms, the use of HPLC to g ive f a s t , r e p r o d u c i b l e and q u a n t i f i a b l e r e s u l t s is beset with p i t -56 FIGURE 6. Diagrammatic r e p r e s e n t a t i o n of a high p r e s s u r e l i q u i d chromatograph system. SOLVENT A SAMPLE INJECTION SOLVENT B SOLVENT PROGRAMMER PUMP COLUMN CHART RECORDER UV DETECTOR OUT 57 f a l l s . Speed of s epa ra t i on a f f e c t s the r e s o l u t i o n as in any chromatography. Re so lu t i on is a l so a f f e c t e d by the f low-ra te and so lvent/pack ing/compound i n t e r a c t i o n s . These e f f e c t s lead d i -r e c t l y to problems o f r e p r o d u c i b i l i t y , v i t a l to any q u a n t i t a t i v e s tudy . Whereas a p a r t i t i o n column of A v i c e l ( m i c r o c r y s t a l 1 i n e c e l l u l o s e ) can be c leaned and d i s c a rded a f t e r use , HPLC columns are much more permanent (and e x p e n s i v e ! ) . I n t e r a c t i o n s that cause i r r e v e r s i b l e b i nd ing between pack ing and compound mixtures (or part t he r eo f ) or decompos i t ion of the packing mate r i a l by poor cho i ce of so l v en t s r e s u l t in a gradual d e t e r i o r a t i o n of r e s o l u t i o n ; peaks become lower and broader and consequent l y tend to merge i n t o each o t h e r . R e p r o d u c i b i l i t y a l so depends on the use of s t anda rd i zed c y c l e s of sample i n j e c t i o n s , so l v en t g r ad -i en t s and column washings . F i n a l l y , problems with q u a n t i f i c a t i o n e x i s t . Compounds having d i f f e r e n t e x t i n c t i o n c o e f f i c i e n t s at the d e t e c t o r wavelength may show s i m i l a r peak he ights even though t h e i r concen t r a t i ons d i f f e r . U ? ! r ? _ ° f _ f l ^vono i ds Normal-phase ( adso rp t i on ) chromatography on s i l i c a gel has been used by Kingston (1979) to separa te f l a v o n e s . A l s o , p o l y -amide columns e l u t ed with water/methanol g r ad i en t s have been used to separate anthocyan ins (Manley & Shubiak , 1975 ) and f l a vanone/ cha lcone mixtures (Ward & P e l t e r , 1974 ; C o l l e t ejt aj_. , 1975). However, most of the s u c c e s s f u l s epa ra t i ons of f l a v o n o i d s come from the use o f reverse-phase p a r t i t i o n chromatography. Reverse-phase means that the s t a t i o n a r y phase is l e s s po la r than the mo-b i l e phase; in consequence , compounds are e l u t ed in o rde r o f dec reas ing p o l a r i t y . 58 Non-polar s t a t i o n a r y phases are bonded c h e m i c a l l y to an i n e r t s u p p o r t , u s u a l l y s i l i c a g e l . The s i l o n y l groups (=Si-0H) are e s t e r i f i e d with f u n c t i o n a l groups such as o c t y l , oc tadecy l , pheny l , a lky lamine or a l k y l n i t r i l e (Johnson & S tevenson , 1978) with the f i r s t two most w ide ly used f o r the s e p a r a t i o n of phe-n o l i c compounds. Severa l so l ven t systems .have been employed f o r f l a v o n o i d s epa ra t i on in reverse-phase HPLC. Methanol/water g rad i en t s a c i d -i f i e d to 5% a c e t i c a c i d s u c c e s s f u l l y r e so l v ed the f l a v o n o i d s of wine (Wulf & Nagel , 1976 ), buckwheat (S t rack & Krause , 1978) and oats ( S t rack et al_. , 1979). W a t e r / a c e t o n i t r i 1 e g rad ien ts were used to separa te i somer i c g l y c o f l avones by Becker et a]_. ( 1977) and i somer i c i s o f l a v o n e s by West ejt aj_. ( 1978). L a s t l y , a meth-anol/water g r ad i en t with a phosphate b u f f e r has been used to r e s o l v e g l y co f l a vones and t h e i r g l y c o s i d e s by Niemann & Brederode (1978) , and tobacco pheno l i c s by Court (1977) . HPLC o f Lupinus s e r i c e u s f l a v o n o i d s The pheno l i c e x t r a c t s of each 1 g sample of I. s e r i c e u s l e a f l e t s were d i s s o l v e d in 1 ml a l i q u o t s of methanol . One micro-l i t r e of each sample was i n j e c t e d in to the HPLC system. The chromatography was performed on a T raco r 900 s e r i e s ins t rument f i t t e d with a 25 cm X 4.6 mm ID column of P a r t i s i l 10 ODS (5% C-^ g oc tadecy l s i 1 ane , Whatman L t d . ) . A g rad i en t of methanol/water with 0.033M monobasic potass ium phosphate (KH^PO^) was employed. The so l v en t programmer was set to d e l i v e r 10% MeOH fo r 10 m i n . , then a concave (80% n o n - l i n e a r i t y ) g rad ien t to 90% MeOH l a s t i n g 30 min. The 90% MeOH was cont inued fo r 5 min. before a 10 min . column wash with pure MeOH. The system was r e - e q u i l i b r a t e d with 10% MeOH f o r 5 min. b e f o r e i n j e c t i o n of the next sample. T o t a l time per sample was 1 hour. The procedure was run a t room tem-p e r a t u r e and at a f l o w r a t e of 1 ml/min. A p r e s s u r e of 800-1400 p s i was m a i n t a i n e d depending upon the s o l v e n t . Compound d e t e c -t i o n i n v o l v e d a 254 nm UV d e t e c t o r connected to a c h a r t r e c o r d e r . A t y p i c a l chromatogram i s shown i n F i g u r e 7. B?§ul t s _ a ^ d _ a ^ a j ^ s i s F i f t e e n f l a v o n o i d peaks were i d e n t i f i e d to s p e c i f i c com- • pounds by comparison of t h e i r r e t e n t i o n times w i t h those o f known f l a v o n o i d s . These had been i s o l a t e d p r e v i o u s l y from a bulk sample of Lupinus s e r i c e u s c o l l e c t e d from Lumby, BC. Two a d d i t i o n a l peaks were noted but remain u n i d e n t i f i e d . Q u a n t i f i -c a t i o n of each compound was a c h i e v e d by measuring the h e i g h t of each peak. T h i s method was c o n s i d e r e d s a t i s f a c t o r y s i n c e a l l the e x t i c t i o n c o e f f i c i e n t s , a t 254 nm, o f the f l a v o n o i d s o f L^ . s e r -i c e u s are almost i d e n t i c a l ( J u r d , 1964). Of the 17 peaks o b s e r v e d , 10 were c o n s i d e r e d major or minor c o n s t i t u e n t s . Nine o f these were p r e s e n t i n a l l p o p u l a t i o n s and over 90% of i n d i v i d u a l s , but one compound, i s o o r i e n t i n , v a r i e d from co-dominance i n many p r o f i l e s to absence from 63 o t h e r s and 4 p o p u l a t i o n s . The r e m a i n i n g 7 f l a v o n o i d s were i n t e r m i t t e n t i n t h e i r d i s t r i b u t i o n , b u t , when p r e s e n t , were u s u a l l y i n t r a c e q u a n t i t i e s o n l y (Appendix I V ) . The 181 chromatograms, when com-pared v i s u a l l y , showed no obvious groupings or d i s c o n t i n u i t i e s . Q u a n t i t a t i v e l y , every compound v a r i e d c o n t i n u o u s l y . However, i t was obvio u s t h a t some compounds v a r i e d more than o t h e r s . In p a r t i c u l a r , o r i e n t i n (peak 2 ) , a major component t h r o u h o u t , ranged from a s i t u a t i o n where i t co-dominated 60 FIGURE 7. HPLC of a t y p i c a l Lupi nus s e r i c e u s i n d i v i d u a l l 2 i I 1 i I 1 = o r i e n t i n 3 ' - 0 - g l u . I i 2 = o r i e n t i n 3 = i s o o r i e n t i n 4 = o r i e n t i n acy l I 5 = v i t e x i n 6 = v i t e x i n acy l I 7 = l u t e o l i n 7-0-g lu . 8 = ap igen in 7-0-g lu . 9 = l u t e o l i n 10= v i t e x i n acy l II 11= unknown b i o s i d e 12= o r i e n t i n 4 ' -0-g1u . 13= o r i e n t i n acy l II . 14= ap igen in 6 15 = i s o v i t e x i n the p r o f i l e with other compounds to where i t was in such l a rge q u a n t i t i e s as to dwarf the peaks of the o ther f l a v o n o i d s . A l s o , as ment ioned, i s o o r i e n t i n was extremely v a r i a b l e . N e v e r t h e l e s s , by v i s u a l assessment , no c l e a r - c u t d i v i s i o n s cou ld be made. The accumula t ion of such o b j e c t i v e data from HPLC made an o b j e c t i v e a n a l y s i s o f the data h i g h l y d e s i r a b l e . It was t h e r e -fo re dec ided to use a computer-aided numerical taxonomic method of a n a l y s i s . Many cho ices of c l a s s i f i c a t o r y programmes were a v a i l a b l e and ranged from s imple c l u s t e r i n g a n a l y s i s to more complex m u l t i v a r i a t e methods. Parker (1976) compared var ious numerical ana lyses of f l a v o n o i d data from the L imnanthaceae. He found that c l u s t e r a n a l y s i s generated ra the r u n s a t i s f a c t o r y two-dimensional dendrograms whereas a m u l t i v a r i a t e o r d i n a t i o n more f u l l y expressed the r e l a t i o n s h i p s between the d i ve rgen t e v o l u t i o n a r y l i n e s of the f a m i l y . S i m i l a r l y , C l i f f o r d & Stephen son (1975) note that in the p roduc t ion of dendrograms, minimum spanning t r e e s , e t c . there was c o n s i d e r a b l e l o s s of i n fo rma t i on dur ing the genera t ion of such s i m p l i f i e d two-dimensional f i g -u r e s . They go on to suggest that much of t h i s i n f o rma t i on l o s s is avoided by examining the data in three or more d imens ions . S ince the present study was a phytochemical one and not an e x c e r c i s e in s t a t i s t i c s i t was dec ided to take the adv ice of the e a r l i e r workers and use a m u l t i v a r i a t e a n a l y s i s of the da t a . P r i n c i p a l components a n a l y s i s (PCA) was chosen s i n ce i t was a v a i l a b l e through the UBC computing f a c i l i t y (an Amdhal 470V/6 •Model II) us ing MIDAS. Th is i s a package of programmes d i s t r i b u ted by the S t a t i s t i c a l Research Labora tory of the U n i v e r s i t y o f Michigan (Fox & G u i r e , 1976) and run under the Michigan Terminal System (MTS) . 62 P r i n c i p a l components a n a l y s i s (PCA) P r i n c i p a l components a n a l y s i s has an im p o r t a n t p l a c e i n p l a n t taxonomy ( C l i f f o r d & Stephenson, 1975); r e c e n t a p p l i c a t i o n s to the study o f i n f r a s p e c i f i c v a r i a t i o n i n c l u d e A c a c i a harpo-phyl 1 a ( C o l d r a k e , 1971), Eucal yptus carnal dul ens i s ( B u r l e y e_t a l . , 1971) and the Limnanthaceae ( P a r k e r , 1976). I t i s an o r d i n a t i o n method t h a t makes no assumptions about the e x i s t e n c e of groupings among the e n t i t i e s being s t u d i e d . In general terms, PCA reduces the d i m e n s i o n a l i t y of a problem by c a l c u l a t i n g axes t h a t summa-r i z e s i m i l a r i t y r e l a t i o n s among e n t i t i e s more e f f i c i e n t l y than the raw a t t r i b u t e s . C o n s i d e r x p l a n t s , t a x a , e t c . ( c a l l e d OTU's i . e . o p e r a t i o n a l taxonomic u n i t s , a f t e r Sokal & Sneath, 1963) sco r e d f o r y a t t r i b u t e s . Each OTU can be p l o t t e d a c c u r a t e l y i n two dimensions f o r each p a i r of a t t r i b u t e s ( F i g u r e 8). P o i n t s f a r a p a r t on the o r d i n a t i o n are q u i t e d i s s i m i l a r , b ut, o n l y f o r those two a t t r i b u t e s . A t h i r d a t t r i b u t e may be shared by the two taxa t h a t are f a r a p a r t . In or d e r to see a c c u r a t e l y the r e l a -t i o s h i p s between OTU's f o r t h r e e a t t r i b u t e s a t h i r d o r t h o g o n a l a x i s must be c o n s t r u c t e d and so on, r e s u l t i n g i n a y - d i m e n s i o n a l c l o u d of p o i n t s . The a t t r i b u t e t h a t most w i d e l y spreads the OTU's ( i . e . accounts f o r the most v a r i a t i o n ) would l o g i c a l l y b e the best c h a r a c t e r on which to base an i n i t i a l d i v i s i o n . T his i s , o f c o u r s e , e q u i v a l e n t to cho o s i n g a key c h a r a c t e r . As we have seen i n L u p i n u s , i s o l a t e d c h a r a c t e r s do not s a t i s f a c t o r i l y s u b d i v i d e the genus. P r i n c i p a l components a n a l y s i s p l o t s the OTU's i n y dimen-s i o n s and then chooses a new a x i s , i n m u l t i d i m e n s i o n a l space, a l o n g which t h e r e i s g r e a t e s t v a r i a n c e C s e e F i g u r e 9 ) . The new FIGURE 8. The r e l a t i o n s h i p between OTU's compared with respec t to two a t t r i b u t e s (A &; B) . -t-> « data c e n t r o i d o| , • CU i £ ro S-^ _ A range of a t t r i b u t e A FIGURE 9. O rd ina t i on of the F i g . 8 OTU's with 1st and 2nd component axes . Component axes are unco r r e l a t ed and account fo r a g rea te r p r o p o r t i o n of the va r i ance among OTU's than was exp la ined by the o r i g i n a l a t t r i b u t e s s e p a r a t e l y . ax is (or component) does not cor respond to any s i n g l e cha rac t e r but has c o n t r i b u t i o n s from a l l a t t r i b u t e s . U s u a l l y , some c h a r -a c t e r s c o n t r i b u t e more s t r o n g l y to c e r t a i n components than o t h e r s . Such components may be i n t e r p r e t e d as summarizing c o r -r e l a t e d pa t te rns of small groups of c h a r a c t e r s . Fo l low ing the e x t r a c t i o n of the f i r s t component and i t s v a r i a n c e , a second component i s c a l c u l a t e d o r t h o g o n a l l y that again shows g rea tes t sp read . In a s tepwise f a sh ion . t h i r d , f o u r t h , f i f t h , e t c . , or thogonal axes are ex t r a c t ed which account fo r p r o g r e s s i v e l y l e s s v a r i a n c e . An excep t ion to t h i s s i t u a t i o n would occur i f the data were random. Mu l t i d imens iona l o r d i n a t i o n would be a sphere and consequen t l y , each component would account fo r an equal (100/y%) share of the o v e r a l l v a r i a n c e . For more d e t a i l e d mathematical exp l ana t i ons of PCA see Seal (1964) , B l a c k i t h & Reyment (1971) and Mor r i son (1967) . Resu l ts and d i s c u s s i o n of PCA on Lupinus s e r i c e u s f l a v o n o i d s The o r d i n a t i o n produced by PCA on the HPLC data from Lupi nus  s e r i c e u s is shown in F igure 10. The f i r s t component ax i s a c -counts fo r 72% of the t o t a l va r i ance whi le the second ax is a c -counts fo r 12%. The e igenvec to r s of the f l a v o n o i d cha rac t e r s are shown in Table IX. From the t ab l e i t can be seen that ax i s I i s dominated by one compound, o r i e n t i n ( v a r i a b l e No .2 ) . I ts high negat i ve c o e f f i c i e n t means that i n d i v i d u a l s f a r to the l e f t of the data c e n t r o i d have h igher l e v e l s of the compound. (The p o s i -t i v e or nega t i ve s ign i s meaningless except fo r g i v i n g s p a t i a l r e f e r ence to the data c e n t r o i d . ) Ax is II i s dominated by i s o -o r i e n t i n ( v a r i a b l e N o . 3 ) ; i n d i v i d u a l s r i c h in t h i s compound have been o rd ina t ed to the top of F igure 10. It i s i n t e r e s t i n g to FIGURE 10. Ord ina t ion of 181 i n d i v i d u a l s of Lupinus s e r i c e u s 1 1 by PCA on HPLC da ta . • Component I accounts fo r 72% t o t a l va r i ance • Component II accounts fo r 12% t o t a l va r i ance • • • • • • • • • • • • • • • • • • •• * •. S ! . * -4 ON 6 7 TABLE IX. Table of e i g e n v e c t o r s o f the 17 f l a v o n o i d v a r i a b l e s f o r the f i r s t t h r e e component axes of PCA. <PRINC0M VAR=1-17 CASES=1-181 OPTION=UNSCALED MAX=3> PRINCIPAL COMPONENTS CASES=CASE/f : 1 - 18 1 TEST STATISTIC DF SIGNIF N= 181 OUT OF 181 SPHERICITY 7516.G 152 (1) (2) (3) COMPONENT 7877.3 132 1.4 1124.5 % VARIANCE 71.79 83.84 94.09 SPHERICITY 4496.5 3513.2 1559.1 DF 135 119 104 SIGNIF 0. 0. 0. 1 V1 .23858 -1 -- .25553 .95647 2 V2 -.99086 .10748 .54483 -1 3 V3 .12088 .90839 .22244 4 V4 .90960 -3 .18869 .114 19 5 V5 .54576 -2 .24231 .39062 - 1 6 V6 - . 44475 -2 .13746 - 1 .38776 -1 7 V7 -. 12235 - 1 .60957 -2 .21094 -1 8 V8 -.41529 -3 .42099 -2 . 11980 - 1 9 V9 -.10792 - 1 • - .99948 -3 .21 188 -1 10 V10 - . 2284 1 -1 .4066 1 -2 -- . 13342 -1 1 1 V1 1 .15624 -1 . 16776 - 1 . 14057 - 1 12 V12 .24344 -1 • - .25529 -2 .58985 -1 13 V13 -.61093 -2 .15100 - 1 .38988 -1 14 V14 .20666 -2 -.12117 -2 .27373 -1 15 V15 .33415 -1 .23834 - 1 .7087 1 -1 16 V16 -.45330 -3 .14478 - 1 .55544 -1 17 V17 .13489 - 1 .45901 -1 .37289 -1 68 note t h a t PCA o b j e c t i v e l y e x t r a c t e d the same two compounds f r o m , the data (as b e i n g o f h i g h e s t v a r i a b i l i t y or i mportance) as the present author d i d s u b j e c t i v e l y . F i g u r e 10 t h e r e f o r e shows a p o i n t spread from those i n d i v i d u a l s having high o r i e n t i n c o n t e n t which t o t a l l y dominates the o t h e r c o n s t i t u e n t s ( f a r l e f t ) to those i n which o r i e n t i n and i s o o r i e n t i n occur i n more s i m i l a r q u a n t i t i e s (top r i g h t ) . S i n c e a x i s I I o n l y accounts f o r 12% o f the t o t a l v a r i a n c e compared to 72% i n a x i s I , the v e r t i c a l spread of p o i n t s may be an e x a g g e r a t i o n of the r e a l s i t u a t i o n . An obvious f e a t u r e of F i g u r e 10 i s the l a c k o f d i s c r e t e groupings ( a g a i n a g r e e i n g w i t h the i n i t i a l v i s u a l a s s e s s m e n t ) . I t appears t h a t i n d i v i d u a l s of j . . s e r i c e u s vary c o n t i n u o u s l y i n t h e i r f l a v o n o i d , or more s p e c i f i c a l l y , o r i e n t i n a c c u m u l a t i o n . However, i f p l a n t numbers were i n s e r t e d onto F i g u r e 10 i t would be seen t h a t a d e f i n i t e p a t t e r n of v a r i a t i o n i s e v i d e n t . To the l e f t of the o r d i n a t i o n are i n d i v i d u a l s of s outhern d i s t r i b u t i o n ; t o the r i g h t are m o s t l y n o r t h e r n p l a n t s . The gradual change i n o r i e n t i n l e v e l s i s a p p a r e n t l y r e l a t e d to some aspect of l a t i t u d e . Huxley (1938) c o i n e d the name ' c l i n e ' f o r a c h a r a c t e r g r a -d i e n t , t h a t i s , any v a r i a t i o n a l t r e n d i n space a p p e a r i n g i n a p o p u l a t i o n or s e r i e s o f p o p u l a t i o n s o f a s p e c i e s . To i n v e s t i g a t e the apparent v a r i a t i o n t r e n d i n j . . s e r i c e u s p o p u l a t i o n s another m u l t i v a r i a t e a n a l y s i s ( c a n o n i c a l v a r i a t e s a n a l y s i s ) was under-taken . C a n o n i c a l v a r i a t e s a n a l y s i s (CVA) U n l i k e PCA, CVA r e q u i r e s t h a t the data be i n s e t s or groups; i t then i n v e s t i g a t e s the r e l a t i o n s h i p s between the groups. (See 69 S e a l , 1964 f o r a f u l l mathematical e x p l a n a t i o n . ) S i n c e CVA be-gins w i t h a l r e a d y d e f i n e d c l u s t e r s , i t i s not p r o p e r l y a c l a s s i -f i c a t i o n s t r a t e g y and has not been so w i d e l y used i n p l a n t t a x -onomy ( C l i f f o r d & Stephenson, 1975). However, s i n c e our data are i n 32 n a t u r a l p o p u l a t i o n a l g r o u p i n g s , CVA would seem an a p p r o p r i a t e a n a l y t i c a l method to determine i f the p o p u l a t i o n s are i n a c l i n a l or a more d i s c o n t i n u o u s d i s t r i b u t i o n . R e s u l t s and d i s c u s s i o n of CVA on Lupinus s e r i c e u s p o p u l a t i o n s F i g u r e 11 shows the o r d i n a t i o n o f the 32 p o p u l a t i o n s o f Lupinus s e r i c e u s . The p o i n t s are the data c e n t r o i d s o f each p o p u l a t i o n . A x i s I accounts f o r 42% o f the t o t a l v a r i a n c e and a x i s II accounts f o r 12%. I t i s c l e a r t h a t the v a r i a t i o n p a t t e r n shown i n the PCA o r d i n a t i o n o f i n d i v i d u a l p l a n t s i s l e s s c o n t i n -uous i n the CVA o r d i n a t i o n o f p o p u l a t i o n s . A more u s e f u l diagram i s F i g u r e 12 i n which the p o p u l a t i o n c e n t r o i d s have +/-1 s t a n d a r d d e v i a t i o n p l o t t e d a l o n g each a x i s to g i v e some i n d i c a t i o n of i n f r a p o p u l a t i o n a l v a r i a t i o n . Three groups are apparent from t h i s o r d i n a t i o n . P o p u l a t i o n s 1-6 form a very t i g h t grouping and have r e l a t i v e l y low i n f r a -p o p u l a t i o n a l v a r i a b i l i t y . The group shows l i t t l e o v e r l a p w i t h p o p u l a t i o n s 7-10 which form the middle group i n F i g u r e 12. Pop-u l a t i o n s 11-32 have much h i g h e r i n t e r n a l and e x t e r n a l v a r i a t i o n with, so much o v e r l a p t h a t a s u b d i v i s i o n of the group i s unwar-r e n t e d . The d i s c o n t i n u i t i e s shown by CVA appear q u i t e d i f f e r e n t from the c o n t i n u o u s v a r i a t i o n we saw from the PCA. The r e s u l t of the CVA suggests more of an e c o t y p i c type of v a r i a t i o n to be p r e s e n t i n Lupinus s e r i c e u s . F u rthermore, the p o s i t i o n s o f the FIGURE 11. O rd ina t i on of 32 popu la t ions of Lupinus s e r i c e u s by CVA on HPLC da t a . Axis I accounts fo r 42% t o t a l va r i ance Axis II accounts for 12% t o t a l va r i ance 5 * 2 1 11 10 II 16 27 30 13.. 14 20 1 2 2 8 1 8 32 29 3119 23 25 26 21 22 24 o FIGURE 12. Ord ina t ion of 32 popu la t ions of Lupinus s e r i c e u s from CVA. Popu la t ion cen t ro ids are drawn with +/-1 standard d e v i a t i o n to show i n f r a p o p u l a -t i onal va r i a b i1 i t y . 72 p o p u l a t i o n s i n the o r d i n a t i o n can be p a r t l y e x p l a i n e d on a geo-g r a p h i c a l b a s i s . P o p u l a t i o n s 1-6 occur south of Great S a l t Lake, Utah and nor t h of the Grand Canyon, A r i z o n a . The d i s t r i b u t i o n of Lupinus  s e r i c e u s i n t h i s area i s almost c o n t i n u o u s and the p o t e n t i a l f o r g e n e t i c exchange i s very h i g h . Such a s i t u a t i o n c o u l d r e s u l t i n the l a c k of p o p u l a t i o n a T " d i f f e r e n t i a t i o n t h a t i s apparent i n these s o u t h e r l y l u p i n e s . P o p u l a t i o n s 7-10 are i n the Snake R i v e r area and a j o i n i n g v a l l e y s . These p o p u l a t i o n s are separated from the s o u t h e r l y group by Great S a l t Lake and Great S a l t Desert a c r o s s which l i t t l e gene f l o w would be ex p e c t e d . They are sep-a r a t e d from many o f the n o r t h e r l y p o p u l a t i o n s by the high montane areas of the Sawtooth Range and the C o n t i n e n t a l D i v i d e . In the nor t h we see t h a t many p o p u l a t i o n s , p a r t i c u l a r l y Nos. 11-18, 32 are c l o s e l y grouped. These m o s t l y r e p r e s e n t c o l l e c t i o n s from the almost c o n t i n u o u s t r a c t s o f L. s e r i c e u s i n the^ g r a s s l a n d s of Montana, Idaho and A l b e r t a . Less s i m i l a r are p o p u l a t i o n s 19-31 t h a t were c o l l e c t e d from the more i s o l a t e d s i t e s i n the e a s t e r n Cascades and Rocky Mountains where t h e r e i s f a r l e s s c o n t i n u i t y i n the d i s t r i b u t i o n of j . . s e r i c e u s . This may have r e s u l t e d i n a l a c k o f gene fl o w and the development of d i f f e r e n c e s between p o p u l a t i o n s . In summary, we see t h a t PCA r e v e a l e d t h a t Lupinus  s e r i c e u s i n d i v i d u a l s v a r i e d c o n t i n u o u s l y i n t h e i r a c c u m u l a t i o n of f l a v o n o i d s ; at the p o p u l a t i o n a l l e v e l , CVA shows the s p e c i e s to be i n more of a d i s c o n t i n u o u s p a t t e r n t h a t may be regarded as e c o t y p i c i n n a t u r e . In both m u t i v a r i a t e a n a l y s e s (PCA and CVA) a l l 17 f l a v o n o i d c h a r a r a c t e r s were, used. 0 n e compound however, o r i e n t i n , dominated 73 the a n a l y s e s so much t h a t the f i r s t axes were b a s i c a l l y measures of o r i e n t i n a c c u m u l a t i o n i n the i n d i v i d u a l s and p o p u l a t i o n s . B e a r i n g i n mind the statement o f S t e b b i n s (1944) t h a t "too much emphasis on- g r a d i e n t s i n s i n g l e c h a r a c t e r s may ser v e to confuse r a t h e r than c l a r i f y the whole p i c t u r e o f v a r i a t i o n " , i t was de-c i d e d to i n v e s t i g a t e f u r t h e r the v a r i a t i o n o f j . . s e r i ceus popu-l a t i o n s i n terms of o r i e n t i n a c c u m u l a t i o n o n l y . The a n a l y s i s o f o r i e n t i n a c c u m u l a t i o n i n Lupinus s e r i c e u s p o p u l a t i o n s One-way a n a l y s i s of v a r i a n c e and Duncan's M u l t i p l e Range Test ( S t e e l & T o r r i e , 1960) was used to compare o r i e n t i n accumu-l a t i o n means f o r each p o p u l a t i o n . Table X shows the r e s u l t s o f the a n a l y s i s of v a r i a n c e . The F-value i n d i c a t e s t h a t t h e r e i s a s i g n i f i c a n t d i f f e r e n c e between the p o p u l a t i o n means f o r o r i e n t i n a c c u m u l a t i o n . As i n the CVA, Table XI shows t h a t p o p u l a t i o n s 1-6 are not s t a t i s t i c a l l y d i f f e r e n t from each o t h e r at the 1% l e v e l . Every o t h e r p o p u l a t i o n , except f o r No.16 i s s i g n i f i c a n t l y d i f f e r e n t from the southern s e x t e t but not s i g n i f -i c a n t l y d i f f e r e n t from each o t h e r . P o p u l a t i o n 16 forms a b r i d g e ( s t a t i s t i c a l l y ) between the two groups. G e o g r a p h i c a l l y t h i s p o p u l a t i o n i s f a r removed from Nos. 1-6; the i n t e r m e d i a t e o r i e n t i n l e v e l may s i m p l y be a chance oc c u r e n c e . I t sh o u l d be remembered t h a t when the o t h e r f l a v o n o i d c h a r a c t e r s were i n v o l v e d i n the a n a l y s i s of p o p u l a t i o n a l d i f f e r e n c e s , No.16 was o r d i n a t e d wel1 away from Nos. 1-6. The o v e r a l l p i c t u r e of o r i e n t i n a c c u m u l a t i o n i n J_. s e r i c e u s i s one of a sharp d i s c o n t i n u i t y between the p o p u l a t i o n s south of 74 TABLE X. A n a l y s i s o f v a r i a n c e f o r o r i e n t i n a c c u m u l a t i o n i n Lupinus s e r i c e u s p o p u l a t i o n s Source of v a r i a t i o n df Sum of squares Mean square F among ~, p o p u l a t i o n s 980062.08 31614.96 11.34** w i t h i n p o p u l a t i o n s 149 415493 .07 2788. 54 T o t a l 1395555 .15 ** denotes s i g n i f i c a n c e at the 1% l e v e l 75 TABLE XI. O r i e n t i n a c c u m u l a t i o n means a n a l y s e d by Duncan's M u l t i p l e Range t e s t f o r 32 p o p u l a t i o n s of L_. s e r i c e u s . (Shown a l s o are the i s o o r i e n t i n a c c u m u l a t i o n means.) P o p u l a t i o n mean peak mean peak number h e i g h t o f h e i g h t o f o r i e n t i n i s o o r i e n t i n 4 266 0 5 259 0 2 231 0 1 209 10 3 208 0 6 201 2 16 148 88 17 109 52 14 106 45 11 104 32 31 96 74 15 95 55 30 91 77 29 83 72 19 83 57 10 81 48 13 79 55 8 76 19 32 76 54 28 72 36 24 69 53 18 57 21 9 55 19 7 43 22 27 43 52 20 41 63 21 38 43 12 3 5 43 23 31 59 22 29 25 26 26 21 25 19 24 D i f f e r e n c e s between p o p u l a t i o n s j o i n e d by the same l i n e are not s i g n i f i c a n t at the 1% 1evel . 76 Great S a l t Lake and those n o r t h . At the very l e a s t , t h e r e i s a s u g g e s t i o n of e c o t y p i c d i f f e r e n t i a t i o n . Fleak (1971) notes t h a t I. s e r i c e u s i n t h i s same area has heavy a n t h o c y a n i n pigment-a t i o n i n the lower p a r t of the stems. I t i s a c h a r a c t e r t h a t i s f a r l e s s apparent i n n o r t h e r l y p o p u l a t i o n s .and has l e d Fleak & Dunn (.1971) to d e l i m i t these s o u t h e r l y l u p i n e s as L_ s e r i c e u s s s p . huffman1i r a n g i n g from n o r t h - c e n t r a l Utah south to the Kaibab P l a t e a u of A r i z o n a . S t e b b i n s (1944) s t a t e s : " i f a number o f c l i n e s run p a r a l l e l and are p a r t l y d i s c o n t i n u o u s , w i t h a steep g r a d i e n t i n some r e g i o n s and a moderate one or a c o n s t a n t l e v e l i n o t h e r s , then the d i f f e r e n t l e v e l s o f v a r i a t i o n i n the c h a r a c t e r s f o r m i n g the c l i n e s may con-s t i t u t e p a r t of the b a s i s of l o c a l races or s u b s p e c i e s . " I t i s c l e a r t h a t o r i e n t i n a c c u m u l a t i o n p a r a l l e l s the d i s c o n t i n -uous c l i n e of a n t h o c y a n i n p i g m e n t a t i o n of I. s e r i ceus; thus i t s u p p o r t s the s u b s p e c i f i c r a n k i n g o f I. s e r i ceus s s p . huffmani i i n t h i s a r e a . Table XI a l s o shows the p o p u l a t i o n means of i s o o r i e n t i n . Although a n a l y s i s of v a r i a n c e c o u l d not be per-formed ( s i n c e some p o p u l a t i o n s were i n v a r i a b l e and d e v o i d o f the compound) the d i s c o n t i n u i t y between the s o u t h e r l y p o p u l a t i o n s and those to the n o r t h i s again e v i d e n t . U n f o r t u n a t e l y , i n the n o r t h , where Fleak (1971) r e c o g n i z e s t h r e e more s u b s p e c i e s o f J_. s e r i c e u s , f l a v o n o i d s t u d i e s show no d i s c o n t i n u i t y and can l e n d no support f o r t a x a d e l i m i t a t i o n here. S i n c e a l l the i n d i v i d u a l s i n t h i s study were c o l l e c t e d from t h e i r n a t i v e a r e a s , we have no e v i d e n c e to determine how much of t h i s v a r i a t i o n i n f l a v o n o i d content i s g e n o t y p i c . I t may be t h a t the d i f f e r e n c e s are s i m p l y examples of the p h e n o t y p i c p l a s t i c i t y t h a t H i t c h c o c k e_t a l _ . (1961) noted to be so common in J_. s e r i c e u s . In order to e s t a b l i s h the bas i s f o r the v a n t i o n in t h i s s p e c i e s , another a n a l y s i s (Experiment III.) was c a r r i ed ou t . 78 Experiment I I I : The d e t e r m i n a t i o n of the g e n o t y p i c or p h e n o t y p i c  nature of o r i e n t i n v a r i a t i o n i n Lupinus s e r i c e u s The q u e s t i o n o f g e n e t i c or e n v i r o n m e n t a l l y induced m o d i f i -c a t i o n s of a p l a n t s p e c i e s was f i r s t posed and answered by Turesson (1922). He e x c l u d e d e n v i r o n m e n t a l d i f f e r e n c e s by growing p l a n t s under u n i f o r m c o n d i t i o n s and r e - a s s e s s i n g t h e i r d i f f e r -ences. In a s i m i l a r manner, Experiment I I I i n v o l v e d the c o l l e c -t i o n of J.. s e r i ceus seed from throughout the n a t u r a l range of the s p e c i e s f o l l o w e d by g e r m i n a t i o n and growth of the p l a n t s i n a u n i f o r m environment. The s e e d l i n g s were then e x t r a c t e d and a n a l y s e d by HPLC. The d i f f i c u l t y i n growing j . . s e r i ceus to h e a l t h y mature p l a n t s n e c e s s i t a t e d t h e i r a n a l y s i s at the seed-l i n g s t a g e . Many i n d i v i d u a l s were noted to damp-off even though numerous growing c o n d i t i o n s were attempted. However, s i n c e i n an e a r l i e r study i t . was found t h a t t h e r e was l i t t l e d i f f e r e n c e i n the f l a v o n o i d c o n t e n t of s e e d l i n g s and mature p l a n t s of J_. a r b o r e u s , i t was d e c i d e d to proceed and use I. s e r i c e u s s e e d l i n g s f o r t h i s s t u d y . (Two-dimensional a n a l y s i s was per-formed on some s e e d l i n g s and the r e s u l t s were q u a l i t a t i v e l y i d e n t i c a l to those a t t a i n e d w i t h mature p l a n t s . ) A t o t a l o f twenty s e e d l i n g were s u c c e s s f u l l y r a i s e d from s i x p o p u l a t i o n s . The f r e s h l e a v e s were weighed and then e x t r a c t e d by p l a c i n g them i n methanol. The young t i s s u e s were r e a d i l y e x t r a c t e d by t h i s method; h e a t i n g and butanol e x t r a c t i o n s were c o n s i d e r e d unnecessary. The m e t h a n o l i c e x t r a c t s were i n j e c t e d i n t o the HPLC i n a s t a n d a r d i z e d way. For example, i f the e x t r a c t o f 0.05 g o f p l a n t m a t e r i a l was d i s s o l v e d i n 1 ml methanol, 10 ul was i n j e c t e d . In the case of 0.25 g of p l a n t m a t e r i a l i n 1 ml 7 9 methanol, 2 ul of e x t r a c t was i n j e c t e d . The very s m a l l amount of p l a n t m a t e r i a l used made the d e t e c t i o n of f l a v o n o i d s d i f f i c u l t . The u s u a l l y dominant peaks of o r i e n t i n , o r i e n t i n 3 1 - 0 - g l u c o s i d e and i s o o r i e n t i n were present but l e s s e r compounds appeared as small p o o r l y r e s o l v e d peaks. C o n s e q u e n t l y , o n l y the o r i e n t i n peak was measured and the r e s u l t s are shown i n Table X I I . From the t a b l e i t can be seen t h a t the o r i e n t i n peak h e i g h t s f o r a l l the s e e d l i n g s range between 42-66. As shown by the i n s i g n i f i c a n t F - v a l u e , t h e r e i s no evidence o f p o p u l a t i o n a l d i f -f e r e n c e s , c l i n a l or e c o t y p i c . T h i s r e s u l t suggests t h a t the v a r i a t i o n i n the a c c u m u l a t i o n o f o r i e n t i n i n Lupinus s e r i c e u s i s a r e f l e c t i o n o f some en v i r o n m e n t a l response t h a t i s r e l a t e d to some as p e c t of l a t i t u d e . G e n e t i c a l l y , a l l the J_. s e r i c e u s p l a n t s appear to have s i m i l a r c a p a c i t i e s f o r the p r o d u c t i o n and accumu-1 a t i o n of f1avonoi ds. TABLE XII . HPLC peak he ights of s e r i c e u s i n d i v i d u a l s c o n d i t i o n s . o r i e n t i n from 20 Lupinus grown from seed in uni form Popu la t i on and p lan t No. Seed source l o c a t i o n O r i e n t i n peak he ight 1.1 1.2 M a r y s v i l i e , Utah 50 46 2.1 2.2 2.3 2.4 Joe ' s V a l l e y Utah 47 66 46 53 3.1 3.2 3.3 Tony Grove, Logan, Utah 57 42 53 4.1 4.2 Shoshone, Idaho 45 48 5.1 5.2 5.3 5.4 L i v i n g s t o n e , Montana 58 42 49 48 6.1 6.2 6 . 3 6.4 6.5 Spokane, Washington 47 66 56 58 42 One-way a n a l y s i s of v a r i ance Source of v a r i a t i o n d . f Sums of squares Mean squares F Between . populat ions 5 126.23 25.25 0.40 w i th in popu la t i ons 14 874.72 62 .48 t o t a l 19 1000 .95 Duncan's m u l t i p l e range t e s t . 4 1 5 3 2 6 Popu la t ion means 46 48 49 51 53 54 No s i g n i f i c a n t d i f f e r e n c e between popu l a t i on means. 81 IV. INTERSPECIFIC FLAVONOID VARIATION IN LUPINUS M a t e r i a l s and methods Small amounts of d r i e d l e a f l e t m a t e r i a l (0.05-0.1 g) were removed from herbarium sheets t h a t r e p r e s e n t e d 73 d i f f e r e n t taxa of L u p i n u s . A p p r o x i m a t e l y 60 tax a are c o n s i d e r e d s e p a r a t e s p e c i e s (depending upon the taxonomic system) and 13 are c o n s i d e r e d sub-s p e c i e s and v a r i e t i e s . For c o n v e n i e n c e , and to a v o i d b i a s , each specimen was r e f e r r e d to as a numbered " o p e r a t i o n a l taxonomic u n i t " (OTU) a f t e r Sokal & Sneath (1963). Each sample was ground i n 80% methanol, f i l t e r e d and the e x t r a c t evaporated to dryness i n vacuo at 40°C. The r e s i d u e was r e d i s s o l v e d i n 0.5 ml methanol and then s p o t t e d at the c o r n e r s o f 15 X 15 cm polyamide TLC p l a t e s . These were developed i n two d i m e n s i o n s : f i r s t i n the aqueous s o l v e n t system, and, a f t e r thorough d r y i n g , i n the o r g a n i c s o l v e n t system. I n d i v i d u a l f l a v o n o i d s were i d e n t i f i e d by com-p a r i n g t h e i r Rf's and c o l o u r r e a c t i o n s ( w i t h boronate spray and ammonia vapours) w i t h those of known f l a v o n o i d s . Whenever new sp o t s were n o t i c e d or s u s p e c t e d the compounds were i s o l a t e d and c h a r a c t e r i z e d i n the normal manner (see Chapter I I ) i f s u f f i -c i e n t amounts of p l a n t m a t e r i a l were a v a i l a b l e . Data g e n e r a t i o n and a n a l y s i s Two-dimensional TLC of each OTU produced 73 maps (Appendix V) from which a t o t a l of 56 d i f f e r e n t f l a v o n o i d s were r e c o g n i z e d . S e v e r a l data t r e a t m e n t s were attempted. I n i t i a l l y , an attempt was made to s o r t the maps i n t o groups o f s i m i l a r p r o f i l e s but problems soon arose i n d e t e r m i n i n g which c h a r a c t e r s C f l a v o n o i d s ) 82 were s u f f i c i e n t l y " i m p o r t a n t " upon which to base d i v i s i o n s . Some obvious groupings d i d appear s i n c e they accumulated unusual com-pounds; c y t i s o s i d e o c c u r r e d i n j u s t e i g h t taxa and f l a v o n e 5-0-g l u c o s i d e s i n f i v e s p e c i e s . There were, however, many i n f r e -q u e n t l y seen compounds such as a c a c e t i n 7 - 0 - g l u c o s i d e which o c c u r r e d i n a few taxa t h a t o t h e r w i s e had very d i f f e r e n t p r o f i l e s , Should such taxa be lumped t o g e t h e r thus r a n k i n g a c a c e t i n 7-0-g l u c o s i d e as an i m p o r t a n t c h a r a c t e r ? Or sho u l d the group be s p l i t u p t h u s i g n o r i n g the d i s t r i b u t i o n o f a c a c e t i n 7-0-gluco-s i d e ? Such problems made the s u b j e c t i v e s o r t i n g of maps i n t o groups an u n s a t i s f a c t o r y method. Moreover, the grouping o b t a i n e d by v i s u a l l y s o r t i n g the maps had very low r e p r o d u c i b i l i t y . Thus, the 73 p r o f i l e s c o u l d be s p l i t i n t o as few as e i g h t groups and as many as 28 groups (many of them s i n g l e t a x a ) depending on the amount of i n f r a g r o u p v a r i a b i l i t y t h a t was t o l e r a t e d . T h e r e f o r e , i t was de c i d e d to a n a l y s e the data as o b j e c t i v e l y as p o s s i b l e u s i n g a computer-aided numerical c l a s s i f i c a t i o n method; once a g a i n p r i n c i p a l components a n a l y s i s was chosen. I t was hoped t h a t the a n a l y s i s would d e l i m i t the groups t h a t I had p r e v i o u s l y r e c o g n i z e d by m a p - s o r t i n g , and p o s s i b l y , expose o t h e r s t h a t I had fa i l e d to see. Data S e t _ I A data m a t r i x (73 OTUs X 56 f l a v o n o i d s ) c o n s i s t i n g o f 9.'s, 5 r s , l ' s and O's was c o n s t r u c t e d (see Table X I I I ) . Each v a l u e was an assessment o f the r e l a t i v e c o n c e n t r a t i o n of each compound w i t h i n any one OTU. Thus, a 9: denoted a f l a v o n o i d p r e s e n t i n major q u a n t i t i e s ; 5, a minor c o n s t i t u e n t ; 1 denoted a f l a v o n o i d p r e s e n t i n t r a c e q u a n t i t i e s and 0 meant t h a t the compound was TABLE X I I I . Data Set I. The d i s t r i b u t i o n of f l a v o n o i d s tn 73 Lupinus t a x a , (9=maJor c o n s t i t u e n t , 5=minor, l ' t r a c e . ) Fl avonolds Nos. compounds Taxa (OTU's) 11111111112222222222333333333344444444445555555555666666666677 77 Nos. 12345678901234 56789012345678901234567890123456789012 3456 789012 3456 789012 3 1 o r i e n t i n 2 v i t e x i n 3 o r i e n t i n a c y l I 4 v i t e x i n a c y l I 5 o r i e n t i n a c y l I I 6 v i t e x i n a c y l I I 7 l u t e o l i n 7 - 0 - g l u c o s l d e 8 a p i g e n i n 7 - 0 - g l u c o s l d e 9 l u . 7-0-glu. a c y l I 10 unknown 'green' 11 l u . 7-0-glu. a c y l I I 12 ap. 7-0-glu. a c y l I I 13 l u t e o l i n 14 a p i g e n i n 15 t s o o r l e n t l n 16 t s o v l t e x i n 17 pink f l a v a n o n e ( h i g h Rf) 18 pink f l a v a n o n e (mid Rf) 19 pink f l a v a n o n e (low Rf) 20 c y t i s o s i d e 21 c y t i s o s i d e a c y l I 22 c y t i s o s i d e a c y l I I 23 4 ' - 0 - m e t h y l o r t e n t 1 n 24 c h r y s o e r t o l 25 a c a c e t i n 26 c h r y . 7-0-glu. 27 c h r y . 7-0-glu. a c y l I 28 c h r y . 7-0-glu. a c y l I I 29 ac. 7-0-glu. 30 ac. 7-0-glu. a c y l I 31 l u . 7 - 0 - d l g l u . 32 ap. 7 - 0 - d i g l u . 33 c h r y . 7 - 0 - d l g l u . 34 ac. 7 - 0 - d l g l u . 35 kaempferol 36 kaem. 3-0-glu. 37 kaem. 7-0-glu. 38 q u e r c e t l n 39 quer. 3-0-glu. 40 quer. 7-0-glu. 41 I s o o r l e n t f n a c y l I 42 g e n l s t e i n 43 g e n l s t l n 44 gen. 7-0-glu. a c y l 45 gen. 7-0-glu. a c y l 46 o r i e n t i n X'-O-glu. 47 v i t e x i n X'-O-glu. 48 o r i e n t i n 3 '-0-glu. 49 l u . 4 ' - 0 - g l u . 50 l u . 4'-0 - g l u . a c y l 51 ap. 4'-0- g l u . 52 ap. 4' - 0 - g l u . a c y l 53 ap. 4' - 0 - g l u . a c y l 54 I s o v l t e x t n a c y l I 55 l u . 5-0-glu. 56 ap. 5-0-glu. 999999999999999959999999959199999999999 999995 915595959595559555995595155155 555 595955 15555 55555555555 5 5555555515 55995995555 15555 515551515 5 5 5555555 15 555 5511 1 15555 1951955555515555515955555 55 5555551555 55155 11519555555 515551595555 51 5555551551 1 55 55151151 5511115 15 55 1 191991 191951 1 111 111  599 111 1 1559 1 55 1555 55 5 955 555 1115999999599599 911 111555555555555 555 111 55555 5 5 111 55555 5 5 15 59 5 55 5 5 15 59 1 5 5 59599995 55559 5955555 555 555555 155555555511 559 55115 5 5 55 555 15 5 1 5 5 11 11195 1 15 1 1 55 59955555555 55 5555555 55 5155 5555 15 555 55 555 55 5 1 151 999959 5 55 5 995555 15111 5 5 5 5 55 5 5 55 1 5 55 1 5 5 5 999 51 1 555 155 5 555 555 55 55 5 1 555 5 5 5 55 5 1 1 115 115 111 11 1 1 1 1 1 1 1 1 1 111 55 5 55 1 5 5 55 95 55 5 55 1 1 1 5 55 55 55 1 11 11 11 1 1 1111 55 55 5 55 5 5 5 I I I II I II 55 5 5 9999 5 555 555 11 1115 1 11 1 15111 11151 11151 11 11 1 11 5 1 5 1 55555 55555 1 1 1 11 11 111 111111 1 1 555 55 5 515 5 5 555 absen t . A l though s u b j e c t i v e , the r a t i n g of each compound as major , m inor , e t c . , was deemed necessary s ince some taxa had i d e n t i c a l compounds but in wide ly d i f f e r e n t c o n c e n t r a t i o n s . For example, L. s e l l u l u s s s p . s e l l u l u s va r . 1o b b i i (OTU No. 53) had a p r o f i l e dominated by l u t e o l i n 7-0-glucos ide and a c y l a t e d d e r i v -a t i v e s ; they were in such concen t r a t i ons as to almost obscure the presence of small q u a n t i t i e s of C - g l y c o f l a v o n e s . On the other hand, L^ . perennis (No. 63) had a C-gl y c o f l avone-domi nated p r o f i l e wi th on ly minor or t r a ce amounts of f l a vone 7-0-glu-c o s i d e s . The c r i t e r i a fo r d e l i m i t i n g the c o n c e n t r a t i o n score fo r each compound should be e x p l a i n e d . From the matr ix i t can be seen that the ma jo r i t y o f the compounds occur as minor c o n s t i -t u e n t s . Th is means that they are p l a i n l y v i s i b l e under UV l i g h t before s p r a y i n g . Major c o n s t i t u e n t s were those tha t appeared as l a rge and i n t e n s e l y co loured s p o t s . Compounds cons ide red to be in t r a ce amounts were those that were not v i s i b l e under UV l i g h t u n t i l a f t e r s p r a y i n g ; they then appeared as r a the r d u l l , d i f f u s e s p o t s . Resu l t s of_PCA_on_Data_Set_ I The o r d i n a t i o n s produced by PCA on Data Set I are shown in F igures 13 and 14. These f i g u r e s are two-dimensional r e p r e s e n t -a t i ons of combinat ions of the f i r s t three component axes which c u m u l a t i v e l y account fo r 54% of the t o t a l va r i ance in the da t a . The e igenvec to r s c o n t a i n i n g the c o e f f i c i e n t s fo r the f i r s t th ree axes are shown in Table XIV. From t h i s t ab l e i t can be seen tha t ax i s I o rd i na t e s the OTU's mainly on the bas i s of two v a r i a b l e s (1 & 7 ) . V a r i a b l e No. 1 is o r i e n t i n ; v a r i a b l e No. 7 FIGURE 13. O r d i n a t i o n of 73 l u p i n e taxa from PCA on Data Set I. Components I/11 35 34 58 31 32 20 9 21 47 19 3 0 46 23 11 27 2 2 3 9 44 24 57 1 63 66 60 55 4 59 18 2 45 10 37 33 8 38 62 29 36 25 43 /15 s. 28 70 A 16 x y 56, / 14 61 65 54 26 48 71 17 43 a x i s I I 10% 64 53 42 41 51 67 68 69 a x i s I 36% 86 FIGURE 14. O rd ina t i on of 73 l up ine taxa from PCA on Data Set I Components 11/ 111 ,4fr i /« L42_ 41 J3 ^ 13 1 4 A 16. 70. ~" ~ - - - 56. ax is II-1 0 % 73 18 72 57 a ^ ° 3 2 7 .35 5 28 38 837 3 ^ 4 6 6 60 5 8 „ ,K51 44., * 59q O 43 26 3 629 4 5 24 J 25 ax is III 7% '54~ • '52 47 ^8 \ 55 50 \ 67 i 6 9 49 V_62 53 64 \ 63 \ \ 61\ — —J 87 TABLE XIV. Table of e igenvec to r s of the 56 f l a v o n o i d v a r i a b l e s fo r the f i r s t three component axes of PCA on Data Set I. <PRINCOM VAR=1-56 CASES=1-73 OPTION=UNSCALED MAX=3> PRINCIPAL COMPONENTS CASES=CASE#:1-73 TEST STATISTIC DF SIGNIF N= 73 OUT OF 73 SPHERICITY 10808. 1595 O. (1) (2) (3) COMPONENT 54.824 15.34 1 11.249 % VARIANCE 3 6 . 2 0 46 .32 53.75 SPHERICITY 9 3 1 7 . 0 8872 .3 8520.1 DF 1539 1484 1430 SIGNIF 0. 0 . 0. 1 V1 - .41739 .37867 - 1 .44374 - 1 2 V2 - .2801 1 .84 120 - 1 .42551 - 1 3 V3 - .26804 .38193 .23421 - 1 4 V4 - . 18390 .33931 . 1221 1 -1 5 V5 - .28064 .88388 - 1 .18307 -1 6 V6 - . 26440 .13198 .10830 7 V7 .33874 .2304 7 - .48522 - 1 8 V8 .22168 . 134 18 .87906 - 1 9 V9 .75379 - 1 .27102 - 1 .66963 -1 10 V10 - .15355 - 1 .54786 - 1 .37763 - 1 1 1 V1 1 .69381 - 1 .97713 -2 .10132 12 V12 .31920 - 1 .12873 - 1 .11502 13 V13 .28418 . 13676 -.22019 - 1 14 V14 .22414 .13254 -.10653 15 V15 - .10373 .18508 -.78851 -2 16 V16 -.94921 -1 . 1337 1 .15205 - 1 17 V17 .13563 - 1 . 144 15 - 1 -• .27428 - 1 18 V18 - .92028 -2 .27190 -2 .25431 -2 19 V19 - .77377 -2 - .34469 -3 .20531 -2 20 V20 - . 50930 -1 - .55009 .47818 - 1 21 V2 1 -.15673 -1 -. 177 15 .18798 - 1 22 V22 - .402 21 - 1 - .38928 .36289 - 1 23 V23 - .69334 -2 - .71074 - 1 --.17457 - 1 24 V24 .14088 .53087 - 1 .10742 25 V25 .66533 - 1 .44571 -1 .36931 -1 26 V26 .16232 .10545 .46185 27 V27 .70220 -1 .54546 -1 .29599 28 V28 .89427 -1 .52130 - 1 .30274 29 V29 .94983 - 1 .42876 - 1 .30664 30 V30 .27660 - 1 - .12548 -2 .56225 - 1 31 V31 .22606 -1 .38738 - 1 --.37890 - 1 32 V32 - .93599 -3 .14664 - 1 . 12148 - 1 33 V33 .20373 - 1 - .16787 - 1 .20446 - 1 34 V34 .39786 -2 .66016 -3 .13880 - 1 35 V35 .86177 - 1 .28485 - 1 --.15848 - 1 36 V36 .13836 .30423 - 1 .66157 - 1 37 V37 .10360 - 1 .61552 -2 .53200 - 1 38 V38 .17997 - 1 .13896 -1 --.14064 - 1 39 V39 .50844 -1 .89844 -2 .55538 - 1 40 V40 .11463 - 1 .28452 - 1 .10442 4 1 V4 1 - .13497 - 1 .24405 - 1 -- .24 144 -3 42 V42 .11575 .64764 -1 --.32429 43 V43 . 13261 .11641 -.51324 44 V44 - .31056 -3 . 19675 -1 --.37922 - 1 45 V45 .11091 - 1 .38121 -1 • -.12968 46 V46 - .33244 - 1 .37991 -2 -.26040 -2 47 V4 7 - .26615 - 1 .21068 - 1 .51248 -2 48 V48 - . 144 10 - 1 .28678 -1 - .26443 -2 49 V49 .11722 .31882 -2 .66923 - 1 50 V50 .14910 - 1 - .30043 -2 -.33308 -1 51 V51 .98737 - 1 - .88075 -2 .75608 - 1 52 V52 . 1837 1 - 1 .31857 -2 -.28801 - 1 53 V53 .13054 - 1 -.2751 1 -3 -.32492 - 1 54 V54 - .10702 - 1 . 15740 -1 .36619 -2 55 V55 - . 73820 -2 - .20254 -1 -.10975 -1 56 V56 - .73820 -2 - .20254 - 1 -.10975 - 1 88 i s l u t e o l i n 7 - 0 - g l u c o s i d e . T h e r e f o r e , c o m p o n e n t a x i s I d i s t r i -b u t e s OTUs f r o m h i g h o r i e n t i n a n d l o w l u t e o l i n 7 - 0 - g l u c o s i d e - c o n -t a i n i n g t a x a t o t h o s e h a v i n g l o w o r i e n t i n a n d h i g h l u t e o l i n 7 - 0 -g l u c o s i d e l e v e l s i n a l e f t t o r i g h t d i r e c t i o n . I t s h o u l d be n o t e d t h a t t h e s e t w o c o m p o u n d s a r e e x t r e m e l y common i n L u p i n u s s o i t s h o u l d n o t be s u r p r i s i n g t o f i n d t h a t t h e y d o m i n a t e t h e f i r s t c o m p o n e n t o f t h e a n a l y s i s . C o m p o n e n t a x i s I I i s s i m i l a r l y d o m i n a t e d b y C - g l y c o f l a v o n e s i n t h e p o s i t i v e d i r e c t i o n ( v a r i a b l e s 3 & 4 ) b u t t h e n e g a t i v e e n d o f t h e a x i s g r o u p s OTUs i n w h i c h 4 1 - m e t h y l a t e d C - g l y c o f 1 a v o n e s a r e f o u n d ( v a r i a b l e s 2 0 & 2 2 , c y t i s o s i d e a n d a n a c y l a t e d d e r i v -a t i v e , r e s p e c t i v e l y ) . T h i s g r o u p ( c o n t a i n i n g OTU N o s . 1 2 - 1 7 , 56 a n d 7 0 ) w i l l be r e f e r r e d t o a s G r o u p A . A x i s I I I i s d o m i n a t e d b y v a r i a b l e s 2 6 - 2 9 ( a c a c e t i n , c h r y s o -e r i o l a n d t h e i r 7 - 0 - g l u c o s i d e s ) a n d v a r i a b l e s 4 3 a n d 4 4 ( t h e i s o -f l a v o n e s g e n i s t e i n a n d g e n i s t i n ) i n a p o s i t i v e a n d n e g a t i v e d i -r e c t i o n f r o m t h e d a t a c e n t r o i d , r e s p e c t i v e l y . M o r e g r o u p i n g s a r e a p p a r e n t f r o m t h e o r d i n a t i o n i n v o l v i n g a x e s I I a n d I I I ( F i g . 1 4 ) . G r o u p B c o n s i s t s o f f i v e t a x a ( O T U N o s . 4 0 - 4 2 , 6 5 & 6 6 ) t h a t p r o d u c e h i g h l e v e l s o f 3 ' - a n d 4 1 - 0 - m e t h y l f l a v o n e s . G r o u p C i s m o r e d i f f u s e b u t a p p e a r s t o be c o m p o s e d o f N o s . 4 9 , 5 0 , 5 3 , 6 1 - 6 4 , 6 7 - 6 9 a n d p o s s i b l y N o s . 4 7 , 5 2 , 54 a n d 5 5 . F i g u r e 14 s h o w s t h r e e g r o u p s m o s t c l e a r l y e v e n t h o u g h t h e t w o a x e s o n l y a c c o u n t f o r a c o m b i n e d t o t a l o f 18% o f t h e v a r i a n c e . A x i s I a l o n e a c c o u n t s f o r 36% b u t o b s c u r e s g r o u p i n g s s i n c e i t i s d o m i n a t e d b y t h e a l m o s t u b i q u i t o u s c o m p o u n d s , o r i e n t i n a n d l u t e o l i n 7 - 0 - g l u c o s i d e . C h a r a c t e r w e i g h t i n g a n d d a t a s c a l i n g c o u l d h e l p t o o f f s e t t h e d o m i n a t i n g e f f e c t o n c e r t a i n a x e s b y u n i v e r s a l l y a b u n d a n t c o m p o u n d s , b u t t h e e a s e w i t h w h i c h t h e o r d i n -89 at ions cou ld be r e l a t e d back to the o r i g i n a l data might wel l dec rease . Data_Set_I I Data Set II was s imply a t r ans fo rma t i on o f the data from set I to presence/absence scores ( 0 / 1 ' s ) . The change to pure l y q u a l i t a t i v e data avo ids the s u b j e c t i v i t y o f Data Set I but at the same t ime , o b v i o u s l y 1 oses: consi derabl e i n f o r m a t i o n ; taxa such as OTU 53 and 63 now become almost i d e n t i c a l . R e s u l t s _ o f PCA_on_Data_Set II The f i r s t three component axes accounted fo r 4 4 % . o f the . . t t o t a l v a r i a n c e . The drop in t h i s f i g u r e , compared to that from Data Set I, can be a t t r i b u t e d to the l o s s o f v a r i a b i l i t y in the raw data s e t . As expec t ed , the o v e r a l l po in t spread was small caus ing much crowding of OTUs in a l l o r d i n a t i o n s i n v o l v i n g the f i r s t three component axes . The only noteworthy po in ts were a s l i g h t s epa r a t i on of the Group A taxa and the i s o l a t i o n o f taxon No. 55. Data_Set_111 Each OTU was scored for i t s b i o s y n t h e t i c r e p e r t o i r e of f l a v -o n o i d : p roduc t i on and m o d i f i c a t i o n c a p a b i l i t i e s . Th is method is a s i m p l i f i e d v e r s i on of that prepared by Levy ( 1977 ) . i n which chemical a f f i n i t i e s were based on the number of b i o s y n t h e t i c s teps shared by two taxa i ns tead of the number of compounds in common. For example, cons ide r three h y p o t h e t i c a l p l an t s and t h e i r f l a v -onoids : 90 A B C l u t e o l i n + + l u t e o l i n 7-0-glucos ide + + + l u t e o l i n 4 1 - 0 - g l u c o s i d e + + Both B and C d i f f e r from A by the absence of one compound. Using a b a s i c , unweighted s t a t i s t i c a l method, they would be equa l l y d i s s i m i l a r from A. However, s i nce l u t e o l i n i s a necessary i n t e r -mediate in the p roduc t ion of i t s g l u c o s i d e s , C must have syn the -s i z e d the ag l y cone ; t h e r e f o r e , i t s on ly d i f f e r e n c e from A is C's accumula t ion of l u t e o l i n . Spec ies B, however, makes no l u t e o l i n 4 1 - 0 - g l u c o s i d e , an end po in t in a b i o s y n t h e t i c pathway. Conse-q u e n t l y , B can be cons ide red more d i s s i m i l a r from A than C i s from A. Th is argument does assume that we know the b i o s y n t h e s i s of-f l a v o n o i d s with su re t y and that B l acks the b i o s y n t h e t i c capa -b i l i t i e s to make l u t e o l i n 4 ' - 0 - g l u c o s i d e . The 56 f l a v o n o i d s were condensed in to 11 b i o s y n t h e t i c a l l y d i f f e r e n t types of compounds, that i s , groups of compounds known to share i d e n t i c a l t e rmina l steps in the b i o s y n t h e s i s , f o r example, 5 - 0 - g l u c o s y l a t i o n . F igure 15 shows the c u r r e n t l y a c -cepted h i o s y n t h e t i c r e l a t i o n s h i p s between the types of compounds found i n Lup inus . As an example, a l l f l a vone 7-0-glucos ides were cons ide red as one compound type s ince the 7-0-g lucosy l a t i on s tep i s subs t r a t e n o n - s p e c i f i c , that i s , the same enzyme i s r e s p o n -s i b l e fo r the 7-0-g lucosy l a t i on of a p i g e n i n , l u t e o l i n , a c a c e t i n , e t c . ( Su t te r & G r i s e b a c h , 1 973 ; Su t t e r e_t al_. , 1 972) . 0-Gluco-s y l a t i o n at another p o s i t i o n r e q u i r e s a d i f f e r e n t enzyme; t h e r e -f o r e , such compounds are t r e a t ed as another f l a v o n o i d t ype . Oata Set III consequent l y was a 73 X 11 matr ix of presence/ FIGURE 15. B i o s y n t h e t i c i n t e r r e l a t i o n s h i p s of l up ine f l a v o n o i d s . (Under l ined compound-types are those used in Data Set I I I , v a r i a b l e numbers are a l so shown.) 3' -0-g lucos ides 4'-0-methyl  C-g l yco f l avones 1. i s o f l a v o n e s 3. 8-C-g lycof lavones f1 avonol s f^1 avanones 6-C-g lyco f lavones 4 8 5-0-glucos ides f l avones 4 ' - 0 - g l u c o s i d e s io 3'-0-methyl ' f l a vones 4'-0-methyl n f l a vones 7-0-glucos ides a - i r r (Diagram adapted from Hahlbrock & G r i s e b a c h , 1975) 92 TABLE XV. Data Set I I I : the d i s t r i b u t i o n o f 11 f 1 a v o n o i d - t y p e s i n 73 Lup inus t axa OTU Lup inus taxon No. 1 e l a t u s 2 b a r b i g e r 3 s e r i c e u s s e r . 4 s e r . h u f f m a n i i 5 s e r . e g g l e s t o n i a n u s 6 s e r . f l e x u o s u s 7 bi co l or 8 p o l y c a r p u s 9 neomexicanus 10 lemmoni i 11 t e x e n s i s 12 d e n s i f l o r u s s c o p u l o r u m 13 dens . au reus 14 r ube r 15 a r b o r e u s 16 h o r i z o n t a l i s 17 f l a v o c u l a t u s 18 benthami i 19 c o n c i nnus 20 s p a r s i f l o r u s 21 a r i z o n i c u s 22 s t o l o n i f e r u s 23 w y e t h i i 24 h i l l i i 25 h o l o s e r i c e u s 26 parv i f l o r u s 27 c auda tus X h i l l i i 28 n e v a d e n s i s 29 1eucophyl1 us 30 a l p e s t r i s 31 g r e e n e i 32 p s u e d o p a r v i f1o r u s 33 1utescens 34 s u l p h u r e u s 35 p a l m e r i 36 a r g e n t e u s a r g . 37 a r g . r u b r i c a u l i s 38 cauda tus 39 a r b u s t u s 40 g r a y i i 41 a l b i f r o n s 42 c h a m i s s o n i s 43 humi co l a 44 a r c t i c u s a r c t . 45 a r c t . c a n a d e n s i s 46 a r c t . s u b a l p i n u s 47 l a t i f o l i u s 48 . 1i t t o r a l i s 49. a.r idus 50 c o n f e r t u s 51 mi niraus 5 2 l y a l l i i 53 s e l1u l u s 54 k u s c h e l 55 d i f f u s u s 56 u n c i a 1 i s 57 c u s i c k i i 58 c a e s p i t o s u s 5 9 l e p i d u s 60 cu l b e r s o n i i 61 n o o t k a t e n s i s 62 b u r k e i 63 p e r e n n i s 64 p o l y p h y l l u s 65 e x c u b i t u s 66 l o n g i f o l i u s 67 a l b i ca u l i s 68 a n d e r s o n i i 69 formosus 7 0 k i n g i i 71 p u s i l l u s rubens 72 pusi11 us p u s i l1 us 73 h i r s u t t s s i m u s 1 2 3 4 5 CO «/) Qi OI > C c ra o o c— > > M-ta to o </> u <u 4- <*- >> c CO o o r— o I — u u cn > O >> >. t to c r— o o> en >> 4- > 1 • O re u •*-> ts> i O) «*- oo ca E 1 1 0 1 =3 i — +J 93 absence scores fo r f l a v o n o i d types (Table X,V)..' The o r d i n a t i o n s produced by PCA on Data Set III are shown in F igures 16 and 17. The f i r s t three component axes accounted fo r 59% of the t o t a l v a r i a n c e ; the e igenvec to rs appear in Table XV;I . From the t ab l e i t can be seen that ax is I main ly separa tes the e f f e c t s of f i v e types of f l a v o n o i d s . The OTUs are d i s t r i b u t e d in such a way as to show a g rada t ion from those that s y n t h e s i z e f l a v o n o l s , f l a vone 4 1 - 0 - g l u c o s i d e s and B-ring methy lated f l avones at the p o s i t i v e end of the ax is to those that s y n t h e s i z e most ly 8-C-g lyco f l avones at the negat i ve end. Axis II spreads taxa that 0 con ta in 3 1 - 0-gl ucos i des of C-g l yco f l avones and, to a l e s s e r ... . e x t e n t , i s o f l a v o n e s , 6-C-g lyco f lavones and f l avone 7-0-g lucos ides , toward the negat i ve end o f the a x i s . Taxa r i c h in f l a vone 5-0-g lucos ides and 4 ' - 0 - g l u c o s i d e s are found in the p o s i t i v e reg ions of ax i s I I I , wh i le those that con ta in methy lated C-g lyco f l avones are found at the negat i ve end. More groupings are apparent from F i g s . 16 and 17. Taxa Nos. 2-6 comprise Group D and are c h a r a c t e r i z e d by having p r o f i l e s c o n t a i n i n g 8- and 6-C-g l y co f l a vones , o r i e n t i n 3 1 - 0 - g l u c o s i d e , s imple f l avone 7-0-glucos ides and i s o f l a v o n e s . Group E i s made up o f OTU Nos. 24-28 which are p e c u l i a r in t h e i r p roduc t ion of f l avone 5-0-glucos ides . In a d d i t i o n , a l l group E taxa con ta in 8-C-g l y co f l a vones . Group A cont inues to be a d i s c r e t e chemical o c t e t c h a r a c t e r i z e d by 8-C-g lycof lavones and methy lated de r i v- . a t i v e s bu t , in F i g . 17, taxa Nos. 71 and 72 appear c l o se to Group A. Group B is a l so r e a d i l y r e c o g n i z a b l e in F i g . 16 a l though i t i s l e s s c l e a r c u t than in o r d i n a t i o n s a r i s i n g from Data Set I. FIGURE 16. O rd ina t i on of 73 lup ine taxa from PCA on Data Set II Components I/11 ax i s II 17% 28 26 25 27 46$% ,15 59V C66 65 42 - - ' 71 72 73 ax is I 30% 33^20 50 j27°31 63 6247 61 19 67 6968 64 4 9 52 53 \2\ \ \ \ \ \ FIGURE 17. O rd ina t i on of 73 l up ine taxa from PCA on Data Set III Components 11 /111 '26 / 28/ < 27 x 24 / s 25/ N/ C 2 /V. ' 5 / 3 4 6 34 T33] 5(M -3i, 19 31 I S 49 52 53 64 42 66 40 41 37 36 54 55 2 2°73 48 46456544 ax is II 17% 57 I860 58 71 72 \ 11 70 cit ' A ax is III 11% TABLE XVI. Table o f e i g e n v e c t o r s o f the 11 f l a v o n o i d - t y p e v a r i a b l e s f o r the f i r s t t h r e e component axes from PCA on Data Set I I I . <PPINC0M VAR=1-11 CASESM-73 MAX = 3> PRINCIPAL COMPONENTS CASES=CASE#:1-73 TEST STATISTIC DF SIGNIF N= 73 INDEPENDENCE 236 .05 55 0 . EOUICORRELATION 299.4 1 54 0 . (D (2 ) (3) COMPONENT 3.2577 2.0101 1.2701 % VARIANCE 29 .62 47 .89 59 .44 INDEPENDENCE 141.64 87 .087 61 .889 DF 54 44 35 SIGNIF .oooo .0001 .0034 1 . V1 . 15589 - .43605 .78507 2 . V2 .40326 .49684 -1 • - .11481 3 . V3 - .42036 -.14801 -1 • -.16456 4 . V4 - .18164 - .4434 1 .20653 5 . V5 -. 15543 .27097 - .37627 6 . V6 .29159 - .4 1740 - .22484 7 . V7 - .91696 -1 .24830 .69058 8 . V8 .39544 .14013 .39942 9 . V9 - .75475 -1 - .52246 .15109 10 . V10 .38985 .61103 -1 • -.23935 1 1 . . V 1 1 .41442 .70227 -1 .47217 Group C i s a l so d i f f u s e but OTUs 67-69 form a c l o se grouping near the pe r iphe ry of the o r d i n a t i o n s and w i l l be r e f e r r e d to as Group C2. F i n a l l y , taxon No. 55 stands a lone in both o r d i n a t i o n s i t i s t h e r e f o r e c l a s s e d as a monospec i f i c group. Ha l f the taxa have now been p laced in s i x groups . The remainder are c e n t r a l l y clumped in a l l o r d i n a t i o n s . These taxa (Group F) have r a the r t y p i c a l l u p i n e f l a v o n o i d s with no unusual s u b s t i t u t i o n s . They appear in t i g h t d i s c r e t e groups in F i g s . 16 and 17 s ince members of each group con ta in i d e n t i c a l types of f l a v o n o i d s ( a l b e i t in d i f f e r e n t c o n c e n t r a t i o n s ) . Between-group d i f f e r e n c e s are based on j u s t one or two types of very common compounds. T h e i r p r o f i l e s a l l tend to be dominated by 8-C-glyco -f l avones and many have sma l l e r q u a n t i t i e s of s imple f l a vone 7-0-g l u c o s i d e s . The s i m p l e s t f l a v o n o i d compliments are shared by OTUs 9 ,11 ,23 ,29 ,36-38 and 43-46, having only 8-C-g l y co f l a vones . Taxa Nos. 10,20,33 and 34 accumulate both 8- and 6-C-glyco-f l a v o n e s . 0-G1ucosy la t ion at the 7-pos i t i on and 8-C-g lyco -f l a vones occur in OTUs 1 , 8 , 2 2 , 5 1 , 5 4 , and 59 and Nos. 7 , 2 1 , 3 0 , 3 2 , 35 and 39 s yn thes i ze 7-0-g lucos ides plus both 8- and 6-C-glyco-f l a v o n e s . The three remain ing taxa are Nos. 19 and 48, which produce 4 '-methy la ted f l a vone 7-0-glucos ides and No. 73 which accumulates 3 '-0-methy lated f l a vone 7-0-glucos ides . A summary t a b l e of the groups i s shown in Table XVII . 98 TABLE XVII. Summary of Lupinus groupings from p r i n c i p a l . ' c o m -ponents a n a l y s i s on Data Sets I and I I I . Group OTU's C h a r a c t e r i s t i c f l a v o n o i d s and remarks A 12-17,56,70 c y t i s o s i d e ; p o s s i b l e o u t l i e r s 71 & 72 B 40-42,65,66 a c a c e t i n and c h r y s o e r i o l 7-0-glucos ides as major c o n s t i t u e n t s . C l ( i ) 47,61-64 g e n i s t e i n • C l ( i i ) 49-54,57-60 v a r i a b l e ; group based on morphology (see d i s c u s s i o n ) . C2 67-69 f l a vone 4 ' - 0 - g l u c o s i d e s D 2-6 o r i e n t i n 3 ' - 0 - g l u c o s i d e s , major amounts of 6-C-g lyco f lavones E 24-28 f l avone 5-0-glucos ides F 1,7-11,18-23 most ly s imple f l a v o n o i d s ; i n t e r n a l 29-39,43-46 groupings to be based on morphology 48,73 (see d i s c u s s i o n ) . 55 s i n g l e taxon with unusua l l y complex p r o f i l e o f s imple compounds; f l avones and f1 a v o n o l s . 99 D i s cus s i on GROUP A OTU 12 L. d e n s i f l o r u s Benth. v a r . aureus (Kel . l . ) Munz 13 L. d e n s i f l o r u s va r . d e n s i f l o r u s 14 L. ruber H e l l e r 15 L. arboreus Sims. 16 L . h o r i z o n t a l i s H e l l e r 17 1. f l a v o c u l a t u s H e l l e r 56 L. u n c i a l i s Wats. 70 L. k i n g i i Wats. The taxa in t h i s group are c h a r a c t e r i z e d by t h e i r possess ion of C-g lyco f l avones i n c l u d i n g the 4 '-methy la ted d e r i v a t i v e of v i t e x i n known as c y t i s o s i d e . Th is compound is p e c u l i a r to t h i s group, o c c u r r i n g nowhere e l se in the s tudy . Seven of the e igh t taxa are annual 1^ u p i n e s , whi le L_. arboreus i s a p e r e n n i a l . The i r chemical a f f i n i t i e s are o b v i o u s , but are there any morpho log i ca l f a c t o r s l i n k i n g the e igh t ? Watson (1873) f i r s t e rec ted the subgener i c group P Iatycarpus to accomodate taxa tha t had connate c o t y l e d o n s . It was a group of annual l up ines to which s i x of the seven taxa in Group A would be long . Lupinus u n c i a l i s l i k e w i s e has connate co t y l edons but Watson (1873) p laced i t in i t s own subgener i c group, Lup ine ! 1 us , as i t has s o l i t a r y , a x i l l a r y f l o w e r s . Smith (.1946) extended the subgenus P Iatycarpus to i n c l ude I. unci a l i s , t r e a t i n g i t as a monospec i f i c subgroup. In a d d i t i o n , Smith (.1944) d i v i d e d the racemose p i a t y ca rps i n to the M i c r o c a r p i . a n d /Pus i11i•based on 100 keel c i l i a t i o n and s t r o n g l y v e r t i c i l l a t e f lowers (both present in the Micro car p i ) . Lupinus: dens i f l o r us , J.. ruber and t . hor i zon^ t a l i s were p laced in the Mi c r o c a r p i whi le r_. f l a v o c u l a t u s and L. k i n g i i became members of the P u s i 1 1 i . The na tu ra lness of t h i s s u b d i v i s i o n of the P Iatycarpus i s d e b a t a b l e , but the bas ic seed -l i n g morphology shared by the seven annuals of Group A suggests a very c l o se r e l a t i o n s h i p , and t h i s i s r e f l e c t e d in t h e i r s i m i l a r f 1 avonoid p r o f i l e s . The a f f i n i t y of j . . arboreus to t h i s group is more problem-a t i c . A shrubby pe renn ia l taxon with p e t i o l a t e c o t y l e d o n s , I. arboreus is o b v i o u s l y an a t y p i c a l member of Group A. Morpho-l o g i c a l l y , i t has been p laced c l o s e to o ther shrubby l up ines of coas t a l C a l i f o r n i a such as I. 1ongi f o l i us ( Smi th , 1944) and a l k a l o i d s t ud i e s by Nowacki & Dunn (1964) have had s i m i l a r con -c l u s i o n s . Given the unexpected f l a v o n o i d s i m i l a r i t i e s between L^ . arboreus and the p l a t y c a r p s , can we remove the b l i n k e r s that lead us to immediate ly d i v i d e annual from perenn ia l taxa and see i f any other s i m i l a r i t i e s e x i s t ? Most p l a t y ca rps are small l up ines with v a r i o u s l y co loured f lowers and commonly a v i l l o u s indumentum. Most occupy d r y , sandy hab i t a t s of southwestern Uni ted S t a t e s . An obvious excep-t i o n i s I. dens i f ! o r u s . Some v a r i e t i e s are t y p i c a l l y small whi le o thers are much more r o b u s t . Lupinus dens i f1orus va r . aureus i s an e r e c t , appressed l up ine that may reach a he ight of 4 dm, with i t s racemes adding another 2 dm (Munz, 1959). The f lowers are v e r t i c i l l a t e to s u b v e r t i c i l 1 ate (an i m p e r f e c t i o n in Smi th ' s M i c r o c a r p t c h a r a c t e r i s t i c s ) , 14-17 mm in l e n g t h , b r i gh t y e l l o w , scented and possess a d i l a t e k e e l . It i s found in mesic to dry coas ta l meadows. A very s t r i k i n g f ea tu re i s the s tout woody stem-base tha t l ed two taxonomtsts ( u n f a m i l i a r with the genus) to r e f e r to i t as a pe renn ia l l u p i n e . Lupinus a r b o r e u s , on the other hand, i s undoubtedly a peren n i a l , bu t , l i k e j . . dens i f 1 orus v a r . a u reus , i t too has appressed pubescent stems, f lowers that are b r i gh t y e l l o w , heav i l y scented and 14-17 mm l o n g . Fur thermore , the keel i s c i l i a t e and the i n f l o r e s c e n c e s c a t t e r e d to s u b v e r t i c i l 1 a t e . The s i m i l a r i t i e s between I. arboreus and 1 . densi f1orus va r . aureus are numerous and s u r p r i s i n g but may s imply be the r e s u l t of p a r a l l e l e v o l u -t i o n in two taxa that have such a bas i c d i f f e r e n c e in s e e d l i n g morphology. However,- i f we agree with Hutchi nson ' s (1959) p r e -mise that the annual hab i t i s de r i ved from perenn ia l a n c e s t o r s , the s t r i k i n g chemical and morpho log i ca l s i m i l a r i t i e s between the t r e e - l u p i n e and the p l a t y ca rps can lead to i n t e r e s t i n g specu -l a t i o n s concern ing p o s s i b l e e v o l u t i o n a r y a f f i n i t y . OTU Nos. 71 and 72 (L . p u s i 1 1 u s Pursh s s p . rubens (Rydb.) Dunn and s s p . pus i11us r e s p e c t i v e l y ) are a l so members o f the s e c t i o n P Iatycarpus but were c h e m i c a l l y r a the r d i f f e r e n t from the Group A t a x a . Both l ack c y t i s o s i d e and have p r o f i l e s domin-ated by 7-0-glucos ides of a p i g e n i n , l u t e o l i n and c h r y s o e r i o l . In a d d i t i o n , small q u a n t i t i e s of C-g lyco f l avones and f l a v o n o l s were present making t h e i r o v e r a l l chemical p r o f i l e s u n l i k e any o ther taxon in the s tudy . Dunn (1959) notes tha t t h i s spec i es complex, whi le sha r ing f ea tu res with the p l a t y c a r p s , have such shor t peduncles and unusual seeds as to make i t unmistakably d i f f e r e n t from a l l o ther l u p i n e s . Evert so , i t can be seen in F igures 14 and 17 that these two' taxa do appear ( p o s s i b l y c o i n -c i d e n t a l l y ) very c l o se to Group A. 102 GR0UP_B OTU 40 L. g ray i CWats.) Wats. 41 L. a l b i f rons Benth. ex L ind l . 42 I. cha misson is E schsch . 65 JL. excub i tus Jon.es 66 j . . 1 ongi f o l i us (Wats.) Abrams This group is c h a r a c t e r i z e d by the p roduc t ion of major q u a n t i t i e s of a c a c e t i n and c h r y s o e r i o l 7-0-g lucos ides . A l l are pe renn ia l spec i es of coas t a l C a l i f o r n i a . Smith (.1944) p laced the f i r s t four taxa i n to the l a rge s e c t i o n S e r i c e i as a l l have abax-i a l l y pubescent banners . Lupinus 1 o n g i f o l i u s was grouped with _L. arboreus in the A rbo re i . However, Nowacki & Dunn (.1964) cons ide red the l a t t e r taxa c l o se r e l a t i v e s of I. a l b i f r o n s , J_. chamissoni s and I. excub i tus on the bas i s of a l k a l o i d s t u d i e s . As ment ioned, the f l a v o n o i d s of I. arboreus were seen to be qu i t e d i f f e r e n t from the Group B taxa and t h i s d i s s i m i l a r i t y i s f u r t h e r supported by Kinghorn et aj_'. ( 1980) who found that _L. arboreus and J_. chamissonis had but one a l k a l o i d in common. Th is con -t r a s t s d r a m a t i c a l l y with Nowacki & Dunn's (1964) a s s e r t i o n that these sympat r i c spec i es were c h e m i c a l l y s i m i l a r . The morpho log i ca l s i m i l a r i t y among the taxa of Group B can be i l l u s t r a t e d by o u t l i n i n g a - L i t t l e of t h e i r taxonomic h i s t o r i e s . Watson (1873) o r i g i n a l l y des c r i bed I. gray i as a v a r i e t y of I. ande r son i i before he r a i s e d i t to s p e c i f i c rank in 1876. The d i s s i m i l a r f l a v o n o i d s of the two taxa tend to support t h e i r sep -a r a t i o n . Jepson ( 1936) des c r i bed J.. a l b i f rons v a r . medi us , which Smith (1938) t r e a t e d as a v a r i e t y of I. gray i before Munz (1959) placed the taxon i n t o I. excubi tiis var m e d i u s ( Jeps. ) Munz. Lupinus 1ongi f o l i us has had a . s i m i l a r h i s t o r y , f i r s t be ing viewed as a v a r i e t y of I. chamissonis by Watson (1876) before being r a i s e d to s p e c i f i c rank by Abrams (1904) . GR0UP_C1 Group CI i s a r a the r d i f f u s e aggrega t ion of t a x a . A l l are pe renn ia l l up ines na t i ve to western North America except fo r No. 64, J_. p e r e n n i s , which extends from eas tern Canada to Geo rg i a . The i n t e r r e l a t i o n s h i p s between the taxa are con fus ing and d i s c o n -t i n u o u s . Group CI can be d i v i d e d in to two m o r p h o l o g i c a l l y i d e n t i f i a b l e u n i t s . The two subgroups are t r e a t e d s e p a r a t e l y f o r reasons of c l a r i t y , bu t , s i nce they do form a : s i n g l e , _ a l b e i t 1'ose chemical group, the p o s s i b l e c r o s s - r e l a t i o n s h i p s between C l ( l ) and C l ( i i ) taxa w i l l be d i s c u s s e d . GR0UP_C1(|) OTU 61 J_. noo tka tens i s Don.n.'.e.x Sims. 62 I. burkei Wats. 6 3 I. perennis L. 64 I. polyphyl1 us L ind l . 47 L. 1 at i f o l tus Agardh These f i v e taxa a l l accumulate the i s o f l a v o n e g e n i s t e i n and/ or i t s 7-0-g lucos ide , g e n i s t i n . They have been regarded by many workers as c l o s e l y i n t e r r e l a t e d and the taxa have undergone c o n s i d e r a b l e changes in rank and name, t y p i f y i n g the nomenclat-104 ura l and taxonomic problems in Lup inus . Lupinus burke i was reduced to a v a r i e t y of I. po l yphy l1us by H i t chcock ejt al_. (1961) whereas P h i l l i p s (1955) d i spensed with the name a l t o g e t h e r , t r e a t i n g i t as a synonym o f I. po l yphy l1 us s s p . po l yphy l1 us . Dunn & G i l l e t t (1966) , however, have main-t a i ned the s p e c i f i c s t a tus of t h i s taxon whi le i n d i c a t i n g i t s c l o se r e l a t i o n s h i p with IL. pol yphyl 1 us as d id Smith ( 1944) , who p laced i t in the s e c t i o n P o l y p h y l 1 i . M o r p h o l o g i c a l l y , the two d i f f e r by keel c i l i a t i o n (present in _L. burke i ) and f lower s i z e , burke i having the s m a l l e r . Lupinus .nootka tens i s was t r e a t ed as a separa te spec i e s by Watson C187 3) but P h i l l i p s (.1955) cons ide red i t as a subspec ies of I. p e r e n n i s . Dunn (1965) found t h i s t r a n s f e r of rank unten -able s i n ce P h i l l i p s had examined so few spec imens. Dunn (1965) suggested tha t morpho log i ca l t r a i t s in I. noo tka tens i s may have been de r i ved from 1 . pol yphyl 1 us , L. 1 a t i f o l i u s , J_. arboreus and L. a r c t i c u s , the l a t t e r of which he views as being part o f the La t i f o l i u s complex. Meanwhi le, L . 1a t i f o l i u s , which P h i l l i p s (1955) reduced to a subspec ies of I. perennis has r e c e n t l y been t r e a t e d by Kenny & Dunn (1977) who recogn ize i t as a separate spec i es and l i s t e igh t s u b s p e c i e s . The above taxonomic c o n t r o -v e r s i e s ev idence the morpho log i ca l s i m i l a r i t i e s between the taxa of Group C l ( i ) » s i m i l a r i t i e s that are f u r t h e r supported by the present f l a v o n o i d s tudy . 105 G R Q U P _C l ( i i ) OTU 49 I. a r i d u s Dougl . 50 _L. con fe r tus Kel 1 . 52 L. l y a l l i i Gray 53 J_. sel 1 ul us s s p . sel 1 ul us va r . 1 o b b i i (Gray ex Wats.) Cox 54 L_. kuschei Eastw. Group C l ( i i ) comprises taxa that are m o r p h o l o g i c a l l y qu i t e d i f f e r e n t from those of Group C l ( i ) . They are v a r i a b l e c h e m i c a l l y and by examinat ion of the raw data or the two-dimensional maps (Appendix V) i t can been seen that t h e i r placement in the o r d i n -a t i ons i s , in a l l l i k e l i h o o d , merely because of t h e i r common accumula t ion of i s o f l a v o n e s in severa l of: : the t a x a . Otherw ise , Groups C1 ( i ) and C l ( i i ) appear r a the r d i s s i m i l a r se ts of t a x a . Except f o r kuschei , these ;.taxa belong to the pe renn ia l c a e s p i t o s e group of l up ines t r e a t e d by D e t l i n g (1951) and Cox (1972) . Over 80 names have been pub l i shed fo r t h i s v a r i a b l e group but D e t l i n g (1951) cons ide red a l l of them to belong to seven subspec ies of _L. 1 e p i d u s . P h i l l i p s (1955) s i m i l a r l y grouped a l l the c a e s p i t o s e l up ines i n to j u s t four s u b s p e c i e s . Cox (1972 ) , ' howeve r , dec ided that the high v a r i a b i l i t y i n ' t h e 1epidus-caes pi tos us complex deserved r e c o g n i t i o n and r e i n s t a t e d many s p e c i f i c e p i t h e t s . s o that the complex then held 30 v a l i d t a x a . Dunn ( in Kar tesz & K a r t e s z , 1980) s i m i l a r l y l i s t s 11 spec i es of perenn ia l c a e s p i t o s e l up ines with 16 subspec ies and 14 v a r i e t i e s . The most c en t r a l taxa in the o r d i n a t i o n s a r i s i n g from Data Set I ( J i g s . 13 &114) were Nos. 49 and 5.0. T h e i r p o s i t i o n i n g so 106 c l o se to the polyphyl1 us group i s c l e a r l y owing to the presence of the i s o f l a v o n e g e n i s t e i n . Otherw ise , they produce none of the C-g lyco f l avones that dominate the p r o f i l e s of Group C l ( i ) . De t l i ng (1951) t r e a t e d j . . a rd i us and L.. con fe r tus (Nos. 49 & 50, r e s p e c t i v e l y ) as separa te subspec ies of I. 1 epidus whi le P h i l l i p s ( 1955) p laced I. a r i dus in to synonomy with L. 1e p i d u s s s p . 1e p i d u s . It i s i n t e r e s t i n g to note that in 1835 Agardh p laced L_. 1 e p i d u s in the t r i b e Po l yphy l1 i , members of which make up the r es t of Group CI . Taxon No. 53, I. se l 1ulus va r . 1obbi i i s a l so c l o se to the CICi ) taxa and in t h i s case appea r s : t o be c h e m i c a l l y qu i t e s i m i l a r . O r i g i n a l l y , t h i s taxon was desc r i bed as I. 1 o b b i i Gray ex Greene but D e t l i n g (1951) and:-Phi H i ps:.(1955 ) cons ide red i t c o n s p e c i f i c with I. 1 epidus s s p . l y a l l i i . C h e m i c a l l y , J_. l y a l l i i COTU No. 52) has e x a c t l y the same b i o s y n t h e t i c r e p e r t o i r e as L. s e l l ul us va r . Tobb i i (see Fig.: 16) thus suppo r t i ng D e t l i n g ' s and P h i l l i p ' s t r ea tmen t s . As we get p r o g r e s s i v e l y f u r t h e r from the c en t r a l group C taxa we encounter OTU 51, L_. minimus, another c a e s p i t o s e l u p i n e . Th is taxon produces no i s o f l a v o n e s but i s o therwise c h e m i c a l l y very s i m i l a r to va r . 1 obbi i and j . . l y a l l i i . The s imple 8-C-gl y co f 1 a vone , 7-0-gl ucof l avone p r o f i l e of I. minimus i s shared by a number of o ther taxa that are thought to belong to the same morpho log i ca l group. One of these i s L. 1epidus (OTU No. 59 ) , the taxon in to which a l l the o ther c aes -p i t o se l up ines have been i n c l uded at some t ime . Lupi nus c u s i k i i , I. caes pi tosus and L. c u l b e r s o n i i complete the l i s t of pe renn ia l c a e s p i t o s e l up ines that on morpho log ica l grounds w i l l be added to Group CI ( i i ) . Cox (19.73) found many isozyme d i f f e r e n c e s in t h i s group and c i t e d t h e i r geographic i s o l a t i o n as a c o n t r i b u t i n g f a c t o r in 107 t h e i r i n t e r s p e c i f i c d i f f e r e n t i a t i o n s i n ce they grow at high e l e v a t i o n s from the S i e r r a Nevada to the Rocky Mountains of Canada . Taxon No. 54, I. kuschei , has s i m i l a r f l a v o n o i d s to both the C l ( i ) and C l ( i i ) t a x a . It i s an endemic l u p i n e in the Yukon, A laska and nor thern BC, Dunn & G i l l e t t (1966) suggest t h i s spec i es to have r e s u l t e d from past i n t r o g r e s s i o n between _L. a r c t i cus and I. s e r i ceus . Its o v e r a l l morphology appears qu i t e s i m i l a r to the 1epi dus-caes pi tosus complex, being a. low p lant with a woody caudex from which long p e t i o l e d leaves a r i s e . The f lowers occur in dense racemes and the banners are g labrous but r a the r longer than in many 1ep idus-1 i ke t a x a . C h e m i c a l l y , the taxon i s qu i t e s i m p l e . The l ack of 6-C-g lyco f l avones or 0-g lucos ides of the C-g lyco f l avones makes the suggested a f f i n i t y with L .' s e r i c e u s l e s s than c o n v i n c i n g . Lupinus a r c t i c u s has much s imp l e r f l a v o n o i d s , on l y C - g l y c o f l a v o n e s , and i s qu i t e s i m i l a r to L. kuschei , bu t , o v e r a l l , the f l a v o n o i d s of j _ . kuschei f a i l to s u b s t a n t i a t e the a f f i n i t i e s that Dunn & G i l l e t t (1966) have suggested us ing morpho log i ca l c h a r a c t e r s . The growth hab i t and f l a v o n o i d s a l l y i t more c l o s e l y with the taxa of the 1epi dus group. GR0L)P_C2 OTU 67 I. a l b i c a u l i s Dougl . ex Hook. 68 I. a n d e r s o n i i Wats. 69" I. formosus Greene Th is t r i o of taxa d i f f e r from the r es t o f Group C by i t s accumula t ion of f l a vone 4 1 - 0 - g l u c o s i d e s . Munz (1959) desc r i bes J_. ande r son i i as being " c l o s e " to j . . a 1 b i c a u 1 i s in many morpo-l o g i c a l cha r a c t e r s and Dunn (persona l communication) regards I. a 1 b i c a u 1 i s and j . . formosus as c o n s t i t u t i n g a c l o s e l y r e l a t e d complex of s p e c i e s . A l though t h i s group is c h e m i c a l l y d i s t i n c t , one o r d i n a t i o n ( F i g .14 ) r e f l e c t s a c l ose r e l a t i o n s h i p between these taxa and those of the r e s t of Group C, and morpho log i ca l ev idence f o r t h i s r e l a t i o n s h i p appears in the l i t e r a t u r e . The d e s c r i p t i o n s of j . . perennis and I. a 1 b i c a u 1 i s in P h i l l i p s ( 1955) are almost i d e n t i c a l ; a l s o , whi le P h i l l i p s ( 1955 ) cons ide r s L_. r i v u1 a r i s (not examined in t h i s study) to be a synonym of I. a 1b i c a u1i s , Dunn & G i l l e t t (1966) t r e a t i t as a spec i e s c l o se in i t s morphology to J.. noo tka tens i s and j _ . 1 at i f o 1 i u s . OTU No. 55 Lupinus di f fusus Nut t . The placement of Lupinus di f f u sus in the o r d i n a t i o n s is such tha t i t i s d i f f i c u l t to a l l y i t with any o ther t axon . P r i n c i p a l components a n a l y s i s on Data Set I p laced i t c l o se to Group C but in the o r d i n a t i o n s a r i s i n g from the a n a l y s i s o f Data Set III i t is found c l o se to Group D. It i s t h e r e f o r e c l a s s e d as a monospec i f i c group. Lupi nus di f f u sus i s both c h e m i c a l l y and m o r p h o l o g i c a l l y d i f f e r e n t from any other taxon in t h i s study It i s na t i ve to sou theas te rn Uni ted S ta tes and has s imple leaves Dunn (1971) suggests that t h i s taxon has i t s o r i g i n s in South Amer i ca . Long-d is tance d i s p e r s a l o f seed by b i rds which have fed on the shrubby , s imp le- leaved l up ines of nor thern B r a z i l may have i n t roduced _L. di f f usus i n to the F l o r i d a a r e a . It occurs in mesic coas ta l s i t e s that are f requented by m ig r a t i ng b i r d s . A l though the f l a v o n o i d s of t h i s spec i e s are not unusua l , they •109 are numerous and v a r i e d , c r e a t i n g a p r o f i l e un l i ke the western s p e c i e s . T h e r e f o r e , owing to both morpho log ica l and chemical o b s e r v a t i o n s , I. di f fus us i s t r e a t ed as a s i n g l e - t a x o n group. GR0UP_D OTU 2 L^ . barbi ger Wats. 3 L. s e r i c e u s Pursh s s p . s e r i c e u s va r . s e r i c e u s 4 j . . s e r i c e u s s s p . huf fmani i (C.P.Sm.) Fleak & Dunn 5 j . . s e r i c e u s s s p . s e r . v a r . eggl es ton i anus C.P.Sm. 6 L.. s e r i c e u s s s p . ser . v a r . f 1 exuos us ( L i n d l . ex Aga r dh ) C . P . Sm . A l l f i v e taxa in t h i s group produce l a rge amounts of both 8- and 6-C-glycof1 avones. Lupinus ba rb ige r was t r e a t e d as part o f the s e r i c e u s complex by F leak (1971) who undertook a d e t a i l e d sys temat i c a n a l y s i s . o f the group. Acco rd ing to F l e a k , 'the com?: pi ex c o n s i s t s of 17 t a x a , a l l pe r enn i a l s with numerous c a u l i n e leaves and unspurred but sometimes gibbous c a l y c e s ; banners are r e f l e x e d at or near mid-point and are a b a x i a l l y pubescent . Lupinus s e r i c e u s s s p . huf fmani, i. i s cons ide red the southern sub-s p e c i e s and i s d i s t i n g u i s h e d by heavy anthocyan in p igmentat ion of i t s stems. V a r i e t y s e r i ceus has dense racemes of l a rge f lowers (.11-13 mm) and few s ide branches whi le va r . f l exuos us has smal l f lowers (.9-11 mm) and lax racemes. V a r i e t y eggl eston- ianus has dense small f lowers and spread ing r e t r o r s e h a i r s , thought to be de r i v ed from i n t r o g r e s s i o n from _L. 1 eucophyl 1 us (Dunn & Gi 1 1 e t t , 19:66 ; Fl ea k , 1971) . Lupinus barb i ge r , a sep -a ra te spec i es from southern Utah and nor thern A r i z o n a , has white 110 f lowers but i s o therwise very s i m i l a r to I. s e r i c e u s . The c l o se s i m i l a r i t y o f the f l a v o n o i d r p r o f i 1 e s of these taxa would support F l e a k ' s c o n c l u s i o n s that these l up ines are c l o s e l y r e l a t e d . GR0UP_E OTU 24 J_. h i l l i i Greene 25 J_. h o l o s e r i c e u s Nut t . ex T o r r . & Gray 26 j . . parv i f 1 orus Nut t . ex Hook. & A rn . 27 L. hi 1 I i i X L. caudatus K e l l . 28 . nevadens i s H e l l e r These f i v e l up ines are p e c u l i a r in t h e i r p roduc t ion of f l a vone 5-0-g lucos ides . O therw ise , they accumulate the t y p i c a l 8-C-g l y co f l a vones . A l l are smal1 -f1owered perenn ia l taxa with t y p i c a l l a b y r i n t h i n e i n t e r r e l a t i o n s h i p s . In Smi th ' s t reatment of the l up ines o f C a l i f o r n i a (.Abrams, 1944) L. ho ios e r i ceus and J. . nevadens j s were p laced in the Cal c a r i t i with I. caudatus , j^. sul phureus and many of the taxa that are now r e f e r r e d to as L. a rgen teus . Recent ly however, Dunn (persona l communication) and Harmon (1980) have pub l i shed a t reatment of the parv i f1orus complex in which JL. h i 11 i i and I. h o l o s e r i c e u s are p l a c e d . The hybr id o r i g i n of OTU No. 27 was determined by P ro f . D.B.Dunn [persona l communication) a f t e r the present author had i d e n t i f i e d i t as L^ . h i l l i i . Dunn notes that the keel and wing c i l i a t i o n is t y p i c a l of I. caudatus whereas the small f lowers i n d i c a t e i n t r o -g r e s s i o n from L. h i l l i i . The f l a v o n o i d p r o f i l e o f the p u t a t i v e hybr id c e r t a i n l y supports i t s morpho log i ca l a f f i n i t y to I. hi. 1 I i i . I l l The on ly c o n t r o v e r s i a l placement in Group E i s j . . nevadensis . Its a f f i n i t y to Smi th ' s C a l c a r i t i i s unquest ioned but in sub-d i v i d i n g t h i s r a the r l a r g e group o f t a x a , Dunn (persona l commun-i c a t i o n ) p laces nevadensis in the L_. sul ph'ureus complex r a the r than the pa r v i f1orus group of l u p i n e s . Both complexes have pubescent banners , a c h a r a c t e r not shared by J_. nevadens i s . Flower s i z e i s more s i m i l a r to I. su lphureus (10-12 mm) than to _L. hoi os e r i ceus (8-10 mm) but as the f l a v o n o i d s show, the prod^' u c t i on of f l a vone 5-0-glucos ides makes I. nevadensi s a d i s t i n c t chemical a l l y o f the parv i f l o r u s complex. GR0UP_F We now turn to the many taxa that have l e s s ou t s t and ing p r o f i l e s . They a l l accumulate 8-C-g lycof lavones and some in • .: J a d d i t i o n produce 6-C-g lyco f l avones and/or s imple f l avone 7-0-g l u c o s i d e s . The l a c k . o f unusual compounds in t h i s group (Group F) r e s u l t s i n t h e i r being c e n t r a l l y p laced and congested i n : a l l o r d i n a t i o n s . To d e l i m i t chemical groups w i th in these taxa i s not d i f f i c u l t , but e x p l a i n i n g the a f f i n i t i e s between a l l the members of a group i s a problem s ince some are c l e a r l y s i m i l a r to members of o ther groups tha t d i f f e r by j u s t one f l a v o n o i d or compound t y p e . T h e r e f o r e , f o r Group F, the d i s c u s s i o n of p o s s i b l e a f f i n i t i e s i s approached from a d i f f e r e n t po int o f v iew. Sets o f t a x a , cons idered , by morphol ogi s ts to be c l o s e l y a l l i e d are ex t r a c t ed from the group and t h e i r chemical a f f i n i t i e s rev iewed. 112 GR0UP_F1 OTU 23 L. wyethi i Wats . 43 JL. humi col a Ne l s . 44 J.. a r c t i c u s Wats. s s p . a r c t i cus 45 L^ . a r c t i cus s s p . canadens is ( C . P . S R I . ) Dunn 46 L . a r c t i c u s s s p . suba lp inus ( P ipe r & Robinson ) Dunn These f i v e l up ines have the s imp les t f l a v o n o i d p r o f i l e s found in the s t u d y ; on l y 8-C-glycof1 avones are p r e sen t . As a group they have been c l o s e l y a l l i e d with the taxa of Group C l ( i ) but t h e i r l ack o f i s o f l a v o n e s c l e a r l y d i s t i n g u i s h e s them. Lupinus  a r c t i c u s was t r e a t e d as a synonym of J.. 1 a t i fo l i us by Smith ( 1944) whereas P h i l l i p s (1955) t r e a t ed i t as a subspec ies of I. poly- phy 11 us . P h i l l i p s a l so p laced I. wyeth i i i n to synonomy with I. pol yphyl 1 us va r . prunophyl 1 us . Lupinus humi col a was viewed by P h i l l i p s as worthy of s p e c i f i c r e c o g n i t i o n , but H i t chcock et al . (1961) and Dunn & G i11et t (1966) t r e a t e d i t as a synonym;of I. wyethi i , a taxon o f s p e c i f i c rank in t h e i r taxonomies . Lupinus humi col a va r . t e to n e n s i s was cons ide red to be a v a r i e t y o f _ L . wyeth i i by Cox & Dunn ( 196 9) a l though they suggest that i t i s a r e l i c t u a l element of J_. a r c t i cus . Such a s i t u a t i o n a t t e s t s to the morpho log i ca l s i m i l a r i t y between the Group F l t a x a , a s i m i l a r i t y that i s a l so r e f e c t e d in t h e i r f l a v o n o i d p r o f i l e s ' . 113 GR0UP_F2 OTU 10 L. 1emmonii C.P.Sm. 30 L. X a l p e s t r i s N e l s . (argenteus X caudatus) 31 L . greenei N e l s . 32 L. a rbustus Dougl . ex L i n d l . s s p . p s e u d o p a r v i f l o r u s (Rydb.) Dunn 33 L. l u t e s cens C.P.Sm. 34 L. su lphureus Dougl . ex Hook. 35 1. palmeri Wats. 36 1. argenteus s s p . argenteus Pursh 37 1. arqenteus s s p . r u b r i c a u l i s (Greene) Hess & Dunn 38 JL. caudatus Ke l1 . 39 L . a rbustus Dougl . ex Hook. Th i s group of l up i nes may wel l be the most t ang l ed f o r e s t of synonomy in the genus. C h e m i c a l l y , the s imp le s t members are Nos. 36,37 and 38, taxa which accumulate on ly C - g l y c o f l a v o n e s . Taxa 10,33 and 34 have in a d d i t i o n to 8-C-g l y co f l a vones , 6-C-glyco-f l a v o n e s . OTUs 30 ,31 ,32 ,35 and 39 have 8-C-g lyco f l avones plus s imple f l avone 7-0-glucos ides . It i s p o s s i b l e , however, to d i s cuss the a f f i n t i e s of one chemical type without i n c l u d i n g nomenclature o f the o the r two chemical t ypes ; , consequen t l y , they are t r e a t e d as one. A l l are r a the r smal1-f1owered perenn ia l taxa that resemble the l up ines of Group E. The banner peta l i s pubescent in many but almost g labrous in JL. su lphureus and J.. a rgen teus . The ca l yx i s s t r o n g l y spurred in J.. caudatus and gibbous in o t h e r s . Indum-entum may be appressed pubescent to s e r i c e u s throughout except f o r .JL.' argenteus which o f ten has a d a x i a l l y g labrous l e a v e s . Smith (1944) grouped a l l the C a l i f o r n i a n na t i ves of group F2 in to the C a l c a r i t i . S ince that time the taxa have been s p l i t i n to a number of d i f f e r e n t complexes that u n f o r t u n a t e l y i n t r o -gress and merge i n t o each other with l i t t l e r espec t f o r t h e i r taxonomic r a n k i n g ! Hess & Dunn (1970) t r e a t ed the argenteus and caudatus com-plexes in one paper , no t ing t h e i r s i m i l a r i t i e s . F i e l d obse r va -t i o n s and taxonomic d e s c r i p t i o n s revea led that the two complexes vary to such a great extent that they are in f a c t two extremes of a cont inuous range of v a r i a t i o n . At one extreme i s cha r a c -t e r i s t i c j^. caudatus with a spurred ca l yx and heavy pubescence; at the o ther end is the g l a b r a t e , gibbous c a l yx-bea r i ng L^ . a r g - en teus . The ex i s t ence of scores of synonyms ev idences the g rada t ion between the two. A lso i n c l uded in Dunn & Harmon ' : (1980) i s L. X a l p e s t r i s , a pu ta t i v e hybr id between I. caudatus and j . . a rgen teus . Kar tesz & Kar tesz ( 1980) l i s t no fewer than 24 synonyms of L. X a l p e s t r i s , s i x of which are v a r i e t i e s of I. a rgen teus . "The f l a v o n o i d s of t h i s hybr id are more c o m p l i -cated than those of the two p a r e n t s . Th is may have r e s u l t e d from a breakdown of gene t i c con t ro l through genome i n t e r a c t i o n or a l t e r n a t i v e l y , i t cou ld be because of another l up ine being i nvo l ved in the h y b r i d i z a t i o n . Dunn & G i l l e t t (19.66) suggest that L. a rbustus may be i n vo l ved in the o r i g i n of JL. X al p e s t r i s they are seen to have i d e n t i c a l f l a v o n o i d s . Lupinus arbustus was t r e a t ed by Dunn (.1957) who viewed i t as being c l o s e l y r e l a t e d to L_. argenteus and J.. cauda tus . One synonym of I. a rbustus s s p . arbustus v a r . montanus (Howel l ) Dunn was L. 1u tescens . Th is taxon i s now t r e a t ed by Dunn (per-sonal communication) as a c l o se a l l y of J_. s u l phu reus . Lupinus  greenei i s cons ide red to be part of the morpho log ica l range of L. caudatus (Dunn, personal communication) a l though i t s f l a v -onoids suggest a c l o s e r a l l i a n c e with L. a r b u s t u s . Lupinus  1 emmon i i g r e a t l y resemble j . . pa 1 mer i , a c co rd ing to Kearney & Peebles (1960) ; they a l so i n d i c a t e a s t rong s i m i l a r i t y between j . . greenei and J.. 1 emmoni i . A l l three taxa (and many o the rs ) were t r e a t ed by Smith (1939) as members of the extremely v a r i -ab le argenteus/caudatus complex o f nor thern A r i z o n a . C l e a r l y t h i s group of l up ines i s a m o r p h o l o g i s t 1 s nightmare and there i s l i t t l e chemical foundat ion fo r d e l i m i t i n g taxonomic subgroups . Taxon No.29 i s I. 1eucophyl1 us Dougl . ex L i n d l . It may f i n d a p lace in the Group F2 melee s i n ce i t , t o o , has a very s imple chemical p r o f i l e . M o r p h o l o g i c a l l y i t has a much more robust hab i t than the f o r ego ing taxa and i t i s c h a r a c t e r i z e d by an extremely wooly indumentum. Its f lowers are small (9-11 mm) and the pubescent banner is r e f l e x e d above the mid-point thereby produc ing a. f lower s i m i l a r to J_. su lphureus and L.. a r b u s t u s . It does , however, remain a very d i s t i n c t i v e t axon . GROUP_ F3 OTU 7 L. bi c o l o r L i n d l . 8 1. polycarpus Greene 9 1. t exens i s Hook. 10 L. benthanii H e l l e r 19 L. concinnus Argardh 20 L. s p a r s i f l o r u s Benth. 21 L. a r i z o n i c u s (Wats.) Wats. 2 2 L. s t o l o n i f e r u s L. 73 L. h i r s u t i s s i m u s Benth. 1 1. e la tus Johnston 9 1. neomexicanus Greene 48 L. 1i t t o r a l i s Dougl . The t h i r t e e n taxa that comprise Group F3 are c h e m i c a l l y and m o r p h o l o g i c a l l y d i v e r s e . Some have s imple p r o f i l e s , o thers have B-ring methy la ted f l a vone 7-0-glucos ides . Perhaps a page should be taken from Agardh ( 1835) who e rec ted the Spec ies Inqui rendae in which to p lace l up ines o f . d o u b t f u l r e l a t i o n s h i p s and a f f i n -i t i e s . The group F3 taxa would c e r t a i n l y be prime cand ida tes fo r such a group! Some t a x a , fo r example, J.. h i r s u t i s s i m u s , show f l a v o n o i d p r o f i l e s u n l i k e any o ther l u p i n e in the s t udy , whi le o thers such as I. neomexi canus has a p r o f i l e that i s simpl and s i m i l a r to many other s p e c i e s , but there the s i m i l a r i t y ends For comple teness , the l i t t l e that is known or i n f e r r e d about these l up ines i s r e p o r t e d . Lupi nus cone i nnus , I. a r i z o n i c u s and ^L. spars i f 1 o r u s are three very s i m i l a r annual spec i e s of the deser t reg ions of southwestern Uni ted S t a t e s . 117 C h r i s t i a n & Dunn (1970) undertook c r o s s i n g experiments w i th i n t h i s complex and d i s cove r ed s t e r i l i t y b a r r i e r s between the s p e c i e s . They conc luded that each of these taxa should comprise a separate complex; the d i f f e r i n g f l a v o n o i d p r o f i l e s in the th ree spec i es would tend to support t h e i r s e p a r a t i o n . Lupinus t exens i s has an extremely s imple p r o f i l e and is s i m i l a r to I. neomex-i c a n u s . the a r c t i c u s group and some of the argenteus t a x a . S ince t h e i r c h e m i s t r i e s are so s imple i t seems unwarranted to i n f e r any rea l a f f i n i t i e s between these t a x a . Moreover , I. t e x e n s i s stands out as one of j u s t two l up ines in North America with a h a p l o i d chromosome number of 18 (compared to the t y p i c a l 24 ) . Lupinus b i c o l o r and J_. po lycarpus are both smal 1 -f 1 owered coas ta l annuals that occur from C a l i f o r n i a to B r i t i s h Columbia . Smith C1944) p laced both taxa in to h is s e c t i o n M i c r an th i and Dunn & G i l l e t t (19-6) a l so cons ide r them to be c l o s e l y r e l a t e d . C h e m i c a l l y , they both accumulate f l avone 7-0-glucos ides and 8-C-g l y c o f l a v o n e s ; c h e m i c a l l y s i m i l a r i s I. s t o l o n i f e r u s , a taxon about which there i s l i t t l e i n fo rma t i on a v a i l a b l e . Lupinus  benthamii , which Smith (1944) p laced in to the s e c t i o n S p a r s i f l o r i with I. s pars i f 1 orus and _L . hi rs ut i s s i mu§ , s y n t h e s i z e s 8-C-glyco-f l a v o n e s , pi u s " a p i g e n i n , l u t e o l i n and c h r y s o e r i o l 7-0-glucos-i d e s . The l a s t compound is absent from L^ . s pars i f 1 orus . Lupi nus hi r s u t i ss imus is a t y p i c a l in i t s l ack of C-glycof1 avones. Few workers have t r ea t ed j _ . benthami i or L_. hi r s u t i ss innis; they remain taxa of unknown r e l a t i o n s h i p s . Lupinus el at us is a pe renn ia l that resembles I. andersoni i ( Smi th , 1944 ) . Jepson ( 1936) cons ide red J_. e l a tus to be a v a r i e t y of L. a 1b i cau1 i s and Smith (1944) , a v a r i e t y of ' , 118 j . . formosus before he r e-e l eva ted i t to s p e c i f i c s t a t u s . Chem-i c a l l y , I. el atus is qu i t e d i f f e r e n t from the group C2 taxa a l -though i t s morpho log i ca l s i m i l a r i t i e s do suggest a p o s s i b l e a f f i n i t y . L a s t l y , J.. 1 i t t o r a l i s should be d i s c u s s e d . Smith ( 1944) p laced i t with L_. arboreus and L.. r i v u 1 a r i s in h is A r b o r e i . Dunn & G i l l e t t ( 1966) a l so cons ide r L^ . arboreus to be i t s c l o s e s t a l l y . The l a t t e r produces the 4 1 -0-methy la ted C-glyco-f l avone of a c a c e t i n ( c y t i s o s i d e ) whi le I. 1i 11 o r a 1i s accumulates the 7-0-glucos ide of a c a c e t i n . Th is compound i s a l so present in I. chamisson is and I. a l b i f rons , o ther sea-shore l up ines to which I. 1i t t o r a l i s amy well be r e l a t e d . As a summary of the l u p i n e groupings that have been d i s -c u s s e d , Table XVIII should be r e f e r r e d t o . TABLE XVIII Group Summary t ab l e morpho log ica l of the groups ev idence . of Lupinus taxa d e l i m i t e d by f l a v o n o i d data and Lupinus taxa C h a r a c t e r i s t i c ' f l a vono ids ( i f any) Remarks d e n s i f l o r u s dens i f1 or us ruber h o r i z o n t a l i s f l a v o c u l a t u s unci a l i s k i n g i i arboreus var var dens . aureus c y t i so s i de Watson's (1873) s e c t i o n P I a t yca rpus . Annual taxa with s e s s i l e , connate co ty l edons . I. arboreus i s an e x c e p t i o n . It i s a shrubby pe renn ia l with p e t i o l a t e c o t y -l e d o n s . Cons idered unre la ted , by o ther workers . C y t i s o s i d e accumula t ion and morpho log i ca l s i m i l a r i t i e s to J.. dens i -f l o r u s v a r . aureus suggest p o s s i b l e l i n k . Group A o u t l i e r s : pusi11us s s p . rubens and pusi11us . P l a t y ca rps but wi thout c y t i sos i de. g r a y i i a l b i f rons chamissonis excub i tus 1ongi f o l i us major amounts of a ca ce t i n and c h r y s o e r i o l 7-0-glucos ides C a l i f o r n i a n coas t a l p e r e n n i a l s . Nowacki & Dunn (1965) cons ide r them to be r e -l a t ed (except j . . g ray i i ) . to each o the r , and to I. a rboreus ( chem i ca l l y d i s t i n c t ) J.. g ray i i cons ide red m o r p h o l o g i c a l l y s i m i l a r to I. a l b i f rons by Munz (1959) ; chemis t r y supports t h i s c o n t e n t i o n . noo tka tens i s burke i C l ( i ) perennis g e n i s t e i n p o l y p h y l l u s 1at i f o l i us M o r p h o l o g i c a l l y s i m i l a r to each other and to the C2 and F l t a x a . G e n i s t e i n accumula t ion i s d i s t i n g u i s h i n g f ea tu re A l l have been s u b s p e c i e s , v a r i e t i e s or synonyms of each other in the pas t . C l ( i i ) an ' dus con f e r tu s 1ya 11 i i s e l1u lus 1 e p i d u s c aesp i t o sus minimus c u s i c k i i c u l b e r s o n i i kuschei g e n i s t e i n in some, others more simple Group d i s t i n -guished by morphology. Some are c h e m i c a l l y s i m i l a r to C1 ( i ) but m o r p h o l o g i c a l l y t h i s group i s d i s -t i n c t . A l l are pe renn ia l c a e s p i t o s e l u p i n e s . D e t l i n g (1951) & P h i l l i p s (1955) t r e a t ed them a l l as s s p . o f L.  1epidus . Cox (1972 ) regards them as having s p e c i f i c s t a t u s , j . . kuschei i s an excep t ion and has not been cons ide red part of the c a e s p i t o s e group. However, morpho log i ca l s i m i l a r i t i e s and chemical a f f i n i t i e s (to some) suggest a p o s s i b l e r e l a t i o n s h i p . a 1b i c a u1 i s C2 ande r son i i formos u s f1 avone 4 ' • g lucos ides 0- A d i s t i n c t t r i o f o l l o w i n g PCA o f Data Set I I I . M o r p h o l o g i c a l l y s i m i l a r to each other and to C l ( i ) t a x a . F lavone 4 1 - 0 - g l u c o s i d e s are p e c u l i a r to C2 taxa ba rb ige r Major amounts of s e r i c e u s var . s e r i c eus both 8-C- and 6-C-s e r i c e u s var-. f l exuosus gl y c o f l avones plus s e r i c e u s va r . egg les ton ianus 3 ' -0-g lucos ide of s e r i c e u s s sp . huf fmani i o r i e n t i n A l l are members of the s e r i c e u s complex of F leak (1971) . Chemica l l y uni form q u a l i t a t i v e l y but high c o n c e n t r a t i o n s of o r i e n t i n in s s p . huffmani i d i s t i n -guishes t h i s t axon . h i l l i i 5-0-glucos ides d i s t i n g u i s h t h i s group. E parv i f1orus h o l o s e r i c e u s f lavone 5-0-g lucos ides M o r p h o l o g i c a l l y s i m i l a r to F2 t a x a . A l l are members of Smi th ' s (1938-1952) h i11 i i X caudatus s e c t i o n C a l c a r i t i . Except f o r L. neva-nevadensis dens is a l l taxa have extremely smal l f1owers . di f fusus Monospec i f i c group. Complex p r o f i l e of s imple compounds; un l i k e any o ther l up ine in t h i s s tudy . M o r p h o l o g i c a l l y d i s t i n c t ; a s imple-1eaved taxon of s.e U.S. Pu ta t i ve ances to rs in S. America (Dunn, 1971). Fl wyethi i h u mi c o1 a a r c t i cus a r c t i cus a r c t i c u s s sp . a r c t i cus ssp . canadensis ssp . suba lp inus 8-C-glycof1avones only C l o s e l y i n t e r r e l a t e d (Dunn & G i l l e t t , 1966) and s i m i l a r to C l ( i ) t a x a . The i r l ack o f g e n i s t e i n and f l avone 7-0-glu-cos ides makes t h i s group d i s t i n c t from C1 ( i ) . 1emmoni i X a l p e s t r i s g r e e n e i 1utescens arbustus F2 p seudopa r v i f l o ru s suphureus p a 1 m e r i argenteus a r g . s s p . caudatus s sp . argenteus r u b r i c a u 1 i s v a r i ed s imple but p r o f i l e s A l l members of s e c t i o n C a l c a r i t i (Smith , 1938-1952). Group d e l i m i t e d by morph-o logy and p a r t l y by chemical s i m p l i c i t y . j . . 1 eucophyl 1 us not cons ide red a part o f the C a l c a r i t i but f l o r a l c h a r a c t e r -i s t i c s a l l y i t with l up ines in t h i s group. F3 b i c o 1 o r po lyca rpus t exens i s b e n t h a m i i concinnus s pa rs i f1orus a r i z o n i c u s s t o l o n i ferus e l a tus h i. r s u t i s s i m u s 1 i t t o r a l i s neomexi canus va r i ed but most ly s imple p r o f i 1 e s Assortment of annuals and p e r e n n i a l s . R e l a t i o n s h i p s d i f f i c u l t to a s c e r t a i n us ing e i t h e r chemical or morpho log i ca l c h a r a c t e r s ; a l though some have been a l l i e d with members o f o ther groups (see d i s c u s s i o n ) . 122 •V. OVERVIEW F lavono ids as taxonomic cha r a c t e r s in Lupinus Cain & Ha r r i son (1958) de f ine cha ra c t e r s as "any a t t r i b u t e (or d e s c r i p t i v e phrase) r e f e r r i n g to the fo rm, s t r u c t u r e or be-hav iour which the taxonomist separa tes from the whole organism fo r a p a r t i c u l a r purpose such as comparison or i n t e r p r e t a t i o n . " Davis & Heywood (1963) view cha rac t e r s more s imply as any f e a -ture whose express ion can be measured, counted or o therwise as -s e s s e d . Any cha r a c t e r i s t h e o r e t i c a l l y use fu l in c l a s s i f i c a t i o n bu t , depending on the group being a s s e s s e d , some cha rac t e r s are more use fu l than others in d e l i m i t i n g t a x a . Davis & Heywood (1963) d i s t i n g u i s h between "good" and "bad " c h a r a c t e r s ; they des c r i be "good" cha rac t e r s as those b e i n g : 1) not sub jec t to wide v a r i a t i o n w i th in the samples being c o n s i d e r e d ; 2) not high in i n t r i n s i c gene t i c v a r i a b i 1 i t y ; . 3) not r e a d i l y s u s c e p t i b l e to env i ronmenta l m o d i f i c a t i o n ; 4) c o n s i s t e n t , that i s , they agree w i t h - c o r r e l a t i o n s of cha ra c t e r s e x i s t i n g in a p r e v i o u s l y con -s t r u c t e d na tu ra l system of c l a s s i f i c a t i o n . One a d d i t i o n a l r e -quirement s h o u l d , I f e e l , be i n c luded here : 5) that the cha r -a c t e r s be homologous between samples . Each of these r e q u i r e -ments i s d i s cussed in turn with r e f e r ence to morpho log i ca l and f l a v o n o i d cha rac t e r s in Lup inus . 1) I n t r a s p e c i f i c v a r i a t i o n of l up ine morphology i s abundant (.Hitchcock et al_. , 1961; P h i l l i p s , 1955 ; Dunn & G i l l e t t , 1966) and has l ed taxonomists to de s c r i be over 1500 separate t a x a . The v a r i a b i l i t y of Lupinus spec i es i s f u r t h e r i l l u s t r a t e d by the range of s i z e , ha i r t y p e , f lower c o l o u r , e t c . that appears 123 in d e s c r i p t i o n s of spec i es in f l o r a s . Lupine f l a v o n o i d s , however, are compara t i ve l y i n v a r i a b l e i n f r a s p e c i f i c a 1 1 y a l though q u a n t i t a t i v e v a r i a t i o n of c o n s t i t u e n t s i s commonly seen . Where q u a l i t a t i v e d i f f e r e n c e s e x i s t , they are u s u a l l y r e s t r i c t e d to minor or t r a ce compounds. Moreover , the q u a l i t a t i v e d i f f e r e n c e s represen t no v a r i a t i o n in the t axon 1 s b i o s y n t h e t i c r e p e r t o i r e of g l y c o s y l a t i o n and s u b s t i t u t i o n c a p a b i l i t i e s . 2) The i n t r i n s i c gene t i c v a r i a b i l i t y of Lupinus cha rac t e r s has had very l i t t l e a t t e n t i o n . R o l l i n s (1958) , in de te rmin ing which cha rac t e r s were use fu l in a taxonomic t reatment of D i thyrea ( C r u c i f e r a e ) , s t a ted that we should r e l y only upon cha rac t e r s that are so deeply rooted in the gene t i c c o n s t i t u t i o n of a spec i e s that they cannot be o b l i t e r a t e d e a s i l y or g r e a t l y mod-i f i e d by any s imple seg rega t ing f a c t o r . In o ther words, cha r -a c t e r s on which taxonomic deMsTons- are to be based should not be governed by a one- or t w o - f a c t o r i a l system of i n h e r i t a n c e , , f o r example, seed c o l o u r in peas. However, Davis & Heywood (1963) d i s t i n g u i s h between cha r a c t e r s that vary by s imple gen-e t i c mechanisms and those that are s imply based g e n e t i c a l l y but remain s t a b l e . They conc lude that "a taxonomic cha r a c t e r i s on ly as good as i t s c o n s i s t e n c y , no matter what i t s gene t i c bas i s . " Smith. ( 19 38-52) d i s t i n g u i s h e d , : in Lupinus , taxonomic e n t i t i e s based on morpho log i ca l cha rac t e r s that Hi tchcock et a l . C1961) view as va r y ing . so-g rea t l y as to render the cha rac t e r s use l ess fo r d e l i m i t i n g t a x a . They add that many spec i es have been desc r i bed on the bas i s of cha ra c t e r s that have been shown to behave as s imple Mendel ian. t r a i t s in o ther genera , f o r exam-p l e , f lower c o l o u r . F l avono id gene t i c s in Lupinus is as yet 124 unknown but t h e i r i n f r a s p e c i f i c constancy seems to s a t i s f y our requi rements for cha rac t e r s of low i n t r i n s i c v a r i a b i l i t y . 3) Enviromental m o d i f i c a t i o n of l u p i n e morphology has not been t e s t ed per s e . The- 'Var ia t ion that we do see in the genus has not been i d e n t i f i e d as phenotypic or g e n o t y p i c . In the case of f l a v o n o i d s , the present study r- •• s uggesteci that q u a n t i t a t i v e v a r i a t i o n in c e r t a i n compounds in Lupinus s e r i c e u s i s a phenotyp ic r esponse . Q u a l i t a t i v e l y (with the except ion of i s o -o r i e n t i n p roduc t ion ) the f l a v o n o i d s of L_^_ s e r i c e u s are una f f ec t ed by env i romenta l f a c t o r s . 4) A cha r a c t e r that i s cons ide red "good" must be c o n s i s -t e n t , r e f l e c t i n g a na tura l c l a s s i f i c a t i o n that was cons t ru c t ed wi thout the use of the cha ra c t e r s in ques t ion (Davis & Heywood, 1963). Th i s statement* l e a d s , as the authors admi t , to c i r c u l -a r i t y and a measure of a_ p o s t e r i o r i we i gh t i ng . If a p a r t i c u l a r cha r a c t e r subd i v ides a group in a way con t r a r y to a former taxonomy then that cha r a c t e r i s cons ide red " b a d " . I f the cha r -a c t e r agrees with the pre-set boundar ies and thereby s t rengthens that taxonomy then the cha rac t e r i s v a l u a b l e . This j udge -ment of cha r a c t e r use fu lness by comparing them to past taxonomies is a r a the r b l i n k e r e d approach and can lead to d i shones t y i f cha r a c t e r s are ignored s imply because they do not f i t i n to the pre-set system of taxonomic g roup ings . S ince there is no wide ly accepted subgener i c c l a s s i f i c a t i o n in Lupinus t h i s d i f f i c u l t y was a v e r t e d . In consequence, any f l a v o n o i d data that agreed or d i sag reed with va r i ous taxonomies were t r e a t ed as equa l l y v a l i d and use fu l (as any new data should be even i f they cut across p r e v i o u s l y e s t a b l i s h e d b o u n d a r i e s ) . The f a c t that a l l the 125 p r o f i l e s of a na tura l group such as the s e c t i o n P l a t yca rpus had the same f l a v o n o i d s does lend weight to that g roup ' s i n t e g r i t y and a l so to the c r e d i b i l i t y that f l a v o n o i d s are "good" taxonomic markers in the genus. 5} The ques t ion of cha rac t e r homology i s of extreme impor t -ance when samples are compared. When d e a l i n g with a c l o s e l y r e l a t e d group of p lan ts such as Lupinus there i s , however, l e s s of a problem at the morpho log i ca l l e v e l . Chemica l l y one must assume, fo r example, that l u t e o l i n 7-0-glucos ide in two l up ines is homologous, we assume that both l up ines b i o s y n t h e s i z e the compound in the same way. We may fur thermore s t a t e that the pos e ss ion of the compound r e f l e c t s a measure of gene t i c i d e n t i t y . It i s an assumption that has been made throughout the h i s t o r y of b iochemica l s ys temat i c s but i t has been ques t ioned r e c e n t l y by Brederode e_t aj_. (1974) and Crawford & Levy ( 1978) who document i n s t ances where va r ious genotypes g ive r i s e to the same f1avonoi d . An example of a case where a compound i s not homologous and g ives f a l s e ev idence of s i m i l a r i t y i s in the p roduc t ion of l y s i n e by the Oomycetes compared to most o ther f u n g i . The former s yn -t h e s i z e l y s i n e from pyruvate and L-aspartate -semialdehyde whi le the l a t t e r make l y s i n e from - k e t o g l u t a r a t e and ace t y l CoA ( Luckner , 1972 ) ; there are no b i o s y n t h e t i c steps in common. The same compound, l y s i n e , being manufactured v i a two d i f f e r e n t pathways cannot be c o n s i d e r e d , f o r taxonomic purposes , the same c h a r a c t e r . In Lu pi nus , our knowledge o f the gene t i c s o f f l a v -onoid b i o s y n t h e s i s i s n e g l i g i b l e , bu t , s i nce they are a group of o b v i o u s l y c l o s e l y r e l a t e d s p e c i e s , we can cont inue to assume that 126 each compound represen ts a homologous cha rac t e r throughout the genus u n t i l proven o the rw i se . It i s the a u t h o r ' s view that the f i v e requi rements of a taxonomic cha rac t e r have been s a t i s f i e d by the f l a v o n o i d s of Lup inus . They are comparable and in some cases s u p e r i o r to morpho log ica l cha r a c t e r s in terms of r e l i a b -i l i t y . R e l a t i o n s h i p s in Lupinus The groupings produced by the a n a l y s i s o f the f l a v o n o i d s of 73 taxa of Lupinus r e s u l t s in a pure l y phenet ic c l a s s i f i c a t i o n . Fur ther i n t e r p r e t a t i o n of the groups and cha rac t e r s are neces -sary i f an e v o l u t i o n a r y c l a s s i f i c a t i o n i s to be a t tempted . Stages in the e v o l u t i o n of morpho log ica l cha rac t e r s have been i l l u s t r a t e d by d e s c r i b i n g sequences of form in e x i s t i n g p lan t groups and the f o s s i l r e c o r d . Some workers , no tab ly Harborne (.1967 , 1 972 , 1 977 ) and Swain ( 197 5 ) , have attempted to arrange f l a v o n o i d s i n to an e v o l u t i o n a r y sequence by d e s c r i b i n g s u b s t i t -u t ion pa t te rns as p r i m i t i v e or advanced. Acco rd ing to Harborne [1975) a s imple f l a v o n o i d i s the r e s u l t of a few enzymatic steps and i s cons ide red p r i m i t i v e . An organism that s yn thes i z e s many h igh l y methy lated f l a v o n o i d s that are the r e s u l t of numerous b i o s y n t h e t i c s t e p s ' i s t h e r e f o r e advanced. As Gorna l l & Bohm (.1978) po in t o u t , these are o v e r - s i m p l i s t i c views that do not account f o r the r educ t i on processes in e v o l u t i o n l e ad ing to secondary s i m p ! i f i c a t i o n of morphology and/or chemis t r y . As examples, Gorna l l & Bohm (1978) c i t e the presence of C-g l yco -f l avones in both the bryophytes and the Cyperaceae and aurones in the bryophytes and the Composi tae . 1 2 7 The f l a v o n o i d s of Lupinus show i n s u f f i c i e n t v a r i a t i o n to serve as a bas i s f o r an e v o l u t i o n a r y c l a s s i f i c a t i o n s ince most i n t e r g r o u p d i f f e r e n c e s represen t d i f f e r e n c e s in type and p o s i t i o n of g l y c o s y l a t i o n and B-ring m e t h y l a t i o n . Fur thermore , the morpho log ica l c l a s s i f i c a t i o n s of l up ines have a l so been phenet i c s ince d i f f e r e n c e s in f lower s i z e , indumentum, banner r e f l e x i o n e t c . cannot be p laced in to an e v o l u t i o n a r y sequence. Perhaps the on ly e v o l u t i o n a r y sequence that can be no t i c ed i s a t rend from a woody pe renn ia l hab i t to an annual type of l i f e - c y c l e (David & Heywood, 1963). The l o s s of woodiness through e v o l u t i o n i s apparent in the case of I. di f f usus which Dunn .(1971) c o n s i d -ers to be de r i ved from South American shrubby a n c e s t o r s . Lupinus  d i f f u s us i s a herbaceous pe renn ia l with s imple l e a v e s , i t s pu t a t i v e ances to rs are a l so s imp le-1eaved ; i t would be i n t e r -e s t i n g to compare the f l a v o n o i d s of the herb with some of the South American s p e c i e s . The annual spec i es tha t Watson (1873) p laced i n t o sec t ion P Ia tycarpus are m o r p h o l o g i c a l l y and chem-i c a l l y d i s t i n c t from a l l o ther l up ines s t ud i ed (with the notab le excep t ion of _L. a rboreus ) . S ince the p l a t y ca rps are annua l s , we may specu l a t e t h a t ' t h e y represen t a c l o s e l y r e l a t e d group de r i ved from a common a n c e s t o r . The i n t e r e s t i n g a f f i n i t y with L_. a r b -oreus , a woody p e r e n n i a l , >provides ev idence that tempts one to suggest a d i r e c t r e l a t i o n s h i p between the t r e e . l u p i n and the p l a t y c a r p s , e s p e c i a l l y in the l i g h t . o f the numerous morpho log i ca l s i m i l a r i t i e s between I. arboreus and L_. densi f 1 orus va r . au reus . A l l o ther annuals in the study belong to Watson's Lupi nus proper and show u n s p e c i a l i z e d f l a v o n o i d p r o f i l e s c o n t a i n i n g no unusual compounds. They e x h i b i t s t rong chemical resemblances to many perenn ia l l u p i n e s but s ince t h e i r c h e m i s t r i e s are so unspec^ i a l i z e d , a d i r c t e v o l u t i o n a r y r e l a t i o n s h i p between any annual and perenn ia l l up ine i s unwarranted at t h i s t ime . The f a c t that both groups do have such s i m i l a r c h e m i s t r i e s , however, makes the i n i t i a l d i v i s i o n between annuals and pe renn i a l s even more unnat -ura l and p h e n e t i c . It i s hoped that fu tu re r e s e a r c h , be i t chemical or m o r p h o l o g i c a l , w i l l f i n d some common cha r a c t e r be-tween small numbers of annuals and pe renn i a l s and thereby lead to the r e c o g n i t i o n of sma l l e r more na tura l g roups . Convergence A f i n a l po in t to d i s cuss is the p o s s i b i l i t y that the chem-i c a l groups seen in t h i s study are the r e s u l t of convergence . I f our u l t ima te goal i s a phenet ic c l a s s i f i c a t i o n then conver -gence i s i r r e l e v a n t . I f we are i n t e r e s t e d in forming an e v o l u -t i o n a r y c l a s s i f i c a t i o n then the problem of convergence is more impor t an t . Cain & Ha r r i son (1960) suggest that convergence or p a r a l l e l i s m r e s u l t i n g in s i m i l a r i t y i s more l i k e l y to occur be-tween c l o s e l y r e l a t e d taxa than between d i s t a n t l y r e l a t e d forms. Consequent l y , convergence w i l l take p lace in p r e c i s e l y those groups in which i t i s most d i f f i c u l t to de tec t (Davis & Heywood, 1963). Our on ly s o l u t i o n to t h i s problem i s to make comparisons between taxa us ing as many d i f f e r e n t types of cha ra c t e r s as pos -s i b l e . The p r o b a b i l i t y of convergence being r e s p o n s i b l e fo r every c h e m i c a l , morpho log i ca l and c y t o l o g i c a l cha r a c t e r being simi. l a r m u s t be lower than the p r o b a b i l i t y of them being s i m i l a r by an assumed common o r i g i n . The f a c t that l up ine f l a v o n o i d s r e f l e c t s i m i l a r i t i e s p r e v i o u s l y noted by morpho log i s t s s t r e n g -thens the argument that w i th in-group members are more 129 c l o s e l y r e l a t e d than members of d i f f e r e n t groups . In s h o r t , the f l a v o n o i d ev idence tends to support the no t ion that the subgen-e r i c groupings that were cons t ru c t ed are na tura l groups and not un re l a t ed l up ines having s i m i l a r p r o f i l e s by convergence . VI. SUMMARY 130 At the o u t s e t of t h i s s t u d y , f o u r g o a l s were s t a t e d : 1) a d e t a i l e d a n a l y s i s o f the f l a v o n o i d s o f some North American l u p -i n e s ; 2) assessment of l u p i n e f l a v o n o i d s f o r t h e i r r e l i a b i l i t y as s p e c i f i c c h a r a c t e r s ; 3) c o n s t r u c t i o n o f a c l a s s i f i c a t i o n based on chemical d a t a ; 4) a comparison between t h i s c l a s s i f i c a t i o n and o t h e r c l a s s i f i c a t i o n s based on m o r p h o l o g i c a l o b s e r v a t i o n s . We must now d e c i d e whether these g o a l s have been a t t a i n e d . S e v e n t y - t h r e e i n d i v i d u a l s , r e p r e s e n t i n g 73 d i f f e r e n t t a x a were a n a l y s e d f o r f l a v o n o i d c o n t e n t and 56 d i f f e r e n t compounds noted. T h i s r e p r e s e n t s a p p r o x i m a t e l y one t h i r d of the s p e c i e s of Lupinus c u r r e n t l y r e c o g n i z e d i n North America ( K a r t e s z & K a r t e s z , 1980). S i n c e few new f l a v o n o i d s were d i s c o v e r e d a f t e r ten o r so s p e c i e s had been a n a l y s e d , i t i s c o n c l u d e d t h a t the compounds i s o l a t e d i n t h i s study r e p r e s e n t a r e a s o n a b l y complete l i s t o f the f l a v o n o i d s t h a t e x i s t i n North American l u p i n e s . F o l l o w i n g t h e i r e x t r a c t i o n and i d e n t i f i c a t i o n , the f l a v -o noids were a s s e s s e d as to t h e i r r e l i a b i l i t y as s p e c i f i c c h a r -a c t e r s . T h i s i n v o l v e d m o n i t o r i n g f l a v o n o i d v a r i a t i o n 1) d u r i n g the m a t u r a t i o n of a Lupinus s p e c i e s ; 2) throughout the g e o g r a p h i c range of t h r e e s p e c i e s ; 3) from f r e s h versus d r i e d p l a n t mat-e r i a l . Few q u a l i t a t i v e d i f f e r e n c e s were seen i n these a n a l y s e s a l t h o u g h v a r i a t i o n i n the r e l a t i v e amounts of the c o n s t i t u e n t f l a v o n o i d s was more common i n f r a s p e c i f i c a l l y . In the case o f Lupinus s e r i c e u s q u a n t i t a t i v e d i f f e r e n c e s i n o r i e n t i n accumr u l a t i o n r e f l e c t e d a d i s c o n t i n u o u s p a t t e r n of v a r i a t i o n from the n o r t h to the south o f the taxon's d i s t r i b u t i o n . Subsequent 131 a n a l y s i s o f s e e d l i n g m a t e r i a l , grown under u n i f o r m c o n d i t i o n s , suggested t h a t the v a r i a t i o n r e p r e s e n t e d i n the mature p l a n t s was the r e s u l t o f a p h e n o t y p i c response. Chapter IV d e t a i l e d an i n t e r s p e c i f i c study o f l u p i n e f l a v -o n o i d s . I n i t i a l l y , the d i s t r i b u t i o n o f the compounds appeared r a t h e r u n i f o r m from s p e c i e s to s p e c i e s ; some compounds were almost u b i q u i t o u s , f o r example, o r i e n t i n . S e v e r a l compounds were i n f r e q u e n t and presumably r e f l e c t e d o p e r a t i o n o f novel b i o s y n -t h e t i c r o u t e s . U t i l i z i n g the d i s t r i b u t i o n o f these more unusual compounds, s i x s u b g e n e r i c groups of l u p i n e s were d e f i n e d . A f t e r d i s c u s s i n g w i t h i n - g r o u p chemical a f f i n i t i e s , the groups were as s e s s e d f o r m o r p h o l o g i c a l s i m i l a r i t i e s by r e f e r e n c e to the l i t e r a t u r e and p e r s o n a l o b s e r v a t i o n s . In many c a s e s , c h e m i s t r y and morphology c o r r e l a t e d wel1 , th e r e b y s t r e n g t h e n i n g the i n t e g -r i t y of the groups as n a t u r a l u n i t s . F i n a l l y . w e must ask the q u e s t i o n : are f l a v o n o i d s u s e f u l as taxonomic markers i n Lupi nus? The answer i s r a t h e r i n c o n c l u s i v e . On the p o s i t i v e s i d e , t h e r e were groups t h a t were e a s i l y e x t r a c -t e d from the 73 t a x a a n a l y s e d . In s e v e r a l cases the ch e m i c a l groups appeared to be r e c o g n i z a b l e m o r p h o l o g i c a l groups. In o t h e r c a s e s , chemical a l l i e s were shown to be m o r p h o l o g i c a l l y d i s s i m i l a r a l t h o u g h f u r t h e r study r e v e a l e d i n t e r e s t i n g s i m i l a r -i t i e s . On the n a g a t i v e s i d e , 30 t a x a e x h i b i t e d very s i m p l e and s i m i l a r f l a v o n o i d p r o f i l e s ; to d i s c e r n chemical subgroups: w i t h i n t h e s e l u p i n e s seemed unwarranted a l t h o u g h some morphoT l o g i c a l d i s c o n t i n u i t i e s e x i s t . A p a r t from the t a x a o f P I a t y c a r -pus ( h a v i n g the f l a v o n o i d c y t i s o s i d e ) a l l the annual s p e c i e s c h e m i c a l l y resembled many of the p e r e n n i a l s . T h i s i n d i c a t e s f u r t h e r the a r t i f i c i a l i t y o f the a n n u a l / p e r e n n i a l d i v i s i o n i n the genus. R e g r e t t a b l y , the l a r g e and i n t e r e s t i n g genus Lupinus remains on the whole, t a x o n o m i c a l l y c o n f u s i n g a t the s p e c i e s l e v e l . However, the p r e s e n t study has r e v e a l e d s e v e r a l sub-g e n e r i c groupings t h a t appear to be n a t u r a l . I t i s hoped t h a t f u t u r e r e s e a r c h , u s i n g a d d i t i o n a l c h a r a c t e r s , w i l l expose y e t o t h e r groupings such as to i n c r e a s e our knowledge of r e l a t i o n ? / ; s h i p s w i t h i n the genus L u p i n u s . 133 REFERENCES Abrams, L. 1904. Lupinus 1 ongi f o l i us . F l o ra of Los Ange les . 209. Abrams, L. 1944. I l l u s t r a t e d F lo ra of the P a c i f i c S ta tes 2. S tan fo rd Un iv . P r e s s , S t a n f o r d , C a l i f o r n i a . Adanson, M. 1763. Fami l i e s des P l a n t e s . P a r i s . Agardh , J . G . 1835. Synopsis Gener is L u p i n i . Lundae, B e r l i n . A l s t o n , R.E. 1959. Phys io logy and the i n h e r i t a n c e of anthocyan in p igmen ta t i on . Genet ica 30: 261 Anonymous. 1979. Tarwi . In: T r o p i c a l legumes: Resorces fo r the Fu tu re . Board of Sc ience and Technology fo r I n t e r n a t i o n a l Development. Spec i a l r epor t No. 25. N.A.S . Washington, DC. Aye, T . T . 1969. M.Sc. T h e s i s , Univ . of Read ing . B.ecker, H . , G .Wi lk ing & K. Hostettmann. 1977. Separa t ion of i somer i c g l y c o f l a v o n e s by high performance l i q u i d chroma-tography . J . chrom. 136: 174-175 Bentham, G. & J . D . H o o k e r . 1865. Leguminosae. In: Genera P lant-arum 1 (2 ) . B i s b y , F.A. & K . W.Nichoi 1s . 1978. E f f e c t s of va r y ing cha rac t e r d e f i n i t i o n s on c l a s s i f i c a t i o n of Gen is teae (Leguminosae) . Bot . J . L i n n . Soc. 74: 97-121 B l a c k i t h , R.E. & R.A.Reyment. 1971. M u l t i v a r i a t e morphometr ies . Academic P r e s s , New York. Bragdtf, M.E. 1957. I n t e r s p e c i f i c c rosses in Lup inus ; c y to logy and i n h e r i t a n c e of f lower c o l o u r . Hered i tas 43: 338-356 Brederode , J . V a n , G.H.van Wie l ink-h i11ebrands & G.van N ig teveeh t . 1974. Dominance r e l a t i o n s between i s o v i t e x i n 7-0-g lucosy l-t r a n s f e r a s e s a l l e l e s in Melandr ium. Mo lec . Gen. Genet. 130: 307-314 B rucher , H. 1968. Die genet i schen reserven Sudamerikas fur d ie k u l t u r p f l a n z e n z i i c h t u n g . Theor . A p p l . Genet. 38: 9-22 Bu rkha r t , A. 1952. Las Leguminosas Argent inas s i l v e s t r e s y c u l t i v a d a s . 2nd ed . Acme Agency, Buenos A i r e s . B u r l e y , J . , P.J.Woodward & A . S .Hans . 1971. V a r i a t i o n in l e a f c h a r a c t e r i s t i c s among prov inances of Euca lyptus cama ldu lens i s Dehn. grown in Zambia. Aus t . J . Bot . 19: 237-249 C a i n , A . J . & G . A . H a r r i s o n . 1958. An a n a l y s i s of the t axonomis t ' s judgement of a f f i n i t y . P roc . Zool . Soc. Lond. 131: 85-98 134 C a i n , A . J . & G . A . H a r r i s o n . 1960. P h y l e t i c we igh t i ng . P roc . Zool . Soc. Lond. 135: 1-31 Chop in , J . & M . L . B o u i l l a n t . 1975. C-G l y cosy l f l a vones . In: J . B . Harborne, T . J .Mabry & H.Mabry ( eds . ) The F l a v o n o i d s . Chapman & H a l l , London. C h r i s t i a n , J . A . & D.B.Dunn. 1970. Nomenclature of the Lupinus  a r i z o n i c u s complex. T r ans . Mo. Acad. S c i . 4: 95-98 C h r i s t o f o l i n i , G. & L . F .Ch i a pel 1 a. 1977. S e r o l o g i c a l s ys temat i c s of the t r i b e Genis teae (Fabaceae ) . Taxon 26: 43-56 C l i f f o r d , H.T. & W.Stephenson. 1975. An i n t r o d u c t i o n to numer-i c a l c l a s s i f i c a t i o n . Acadamic P r e s s , New York. C o l l e t , G . , J . L . R o c c a , D.Sage & P . B e r t i c a t . 1976. Polyamides en chromatographie en phase l i q u i d e a haute per formance. J . Chrom. 121: 213 Cour t , W.A. 1977. High pressure reverse-phase l i q u i d chromatog-raphy of n a t u r a l l y o c c u r r i n g pheno l i c compounds. J . Chrom. 130: 287-291 Cox, B.J. & D.B.Dunn. 1969. A change in s ta tus of Lupinus humi col a va r . tetonens i s . T r ans . Mo . .Acad . S c i . 3: 82-83 Cox, B.J. 1973. P ro te in r e l a t i o n s h i p s among the pe renn ia l c a e s p i t o s e l u p i n e s . B u l l . T o r r . Bot . Club 100: 153-158 Cox, B.J. 1974a. Taxonomy of Lupinus a r i dus ( P a p i l i o n a c e a e ; G e n i s t e a e ) . Can. J . Bot . 52: 651-658 Cox, B.J. 1974b. Taxonomy of L u p i. n u s sel l u i us ( Pa pi 1 i onacea e) . Amer. M i d i . Nat. 91 : 214-223 Crawford , D.J . & M.Levy. 1978. F l avono id p r o f i l e a f f i n i t i e s and gene t i c s i m i l a r i t y . S y s t . Bot . 3: 369-373 Dav i s , P.H. & V.H.Heywood. 1963. P r i n c i p a l s of Angiosperm Taxonomy. Van Nos t rand , P r i n c e t o n . De C a n d o l l e , A . P . 1825. Prodromus Systemat ic Natura l i s . 2 . ' P a r i s . D e t l i n g , L .E . 1951. The c a e s p i t o s e l up ines of western North Amer i ca . Amer. M i d i . Nat. 45 : 474-499 Douglas , D.E. 1914. Journal kept by David Douglas dur ing his t r a v e l s in North Amer i ca , 1823-1827. Wesley & Son, London. Dunn, D.B. 1956. Leguminosae of Nevada, Part 11-Lupinus. C o n t r i b u t i o n s toward a f l o r a of Nevada 3.9. Beaureau of P lant I ndus t r y , U .S .Dept . of A g r i c u l t u r e , Washington, DC. Dunn, D.B. 1956a. The breeding system of L u p i n u s, group M i c r a n t h i . Amer. M i d i . Nat. 55: 443-472 135 Dunn, D.B. 1957. A r e v i s i o n of the Lupinus arbustus complex of the L a x i f l o r i . Madrono 14: 54-73 Dunn, D.B. 1959. Lupinus pusi11us and i t s r e l a t i o n s h i p s . Amer. M i d i . Nat. 62: 500-510 Dunn, D.B. 1965. The i n t e r r e l a t i o n s h i p s of the A laskan l u p i n e s . Madrono 8: 1-32 Dunn, D.B. & J . M . G i l l e t t . 1966. The Lupines of Canada and A l a s k a . Queen's P r i n t e r , Ottawa . Dunn, D.B. 1971. A case of l ong-d i s t ance d i s p e r s a l and "Rapid S p e c i a t i o n " in Lup inus . T r ans . Mo. Acad . S c i . 5: 26-38 Dunn, D.B. & W.E.Harmon. 1977. The Lupinus montanus complex o f Mexico and Centra l Amer i ca . Ann. Mo. Bot . Gard. 64: 340-365 Erdtman, H. 1963. Some aspects of chemotaxonomy. In: T.Swain ( e d . ) , Chemical P lant Taxonomy. Academic P r e s s , London. F l e ak , L.S. 1971. B i o sys t ema t i c s of the Lupi nus s e r i ceus complex. Ph.D. d i s s e r t a t i o n , Un iv . of M i s s o u r i , Co lumbia . F l e a k , L.S. & D.B.Dunn. 1971. Nomenclature of the Lupinus s er i ceus complex (Pap i1 ionaceae ) . T rans . Mo. Acad. S c i . 5: 85-88 F l i i ck , H. 1963. I n t r i n s i c and E x t r i n s i c Fac to rs a f f e c t i n g the p roduc t ion of Natura l P roduc t s . In: T.Swain ( e d . ) , Chemical P lant Taxonomy. Academic P r e s s , London. Fox, D.J. & K . E . G u i r e . 1976. Documentation fo r MIDAS. S t a t i s -t i c a l Research L abo ra to r y , Univ . of M i c h i g a n . 203pp. F r a n c i s , C M . & A . C . D e v i t t . 1969. The e f f e c t of wa te r logg ing on the growth and i s o f l a v o n e c o n c e n t r a t i o n in T r i f o l i um s u bter-raneum L. Aus t . J . Agr . Res. 20: 819 G ibbs , P.E. 1966. A r e v i s i o n of the genus Geni s ta L. Not. R. Bot . Gard. Ed inb . 27: 11-99 G o r n a l l , R.J. & B.A.Bohm. 1978. Angiosperm F l avono id E v o l u t i o n : A R e a p p r a i s a l . S y s t . Bot . 3: 353-368 Grant , V. 1971. P lant S p e c i a t i o n . Columbia Un iv . P r e s s , New York Hah lb rock , K. & H .G r i s ebach . 1975. B i o s y n t h e s i s of F l a v o n o i d s . In: J . B . Harborne, T . J .Mabry & H.Mabry ( e d s . ) , The F l avono ids Chapman & H a l l , New York. Harborne , J . B . 1967. Comparative B iochemis t r y of the F l a v o n o i d s . Academic P r e s s , London. 136 Harborne , J . B . 1967. Chemosystematics of the Leguminosae: F l avono id and i s o f l a v o n o i d pa t te rns in the t r i b e Gen i s t eae . Phytochemist ry 8: 1449-1456 Harborne, J . B . 1972. E vo l u t i on and f u n c t i o n of f l a v o n o i d s in p l a n t s . Recent Adv. Phytochem. 4: 107-144 Harborne, J . B . 1975. The B iochemica l Sys temat ics of F l a v o n o i d s . In : J . B . Harborne , T . J .M abry & H .Mabry (eds . ) , The F lavono ids Chapman & H a l l , New York. Harborne , J . B . 1977. F l avono ids and the e v o l u t i o n of the Angio-sperms. Biochem. S y s t . E c o l . 5: 7-22 Harborne, J . B . & C . A . W i l l i a m s . 1975. Flavone and f l a v o n o l g l y -c o s i d e s . In : J . B . Harborne , T . J .Mabry & H.Mabry ( e d s . ) , The F l a v o n o i d s , Chapman & H a l l , New York. Harborne, J . B . , J . L . I ngham, L .King & M.Payne. 1976. Luteone as a p r e - i n f e c t i o n a l a n t i f u n g a l agent in the genus Lupi nus. Phy tochemis t ry 15: 1485-1487 Ha rd ing , J . , CB.Manki .nen & M . H . E l l i o t . 1974. Genet ics of Lupinus V I I I : o u t c r o s s i n g , a u t o f e r t i 1 i t y and v a r i a b i l i t y in na tu ra l popu l a t i ons of the Nanus group. Taxon 23: 729-738 H a r r i s o n , G.A. 1964. In: V.H.Heywood & J . M c N e i l l ( e d s . ) , Phenet ic and Phy logene t i c c l a s s i f i c a t i o n . S y s t . A s s . Pub l . No . 6 : 115. London. Hawkes, J . G . 1978. The Taxonomis t ' s Role in the conse r va t i on of gene t i c d i v e r s i t y . In: H . E . S t r ee t ( e d . ) , Essays in P lant Taxonomy. Academic P r e s s , London. He re ra , F .L . 1942. El Tarhui o chochos , Lupinus m u t a b i l i s Sweet. Acad. Mac. C i e n c . Exac t . F i s . y Nat: 87-96. L ima. Hess , L.W. & D.B.Dunn. 1970. Nomenclature of the Lupinus a rgen -teus and I. caudatus complexes . Rhodora 72: 109-114 Heywood, V .H . 1972. The Role of chemis t ry in P lant S y s t ema t i c s . In: T.Swain ( e d . ) , Chemistry in E vo l u t i on and S y s t ema t i c s . Bu t t e rwor th , London. H i t c h c o c k , C . L . , A . C r o n q u i s t , M.Ownbey & J .W.Thompson. 1961. Vascu l a r P lants of the P a c i f i c Northwest . V o l . 3. Un iv . of' Washington- P r e s s , S e a t t l e . 'Hb'rhammer, L. & H.Wagner. 1962. I s o l a t i o n o f the e s t r o g e n i c 4 1 , 5 , 7- t r i h yd roxy i so f1avone 7-0-glucos ide ( g e n i s t i n ) from m u l t i f o l i a t e d l up ine (Lupinus p o l y p h y l l u s ) and broom (Cy t i sus s copa r i us . A r z n e i m i t t e l Forserr. 12: 1002-1005 Hu tch inson , J . 1959. The Fami l i e s of F lower ing P l a n t s . 2nd ed . Oxford Univ . P ress . 137 Hutch inson , J . 1964. The Genera of F lower ing P l a n t s . V o l . 1. Oxford Un iv . P r e ss . Hooker, W.J. & G .A .W.Arnot t . 1841. The Botany of Capta in . Beechey 's voyage. J . C r amer , Weinheim ( r e p r i n t , 1965). J epson , W.L. 1936. F l o r a of C a l i f o r n i a II: 246-283. C a l i f o r n i a School Book Depos i t o r y , San F r a n c i s c o . Johnson, E.L. & R .Stevenson. 1978. Bas ic L i q u i d Chromatography. V a r i a n , Palo A l t o , C a l i f o r n i a . J u r d , L. 1962. S t r u c t u r a l P r ope r t i e s o f f l a v o n o i d compounds. In: T .A .Ge issman (ed .) , The Chemistry of F l avono id Compounds MacMi11 an , New York. Kearney, T . H . & R .H .Peeb les . 1960. A r i zona F l o r a , 2nd ed . Un iv . o f C a l i f o r n i a , B e rk l e y . K a r t e s z , J . T . & R .Ka r t e sz . 1980. A synonomized c h e c k l i s t of the v a s cu l a r f l o r a of the Uni ted S t a t e s , Canada and Green land . 223-230. Un iv . North C a r o l i n a P r e s s , Chapel H i l l . K a z i m i e r s k i , T . 1963. S tud ies on the hybr id Lupinus hartwegi L i n d l . X _L. hybr i dus Now. Genet ic a f f i n i t y o f fou r teen American spec i es of l u p i n e s . Genet. P o l . 4: 233-268 K inghorn , A . D . , M.A.Se l im & S . J . Smolenski . 1980. A l k a l o i d d i s -t r i b u t i o n in some New World Lupinus s p e c i e s . Phytochem. 19: 1705-1710 K ings ton , D.G. I . 1979. High pressure l i q u i d chromatography of na tu ra l p r o d u c t s . L l o y d i a 42: 237-260 Laman, N.A. 1978. Ves t i Acad . Nuvuk BSSR, Ser . B i y a l . Navuk 1: 99-101 . Levy, M. 1977. Minimum b i o s y n t h e t i c - s t e p i n d i c e s as measures o f comparat ive f l a v o n o i d a f f i n i t y . S y s t . Bot . 2: 89-98 L innaeus , C. 1753. Spec ies Plantarum II. Facs . Ed. Roy. Soc. London. Luckner , M. 1972. Secondary metabol ism in P lants and An ima l s . Academic .Press , London. Mabry, T . J . , K.R.Markham & M.B.Thomas. 1970. The sys temat i c i d e n t i f i c a t i o n of f l a v o n o i d s . S p r i n g e r - V e r l a g , New York. Mankinen, C . B . , J . Harding & M . E l l i o t t . 1975. Genet i cs of Lupinus V I I I . V a r i a t i o n s inhte occur rence of a l k a l o i d s in na tura l popu l a t i ons of Lupinus nanus. Taxon 24: 415-429 Manley, C .H. & P .Shubiak. 1975. High pressure l i q u i d chromatog-raphy of an thocyan ins . J . Can. I ns t . Food S c i . Tech . 8: 35-39 Markham, K.R. 1975. I s o l a t i o n techn iques fo r f l a v o n o i d s . In : J . B . Harborne , T . J .Mabry & H.Mabry ( e d s . ) , The f l a v o n o i d s . Chapman & H a l l , New York. Moore, D.M. 1972. In: D .H .Va l en t i n ( e d . ) , Taxonomy, Phytogeog-raphy and E v o l u t i o n : 115-138. Academic P r e s s , London. M o r r i s o n , D.F. 1967. M u l t i v a r i a t e S t a t i s t i c a l Methods. McGraw H i l l , New York. Munz, P.A. 1959. A C a l i f o r n i a n F l o r a . Un iv . C a l i f o r n i a P r e s s , Berkl ey.' N i c h o l l s , K.W. & B.A.Bohm. 1978'. F l avono ids of Lupinus arboreus Phytochemist ry 18: 1078 Niemann, G. J . & J . van Brederode. 1978. Sepa ra t ion of g lyco-f l avones by high pressure l i q u i d chromatography. J : Chrom. 152: 523-527 N i k l a s , K.J . & D. E . G i anass i . ' 1977 . F l avono ids and o ther chemical c o n s t i t u e n t s of f o s s i l Miocene Zelkova (Ulmaceae) . Sc ience 196: 877-878 Nowacki, E. 1960. Sys temat ics of Genis teae in the l i g h t of chemical a n a l y s i s . Genet. P o l . 1 :119-143 Nowacki, E. & D.B.Dunn. 1965. Shrubby C a l i f o r n i a l up ines and r e l a t i o n s h i p s suggested by a l k a l o i d con t en t . Genet. P o l . 5: 47-56 Nowacki, E. & W.Prus-Glowacki . 1971. D i f f e r e n t i a t i o n of p ro t e i n f r a c t i o n s in spec i es and v a r i e t i e s of the genus Lupinus with the use of s e r o l o g i c a l methods. Genet. P o l . 12: 245-259 Nutta l , T. 1818. The genera of North American p l a n t s . D. Hear t t Ph i l a del phi a . Ove r ton , E. 1899. J ahrb . Wiss . Bot . ( L e i p z i g ) 33: 171 P a r d o d i , L.R. 1938. P lantas psamof i l as ind igenas que pueden ser c u l t i v a d a s para c o n s o l i d a dunas. J . Agronom. y Ve t e r . 1937: 320. B u e n o s A i r e s . Pa rke r , W.H. 1976. Comparison of numerical taxonomic methods used to es t imate f l a v o n o i d s i m i l a r i t i e s in the Limnanthaceae B r i t t o n i a 28: 390-399 P h i l l i p s , L .L . 1955. A r e v i s i o n of the pe renn ia l spec ies of Lu pi nus of North America e x c l u s i v e of southwestern Uni ted S tates and Mexico . Res. S tud . S t . C o l l . Wash. 23: 161-201 P h i l l i p s , L .L . 1957. Chromosome numbers in Lup inus . Madrono 14: 30-36 P i p e r , C.V. 1906. F l o r a of the S tate of Washington. Con t r . Nat. Herb. 11 : 350-358 139 Planchero de Rave lo , A. 1978. A monograph of Lupinus fo r Argen -t i n a . Ph.D. d i s s e r t a t i o n . Univ . M i s s o u r i , Co lumbia . P o l h i l l , R.M. 1971. Gen is teae (Adans . )Ben th . and r e l a t e d t r i b e s (Leguminosae) . Ph.D. t h e s i s , Univ . Reading. Pu rsh , F. 1814. F l o r a Americae S e p t e n t r i o n a l i s . Whi te , Cochrane & Co. London. R o l l i n s , R.C. 1958. The gene t i c e va l ua t i on of a taxonomic c h a r -ac te r in Di thyrea ( C r u c i f e r a e ) . Rhodora 60: 145-152 Rothmaler , W. 1944. Die G l i ederung der Gattung Cy t i sus L. Feddes Reper t . 53: 137-150 Rydberg, P.A. 1917. F l o r a of the Rocky Mountains and Ajacent P l a i n s . S c o g i n , R. 1973. Leucine aminopept idase polymorphism in the genus Lup inus . Bot . Gaz. 134: 73-76 S e a l , H.L. 1964. M u l t i v a r i a t e S t a t i s t i c a l A n a l y s i s fo r B i o l o g i s t s Methuen , London . Smi th , C.P . 1938-1952. Spec ies Lupinorum S i g n a t u r e s . P r i v a t e l y p u b l i s h e d . Smi th , C .P . 1938. A p r e l i m i n a r y c a t a l o g o f l up ines of Baja C a l i f o r n i a and Sonora . Spec ies Lupinorum, paper 2: 28-32 Smi th , C.P . 1939. A d i s t r i b u t i o n a l c a t a l o g of l up ines in A r i z o n a . Spec ies Lupinorum, paper 10: 119-143 Smi th , C P . 1944. Lupinus . In: L. Abrams, I l l u s t r a t e d f l o r a of the P a c i f i c S t a t e s , vol . 2. S tan fo rd Un iv . P r e s s , C a l i f o r n i a Smi th , C P . 1946. Lupinus in North America- Bas ic Taxonomy. Spec ies Lupinorum, s i gna tu re 32: 545-551 S o k a l , R.R. & P .H .A .Snea th . 1963. P r i n c i p l e s of Numerical Tax-onomy. Freeman & C o . , San F r a n c i s c o . S t e b b i n s , G .L . 1944. V a r i a t i o n and E vo l u t i on in P l a n t s . Columbia Univ . P r e ss , New York. S t e e l , R.G.D. & J . H . T o r r i e . 1960. P r i n c i p l e s and Procedures of S t a t i s t i c s . McGraw-Hi l l , New York. S t r a c k , D. , K . F u i s t i n g & G . P o p o v i c i . 1979. High pressure l i q u i d chromatography a n a l y s i s of oat (Avena s a t i va) f l a vone d e r i v -a t i v e s . J . Chrom. 176: 27Q-273 S t r a c k , D. & J . K r a u s e . 1978. Reversed-phase HPLC s epa r a t i on of n a t u r a l l y o c c u r r i n g mixtures of f l avone d e r i v a t i v e s . J . Chrom. 156: 359-361 1 4 0 S u t t e r , A . , R.Ortmann & H . G r i s e b a c h . 1972. P u r i f i c a t i o n and p r o p e r t i e s of an enzyme from c e l l suspens ion c u l t u r e s of pa r s l e y c a t a l y s i n g the t r a n s f e r of D-glucose from UDP-D-g lucose to f l a v o n o i d s . Biochem. B iophys . Acta 258: 71-87 S u t t e r , A. & H .G r i s ebach . 1973. UDP-glucose: f l a v o n o l 3-0-g l u c o s y l t r a n s f e r a s e from c e l l suspens ion c u l t u r e s of p a r s l e y . Biochem. B iophys . Acta 309: 289-295 Swain, T. 1975. E vo l u t i on of f l a v o n o i d compounds. In: J . B . Harborne , T . J .Mabry & H.Mabry ( e d s . ) , The F l a v o n o i d s . Chapman & H a l l , New York. T i s s u t , M. & K.Egger . 1972. Les G l y cos ides f l a von iques f o l i a i r e s de quelques a r b r e s , au .cour du c y c l e ve 'getat i . f . Phyto-chemis t ry 11: 631-634 T o u r n e f o r t , J . P . 1719. I n s t i t u t i o n e s r e i h e r b a r i a e . P a r i s . T u r e s s o n , G. 1922. The geno typ i ca l response of the p lant spec ies to hab i t a t and c l i m a t e . Hered i tas 3: 211-350 T u r n e r , B.L. 1957. The chromosomal and d i s t r i b u t i o n r e l a t i o n s h i p s of Lupinus texens i s and L_. subcarnosus . Madrono 14: 13-16 Tu rne r , B.L. 1958. The Legumes of Texas . Un iv . Texas P r e s s , A u s t i n . Vaugn, P. & D.B.Dunn. 1977. The Lupi nus 1at i f o l i us Agardh com-p l e x . T r a n s . Mo. Acad . S c i . .10: 89 r 106 Venkataraman, K. 1962. Methods fo r de te rmin ing the s t r u c t u r e s of f l a v o n o i d compounds. In : T .A .Ge issman ( e d . ) , the Chem-i s t r y of f l a v o n o i d compounds. M a c M i l l a n , New York. W a l l e r , G.R. & E.Nowacki . 1978. A l k a l o i d b i o l ogy and metabol ism in p l a n t s . Plenum, New York. Ward, R.S. & A . P e l t e r . 1974. The a n a l y s i s o f mixtures of c l o s e l y r e l a t e d n a t u r a l l y o c c u r r i n g o rgan i c compounds us ing HPLC. J . Chrom. S c i . 12 : 570 Watson, S. 1873. Rev i s ions o f the e x t r a - t r o p i c a l North American spec i es of the genera Lup inus , Potent i11 a and Oenothera . P roc . Amer. Acad. 8: 517-618 Watson, S. 1876. P roc . Amer. Acad. 12: 250 West, L . G . , P .M.B i rac & D . E . P r a t t . Separa t ion of the i somer i c i s o f l a v o n e s from soybeans by HPLC. J . Chrom. 150: 266-268 W i l k i n s o n , J . G . 1854. A popular account of the anc ien t Egyp t i ans . II. New York. Wulf , L.W. & C.W.Nagel . 1976. A n a l y s i s of pheno l i c ac ids and f l a v o n o i d s by HPLC. J . Chrom. 116: 271-279 141 APPENDIX I. P lants and Sources D e t a i l s o f c o l l e c t i o n s of p lant ma te r i a l used in the s tudy . A l l c o l l e c t i o n housed in UBC except where no ted . A. Bulk samples of Lupinus spec i es used fo r the e x t r a c t i o n , i s o l a t i o n , c h a r a c t e r i z a t i o n and accumulat ion of f l a v o n o i d compounds that were subsequent l y used in comparat ive TLC. Lupinus arboreus Sims. N i c h o l l s 7702, 1 June 1977. Grown from seed c o l l e c t e d on UBC campus, Thunderb i rd Road. 900 g ex t r ac ted ( f r e sh we igh t ) . L_. argenteus Pursh N i c h o l l s 7840, 30 June 1978. Hwy.93, 2 mi les s. D a r l i n g t o n , Butte C o . , Idaho. 15 g .ext rac ted ( d r y ) . I. excub i tus Jones v a r . j o h h s t o n i i C.P.Sm. N i c h o l l s 7817, 15 June 1978. Hwy.2, Cooper Canyon en t r ance , San Gabr ie l M t n s . , Los Angeles Co. C a l i f o r n i a . 25 g ( d r y ) . j . . h o l o s e r i c e u s Nut t . ex T o r r . & Gray N i c h o l l s 7832, 28 June 1978. 2 mi les a long L a n o i l l e Canyon Road, o f f Hwy . l l near E l k o , Elko Co. Nevada. 15 g (dry) e x t r a c t e d . L_. 1 eucophyl 1 us Dougl . ex L i n d l . N i c h o l l s 7708, 20 June 1977. Shuswap Road, 10 mi les east of Lumby, BC. 400 g ( f r e s h ) e x t r a c t e d . . j . . 1 i 11 o r a 1 i s Dougl . N i c h o l l s 7701, 1 June 1977. Roadside near Thunderb i rd Stad ium, UBC campus, Vancouver , BC. 50 g ( f r e sh ) e x t r a c t e d . I. ruber H e l l e r N i c h o l l s 7810, 14 June 1978. h mi le s. P i t t R iver B r i d g e , Hwy. 299, Canby, Modoc Co. C a l i f o r n i a . 20 g (dry) e x t r a c t e d . J_. se l 1 ul us K e l l . va r . 1 obbi i (Gray ex Wats.) Cox N i c h o l l s 7837, 29 June 1978. Elk Meadows, Hwy.21, 50 mi les n. Lowman, Custer Co. Idaho. 12 g dry e x t r a c t e d . j . . s e r i ceus v a r . s e r i c e u s Pursh N i c h o l l s 7615, 14 May 1976. T r i n i t y V a l l e y , Lumby, BC. 1.1 kg ( f r e sh ) e x t r a c t e d . 142 B. Lupinus arboreus specimens, used in Experiment I ,Chapt . I I I . N i c h o l l s 7703, 1 June 1977. Thunderb i rd Road, UBC campus, Vancouver , BC. Franko s . n . , 22 May 1976. So. Pender I s l a n d , BC. Eastham s . n . , 2 June 1938. Beacon H i l l Park, V i c t o r i a , BC. Hebda s . n . , 1 4 June 1975. Point Rober t s , Whatcom Co. Washington. Spe l l enburg s . n . , 17 June 1966. Monroe, King Co. Washington. Stewart 6163, 22 June 1952. Newport, L i n c o l n Co. Oregon. K ra j ina s . n . , 10 June 1953. P i s t o l R i v e r , Josephine Co. Oregon. F inch 315, 6 June 1953. Crescent C i t y , Del Norte Co. C a l i f o r n i a Gorna l l 0090, 10 June 1977. Westpor t , Mendocino Co. C a l i f o r n i a . Frank 92, 8 Ju ly 1965. L i t t l e Sur R i v e r , C a l i f o r n i a . C. Lupinus bi col or L i n d l . specimens used in Experiment I Ch. I I I . Davidson s . n . , 23 May 1914. Savary I s l a n d , BC. ( s sp . b i c o1o r) N i c h o l l s 76513, 13 May 1976. Nanoose H i l l , Nanaimo, BC. ( s sp . b i c o1o r) Davidson s . n . , 15 May 1913. R i f l e Range, V i c t o r i a , BC. ( s sp . b i c o 1 o r ) McCa l la 5322, 24 A p r i l 1939. Mos i e r , Wasco Co. Oregon. Maze et_ aj_. 765 , 4 A p r i l 1971. Shasta C i t y , Shasta Co. C a l i f o r -n i a , ( s sp . t r i d e n t a t u s (Eastw.)Dunn) Maze et aj_. 742 , 4 A p r i l 1971. Oasis Rd. e x i t ' I 5, Shasta Co. C a l i f o r n i a . ( s sp . umbel 1atus (Greene)Dunn) Maze et aj_. 914, 13 A p r i l 1971. Elk Creek, Glen Co. C a l i f o r n i a . T~ssp. umbel 1 atus ) Booras 1, 26 A p r i l 1955. C l e a r l a k e Park, Lake Co. C a l i f o r n i a . Dunn 938, 29 March 1941. Pomona, Los Angeles Co. C a l i f o r n i a , ( s sp . mi c rophy l1 us (Wats. )Dunn Dunn 1278, 10 June 1941. Ha rpe r ' s Ranch, San Diego Co. C a l i f o r -n i a , ( s sp . marginatus Dunn) 143 D. Lupinus s e r i c e u s c o l l e c t i o n s fo r Experiment I I , Ch . I I I . Popu la t ion number 1 N i c h o l l s 7825, 22 June 1978. Mi le 593, Hwy.89 5 mi les s. Jacob Lake, Coconino Co. A r i z o n a . 2 N i c h o l l s 7827, 24 June 1978. North of Esca l en te on Loa Rd. D i x i e l a n d Nat. Fo r . G a r f i e l d Co. Utah. 3 N i c h o l l s 7828, 26 June 1978. Hwy.24, 1 mi le s. L i t t l e Creek t u r n o f f , S ev i e r Co. Utah. 4 N i c h o l l s 7829, 27 June 1978. Mountain road w. Marysva l e , P iu te Co. Utah. 5 N i c h o l l s 7830, 27 June 1978. 3 mi le past campgroud sw. Joe ' s V a l l e y Re sevo i r , Emery Co. Utah. 6 N i c h o l l s 7831, 27 June 1978. Recrea t ion a r e a , w. s ide of S c h o f i e l d R e s e v o i r , Carbon Co. Utah. 7 N i c h o l l s 7835, 29 June 1978. Hwy.21, 27 mi les n. B o i s e , Boise Co. Idaho. 8 N i c h o l l s 7836 , 29 June 1978'. Mountain View campground, Lowman, Bo ise Co. Idaho. 9 N i c h o l l s 7841, 1 mi le w. campground, Pebble Creek Road, Lava Hot S p r i n g s , Bannock Co. Idaho. 10 N i c h o l l s 7851, 5 Ju ly 1978. Banks of Henry 's Fork , 2 mi les n. Ash ton , Fremont Co. Idaho. 11 N i c h o l l s 7853 , 6 Ju ly 1978. M i le 28, Hwy.19.1, 20 mi les n. West Ye l l ows tone , G a l l a t i n Co. Montana. 12 N i c h o l l s 7854, 6 Ju l y 1978. \ mi l e s. j u c t i o n I90/89N, e. L i v i n g s t o n e , Park Co. Montana. 13 N i c h o l l s 7855, 7 Ju l y 1978. Hwy.89, 15 mi les n. White Sulphur S p r i n g s , Meagher Co. Montana. 14 N i c h o l l s 7858, 7 Ju l y 1978. Hwy.89, 5 mi les n. Monach, Cascade Co. Montana. 15 N i c h o l l s 7859, 7 Ju ly 1978. Hwy.287, 9 mi les s. Choteau, Teton Co. Montana. 16 N i c h o l l s 7860, 7 Ju ly 1978. Hwy.89, mi le 88 , between Browning and Choteau, Teton Co. Montana. 1 4 4 D. (contd . ) Popu la t i on number 17 18 19 20 21 22 23 24 25 26 27 28 2 9 30 31 32 N i c h o l l s 7861, 8 Ju ly 1978 Park, F la thead Co. Montana Hwy.2, 8 mi les e. West G l a c i e r N i c h o l l s 7862, 8 Ju ly 1978. Hwy.2, 2 mi les w. Paul Bunyan campground. 10 mi les s. L i bby , L i n c o l n Co. Montana N i c h o l l s 7863, 8 Ju ly 1978. Hwy.2, Moyier R iver Rd Moyier R iver b r i d g e , Boundary Co. Idaho. w N i c h o l l s 7867, 8 Ju ly 1978. Hwy.95a, 2 mi les s.on e. s ide Coeur d 'A l ene Lake, Kootenai Co. Idaho. N i c h o l l s 7872, 9 Ju ly 1978. Washington. N i c h o l l s 7873, 9 Ju l y 1978. Perce Co. Idaho. N i c h o l l s 7874, 9 Ju l y 1978. New Meadows, Adams Co. Idaho N i c h o l l s 7877, 10 Ju ly 1978. Powder, Union Co. Oregon. N i c h o l l s 7879, 10 Ju l y 1978. Yakima Co. Washington. N i c h o l l s 7880, 10 Ju l y 1978. of Yakima R i v e r , Yakima Co. Centra l F e r r y , G a r f i e l d Co. Hwy.95, s. Lew i s ton , Nez Packer John 's Cabin S tate Park, Hwy.237, 4 mi les n. North Hwy.12, 6 mi les nw. Yakima, Hwy.821, mi le 10, f l o o d p l a i n Washi ngton . N icho l1s 7881 , 11 Ju ly 1978 Okanogan Co. Washington. N icho l1s 7882 , 11 Ju l y 1978 Douglas Co. Washington. N i c h o l l s 7905 F r u i t v a l e , BC Hwy.97, 1 mi le s. B rewster , Hwy.151, 7 mi les e. Orondo, 2 Ju l y 1979. Route 3, between Creston and N i c h o l l s 7916, 5 Ju ly 1979 N i c h o l l s 7910 Park. Route 6 2 Ju ly 1979 A l b e r t a . T r i n i t y V a l l e y , Lumby, BC 1 km n. Waterton Lake Nat N i c h o l l s 7914, 3 Ju ly 1979. Route 2, Between Park land and Nanto.n, A l b e r t a . 145 E. Lupinus col 1ec t ions used fo r i n t e r s p e c i f i c v a r i a t i o n study Chapter VI. OTU or taxon No . 1 Lupinus e l a tus Johnston N i c h o l l s 7818, 15 June 1978. Cooper Canyon, o f f Hwy.2, San Gabr i e l Mounta ins , Los Angeles Co. C a l i f o r n i a . 2 I. barbi ger Wats. Holmgren 2461, 14 August 1965. Posy Lake, Aquar ius P l a t e a u , D i x i e l a n d Nat. For . 15 mi les n. E s c a l e n t e , G a r f i e l d Co. Utah 3 L_. s e r i c e u s Pursh va r . s e r i c e u s Kra j ina & F raser 645-37, 23 May 1964. Mount A n a r c h i s t , Osoyo.os, BC. 4 j . . s e r i ceus s s p . huf fmani i (C.P.Sm.) Fleak & Dunn N i c h o l l s 7828, 26 June 1978. Hwy.24, 1 mi le s. L i t t l e Creek t u r n o f f , S e v i e r Co. Utah. 5 L_. s e r i c e u s va r . egg les ton ianus C.P.Sm. N i c h o l l s 7907, 2 J u l y ' l 9 7 9 . Route 3, mi le 35, near Wardner BC. 6 I. s e r i c e u s va r . f 1 exuosus ( L i n d l . ex Agardh) C.P.Sm. N i c h o l l s 7902, 1 Ju l y 1979. Route 3, Johnstone Creek camp-ground, B r i d e s i l i e , BC. 7 _L. b i ' co lor L ind l . N i c h o l l s 76513, 13 May 1976. Nanoose H i l l , Nanaimo, BC. 8 L. po lyear pus Greene N i c h o l l s 76135, 13 May 1976. Nanoose H i l l , Nanaimo, BC. 9 I. neomexicanus Greene Ya tsk ievych 79-448. New Mexico . 10 I. 1emmoni i C . P . Sm. N i c h o l l s 7824, 22 June 1978. Hwy.89, mi le 573, 5% mi les e. Jet .67.. near Jacob Lake, Coconino Co. A r i z o n a . 11 J_. t exens i s Hook. Benson 9328, 24 A p r i l 1966. Brownwood, Brown Co. Texas [Logan) , 12 L. den s i f l o r us Benth. v a r . dens i f T o r u s Eastham 6869, 11 June 1939. Beacon H i l l , V i c t o r i a , BC. 146 13 J.. dens i f 1 orus va r . aureus ( K e l l . ) Munz Bohm & Banek 1413, 9 May 1980. Hwy.101, mi le 7 6 . 1 6 , Mendocino Co. C a l i f o r n i a . 14 j . . ruber H e l l e r N i c h o l l s 7810, 13 June 1978. P i t t R iver B r i d g e , Canby, Modoc Co. C a l i f o r n i a . 15 L_.arboreus S ims. N i c h o l l s 7702, 1 June 1977. Thunderb i rd Rd. UBC campus, Vancouver , BC. 16 L_. h o r i z o n t a l i s H e l l e r Holmgren 7659, 7 A p r i l 1974. Mojave Dese r t , Kern Co. C a l i f o r n i a . (Logan ) . 17 L. f 1avocu l a tus H e l l e r Beatty & Car l 3382, 9 June 1965. P lo t 27, Test S i t e , Nye Co. Nevada. (Logan) 18 L_. b e n t h a m i i H e l l e r Breedlove 2774, 12 May 1962. New Cayama, San Luis Obispo Co . Cal i fo rm 'a . 19 L_. cone i nnus Agardh H i tchcock & Muhl ick 22153, 16 March 1962. Temescal Canyon, R i v e r s i d e Co. C a l i f o r n i a . 20 L_. s pa rs i f 1 or u s Benth. Foust 007 , 21 I. a r i z o n i c u s '(Wats .)Wats . Maguire 10152 , 20 March 1935. Graham Mtn.s. Graham Co. A r i zona . (Logan) 22 I. s t o l o n i f e r u s L. Egg lestone 8904, 13 September 1972. A t h o l , Bonner Co. Idaho (Logan) . 23 L. wyeth i i Wats . K ra j ina & F raser 645-21 , 22 May 1964. O l a l l a , B.C. 24 I.. h i l l i i Greene H igg ins 9072, 9 August 1973. Pine V a l l e y , Washington C o . , Utah. (Logan) 25 I. h o i o s e r i ceus Nut t . ex T o r r . & Gray N i c h o l l s 7832, 28 June 1978. 2 mi les a long L a n o i l l e Canyon Rd. Elko , Elko Co . Nevada . 147 26 j . . parv i f l orus Nut t . ex Hook. & A rn . Morgan 7 , 17 August 1 9 6 3 . I s land Park Resevo i r , Freemont Co. Idaho. (Logan) 27 I. caudatus K e l l . X I. h i l l i i Greene N i c h o l l s 7 8 2 2 , 22 June 1 9 7 8 . South Rim, Grand Canyon, Coconino Co. A r i z o n a . 28 J_ . nevadens is H e l l e r Tiehm 4 2 1 0 , 19 May 1 9 7 8 . Sage Hen H i l l s , Humboldt Co. Nevada. (Logan) 29 j . . 1 eucophyl 1 us Dougl . ex L i n d l . N i c h o l l s 7 7 0 8 , 20 June 1 9 7 7 . Shuswap Rd. 10 mi les e. Lumby BC. 30 L. X a l p e s t r i s Ne l s . Smith 1 9 0 5 , 21 Ju l y 1 9 0 9 . Red Butte Canyon, S a l t Lake C i t y Utah. (Logan) 31 _L . g r e e n e i Ne l s . Galway 2 1 2 5 9 , 7 Ju ly 1 9 4 4 . Panaca, L i n c o l n Co. Nevada. (Logan) 32 j . . pseudoparv i f 1 orus Rydb. Davis 2 3 9 9 , 28 June 1 9 4 0 . R i g g i n s , Idaho Co. Idaho (Logan) 33 I. 1utescens C.P.Sm. N i c h o l l s 7 6 2 2 , 10 Ju ly 1 9 7 6 . East banks of Lake Okanagan, BC. 34 J_. sul phureus Dougl . ex Hook. Eastham s . n . , 8 May 1 9 4 0 . Ka leden, BC. 35 I. palmeri Wats. Gould 4 7 1 5 , 1 June 1 9 4 8 . S i e r r a Ancha Mtns. G i l a Co. Ar i zona . 36 L_. .argenteus va r . argenteus Pursh N i c h o l l s 7 9 1 2 , 30 June 1 9 7 9 . Route 5 , 30 mi les s. l e t h -b r i d g e , A l b e r t a . 3 7 L.. argenteus va r . r u b r i c a u l i s (Greene) H e s s & Dunn Welsh 1 7 5 4 9 , 13 Ju ly 1 9 7 8 . Monroe Mtn. Sev ie r Co. Utah. (Logan) 38 L. caudatus K e l 1 . N i c h o l l s 7 8 4 3 , 3 Ju ly 1 9 7 8 . Hwy. 1-70 , near Idaho Spr ings Co lo r ado . 148 39 j . . a r b u s t u s Dougl . ex L i n d l . G r i e r s o n & Jackson s.n., 16 May 1948. Wenachee V a l l e y 40 j . . g r a y i i (Wats.)Wats. Grown from seed at UBC. Seed s o u r c e : Rancho Santa Anna Gard 41 L.. a l b i f r o n s Benth. ex L i n d l . M c C a l l a 6050 , 9. May 1940. Empire Union High S c h o o l , San Diego Co. C a l i f o r n i a . 42 I. c h a m i s s o n i s Eschs. B r e e d l o v e 4281, 29 March 1963. A n t i o c , Contra Costa Co. C a l i f o r n i a. 43 L.. humi c o l a N e l s . H i t c h c o c k & M u h l i c k 11916, 3 J u l y 1945. H a l f Moon Canyon, Fergus Co. Montana. (Logan) 44 I. a r c t i c u s Wats. s s p . a r c t i cus T a y l o r 375, 18 June 1956. C a s s i a r , BC. 45 JL. a r c t i c u s ssp. c a n a d e n s i s (C.P.Sm.) Dunn M c C a l l a 5051, 30 J u l y 1938. P a r a d i s e , Mt. R a n i e r , Wash. 46 L_. a r c t i cus ssp. s u b a l p i n u s ( P i p e r & Robbins) Dunn Brayshaw s.n., 21 September 1954. Mt. C h e l a n , BC 47 I. 1 a t i f o l i us Agardh M a r t i n 4830, 21 June 1939. S i s t e r s , Deschutes Co. Oregon. (Logan) 48 L. 1 i t t o r a l i s Dougl . N i c h o l l s 7701, 1 June 1977. Near T h u n d e r b i r d Stadium, UBC campus, Vancouver, BC. 49 J_. a r i d u s Dougl. ex L i n d l . H i t c h c o c k 4977, 28 June 1939. Flood p l a i n of Roger R i v e r , F a r e w e l l Bend F o r e s t camp, Jackson Co. Oregon (.Logan). 5 0 J.. conf e r t u s Ke 11 . H e l l e r 15160, 14 June 1938. Feather R i v e r hwy. Gray's F l a t C a l i f o r n i a . (Logan) 51 L. minimus Dougl. Constance 1873, 1 June 1937. Anatone B u t t e , Blue Mtns. A s o t i n Co. Washington. (Logan) 52 L.. 1 ya 11 i i Gray T a y l o r 1330 , 14 J u l y 195.1. C a t h e d r a l Lakes, A s h n o l a , BC. 149 53 JL. s el 1 ul us K e l l . v a r . 1 o b b i i (Gray ex Wats.) Cox N i c h o l l s 7802, 13 June 1978. Hwy.58, n. K lamath, Oregon. 54 _L. kuschei Eastw. Ca lder & G i l l e t t 25144, 9 June 1960. A t l i n , BC 55 J_. di f f usus Nut t . Leonard & Radford 1384, 25 A p r i l 1968. C h e s t e r f i e l d Co. South C a r o l i na . 56 J_. unc ia l i s Wats . Gentry 1600, 21 June 1967. Winnemucha, Humboldt Co. Nevada. (Logan) 57 L_. c u s i c k i i Wats. C ronqu i s t 7332, 30 June 1953. Grant Co. Oregon. 58 I. c ae sp i t o sus Nut t . G i l l e t t & Tay l o r 11292 , 17 June 1 952. Baker , Grant Co. Oregon. 5 9 J_. 1e p i d u s Dougl . ex L i n d l . C ronqu i s t 7318, 28 June 1953. Grant Co. Oregon. 6 0 k- c u l b e r s o n i i Greene Evere t t & Johnson 7419, 18 Ju l y 1935. V e r m i l l i o n V a l l e y , S i e r r a Nevada, Fresno Co. C a l i f o r n i a . (Logan) 61 j . . noo tka tens i s Donn. ex Sims. Eastham 128, 5 September 1954. T h o r n h i l l Mtn. P r ince Rupert , BC. 62 J_. burke i Wats . N i c h o l l s 7904, 2 Ju l y 1979. Route 3 near Nancy Green Lake, BC. 63 L_. perenn is L. McGaha 43, 17 A p r i l 1962. Laxahaw, South C a r o l i n a . 64 _L . pol yphyl 1 us L ind l . Davis s . n . , 12 August 1951. Burnaby, BC. 6 5 J_. excub i tus Jones N i c h o l l s 7817, 15 June 1978. Hwy.2, Cooper Canyon, San Gabr i e l Mtns, Los Angeles Co. C a l i f o r n i a . 66 j _ . 1 ongi f o l i us (..Wats . )Abrams N i c h o l l s 7920 , 9. August 1979. Grown from seed at UBC. Seed c o l l e c t e d at Rancho Santa Anna Bot . Gard . 16 June 1978. 150 67 J.. a 1 b i c a u 1 i s Dougl . ex Hook. H i t chcock 24396, 22 May 1966. Camassia Nature Conservancy A r ea , Multnomah Co. Oregon. 68 L_. a n d e r s o n i i Wats. McCal la 6312, 26 June 1940. Summit Lake, Mt. Lassen V o l -can i c Park, C a l i f o r n i a . 69 J_. formosus Greene Bac i qu lup i 7385, 9 May 1960. Lockwood, Monterey Co. C a l i -f o r n i a . (Logan) 70 J_. k i n g i i Wats. Holmgren 7396, 25 August 1973. Manzano Mtns. B e r n a l i l l o Co. New Mexico . (Logan) 71 J_. pusi 11 us Pursh s s p . rubens (Rydb.)Dunn Cronqu is t 9043, 16 May 1961. H i t e , G a r f i e l d Co. Utah. 72 J.. pusi 11 us s s p . pusi 11 us Pursh Bo i v i n 9931, 27 Ju ly 1952. Agass i z Cou lee , Empress, A l b e r t a . 73 I. hi r s u t i ssimus Benth. H i t chcock 5978 , 9 A p r i l 1940'. Cuyamara Lake, San Diego Co. Cal i f o r n i a . (Logan) 151 APPENDIX II. So lvent systems and media used in the t h i n - l a y e r chromatography of f l a v o n o i d s from Lup inus . Rat ios are by volume. Polyamide DC6.6 a) water 70: n-butanol 15: acetone 10: dioxane 5. b) water 55: methy le thy l ketone 20: i soporpono l 20: a c e t i c a c i d 5 c) benzene 55: methy le thy l ketone 22: methanol 20: water 3. d) d i c h i o r o e t h a n e 50: methy le thy l ketone 25: methanol 21: water 4 e) d i c h l o r o e t h a n e 50: methy le thy l ketone 20: methanol 20: water 5 a c e t i c a c i d 5. S i l i c a gel a) n-butyl ace ta te 60: methy le thy l ketone 25: a c e t i c a c i d 12: water 3. Ce l1u lose a) water 90: a c e t i c a c id 10. b) water 85: a c e t i c a c i d 15. c) e thy l ace ta te 10: p y r i d i n e 3 .2 : water 2 , ( f o r sugars o n l y ) . APPENDIX I I I . UV Spec t ra l abso rp t i on maxima (nm) of some of the f l a v o n o i d s i s o l a t e d from Lupinus ap igen in MeOH: 268 334 NaOMe: 275 326 390 A lC I- : 276 300 348 382 A lC I , /HC l : 275 302 378 NaOAc : 275 302 378 NaOAc/H 3 B0 3 : 268 336 ap igen in 4 ' - 0 - g l u c o s i d e MeOH: 268 300s 329 NaOMe: 275 295s 260 A lC I- : 277 295s 340 384 A lC I , /HC l : 277 292s 336 379 Na OAc: 276 356 NaOAc/H 3 B0 3 : 269 333 i s o v i t e x i n MeOH: 269 303 338 NaOMe: 279 330 396 A 1 C 1 , : 279 306 349 385 AlCI~/HCl : 279 305 344 382 NaOAc : 278 304 386 NaOAc/H 3 B0 3 : 270 344 a c a c e t i n 7-0-glucos ide MeOH: 268 325 NaOMe: 287 359 A1C1-: 277 302 347 386 AlCI^/HCl : 277 299 339 382 NaOAc: 268 324 NaOAc/H 3 B0 3 : 268 326 l u t e o l i n MeOH: 254 268 351 NaOMe: 268 329s 405 Al CI : 274 302s 328 426. AlCI^/HCl : 274 298s 354 388 NaOAc : 269 325s 382 NaOAc/H 3 B0 3 : 259 302s 374 425 ap igen in 7-0-glucos ide MeOH: 268 333 NaOMe: 270 300s 386 Al CI : 277 300 348 386 A l C I , / H C l : 277 298 342 384 NaOAc: 268 366 387 NaOAc/H 3 B0 3 : 268 335 v i t e x i n MeOH: 270 302s 339 NaOMe: 280 331 397 A lC I- : 279 306 350 386 A l C I , / H C l : 279 304 344 384 NaOAc: 278 304 381 NaOAc/H 3 B0 3 : 270 342 a c a c e t i n MeOH: 269 327 NaOMe: 276 364 A lC I- : 276 300 338 382 A lC I ; /HC l : 276 296 334 378 Na OAc: 276 359 NaOAc/H 3 B0 3 : 69 329 c y t i s o s i d e MeOH: 272 342 NaOMe: 278 396 A l C I , : 266 273 300 358 390 A lC I , /HC l 266 273 300 352 380 NaOAc : 277 370 NaOAc/H 3 B0 3 : 272 348 l u t e o l i n 7-0-glucos ide MeOH: 256 269s 350 NaOMe: 265 300s 398 A l C I - : 275 302s 330 429 A lC I ; /HC l : 275 295s 358 390 NaOAc: 260 365s 405 Na0Ac/H,B0, : 260 372 153 l u t e o l i n 4 1 - 0 - g l u c o s i d e MeOH: 243 272 335 NaOMe: 270 295s 376 AlCI : 278 298s 352 386 A l C I ^ / H C l : 278 290s 344 382 NaOAc : 278 320s 370 NaOAc/H 3 B0 3 : 272 344 i s o o r i e n t i n MeOH: 258 271 353 NaOMe: 272 336s 409 A l C I _ : 276 302s 332 429 A lC I | /HC1 : 278 298 357 384 NaOAc: 276 328 392 NaOAc/H 3 B0 3 : 266 377 430s c h r y s o e r i ol MeOH: 244 270 347 NaOMe: 264 274s 329s 405 A 1 C U : 264 274 295 388 AlCI|/HC1 : 275 292 350 384 NaOAc: 271 320 391 NaOAc/H 3 B0 3 : 268 349 4 ' - 0 - m e t h y l o r i e n t i n MeOH: 254 269 290s 343 NaOMe: 274 302s 390 A 1 C U : 270 294s 366 386 AlCIf/HCI : 266 350 378 NaOAc: 281 324 372 NaOAc/H 3 B0 3 : 254 272 346 g e n i s t e i n 7-0-glucos ide MeOH: 263 326s NaOMe: 272 360 A lC I _ : 274 302s 372 AlCI^/HCl : 274 302s 370 NaOAC : 263 327s NaOAc/H 3 B0 3 : 263 327s kaempferol 7-0-glucos ide MeOH: 268 273 283 315 364 NaOMe: 274 283s 424dec. A 1 C U : 269 305s 356 420 A lC I ; /HC l : 269 304s 350 420 NaOAC: 275 283 380 415s NaOAc/H 3 B0 3 : 273 283 318s 366 o r i e n t i n MeOH: 258 271 297s 351 NaOMe: 272 336s 408 AlCI • 276 302s 334 428 A 1 C U / H C 1 : 268 277 298 358 386 NaOAc: 279 330 388 NaOAc/H 3 .B0 3 : 266 378 434s o r i e n t i n 3 ' -0-g lu cos i de MeOH: 275 332s 341 NaOMe: 244 274 304s 386 Al C U : 260 271 348 382 Al CI ,/H-Cl : 260 271 346 °382 NaOAC: 280 322s 378 NaOAc/H 3 B0 3 : 276 338 c h r y s o e r i o l 7-0-g lucos ide MeOH: 254 269 346 NaOMe: 254 270 350 400 A l C Io : 264 275 300 386 AlCI^/HCl : 264 272 296 350 380 NaOAc: 267 385 NaOAc/H 3 B0 3 : 268 347 g e n i s t e i n MeOH: 261 330s NaOMe: 276 330s A 1 C U : 272 305s 372 A lC I f /HC I : 272 305s 372 NaOAc: 271 336s NaOAc/H 3 B0 3 : 262 336s unknown ' p i n k ' ( f l avanone ) MeOH: 287 326s NaOMe: 246 329 A 1 C U : 276 312 367 A lC I , /HC l : 274 308 366 NaOAC: 278 330 NaOAc/H 3 B0 3 : 286 330s que r ce t i n 3-0-glucos ide MeOH: 257 269s 302s 365 NaOMe: 273 330 310 A 1 C U : 275 306s 330s 440 AlCI~/HCl : 268 298s 366 405 NaOAc: 274 324 376 NaOAc/H 3 B0 3 : 262 294s 370 s denotes shou lder dec denotes decompos i t ion of peak a f t e r 3 minutes . 154 APPENDIX IV Raw HPLC d a t a : peak he ights of 17 f l a v o n o i d s from 181 i n d i v i d u a l s of Lupinus s e r i c e u s . (For exp l ana t i on of compound Nos. see t ex t or F i g . 7) P lant Popu la t i on No . Compound Nos c o d e t—1 CM ro "3- LO to 00 cn O x—1 CM CO LO LO No. >—i »—1 I—1 I—1 I-H r*H I—1 t 251 24156 0 20 25 13 11 10 9 6 0 0 8 0 0 0 0 252 34 196 0 25 21 12 15 11 10 12 0 0 9 o o 8 0 253 22252 0 0 19 10 1 1 10 9 11 0 0 7 0 0 7 0 254 13100 80 16 12 9 26 9 9 8 0 0 7 0 0 6 0 255 23240 0 0 23 14 15 1 1 1 1 0 0 0 9 0 0 8 0 257 34360 0 32 32 14 15 13 10 0 0 0 9 0 0 8 0 258 22142 0 22 27 14 19 16 13 13 0 0 9 0 0 8 0 259 24228 0 14 17 10 12 10 9 10 0 0 0 0 0 0 0 271 25260 0 18 25 20 13 10 10 0 0 0 7 0 0 14 0 272 15194 0 18 21 12 14 1 1 10 12 0 0 9 o 0 8 o 273 28102 0 0 37 16 16 12 12 0 0 0 10 0 0 10 0 274 16172 0 20 25 14 13 1 1 13 0 0 0 10 0 0 9 0 275 34228 0 14 25 1 1 12 9 8 12 0 0 0 0 0 0 0 276 763 12 0 16 22 15 13 9 9 15 0 0 7 0 o 7 0 277 36220 O 23 29 15 20 12 1 1 0 0 0 10 0 0 9 o 278 51224 0 28 31 2 1 28 18 16 2 1 o 0 12 0 o 0 o 279 4 1 182 0 23 23 14 14 1 1 1 1 0 0 0 9 0 0 8 0 27 10 46420 0 16 17 10 13 8 8 9 0 0 7 0 0 7 0 28 1 28 176 0 2 1 26 13 13 1 1 1 1 13 0 0 10 0 0 0 0 282 33176 0 27 24 14 15 10 10 13 0 0 9 0 o 8 0 28 3 23268 O 18 2 1 1 1 13 9 9 13 0 o 8 o o 8 0 284 28196 0 30 28 15 15 12 1 1 15 0 0 10 0 0 9 0 285 26204 0 16 18 8 8 7 6 9 0 0 6 0 0 O 0 '286 40252 0 29 29 17 15 13 12 0 0 0 10 o o 9 0 287 34248 0 27 29 14 15 12 1 1 15 0 0 10 0 0 9 0 288 23200 0 25 26 12 14 1 1 1 1 12 0 0 9 0 0 8 0 289 31 158 0 21 24 12 13 10 9 12 0 0 8 0 0 0 0 29 1 19 160 o 18 19 1 t 10 9 1 1 10 0 0 9 0 o O 0 292 37332 0 21 22 10 1 1 9 9 1 1 0 0 9 0 0 7 0 293 39346 0 27 33 19 16 13 14 0 0 0 13 0 0 1 1 0 294 21 188 0 22 25 13 12 10 9 12 0 0 9 0 0 O 0 295 34304 0 19 22 13 1 1 10 9 15 0 0 0 0 0 0 0 301 42368 0 28 30 15 16 12 12 15 6 0 16 0 0 0 0 302 59392 0 0 26 15 15 1 1 12 16 0 o 10 o o 0 0 303 20170 0 15 25 12 14 1 1 16 0 0 o 0 0 o 10 0 305 24 160 0 23 31 20 22 18 15 20 0 0 12 o o 11 0 304 64308 0 31 34 16 15 9 8 14 0 0 7 0 0 9 0 306 20156 0 16 20 1 1 12 9 1 1 1 1 0 0 8 0 0 0 0 3 1 1 7 12 10 O 19 0 6 6 6 0 0 0 O 0 0 O o 312 36300 0 29 26 16 15 13 12 16 0 0 10 0 0 10 0 313 19232 0 25 26 22 18 15 14 20 0 0 11 0 0 IO c 314 33108 0 22 25 18 14 1 1 1 1 17 0 0 10 o 0 0 0' 315 23208 0 21 23 12 14 12 12 13 0 0 10 0 0 0 0 316 53350 0 28 32 21 15 14 37 19 0 0 11 oo 00 10 0 351 61 38 27 0 34 12 9 10 9 10 0 0 0 0 0 0 0 352 24 15 12 0 19 9 8 8 8 0 0 0 0 0 0 0 0 353 77 38 29 10 27 0 8 8 9 0 0 0 0 00 0 0 0 354 63 91 57 23 23 20 1 1 10 10 14 0 0 10 0 0 9 o 355 13102 0 22 37 12 1 1 13 17 0 0 0 11 0 o 0 0 356 47 31 23 14 26 14 10 9 9 10 0 0 0 0 0 9 0 357 31 14 13 10 22 9 8 8 0 0 0 0 o 0 0 0 o .358 16 18 14 14 16 8 8 7 7 0 0 0 0 0 0 0 0 155 8 9 11 12 13 14 15 16 17 36 13 63 23 1 1 12 13 10 10 10 10 0 0 10 00 0 0 0 361 29 64 33 14 19 10 9 9 9 0 0 0 8 0 0 0 0 362 25 37 29 15 19 9 8 7 7 0 0 0 0 o 0 O 0 363 36134 0 14 23 1 1 13 12 1 1 1 1 0 0 9 0 0 0 0 364 44 28 31 19 24 12 12 13 10 12 0 0 9 0 0 0 0 365 8130 0 0 25 9 8 8 10 0 0 0 O 0 0 0 0 4 1 1 1 1. 26 17 10 15 O 8 7 8 0 0 0 0 0 0 0 0 4 12 65 15 16 10 14 9 8 7 7 0 0 0 0 0 0 0 0 4 13 18 36 21 0 17 8 8 6 6 8 0 0 0 0 0 0 0 4 14 71 14 0 14 19 10 9 8 8 0 0 0 12 0 o 0 0 4 15 16 52 35 16 22 1 1 10 9 10 0 0 0 0 o o o 0 4 16 16 84 27 18 23 1 1 16 10 14 10 0 0 8 0 0 8 0 511 124105 64 27 30 26 13 12 1 1 15 0 0 10 0 0^ 10 52 512 38 87 64 22 22 12 10 10 9 0 0 0 8 0 0 0 20 513 41 61 42 39 38 25 18 15 14 0 0 0 0 0 0 0 0 514 4 7 90 43 33 30 19 15 1 1 10 0 0 0 0 0 0 0 20 515 44 47 42 31 28 16 14 14 13 0 0 0 12 0 0 12 31 516 42 94 35 26 28 8 9 8 8 8 0 0 0 0 o 0 0 53 16 66 50 2 1 30 9 8 7 8 0 0 0 7 0 0 0 15 531 13100 84 31 37 10 8 7 7 1 1 0 0 6 0 25 0 8 532 14 92 0 36 24 10 7 7 7 1 1 8 0 7 0 0 6 0 533 14 89 32 22 19 8 7 6 7 10 6 0 6 0 0 0 0 534 162 10 82 32 38 10 8 0 8 10 0 0 6 0 0 0 9 535 13 67 96 16 27 0 8 8 7 1 1 0 0 6 0 0 7 17 54 1 9 32 27 16 28 7 6 6 5 0 0 4 5 0 0 0 0 542 16 28 42 22 43 15 12 12 O 12 10 9 0 0 0 10 0 543 53 59 60 32 42 28 13 12 16 1 1 7 10 0 36 10 0 0 544 22 22 38 17 25 1 1 10 10 9 10 8 0 0 0 0 0 1 1 545 40 54 69 29 38 17 13 12 1 1 14 0 0 10 0 ' 0 9 28 546 10 12 22 18 25 15 13 12 1 1 0 9 10 11 0 27 10 0 55 14 33 24 15 22 10 8 9 9 9 0 7 8 0 0 O 0 551 31 55 0 28 46 18 13 1 1 1 1 13 0 7 11 o 0 9 0 553 27 88 97 38 40 19 17 12 12 0 0 0 15 0 31 14 0 554 46 53 65 78 79 18 12 1 1 10 0 0 0 1 1 o 30 10 0 555 18 79 73 32 4 1 1 1 1 1 9 9 10 0 0 0 0 0 8 0 556 27 168 72 62 49 12 9 0 8 10 7 0 7 0 o 0 O 58 1 83 132 86 60 57 17 "14 13 12 13 0 7 1 1 0 0 1 1 0 582 22124 7 1 42 40 14 13 10 10 12 8 0 9 0 0 9 0 '583 20140110 48 52 15 12 1 1 1 1 13 7 0 10 0 0 9 8 584 26100 0 38 27 13 10 10 9 1 1 0 0 9 0 31 9 8 585 16 88 0 48 36 15 1 1 10 10 12 0 6 0 0 34 9 0 586 9 52 0 24 33 12 10 9 9 1 1 0 0 9 0 0 8 O 59 1 12 57 52 25 38 12 10 9 0 0 8 0 8 0 0 0 0 •592 24110104 40 54 15 13 10 10 0 0 0 10 0 29 9 8 593 38104 0 53 28 13 14 9 9 12 7 10 9 0 0 19 0 594 10 73 0 27 38 14 1 1 53 1 1 12 0 0 10 0 0 9 7 595 35144 7 1 33 40 14 1 1 9 9 12 0 5 8 0 0 7 0 596 11 81100 28 51 14 13 10 12 12 8 0 1 1 0 0 9 0 601" 131OO110 0 65 17 12 12 1 1 14 7 6 10 0 0 9 0 602 49200 77 59 4 1 14 10 9 9 10 9 9 9 0 o 8 0 603 37 130 66. 34 41 16 12 10 10 13 9 7 9 0 24 18 0 604 21 190 90 58 66 16 12 10 10 14 5 0 13 o 0 8 o 605 39130 94 45 51 16 14 14 13 15 0 8 14 0 0 12 0 606 31 138 90 57 77 21 15 14 12 0 0 8 1 1 0 0 10 10 61 1 31204 79 4 1 45 14 1 1 10 9 0 8 O 8 o 0 8 o 612 8 67 42 22 22 1 1 8 0 7 0 6 5 0 0 20 8 0 614 42 60 0134 73 21 16 14 15 0 0 7 12 0 0 14 0 615 21 91 66 32 38 16 13 1 1 1 1 14 1 1 8 13 0 0 1 1 0 616 13124 72 28 36 1 1 9 8 8 0 8 0 0 0 20 8 0 621 7 20 10 18 16 1 1 10 10 0 0 8 7 0 0 13 0 b 622 10 98 0 29 75 17 13 12 0 0 25 8 0 0 O O 0 623 5 33 0 27 24 14 0 0 0 0 14 4 0 0 0 0 0 624 13 50 0 19 24 24 14 13 12 0 0 8 1 1 0 0 0 1 1 625 39100114 52 65 24 19 18 17 0 0 10 15 0 O 14 0 626 1 1 46 0 20 39 0 10 10 10 0 0 8 0 0 0 o 9 631 47108108 50 63 16 14 13 12 0 0 11 1 1 0 0 1 1 20 633 17 110 85 35 48 14 12 12 10 0 0 7 9 0 33 9 13 634 150 54 0 30 27 15 14 12 1 1 0 0 16 1 1 0 0 10 17 635 15104. 89 64 46 0 1 1  1 0 0 0 7 0 0 o 0 10 636 1 1 38 0 26 32 17 13 12 12 0 10 7 12 0 38 o 0 671 14 44 68 31 66 19 14 14 13 0 0 0 0 0 30 0 10 672 14 38 58 19 38 12 12 1 1 10 0 9 0 10 0 19 9 10 '72A 24 55100 26 26 16 14 14 13 14 10 9 12 o 14 0 0 72C 83 35 34. 33 25 17 14 14 14 14 13 15 13 0 29 13 0 72F 1 15 23 30 36 31 27 16 17 17 0 0 14 17 0 0 19 18 72H 128 30 49 35 30 16 14 14 13 0 14 18 13 0 0 12 0 72X 84 50 0 25 21 10 10 7 7 0 0 0 7 0 0 18 0 73E 64" 40 30 24 30 10 9 9 9 8 0 8 8 0 0 7 0 73 47 13 1 1 18 16 8 7 7, 0 0 0 7 7 0 0 7 O 73K 66 24 26 4 1 32 14 1 1  1 0 0 0 13 1 1 0 0 10 12 73J 130 34 23 32 23 14 12 13 1 1 0 0 20 1 1 0 21 10 0 73M 87 34 26 37 26 16 12 12 1 1 12 0 14 10 0 24 10 12 73P 120 26 33 26 25 13 12 13 1 1 0 0 1 1 10 0 31 10 0 74A" 13 19 32 16 23 12 1 1 1 1 10 10 8 8 9 0 0 9 8 74H 25 36 54 26 29 19 16 16 13 15 10 8 12 0 26 1 1 15 74K 14 37 55 23 26 15 14 15 12 0 0 8 1 1 0 19 0 15 74N 14 20 25 17 2 1 13 1 1 1 1 0 0 0 10 10 0 0 10 1 1 74U 19 42 130 27 24 17 14 14 1 1 13 9 1 1  1 0 2 1 11 14 77 A 130 44 19 14 22 13 9 0 8 0 8 5 8 0 2 1 9 0 77B 96 46 19 15 19 9 7 0 7 0 0 -9 6 0 16 8 0 77N 260 67 56 32 28 18 17 15 14 0 22 13 14 22 18 12 0 77R 291 10170 29 26 14 13 13 12 0 1 1 9 1 1 o 28 10 2 1 77X 192 76 0 17 13 13 1 1 10 10 0 0 9 1 1 0 25 10 0 79D 38 17 20 26 26 20 13 13' 12 14 1 1 8 1 1 0 20 13 0 79E 49 17 22 18 24 15 13 14 12 12 1 1 8 12 0 24 18 0 79V 54 23 29 27 22 13 14 13 1 1 0 10 8 10 0 2 1 10 0 "80 39 15 19 19 22 13 1 1 10 10 1 1 0 9 10 0 0 0 0 80A 19 25 32 23 18 12 1 1  1  1 10 13 7 10 0 23 0 0 80L 61 18 21 22 22 14 14 14 12 13 12 14 12 0 23 14 0 80T 45 13 15 14 18 1 1 10 1 1 9 0 10 8 9 0 19 9 0 80X 62 43 19 18 18 9 9 8 8 8 0 8 8 0 18 8 0 80Y 84 42 22 17 18 13 12 10 10 0 0 7 10 0 18 10 0 8'i 19 29 31 25 37 '17 10 1 1 10 1 1 9 8 10 0 O 1 1 0 8 1C 13 27 45 27 45 18 12 1 1 1 1 12 9 7 11 o 0 1 1 o 81L 28 35 50 39 55 2 1 14 13 12 14 O 8 12 0 0 0 o 8 1M 38 53 65 40 55 2 1 15 13 0 15 10 8 0 0 0 o 0 8 1R 23 43 52 35 50 19 14 12 12 14 10 7 12 0 0 0 0 8 1S 49 72 66 39 50 18 14 13 12 15 0 9 1 1 0 0 0 0 82 21 63 43 18 23 12 1 1 9 9 1 1 0 6 9 0 0 0 0 82B 20 17 30 17 24 13 12 1 1 10 1 1 8 6 10 0 0 o o 82D 80140 0 38 32 21 19 17 0 19 0 16 0 0 29 0 0 82F 30 40 37 24 25 18 13 14 13 0 12 9 1 1 0 0 12 o-82J 60100 7 1 39 39 19 16 15 13 0 0 10 12 0 0 1 1 0 C 1A 13 91 58 20 25 0 10 10 0 0 5 0 11 0 0 0 6" C1B 261 10 98 38 38 10 12 10 O O O 0 10 0 12 o 0 C 1C 18 50 60 15 30 17 1 1 10 7 0 15 0 0 0 12 0 0 C 1D 42 82 70 21 60 0 1 1 1 1 9 0 10 0 12 0 1 1 0 10 C2A 28 48 28 28 15 18 12 12 10 0 0 20 . O 0 IO o o C2B 3e 89 90 18 56 0 15 13 9 5 0 15 10 0 0 22 0 C2C 15150100 62 78 20 10 10 5 0 0 0 10 0 10 0 0 C2D 21 78 90 42 30 21 10 10 9 10 0 10 0 0 0 18 0 C3A 48 90 80 18 25 16 0 10 7 0 0 0 0 0 1 1 0 10 C3B 82 130 78 25 35 10 1 1 1 1 0 0 5 0 12 0 1 1 0 5 C3C 28 82 70 33 41 0 1 1 13 10 10 0 5 13 0 1 1 30 7 C3D 47 80 66 42 52 12 1 1 13 7 ' 5 0 7 0 0 0 O 0 "C4A 36 68 38 '18 26 15 12 1 1 0 ' 5 0 0 10 0 O 0 0 C4B 21 58 21 15 30 17 10 1 1 9 0 0 9 11 0 10 10 0 C4C 49 78 80 19 38 21 10 10 10 0 0 0 1 1 0 7 0 0 C4D 55 98 78 29 40 0 1 1 10 0 0 5 0 0 0 9 0 0 APPENDIX V. Two- dimens iona l chromatograms (2- D maps) of the 73 Lupinus taxa that are desc r i bed in Chapter IV. S o l v e n t s : A s c e n d i n g ; the o rgan i c so l ven t system (benzene 55: methy le thy l ketone 22: methanol 20: water 3 ) . Ho r i zon t a l ( l e f t to r i g h t ) ; the aqueous so l ven t sys-tern (water 70: n-butanol 15: acetone 10: dioxane 5 ) . Medium: Polyamide DC6.6 Compound numbering system (as used in Data Set I ) : 1 o r i e n t i n 29 a c a c e t i n 7-0-glucos ide 2 v i t e x i n 30 a c a c e t i n 7-0-glucos ide 'acyl I 3 o r i e n t i n acy l I 31 l u t e o l i n 7-0-d ig lucos ide 4 v i t e x i n acy l I 32 ap igen in 7-0-d ig lucos ide 5 o r i e n t i n acyl II 33 c h r y s o e r i o l 7-0-d ig lucos ide 6 v i t e x i n acyl II 34 a c a c e t i n 7-0-d ig lucos ide 7 l u t e o l i n 7-0-glucos ide 35 kaempferol 8 ap igen in 7-0-glucos ide 36 kaempferol 3-0-glucos ide 9 l u t e o l i n 7-0-glu. acy l I 37 kaempferol 7-0-glucos ide 10 unknown 1 green 1 38 q u e r c e t i n 11 l u t . 7-0-glu . acy l II 39 que r ce t i n 3-0-glucos ide 12 ap. 7-0-g lu , acy l II 40 q u e r c e t i n 7-0-glucos ide 13 1u t e o1 in 41 i soor i en t i n acy l I. 14 ap igen in 42 g e n i s t e i n 15 i s o o r i e n t i n 43 g e n i s t e i n 7-0-glucos ide 16 i s o v i t e x i n 44 gen. 7-0-glu . acy l I 17 pink f lavanone (high Rf) 45 gen . 7-0-glu. acy l 11 18 pink f lavanone (mid Rf) 46 o r i e n t i n X ' -O-g lucos ide 19 pink f lavanone (low Rf) 47 v i t e x i n X ' -O-g lucos ide 20 c y t i sos i de 48 o r i e n t i n 3 ' -0-g lucos ide 21 c y t i s o s i d e acy l I 49 l u t e o l i n 4 ' - 0 - g l u c o s i d e 22 c y t i s o s i d e acy l II 50 1 u t e o l i n 4 ' - 0 - g l u . acy l 11 23 4 ' - 0 - m e t h y l o r i e n t i n 51 ap igen in 4 ' - 0 - g l u c o s i d e 24 c h r y s o e r i o l 52 ap igen in 4 ' - 0 - g l u . scy l I 25 a c a c e t i n 53 ap igen in 4 ' -0-g1u . acy l II 26 c h r y s o e r i o l 7-0-glu . 54 i s o v i t e x i n acy l I 27 ch r y . 7-0-glu . acy l I 55 l u t e o l i n 5-0-glucos ide 28 ch r y . 7-0-glu . acy l 11 56 ap igen in 5-0-glucos ide compounds present in t r a ce q u a n t i t i e s are shown as spots with dot ted p e r i m e t e r s . 158 2-D map of the f l a v o n o i d s of L. e l a t u s , OTU 1 159 2-D map of the f l a v o n o i d s of L. b a r b i g e r , OTU 2 2-D map o f t h e f l a v o n o i d s o f L. s e r i c e u s v a r . s e r i c e u s OTU 3 161 2-D map o f t h e f l a v o n o i d s o f L. s e r i c e u s s s p . h u f f m a n i i OTU 4 162 -D map-of. the f l a v o n o i d s of L. s e r i c e u s v a r . eggl e s t o n i anus O T U 5 -S 8 4 2 ; 1 6 3 2-D map o f t h e f l a v o n o i d s o f L. s e r i c e u s v a r . f l e x u o s u s OTU 6 164 2-D map of the f l a v o n o i d s of L. b i c o l o r , OTU 7 •.46; •'' 165 2-D map of the f l a v o n o i d s of L. p o l y c a r p u s , OTU 8 166 2-D map of the f l a v o n o i d s of L. neomexicanus, OTU 9 2-D maps of the f l a v o n o i d s of L. l emmoni i , OTU 10 168 2-D map of the f l a v o n o i d s of 1 . t e x e n s i s , OTU 11 '.47l, o 169 2-D map of the f l a v o n o i d s of L. d e n s i f l o r u s va r . d e n s i f l o r u s OTU 1 2 170 2-D map o f t h e f l a v o n o i d s o f L• d e n s i f l o r u s v a r . a u r e u s OTU 13 172 2-D map of the f l a v o n o i d s of L. a r b o r e u s , OTU 15 173 2-D map of the f l a v o n o i d s of L. h o r i z o n t a l i s , OTU 16 174 2-D map of the f l a v o n o i d s of L. f l a v o c u l a t u s , OTU 17 2-D.map of the f l a v o n o i d s of L. benthami i , OTU 18 177 -D map of the f l a v o n o i d s of L. spars i f T o r u s , OTU 20 2-D map of the f l a v o n o i d s of 1 . a r i z o n i c u s , OTU 21 \46 ; 2-D map o f the f1avonoids o f 1. s t o l o n i f e r u s , OTU 22 1 8 0 2-D map of the f l a v o n o i d s of L. wyethi i , OTU 2 3 2-D map of the f l a v o n o i d s of L. h i l l i i , OTU 2 4 V 1 8 2 2-D map of the f l a v o n o i d s of 1. h o l o s e r i c e u s , OTU 25 183 2-D map of the f l a v o n o i d s of L. p a r v i f l o r u s , OTU 26 13) 2 4 ) 184 2-D map of the f1avonoi ds of L. caudatus X L ; h i 11 i i , OTU 27 185 map of the f l a v o n o i d s of L. nevadens i s , OTU 28 '••47\ "'46 6 13J 1 8 6 2-D map of the f l a v o n o i d s of L. 1eucophy l Ius , OTU 29 1 4 6 : W w 14 1 8 7 2-D map of the f l a v o n o i d s o f L.X a l p e s t r i s , OTU 30 :A6,' 1 8 8 2-D map of the f l a v o n o i d s of L. g r e e n e i , OTU 31 189 2-D map of the f l a v o n o i d s of L. pseudoparv i f 1 o r u s , OTU 32 map of the f l a v o n o i d s of L. 1u tescens , OTU 3 3 •46 is;' 191 2-D map of the f l a v o n o i d s of L. s u l p h u r e u s , OTU 34 1 9 2 -D map of the f 1 avonoi ds o f 1. paTmer i , OTU 35 :-47, ' : 46 . ; 193 2-D map of the flavonoids of L. argenteus var. argenteus OTU 36 • \ 4 ; i'5 /' 1 9 4 2-D map of the f l a v o n o i d s of 1 . argenteus va r . r u b r i c a u l i s OTU 37 195 2-D map of the f l a v o n o i d s of 1. cauda tus , OTU 38 1 9 6 2-D map of the f l a v o n o i d s of L. a r b u s t u s , OTU 39 •47 :32 •46 198 Oi 199 200 2-D map of the f l a v o n o i d s of L. humocola, OTU 43 ' ,46 ; 201 2-D map o f t h e f l a v o n o i d s o f L. a r c t i c u s s s p . a r c t i c u s OTU 44 202 2-D map o f t h e f l a v o n o i d s o f L. a c t i c u s s s p . c a n a d e n s i s , OTU 45 203 2-D map of the f l a v o n o i d s of L. a r c t i c u s s s p . s u b a l p i n u s , OTU 46 205 2-D map of the f l a v o n o i d s of L. 1 i t t o r a 1 i s , OTU 48 '.34.:' 207 2-D map of the f l a v o n o i d s of L. c o n f e r t u s , OTU 50 35^7^) 14>42) 2\) 25) 208 2-D map of the f l a v o n o i d s of L. minimus, OTU 51 209 2-D map of the f l a v o n o i d s of L. l y a l l i i , OTU 52 210 2-D map of the f l a v o n o i d s o f L. s e l l u l u s v a r . l o b b i i , OTU 53 211 2-D-map of the f l a v o n o i d s of L. k u s c h e i , OTU 54 46 ; 212 2-D map of the f l a v o n o i d s of L. d i f f u s u s , OTU 55 213 2-D map of the f l a v o n o i d s of L. unc i a l i s O T U 56 214 215 2-D map of the f l a v o n o i d s of L. c a e s p i t o s u s , OTU 58 V 3 1' { f c •46 ) 216 2-D map of the f l a v o n o i d s of L. 1ep idus , OTU 59 (3i; '-32; 218 219 2 - D map o f t h e f l a v o n o i d s o f L . b u r k e i , OTU 62 220 2-D map of the f l a v o n o i d s of L. p e r e n n i s , OTU 63 221 2-D map of the f l a v o n o i d s of L. p o l y p h y l ! u s , OTU 64 222 2-D map of the f l a v o n o i d s of L. e x c u b i t u s , OTU 65 2231 2-D map o f the f l a v o n o i d s of L. 1 o n g i f o l i u s , OTU 66 224 2-D map of the f l a v o n o i d s of L. a l b i c a u l i s , OTU 67 225 2-D map of the f l a v o n o i d s of 1. a n d e r s o n i i , OTU 68 i \ •31 •• 13) 1?) 226 2-D map of the f l a v o n o i d s of L. formosus, OTU 69 ( 3 0 / • 8 ; 227 2-D map of the f l a v o n o i d s of 1. k i n g i i , OTU 70 228 2-D map of the f l a v o n o i d s of L. p u s i l l u s s s p . rubens , OTU 71 \ 31> / T N 26 229 2-D map of the f l a v o n o i d s o f L. p u s i l l u s s s p . p u s i l l u s , OTU 72 3> 35) l O 24) 230 2-D map of the f l a v o n o i d s of L. h i r s u t i s s i m u s , OTU 73 

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