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

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FLAVONOID SYSTEMATICS LUPINUS SPECIES  OF NORTH AMERICAN (LEGUMINOSAE)  by KEVIN B.Sc.(Hons . ) ,  WILLIAM NICHOLLS  U n i v e r s i t y C o l l e g e of Wales,  A THESIS  SUBMITTED  IN  THE REQUIREMENTS DOCTOR OF  PARTIAL  A b e r y s t w y t h , 1972  FULFILMENT OF  FOR THE DEGREE OF PHILOSOPHY  in THE  FACULTY OF GRADUATE STUDIES (Department  We a c c e p t t h i s to  thesis  Botany) as c o n f o r m i n g  the r e q u i r e d s t a n d a r d  THE UNIVERSITY  OF"BRITISH COLUMBIA  August ©  of  Kevin W i l l i a m  1981 Nicholls,  1981  In  presenting  requirements of  British  it  freely  agree for  this f o r an  available  that  I agree  understood  that  that  may  be  copying  f i n a n c i a l gain  The University of British 2075 W e s b r o o k P l a c e Vancouver, Canada V6T 1W5  c  i i  / -in  \  shall  and  I  study.  copying by  or publication  shall  n o t be  allowed  Columbia  of the  University make  further  of this  t h e head  h i s or her representatives.  Department  I-»TT>  at the  granted  permission.  Date  fulfilment  the Library  f o r extensive  purposes  o r by  degree  f o r reference  permission  scholarly  in partial  advanced  Columbia,  department  for  thesis  thesis  of  my  It i s  of this without  thesis my  written  11  ABSTRACT  This onoids  study  as  The genus specific result  was  itself  readily  recognizable,  the  but was the  in is  North  America,  probably  the  plasticity  outcrossing  Fresh  onoid c o n t e n t , tical.  peren-  was  sure  liquid  levels  of  was  or  dried  Utah,  southerly  ferences  to  as  and l e s s  commonly  only  the  sugar  6- or  8-  attached positions  Some f l a v o n o l s  lending  ages  quantitative of  and  L u p i nus  by  to  Fleak  were c h e m i c a l l y  iden-  in  flavonoid  s e r i ceus  north the  by h i g h  and s o u t h  subspecific  (1971).  was flav-  discontinuity  uniform c o n d i t i o n s  have been a p h e n o t y p i c  characters  had i d e n t i c a l  variation  A sharp  support  specific  material  between p o p u l a t i o n s  in  majority  1  varying  an a n a l y s i s  population  grown  the  the  4 -positions.  plant  of  chromatography. seen  Lake,  plants  in  was  flavonoids  and p l a n t s  noted  identified,  flavonoids  identified.  Geographically,  tent  or  Lu p i n u s  luteolin  Glucose  5,7,3'  The r e l i a b i l i t y  of  f o u n d C-bonded at  were a l s o  tested.  analysis  compounds were  and c h r y s o e r i o l .  isoflavones  of  the  flav-  (Leguminosae).  morphological  amongst  based on a p i g e n i n ,  and 0-bonded at  the  but, This  considerable  a detailed  Fifty-six  flavonoids  Salt  of  Lupinus  defined.  (particularly  outset,  flavones  acacetin  next  poorly  a combination  made.  being  to  are  genus  of  taxa). At  was  usefulness  in  is  the  the  markers  boundaries  of  of  taxonomic  and h y b r i d i z a t i o n nial  an a s s e s s m e n t  in of  showed  response.  the  pres-  orientin Great  ranking  Subsequent  con-  of  analysis  flavonoid  dif-  i i i Since  flavonoids  characters,  an  interspecific  cipal  components  these  accumulated  analysis  The s e v e n t h  group  One s p e c i e s  (L_.  classed  as  in  affinities natural basis  contained  (chemical Overall,  characters  support  subgeneric cases, that  in  or it  hitherto  was  stable  made.  groups.  peculiar  to  Prin-  Six each  no unusual  of group.  compounds.  complex p r o f i l e  and  was  group. and p e r s o n a l  observations  affinities the  however,  a collection  integrity  of  divided  into  was  of  paralleled  as was  lupines  the  it  was  seen  morphological  the  groups  two  on  seventh  with doubtful  as  the group affin-  morphological).  is  concluded that  the  groupings  flavonoid  had a v e r y  discontinuities  Lu p i n u s . to  73 t a x a  containing  strengthening  Group C,  of  flavonoids  chemical  morphological  (F).which  lends  the  thereby  units.  of  ities  literature  be i n f r a s p e c i f i c a l l y  study  had p r o f i l e s  d i f f usus)  5 groups  to  exposed 7 chemical  some n o v e l  a monospecific  Using.the that  appeared  data  Their  distribution,  recognition that  flavonoids  of  are  good  interspecifical1y,  some p r e v i o u s l y  were based on m o r p h o l o g y .  suggests  were c o n s i d e r e d  affinities unrelated.  specific  described In  between some  other taxa  iv Table  of  Contents  Abstract List  of  ii Tables  vii  ListofFigures I  ix  INTRODUCTION Tribal  1 position  Subgeneric  of  L u p i nus  taxonomy o f  Some r e a s o n s  for  the  2  L u p i nus  5  c o n f u s i n g taxonomy  of  Lupi nus Previous  7 chemotaxonomic s t u d i e s  Economic a s p e c t s Thesis II  of  on L u p i n u s  .  10  Lupinus  15  aims  17  FLAV0N01D CHEMISTRY OF LUPINUS Introduction M a t e r i a l s and methods andpurif ication  I.  M a t e r i a l s and methods tification  . Flavonoid  II.  18  extraction . . .  Flavonoid  iden-  29  INFRASPECIFIC VARIATION IN  LUPINE  FLAVONOIDS  .  .  35  Introduction  35  The e f f e c t  of  content  a Lupinus  species  of  and s t o r a g e  of  The e f f e c t flavonoid  20 24  Results.. III  18  plant drying  content  of  age on t h e  Experiment  I.  Experiment  II.  Materials  38  a Lupi nus  Geographical v a r i a t i o n o f Lupinus s p e c i e s  in  the  A preliminary Variation  in  and m e t h o d s .  flavonoid on  the  species.  flavonoid  study Lupinus  .  .  .  .  43  content .  . 4 6  s er i ceu s .  53 53  High P r e s s u r e HPLC o f  Liquid  .  . 5 5  flavonoids  HPLC o f - L u p i n u s Results  Chromatography  57  sericeus  flavonoids  .  .  58  and d i s c u s s s i on  Principal  59  components a n a l y s i s  R e s u l t s and d i s c u s s i o n o f sericeus flavonoid data, Canonical  variates  of  analysis  orientin  L u p i n u s sericeus  .  .  62  PCA on L u p i nus _  R e s u l t s and d i s c u s s i o n o f sericeus populations The a n a l y s i s  (PCA)  (CVA).  .  65  .  68  CVA on L u p i n u s . .69 accumulation  populations  .  .  in  .  .  73  Experiment III. The d e t e r m i n a t i o n o f t h e phenot y p i c or genotypic nature of o r i e n t i n variation in L u p i n u s - s e r i c e u s 78 IV  INTERSPECIFIC  FLAVONOID  Materials Data  LUPINUS  .  .  and methods  generation  Discussion. V  VARIATION IN  81  and a n a l y s i s  81  .  .99  OVERVIEW  122  Flavonoids  as  Relationships  taxonomic in  characters  in  L u p i nus  L u p i nus  128  SUMMARY  130  References APPENDIX  I.  APPENDIX  II.  133 .Plants  III.  and S o u r c e s  Sol v e n t , systems  flavonoids APPENDIX  122 126  Convergence VI  81  flavonoids  and media  141 used  i n TLC o f  from L u p i n u s UV s p e c t r a l  a b s o r p t i o n maxima o f  from L u p i nus  151 some  APPENDIX IV. Raw HPLC d a t a : peak h e i g h t s o f 17 f l a v o n o i d s from 181 i n d i v i d u a l s o f L u p i nus s e r i c e u s  152 .  154  APPENDIX V. t h e 73  Two-dimensional l u p i n e taxa that  chromatograms (2-D maps) were a n a l y s e d .  vii List  I.  II  of Tables  A c o m p a r i s o n o f s u b g e n e r i c g r o u p i n g s o f European l u p i n e s by Nowacki & P r u s - G l o w a c k i (1971) w i t h t h a t o f T u t i n e t aj_. ( 1968) u s i n g a l k a l o i d and morphological d a t a , r e s p e c t i v e l y  .  12  Subgeneric groupings o f North American l u p i n e s from a l k a l o i d and s e r o l o g i c a l s t u d i e s by Nowacki (1960) and Nowacki & P r u s - G l o w a c k i (1971) . .  .  13  .  25  III  Colour reactions  IV  The f l a v o n o i d s i s o l a t e d American Lupinus  V  The d i s t r i b u t i o n  VI  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 i n L u p i n u s samples f o l l o w i n g d r y i n g and s t o r a g e  VII  The d i s t r i b u t i o n  of flavonoids  among  individuals  VIII  The d i s t r i b u t i o n  of flavonoids  among  individuals  IX  T a b l e o f e i g e n v e c t o r s o f t h e 17 f l a v o n o i d v a r i a b l e s f o r ' t h e f i r s t t h r e e "component axes o f PCA . .  67  X  Analysis of variance f o r o r i e n t i n Lupinus s e r i c e u s p o p u l a t i o n s  74  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 D u n c a n ' 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 o f L u p i n u s s e r i ceus .  75  HPLC peak h e i g h t s o f o r i e n t i n from 20 L u p i n u s s e r i c e u s i n d i v i d u a l s grown from seed i n u n i f o r m conditions  80  light  of  and r e s u l t a n t  maturity  of  Lupinus  of  of lupine  Lupinus  flavonoids  structural  o f L u p i nus a r b o r e u s  arboreus  inferences  from 73 t a x a  of flavonoids  under UV .  .  o f North  in various  from t e n p o p u l a t i o n s .  b i c o 1 o r from t e n p o p u l a t i o n s  .  34  stages  41  arboreus  .  .  .  .  44  .  48 .  -  XII  XIII  Data  XIV  T a b l e o f e i g e n v e c t o r s o f t h e 56 f l a v o n o i d v a r i a b l e s f o r t h e f i r s t t h r e e component axes o f PCA on Data Set I .  XV  S e t I:  73 L u p i n u s  Data  types  the d i s t r i b u t i o n  accumulation in  taxa  Set III:  of flavonoids in  The d i s t r i b u t i o n  i n 73 L u p i n u s  taxa  o f 11 f l a v o n o i d -  51  83  87 92  vi i i XVI  T a b l e o f e i g e n v e c t o r s o f the 11 f 1 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 of PCA on Data Set III  96  XVII  Summary ponents  98  XVIII  Summary t a b l e o f the g r o u p s o f L u p i n u s t a x a d e l i m i t e d by f l a v o n o i d d a t a and m o r p h o l o g i c a l e v i d e n c e  o f L u p i n u s g r o u p i n g s from p r i n c i p a l coma n a l y s i s on Data S e t s I and III . . .  .  119  ix List  of  1  The numbering s y s t e m o f  2  Wesley-Moser  3 4  5  6  during  Figures  flavonoid  rearrangement  of  prolonged treatment  Two-dimensional  Lupinus  arboreus  C-glycof1avone  with  TLC map o f at  a  the  carbon atoms.  strong  acid.  flavonoids  6 months.  Map to show the l o c a t i o n s o f t h e L u p i n u s and L_. bi c o l o r • c o l 1 e c t i o n s ' m o n i t o r e d . f o r i f i c flavonoid variation  .  from  ,  .  19  .  28  .  40  arboreus infraspec^  Map to show t h e l o c a t i o n s o f 32 p o p u l a t i o n s o f 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 flavonoid variation Diagrammatic liquid  representation  of  a high  54  pressure  chromatograph system, Lupinus  56  7  HPLC o f  8  The r e l a t i o n s h i p  9  to two a t t r i b u t e s O r d i n a t i o n of the ponent axes  10  Ordination of  181 i n d i v i d u a l s  11  O r d i n a t i o n of  32 p o p u l a t i o n s  12  O r d i n a t i o n o f 32 p o p u l a t i o n s o f L u p i n u s s e r i c e u s from CVA. P o p u l a t i o n c e n t r o i d s a r e drawn w i t h +/- 1 s t a n d a r d d e v i a t i o n to show i n f r a p o p u 1 a t i o n a l variabi 1 ity  by  a typical  sericeus  individual  between OTU s compared w i t h 1  (A and B) F i g . 8 OTU's with of  PCA on HPLC d a t a  by CVA on HPLC d a t a .  if  1st  .  . 6 0  respect  and 2nd com-  Lupinus Lupinus  sericeus sericeus  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  14  Ordination  73 l u p i n e  taxa  from PCA on Data  Set  I.  I.  Components I / 1 1 . of  Components 11 /111  15  Biosynthetic  16  Ordination  17  .  of  taxa  III.  Components 1/11  III.  Components 11 / 1 1 1 .  O r d i n a t i o n of  73 l u p i n e  63 64 66 70  71 85  86  interralationships 73 l u p i n e  .47  taxa .  of  lupine  flavonoids  from PCA on Data  .  from PCA on Data .  .  .  .  .  91  Set  .  Set .  94 95  ACKNOWLEDGEMENTS I  would  Bruce A. tance  like  'to e x p r e s s my g r a t i t u d e  Bohm f o r  throughout  generous  his the  financial  advice,  script  is  I Jack  greatly  plant  like  statistics;  me a c c e s s tance  in It  to the  is  present,  Lastly,  Prof.  of I  the  Schofield  thank  Bradfield parts Mr.  of  the for  this  thesis  thank  Mary  for  My o t h e r  reading  E.  Barkworth  of  R. of  Prof.  assis-  his  committee Ganders. this  Prof.  people:  running  manu-  Prof.  Profs.  and  inter-  Tony G l a s s  for  s u p p l y i n g me w i t h  (Utah S t a t e ) David  B.  for  Dunn f o r  allowing assis-  specimens. the the  graduate last  and d u r i n g  her  home ( a m i d s t  in  study;  invaluable  Joy;  following  Moore f o r  name a l l  was  Lesley  trips  on f i e l d  and a l s o  and F r e d  help  this  Dennis  laboratory  work  contributed  writing  too was  constant  her  few y e a r s .  the  as  students,  past  and  Their and  their  final  friendship.  c o m p a n i o n s h i p and  own s t u d i e s )  immesurably  plus  to  the  her  hard  completion  work.  Finally, tantships, is  to  i m p o s s i b l e to  in  e n t h u s i a s m and  work.  and c r i t i c a l  identification  at  this  B.  herbarium m a t e r i a l ;  encouragement  of  field  who have h e l p e d me o v e r  assistance typing  E.  computational  material;  my s t u d i e s  for  Wilfred  interest,  my s u p e r v i s o r ,  appreciated.  Maze and Gary  help with  of  encouragement  should also  preting  course  support  members were P r o f s . Their  continuing  to  graduate  gratefully  Department  financial  support  scholarships  acknowledged.  and the  Faculty  For of  in  the  form o f  and a t r a v e l these  Graduate  I  teaching award to  thank  Studies.  the  UBC  assisthe  U.K.  Botany  1 I.  Lupinus and South  L.  America  Mediterranean of  habitats,  I.  (e.g. the  is  in  of  of  of  The genus  retreat  the  bacteria  herbs  taxonomic  problems w i t h i n  With  few  flowers  generic  exceptions  lupines  determine  precisely  lack  of  the  all  the  that  there  Old W o r l d ,  100 or more"  are  is,  This  thus  (Hitchcock  et  in  able  lupines  to  are  glacial  spread. into  is  no s m a l l  is  In  this  sympatry  especially way  readily  to  so  and in  the  of  recognizable. papi11ionaceous  Both annual  however, species  number o f  about  whereas  As  particularly  quickly  have racemes  number o f  large  the  symbiotic  following  brought  compound l e a v e s . It  have  in  genus.  Lupinus  taxa  exist.  the  in  and  very in  and  difficult  the  genus  10 s p e c i e s ,  North  ajk,  America  1961)  all  It  annuals,  there  and perhaps  are 200  to  owing  p u b l i s h e d names and  modern c o m p r e h e n s i v e m o n o g r a p h i c t r e a t m e n t s .  estimated to  of  often  result.  the  level,  and p a l m a t e l y  existence  they  1 evel  kuschei) .  and are  They are  and c o n t r i b u t e s  perennial  the  are  commonly the  lupines  the  man.  variety  1yal1i i) ,  Consequently,  a l o n g which taxa  I.  species  nodules  soils.  of  perennial  At  Lupinus  in  at ,sea  odoratus) (e.g.  America  a wide  I.  zone ( e . g .  I.  North  distribution  and dunes  (e.g.  root  of  western  on exposed t e r r a i n  activities  is  USA  in  deficient  isolated  hybridization  subalpine  to  be found i n  Yukon and A l a s k a  on r o a d s i d e s  formerly  can  beaches  Leguminosae,  invasive  or  abundant  the  the  native  a second c e n t r e  southwestern  nutrient  important  genus  i n c l u d i n g sandy  nitrogen-fixing  way,  with  Europe.  regions  typical  grow  a large  1i t t o r a l i s) , the  deserts  arctic  is  INTRODUCTION  to  the is native  "probably (Turner,  2  1959). there  For to  the  de R a v e l o ,  infraspecific separate  the  is  (the  obvious level  go so  chaotic  state  position  of  (1976)  estimates  communication  600 t a x a  (1938-1952)  any  say  that  other  with  in  50% at  the  documented o v e r  600  lupines. Lupinus  of  is  to  the  problems  below  Hitchcock  et  al .  "probably  in  a more  be found i n  United  our  area"  States).  Lupinus  last  described  150 y e a r s ,  related  plants  their.distinctive  controversial  many t a x o n o m i c  (genus)  portion  the  closely  are  New World  number o f  group o f  is  there  the  over  it  to  Northwest  steadily  the  suggests  and Smith  the  as  than  Although  Indeed,  Polhill  Dunn ( p e r s o n a l  1978)  that  in  far  Pacific  Tribal  level  a whole,  species .  genus  (1961)  as  be 200 s p e c i e s ,  Planchero  It  Americas  tribal  the  has  species  has  integrity  never  placements  of  of  been i n  morphology t h a t Lupinus  increased Lupinus doubt.  has in  as a  resulted  the  in  '.  Leguminosae. Originally placed  in  including  the  described  Genisteae  clovers).  variability  the  of  (1719),  by Adanson (1763)  Geni s t a , C y t i s us  T r i f o l i um ( t h e  by T o u r n e f o r t  (the It  is  brooms), clear  group t r o u b l e d  Lupinus  L.  w i t h many o t h e r  was genera  Laburnum , M e l i 1 o t i s and  that  the  size  and  the  him:  "J'aurais desire d i v i s e r cette section en 2, s q a v o i r c e l l e des Genets et c e l l e des T r i f l e s ; mais j e n ' a i pu t r o u v e r dans aucune de l e u r s p a r t i e s un c a r a t e r e que marquat des bournes a cette 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 i n t o 2, t h e brooms and the t r e f o i l s , but I have been u n a b l e to f i n d c h a r a c t e r s i n each on which to base such a d i v i s i o n . )  3 In taxa  1862 Bentham  with  & Hooker  a two-lipped  anthers.  Later  restricted  calyx,  research  has  closed  (Hitchcock  revealed  (Polhill, with  1976).  Geni s t a ,  et^ a]_. , 1961) Lupinus  was  Genisteae  stamina! that  chromosome number based on 12 ( G i b b s , clovers  the  placed  all  1966)  and 9 i n in  such t a x a  the  tribe  subtribe  La burnum ,  placed  strophiolate  seed-bearing  and L o d d i g e s i a , This until  in  the  subtribe  classification  Rothmaler  (1944)  group i n  rather  Hooker's  subtribes  "Genistineae"  genera,  ways.  the  stable  for  (1964)  Rothmaler  Cytisineae  (which  Cytiseae  Hutchinson  and S p a r t i e a e ,  Genisteae, Genisteae  to  delimiting  form t h e  the  similarities  for  to  to  respectively.  A r g y r o l o b i um, were  appear  raised  be r a t h e r  Of t h e been a d o p t e d  Five  placed  between  tribal  Laburneae  Lupineae,  .convenience  united  he c o n s i d e r e d as  in  genera  for  classifications cases  available, (Gibbs,  In  subtribes, and  from  two,  and  In  recognized They  partnered,  the  Lupinus  Lupineae.  and A r g y r o l o b i um .  phylogenetic  name  a monogeneric  were removed  (1964)  d i s s i m i l a r genera,  the  Cytiseae  and a f u r t h e r  the  Bentham &  Loddi g e s i a .  the  a new t r i b e ,  century  correct).  & Hooker's  rank,  Hutchinson  Lupi nus  than  i n most  Bentham  a  reinterpreted  (although  s u b t r i b e ) , H y p o c a l y p t i i s , A r g y r o l o b i um and contrast,  the  Hypocalyptus  almost  have been n o m e n c l a t u r a l l y  E x c l u d e d were L u p i n u s  non-arillate  Cytiseae.  and H u t c h i n s o n  into  would  C y t i s us , Ulex ,  remained  different  (1962)  the  Ca1i c o t o m e ,  (estrophiolate)  & Hooker  7 in  Spartieae  by  Bentham  have a  Podalyrieae  P e t t e r i a , S p a 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 seeds.  those  and d i m o r p h i c  compared to  the  A r g y r o l o b i um, A d e n o c a r p u s ,  tube  to  calyx otherwise  perhaps,  more  considerations. Rothmaler's  1966; P o l h i l l ,  system 1976;  has  Christofolini although  the  & Chiapella, taxonomic  controversial.  The  common annual bracteoles  native  to  similarities latter are  two  Rothmalerian  the  flavonoids  Lupinus  the  the  its  bracts, toward  majority  (1976)  temperate  of  species  suggests  views  tribe  evolution  Genisteae  in  the  A r g y r o ! o b i um as  genus  the  rest  Mel o b i um o f  spp.)  by Horhammer  to  by  that (the  Podalyrieae)  as  invasive  herbs  Mediterranean having the  more  Crotalarieae  Harborne  be q u i t e  the  (1976)  exclusion his  survey  the  isolation  ( 1962 ),  and t h e  of  this  of  author  the  the  the (26  compounds conclusion  is  from  isoflavones  Laman ejt aj_.  present  from  from  Lupinus  isoflavones,  However,  & Chiapella  different  determined that  tribe.  & Wagner  its  ( 1969 ), i n  were d e v o i d o f  of  Christofolini  and s u p p o r t e d  following  e_t aj_.  studies  Genisteae,  species)  now d i s c r e d i t e d  Harborne  the  (2  Genisteae  of  unspecified  geniis  including  tendency  that  parallel  African  Furthermore,  common to  of  serological  showed  tribe.  still  Lupinus.  Finally, (1977)  its  Polhill  north  of  (1976)  with  with  the  result  Polhill  is  1977)  by L u p i n u s , B a p t i s i a and Thermo ps i s  divergence  affinities than  the  fact  New W o r l d .  members o f  the  region.  the  Lupinus  predominance o f  leaves,  and the  shared  possibly  before  stipules,  flowers  of  & Nicholls,  has many a n o m o l i e s ,  and m u l t i f o l i a t e  verticillate are  placement  genus  habit,  1977; Bisby  (.1978),  (Nicholls  & Bohm,  1978) . In sheep",  summary, having  similarities North  L u p i n u s may be r e g a r d e d as  obvious  Cperhaps  American  affinities by  to  parallelism)  Thermopsideae).  There  the to  rather  Genisteae the  are,  the  a  "lost  and  Podalyrieae  however*,  too  (or many  5  dissimilarities Subgeneric  its  was  (1753)  next  into  American  (Pursh,  1814; N u t t a l l , 1 8 1 8 ;  Hooker,  1862 ;  classify  them i n t o  Candolle  (1825)  as  distinct  first,  and to  d i v i d e d the Species  genus  type  but,  for  in  twelve  Five  the  with  European in  genus.  Species During  from N o r t h  & Arnott,  the  America  1 8 4 1 ; Bentham &  no a t t e m p t  was. made to from t h a t  annuals  and  * ' of  De  perennials  1 8 3 5 , Agardh p u b l i s h e d t h e -  monograph o f tribes  containing  be c r e d i t e d  appeared  r e c o g n i z e d the  only;,' w o r l d  Inquirendae,  must  groupings apart  Finally,  into  (1719)  literature.  Hooker  1914)  who s i m p l y  date  either.  were d e s c r i b e d  subgeneric  groups.  in  modern taxonomy by  perennial  the  new s p e c i e s  Douglas,  into  Tournefort  L^. al bus as  years  placed  botanical  and one North  eighty  confidently  Lupinus  although  PI antarum w i t h  it  officially  introduction  annuals  include  taxonomy o f  Lupinus Linnaeus  to  taxa  plus of  Lupinus  i n which  a thirteenth,  unknown or  he  the  doubtful  relationships. A second c l a s s i f i c a t i o n the  extratropical  divided  the  annuals  with connate  annual  with  50 a n n u a l s  genus  lupines  Watson's six  genus has  p r o p o s e d by Watson  North  three  America.  sect i o n s :  axillary  and p e r e n n i a l s  his  PIatycarpus ,  he  containing  and L u p i n u s  petiolate  for  revision  L u p i n e11u s , c o n t a i n i n g  flowers;  having  In  (1873)  a  single  proper,  cotyledons  with  and  inflorescences.  (1906),  sections  complexes  of  cotyledons;  solitary,  multif1owered Piper  into  was  in to  his  subgenera  following  suffered  flora  the  the  o f Washington and d i v i d e d  format  consequences  Lupinus  of Agardh. of  State,  raised  proper  into  Subsequently,  over-zealous  the  'splitters'  6 and s p e c i a l i s t s . Rocky (one  Mountains species  appeared  descriptions. flower  over  the of  which  was  the  taxonomic  Watson's  of  Pacific  the  North  Smith  more as  some s p e c i e s  perennial  species  exclusive  of  More r e c e n t l y ,  other  and s e v e r a l  Dunn ( 1 9 5 7 ,  American  in  For  containing South  lupines  recognized  annuals  for  k e y i n g than  in  as  rseen  different  1  in  North by  variation  (1944)  into  in  Rydberg  groups.  (1971)  inherent  in  clarify  taxonomy o f  variety  of  treatments  of  small  r e d u c i n g many e p i t h e t s the  species  evolutionary  to  and s u b s p e c i e s trends  have c a r r i e d  numbers o f  Lupinus.  & Dunn, 1 9 7 0 ; C o x , 1 9 7 4 a , b;  Lupinus.  taxa,  synonymy level  as  they  and a f f i n i t i e s  Through t h e out  detailed  have  within  In  have  Fleak done  use o f a systematic -  termed complexes a result.  for  co-workers  much to  they  16  America  & Dunn, 1 9 7 7 ; Dunn & Harmon, 1977)  techniques  18  statement  & Dunn, 1 9 7 1 ; Vaugn the  Flora  a  Fleak  and h i s  (one  proper  hand, r e c o g n i z e d only  1 9 5 9 , 1965)  & Dunn, 1 9 7 0 ; Hess  as  criticized  the  Abrams genus  variants  his  and  4 groups o f  subspecies  but was  accounting adequately  from North  s u b d i v i d e d the to  1938  specific  volume work  Once a g a i n ,  on t h e  California  T h e n , between  resulted  the  sections  d o c u m e n t i n g minor  groups.  occurred twice,  (1955),  19  L u p i n e l l u s ) , and L u p i n u s  an a i d  affinities.  from  them i n  number o f  containing  section  States,  perhaps,  (Christian  entities  and 13 p e r e n n i a l  Phillips  not  sections!).  Lupinorum, a three  PIatycarpus,  evolutionary  (1917),  two  placing  indumentum and h a b i t  treatment  6 annual  groups, of  His  plains,  preoccupation with  Species  subgenera  with of  His  600 a s s o r t e d  America.  in  d e s c r i b e d 80 s p e c i e s  produced a s t a g g e r i n g  colour,  publishing  (1917)  and a d j a c e n t  and 1 9 5 2 , Smith  in  Rydberg  by t h e m ,  addition,  at  suggested and between  complexes,  7  all  o f which  represents  considerable  improvement  over  previous  work. In  spite  Estimates from t h e species  of  of  this,  the  the  number o f  1500-1700 names is  Kartesz,  genus  now t h e  1980).  distinctive  accepted  figure  Interspecific classifications  and monographs  (Phillips,  the  Dunn & G i l l e t t ,  years  are  of Watson, initially  1873).  supposedly Heywood, evolved  For  segregated a derived  1963),  taxa  example,  vary  North  separate  enormously  approximately America  from modern  absent  Dunn,  mostly  (with the  annuals  et  possible  al . , over  exception been  The annual  habit  1959; Davies  probable that  occasions  unclear  proposed  have always  &  floras  1956 ; H i t c h c o c k have been  150  (Kartesz  are  (Hutchinson,  more  unwieldy.  remain  perennials.  condition  and  affinities  unnatural  is  for  Those t h a t  from the  and i t  on numerous  1955 ;  1966).  obviously  large  p u b l i s h e d , although  and s u b g e n e r i c  1961;  remains  annuals  &  have  from d i f f e r e n t  is  •  perennial  ancestors. Some r e a s o n s  for  the  The taxonomy o f in  North  America.  Lupinus  Specific  obscure,  especially  available  epithets  biological  confusing.taxonomy of  can  evolutionary  are  Many t a x a plasticity.  particularly  species.  to  a)  a number  later,  for  (Phillips,  and  of b) and  i n t e r r e l a t i o n s h i p s .and 1955).  degree  characters,  are  The 1500 or  c a t e g o r i z e d and d e s c r i b e d t a x a  show a b e w i l d e r i n g  Morphological  problems,  based on m o r p h o l o g y  described  no c o n s i d e r a t i o n  ramifications  with  perennial  be a t t r i b u t e d  t a x o n o m i s t s who have s i m p l y with  rife  boundaries  among the  phenomena t h a t  individuals  is  Lupinus:  of  phenotypic  including  indumentum  ';  so  8  type,  flower  variable.  s i z e and c o l o u r ,  Nevertheless,  used' such c h a r a c t e r s hundreds o f state,  these  almost  certain  for  the  between  taxa.  lupines. ted  is  of  p r o d u c e d good seed  lupine  interfertility  fertile  normal  lupines  occur  contact often  attribute  lupine  are  every  did  In  ejt  a]_.  that  (1961)  it  is  be used  by t h e  natural  They  species  the  moderating  lupines  sterility  are  to  the  brought  man Ce.g.  be the  retreat  the  of  they  in  are  (1966) of  the spread  populations as  into  along  result  northward  behind  likely  populations of  inter-  other is  Dunn & G i l l e t t  species  which  turn  climate.  interfertility,  chromosome compl e'ments o f  species  difficulty  left  off-  demonstrated  introgression  that  attemp-  found groups o f  activities  suggest  she  fertile  also  isolated  following  that  w i t h members o f  causes.  taxonomic  cross  perennial  a number o f  general, lupine  hybridization  i n most  (.1963 )  h y b r i d i z e d w i t h more t e m p e r a t e  Wtth such wide  (1938-52),  combination,  (19 57)  Previously  migrations  in  extremely  published  Lupinus  perfectly  discover  Kazimierski  and a l p i n e  moved n o r t h w a r d s  in  low  Phillips  perennial  glaciers .  arctic  in  considerable  and y i e l d e d  sympatry  the  and  As H i t c h c o c k  were i n t e r s t e r i l e  two  into  much o f  PIefstocene  then  but  and t h r o u g h  successive  the  set  (Phi 1 1 i p s , 1 9 5 7 ) .  brought  of  found t h a t  section).  whenever  roadsides)  of  next  result  meiosis.  that  Smith  species  except  barriers  Furthermore,  Csee  groups to  the  (1957)  with  are  taxa."  also  spring  of  so g r e a t l y  cannot,  Sterility  Bragdtf  barriers.  "vary  they  recognition  Variation  recognition  notably  descriptions.  characters that  some w o r k e r s ,  for  new s p e c i e s  b r a n c h i n g , and h a b i t  are  it  is  not  found to  s u r p r i s i n g that be v e r y  uniform.  the  9  Except L_.  for  two  clearly  s u b c a r n o s u s , which  all  North  related  have a h a p l o i d  American taxa A few  s p e c i e s , JL. number o f  have n = 24 ( P h i l l i p s , ,  Gillett,  1966).  they  morphologically  are  annual  populations  18 ( T u r n e r , 1957 ;  have been  indistinguishable  t e x e n s i s and 1957)  Dunn &  found w i t h _n =48 but  from  typical  individuals. Less annual  taxonomical1y  species  Dunn ( 1956) absence  of  found t h a t  Lupinus insect  an i n s e c t  al.  bi c o l or  (1974)  have v e r y  for  or  keel  discontinuities  L..  nanus  very  uniform  but  much more c o n t i n u o u s the  self-pollinating  high s t e r i l i t y  I.  barriers;  succulentus,  1956)  in  as  did  does  L.  I.  thus,  occur,  the  gene  flow,  has  artificial  within  in  the  crosses  heavily  scented,  clearly  a d a p t e d to  sub.carnos us  large-flowered  annuals  were  :  populations  s pa rs i f 1 or us and j . . a r i z o n i c u s  t e x e n s i s X I.  slight  hand,  The g e n e t i c  resulted  nanus  which  other  individuals  Harding  I.  of  exhibit  populations  ( Erbe,  low  were  isolation  formation  of  between failed 1957).  and p e r e n n i a l s  c r o s s - p o l 1 i n a t i o n and show  of  is •  and,  varieties  variation.  annuals  simulation  present  On the  insect  flowers,  b i c o1o r t o  of  the  few  100%).  between  greater  their  attract  (approaching  individuals.  owing to  in  self-pollination  visits  showed more v a r i a b i l i t y  populations,  of  before  found I.  morphological  seed  larger  large-flowered  Dunn (1956)  set  flowers  some c r o s s - p o l l i n a t i o n to  Consequently,  self-pollinated.  simple mechanical  insect  found t h a t  smal1 - f l o w e r e d  are  readily  slightly  high o u t - c r o s s i n g r a t e s  made up o f  the  part,  i n d e e d , the  visitation  for  most  nanus, with  The need  opportunity  the  I.  l a n d i n g on t h e  effected.  et  for  pollinators;  visitors. requires  which,  troublesome are  self-  are  (Dunn, The  compatabi 1 i t y 1955).  This  sympatry the  and t h e  confusing Previous  situation  to  alkaloid  help  protein  that  emphasis  important  World t a x a  appear  group w i l l  O l d World  in  use  and It  is  were u n d e r t a k e n Only  some c a s e s  not emanates  on  recently  the has  any  economically  both  but,  al . ,  serological  chemotaxonomic work  same r e s e a r c h ,  of  Old and New  for  convenience,  separately.  lupines  The c u l t i v a t i o n natural  cultivars  ranges  of  Mediterranean  has  resulted  and c o n s e q u e n t l y Flora  Europea  distinctive  synonymy.  assess  value  the  related  of  lupines the  in  areas  selection  specific  ejt^ al_. , 1968)  alkaloids  ( 1960) in  away  descriptions. lists  only  nine  epithets  a n a l y s e d .'12 t a x a  to  d i s t i n g u i s h i n g groups  of  Seven a l k a l o i d s  from  o f many  and s u b s p e c i e s , w i t h many  ^NDwacki  species.  in  numerous  (Tutin  species  r e d u c e d to  closely  1973)  more numerous but  be ^ d i s c u s s e d  the  1 9 6 0 ; Ki nghorn et  1971).  Old World t a x a .  the  chemical  classifications  involve  (Scogin,  In  '  today.  confusing,  ' (JNowacki ,  studies  New World s p e c i e s .  with  produce the  subgeneric  early  been p l a c e d on t h e  neglected  quite  the  interacted  of  Lupinus  & Prus-Glowacki,  where d e t a i l e d  economically  on  have to  (Bragd/6',  common e x i s t e n c e  taxonomy  available  patterns  most o f  the  taxa  lupine  refute  electrophoresis  ([Nowacki  from Europe  However,  in  The s t u d i e s  d i s t r i but i o n  surprising  their  Lupinus  taxonomical1y  c o n f i r m or  few.  techniques  each  of  is  as  hybridization  evident  Lupinus  self-pollinated  as w e l l  chemotaxonomic s t u d i e s  been  1980),  resultant  variability  Although  have  when a r t i f i c i a l l y  breeding system,  inherent  studies  even  were i s o l a t e d  and  five  identified. sections:  Distribution  Albus,  The r e a l i t y ther  support  by  interferti1ity bers  of  techniques. an  of  groups  taxa  as  (1963)  groups  they  taxa  earlier  own s e c t i o n .  and  The c l o s e  based on c h e m i s t r y  ones,  genus  unit.  were a b l e  groups  units  was  to  left  and m o r p h o l o g y  (1971)  4 of  between  the L_  re-  constitutes  shown  in  of  L u p i nus a r e  and  additional hirsutus,  the  is  mem-  serological  Europe  one t a x o n ,  agreement  of  Combining a l k a l o i d  place  4  fur-  between  using  in  given  levels  & Prus-Glowacki  the  discontinuous  data,  natural  additional  delimit  Luteus.  who found h i g h  Nowacki  and f i v e  and  him to  and i n t e r s t e r i 1 i t y  concluded that  serological  its  the  groups.  They  the  these  Kazimierski  immunologically  in  compounds l e d  Angustifolius  within  these  the  Pilosus,  different  examined  of  in  classifications  Table  I.  New Worl d_l up_i nes Whereas t h e distinct, obscure  specific  (1971)  boundaries  analysed  and s e r o l o g i c a l their  uniform.  (shown fn T a b l e riers  led  tions  Mutabilis  Mutabilis  II)  taxa but  North  (1960)  the  to  revealed  sterility.  and Nowacki  & Prus-  species  they  to of  the  the  section  less  very  Mutabilis  sterility  section  infra-group  complex  showed much  C r o s s i n g experiments high  are  alkaloidal  were a l s o  internal  subdivide  for  them much more  New World l u p i n e s  presence  (1963)  species  finding  were a s s i g n e d  morphologically  American  American  Serologically,  and P e r e n n i s .  Perennis  inter-group  While  relatives,  Kazimierski  and  20 North  variability.  Thirteen  between  Nowacki  affinities.  European  interspecifc  and  species  and p r o b l e m a t i c .  Gl owacki  than  European  bar-  into  with  sec-  sections  interferti1ity  TABLE  I.  A c o m p a r i s o n o f s u b g e n e r i c g r o u p i n g s o f European l u p i n e s by Nowacki & P r u s - G l o w a c k i (1971) w i t h t h a t o f T u t i n e_t aj_. ( 1968) u s i n g a l k a l o i d and m o r p h o l o g i c a l data respectively  Alkaloid group  Lupinus  taxa  Major alkaloids  Taxa a c c o r d i n g to T u t i n et a 1 . 1uteus  1uteus LUTEUS  ALBUS  rothmaleri  hi s pani cus  1upi n i ne  hi s p a n i cus  hispanicus  al bus  albus  pelestinus  1upani ne  a 1 bus s s p .  graecus  hydroxy1upani ne  albus  ssp.  megalospermus  LA4  albus  ssp.  v a r i us s s p . ori entali s  LA4  varius  ANGUSTIFOLIUS  1 i ni f o l i us cryptanthus  HIRSUTUS n.t.  denotes  not  treated  varius  angustifolius ssp. a n g u s t i f o l i us  1upanine hydroxy1 upani n.e a n g u s t i f o l i ne  a n g u s t i f o l i us s s p . reticulatus angustifolius ssp. angustifolius mi c r a n t h u s  hi r s u t u s taxa  ssp.  n .t.  pri ncei a n g u s t i f o l i us  graecus  n . t.  e p i 1 u p a n i ne  d i gi t a t u s vari us  albus  varius ssp. o r i e n t a l is  pi 1 osus p a l e s t i nus  albus  megalos permus angustifolius ssp. reticulatus  jugoslavianus  PILOSUS  ssp .  in  Tutin  et  al-  (1968)  TABLE  II.  S u b g e n e r i c g r o u p i n g s o f North A m e r i c a n l u p i n e s from a l k a l o i d and s e r o l o g i c a l s t u d i e s by Nowacki (1960) and Nowacki & P r u s - G l o w a c k i (1971).  A l k a l o i d group  Lupi nus  Major  taxa  alkaloids  andi col a pubescens MUTABILIS*  mut abi 1 i s ornatus  s p a r t e i ne  douglas i i  1 upani ne  p o l y p h y l 1 us  1upi n i ne  per en n i s arboreus PERENNIS*  nootkatensis ha rt we gi e l e g a ns a l b i frons 1 at i fo1 i us  BARKERI  SUCARNOSUS DENS IFLORUS  MICRANTHUS  barkeri  1upani ne h y d r o x y l u p a n i ne  truncatus  s p a r t e i ne 1upani ne hydro x y l u pa n i n e  subcarnosus texensi s # densiflorus  sparteine 1u pan i ne hydroxylupanine N 4/5  nanus polycarpus  * Both groups c h e m i c a l l y s i m i l a r . Subdivision a b i l i t y s t u d i e s by K a z i m i e r s k i (1963). # A l k a l o i d s not d e t e r m i n e d ; c o u n t s : n= 18.  grouping  is  based on cross-  based on chromosome  Some members o f alkaloid  study  lupines  concluded  that  excubitus  Perennis  by Nowacki  perennial  J_.  the  of  I.  & Dunn (1965)  coastal  and i n t e r f e r t i 1 i t y , t h e  latter  from t h e  of  Recently,  another  published  by  agreement  between t h e  and-the they  alkaloid  saw  taxa.  Kinghorn  very  stable  hand,  Vaugn  characters  Leaflets  collected  locality  had i d e n t i c a l  In lupines their  e_t aj_.  America are  complex i n  He found h i g h that  t h e J..  chemically specific  chemistry  relationships  caespitose  infra-  distinct  entities.  omitted (1971).  Dunn  (1944)  addition,  profiles  different  been  Smith In  the  and  has  poor  of  discovered  of  some  (personal the  alkaloid  collection  found a l k a l o i d s  to  sites. be  location.  ago and r e c e n t l y  from the  conclude,  much more d i f f i c u l t  than  are  the  over  lupines  of  b e l o n g i n g to  80 t a x a  - caespi tosus  populations  to  same  classify  by  in the  an  attempt  perennial  have been d e s c r i b e d . variation  complex i s  rather  the  Europe.  electrophoresis  among t a x a  which  ( 1978)  and i n t e r - s p e c i f i c  1 e p i dus  w§s~  geographical  Wall er'&..Nowacki  (.1973 ) employed p r o t e i n  reveal  affinities  compounds.  of  alkaloid  ( 1980)  100 y e a r s  as  North  chemical  21 s p e c i e s  in  ( 1975)  a given  summary,  Cox to  over  al bi f rons ,  obtained.  & Dunn (.1977 )  at  they  found v e r y  variation  i n f r a s p e c i f i c a 11 y at  other  of  patterns  content On t h e  Although  classification  K i n g h o r n ejt aj_.  vary  shrubby  species  who  communication), to  examined the  & Prus-Glowacki  ( 1980)  seasonal  found by Mankinen  of  survey  subgeneric  considerable  As  pair  alkaloid  distribution  another  showed c l o s e  Nowacki  et_ al_.  in  n o o t k a t e n s i s , I.  chamissonis  classification  who  California.  a r b o r e u s , JL.  and J_.  group were i n c l u d e d  than  and  concluded  composed o f :  recognizable  F i n a l l y , S c o g i n ( 1973) L. n a n u s , L_. 1 e p i dus  and I.  leucine aminopeptidase.  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 y a l l i i)  electrophoretically for  The d i s t r i b u t i o n  bands l e d him t o s p e c u l a t e on a p o s s i b l e taxa.  of three  isoenzyme  phylogeny  between t h e  However, i n s o f a r as o n l y f o u r o u t o f p e r h a p s  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, s u c h s p e c u l a t i o n s  are rather  premature. Economic a s p e c t s o f Lupinus The  ability  o f l u p i n e s t o grow on n u t r i e n t  where o t h e r c r o p s t e n d t o f a i l that  lupines  deplete the s o i l  led early  deficient  soils  farmers to conclude  of i t s f e r t i l i t y .  They were t h u s  g i v e n t h e i n a p p r o p r i a t e name, W o l f B e a n , and hence t h e L a t i n name L u p i n u s . nodules  Nowadays, we a r e aware o f n i t r o g e n  and R h i z o b i um; f a r f r o m r o b b i n g t h e s o i l ,  fixation, lupines  enrich  it. L u p i n e s have been c u l t i v a t e d Regions  and t h e New W o r l d  reported  i n both the M e d i t e r r a n e a n  f o r o v e r 2000 y e a r s .  t h e p r e s e n c e o f l u p i n e s seeds  Wilkinson  (1854)  i n E g y p t i a n tombs and t h e  d o m e s t i c a t i o n o f t h e T a r w i Bean (L_. m u t a b i l ; i s )  i s thought to  go back  America  mous,  t o p r e - I n c a n time i n South  and C e n t r a l  (Anony- •  1979). The  main d r a w b a c k o f L u p i n u s s p p . as c r o p p l a n t s  predominance alkaloids containing  of b i t t e r - t a s t i n g  and, moreover,  i s the  poisonous  i n t h e l e a v e s and more i m p o r t a n t l y , i n t h e p r o t e i n seeds.  fertilizers,  T h i s has n o t h i n d e r e d t h e i r  however.  Mediterranean annuals,  use as g r e e n particularly  L. a l b u s , L_. 1 u t e u s and IL. a n g u s t i f o l i u s , a r e now grown e x t e n sively  not only 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 north-  eastern United  Europe,  has  Brucher seeds,  of  been  alkaloids practised  (1968)  by s o a k i n g t a r w i for  determined  a v a l u e which  centuries  the  Modern s e l e c t i v e  alkaloid-free  lupines  Old World a n n u a l s  requirements  of  highlands  South  though,  of  that  species, as  as  human and animal  in  there  protein  the  has  species  sources  already  uses  of  seedlings  Pardodi  (1938)  hi 1 a r i a n u s  in  lupines  as  cites  the  to  Marchant,  grown  extensively  as  among L_.  mutabil i s being  strains of  of  the  L.  mutabi1is  seem o b v i o u s  perhaps  with  arid  the  150 . .  great  and  /  potential  semi-arid  southwest.  include  natural  and J_.  resulted L_.  in  and the  (Burkart,  pol y p h y l 1 u s , the  world  variants L_. as  as  are  et  al . ,  lupines  w i t h which  many c o l o u r  ground,  1952).  (Hitchcock  Finally,  The ease  grown t h r o u g h o u t  use o f  a sand s t a b i l i z e r  arboreus  communication).  ornamentals.  insecticide  1942)  substitute  1952 )  arboreus,  are  the  as a  use to  does  taxa  mill t i f 1 or us as  personal  be h y b r i d i z e d has  flowers.  I.  beans  the  climatic its  It  many o t h e r  a coffee  (Burkart,  1961;  garden  America.  adapted  soy  the  Indians.  be 46% i n  'sweet'  restricts  by L_. m u t a b i 1 i s ( H e r e r a ,  roasted  j..  the  running  New World  N o r t h : A m e r i c a where t h e r e  Other  cultivars  and t h e  latter  and C e n t r a l  to  importance  Unfortunately,  prevail  can  the  in  Andean  with  breeding of  crops  should e x i s t  beans  content  feed.  that  J_.  and l a t e l y ,  by the  favourably  increased  fodder  conditions  production  protein  compares  food s o u r c e .  as  New Z e a l a n d  States .  Removal water  USSR, A u s t r a l i a ,  are  they and  hartwegi popular  and  Thesis  Aims  This  study  flavonoids are  as  is  t a x o n o m i c markers  particularly  reasons.  It  an a s s e s s m e n t  is  appropriate a large  a conspicuous  part  Collection  plant  genus  of  level,  confusing  hybridization.  material  at  the  moment  making  progress  between  of  the  The aims  flavonoids  of  species of  occurring  of  3)  construction  flavonoids  as of  in  are:  Lupinus;  a tentative  other  been based on m o r p h o l o g y and to data.  data;  forms  the  are  and  classification co-workers  Finally,  are  evolutionary  poorly  known.  makes  the  highly  1) a detailed  analysis  2 ) an a s s e s s m e n t  of  the  characters;  subgeneric 4 ) comparison of  workers  a lesser  Below  sympatry  and h i s  infraspecific  with  and a l k a l o i d  easy.  new t a x o n o m i c e v i d e n c e study  that  America.  interrelationships  classification  of  several  genus  subgeneric  D.B.Dunn  based on f l a v o n o i d those  for  Lupines  taxonomical1y  plasticity,  classification  information  is  and p e r e n n i a l s  of this  reliability possible  Lupinus  of  Lupinus.  North  consequently  such a t r e a t m e n t ) .  and a p p l i c a t i o n  desirable.  genus  recognizable  acceptable  annuals  The u n c e r t a i n t y discovery  is  (although  toward  the  usefulness  of western  phenotypic  No w i d e l y  exists  affinities  flora  the  such a s t u d y  and e a s i l y  o f the  its  in  for  as we have s e e n ,  through  of  whose  extent  systems on  this have  breeding  II.  FLAVONOID  CHEMISTRY OF LUPINUS  Introduction Very  few  flavonoids Harborne  publications  of  Lupinus,  (1969)  investigation intergeneric differences species  the  are  As i s  and l u t e o l i n ,  their  as  Genisteae.  as  too  the  His  of  a  flavonoid  work c o n c e n t r a t e d  interspecific  the  names o f  in  the  rest  contained  the  of  the  and q u e r c e t i n .  lupine Genisteae,  flavones  Horhammer  on  chemical  the  c o r r e s p o n d i n g C - g l y c o f 1 avones  kaempferol  the  chemotaxonomic.  are  case  Lupinus  is  part  and t h e r e f o r e  lacking,  found t h a t  flavonols  tribe  concerning  one s t u d y  20 s p e c i e s  differences  studied.  Harborne  and o n l y  analysed  of  have a p p e a r e d  apigenin and  the  & Wagner  (1962)  r  reported  the  presence  of  the  isoflavone  L_.  po1yphyl1 us and Laman et_ a_l_.  L_.  1 uteus ,  isoflavones  I.  apigenin, with  (see  The l a s t  luteolin,  their  quercetin  group o f  acacetin,  1).  of  Finally,  species  luteone, 12 s p e c i e s  be a p r e - i n f e c t i o n a l  an i s o p e n t y l of  also  Lupinus.  anti-fungal  and  albus,  flavonols  ejt a_l_.  had t h e  flavones  agent.  together  kaempferol,,. at  (1978)  isoflavone, This  the  methylene-  and c h r y s o e r i o l  glucosylated  Harborne  J_.  p o l y p h y l 1 us , found  diosmetin  all  in  investigating  orobol , 5-methylgenistein  and i s o r h a m n e t i n ,  characterized  to  L_.  7 - 0 - g l u c o s i d e s ; and t h e  Figure  seedlings  a n g u s t i f o l i us and  genistein,  dioxyorobol.  (1978),  genistein  the  3-position  isolated from  compound i s  and  the thought  20 Materials  and methods  1.  Flavonoid  extraction  and  purification  E x t a c t i on_p_rocedure The e x t r a c t i o n all  taxa,  plant  but s o l v e n t  material  Leaves  p r o c e d u r e d e s c r i b e d here was  used.  and p e t i o l e s  quantities (See  of  varied  Plants  or 80% f o r  plant  material).  After  30 m i n u t e s  boiling.  through  glass  wool.  methanol  The p l a n t  extract  material  was  dryness  The two  i_n vacuo  at  The green t a r Filter  4 times  with  a separatory  material  was  hot  and t h e  dryness  left.  d i s s o l v e d in  first  slurried  with  was  heated  was  filtered  then  fresh  to  re-heated  with  and  g of  water  Celite  to  filtrate  i_n v a c u o .  layers  Analytical  and f i l t e r e d was  through  extracted  of water-saturated  organic  The m a t e r i a l  methanol  5-10  (90°C)  100 ml a l i q u o t s  to  for  were combined and e v a p o r a t e d  The brown aqueous  funnel  and e v a p o r a t e d  or  was  funnel.  weighed  40°C.  A i d and 10-25 ml o f  a Buchner or  extracts  of  anthesis,  a s e c o n d 200-400 ml of'~'fresh m e t h a n o l , r e - f i l t e r e d discarded.  for  I.)  (100% methanol  The s l u r r y the  amount  Appendix  L u p i n u s were removed at  200-400 ml o f  gentle  the  and S o u r c e s ,  and b l e n d e d w i t h dried  with  standard  3  n-butanol  in  r e m o v e d , combined  A brown  glass  c o u l d be s t o r e d  and s t o r e d , or t a k e n  of  in  phenolic  this  directly  form,  to  the  column p r o c e d u r e .  Thin_Layer_CJ2ro  mato  Most TLC was prepared  in  the  (Macherey-Nagel) 10 g l a s s  plates  Sr^9!2-^  (TLC)  p e r f o r m e d on p o l y a m i d e  laboratory with (.20  by b l e n d i n g 15 g o f  100 ml o f  X 20 cm)  Two main s o l v e n t  plates.  systems  to  50% methanol a depth o f  These were  Polyamid  DC 6.6  and s p r e a d i n g i t  on  0.33 mm.  were used r o u t i n e l y  in  this  study  21 although full  on numerous o c c a s i o n s  list  of  solvents  used were t h e dioxane; ethyl  'aqueous'  70:15:10:5)  ketone, Less  these  occasions,  10% or  Flavonoid  sheets  most  examination solution  ethanolamine  yellow.  Further  were s u b j e c t e d on t h e s e Initial  colour  Separation extract  the  remove  from a l l  generation The c r u d e  of  air  phenolic  0.1  as  solvent.  mm MN300  On  (Macherey-  on TLC p l a t e s  compounds a p p e a r  as  plates  Chemical).  under dark  UV  light  absorbing spots.  were s p r a y e d of  with a  di phenyl b o r i c  Under  UV  light  variously  c o l o u r e d but  reactions  were o b s e r v e d when the  are  Structural  given  in  mostly  green  inferences  Table  acid  or  plates based  III.  separation  of  to  acid  plates  visualization  reactions  slurry  attached  methyl-  used.  ammonia f u m e s .  an aqueous  flask  were  constituent  of  through  solvents  LH20.  phenolic  a column  of  Care was  by p l a c i n g  an a s p i r a t o r .  The removal  bubbles w i t h i n  the  extract  from t h e  A 30 x 4 cm column was  water-swollen the  flavonoids  chromatography  LH20 ( P h a r m a c i a ) .  air  acetic  Polygram  (Aldrich  commenced w i t h  Sephadex  (benzene,  p e r f o r m e d on c e l l u l o s e  methanol: water)  flavonoid  acetone,  55:22:20:3).  (.1:1,  colour  to  system  A  Most commonly  n-butanol,  UV l i g h t ,  appeared  were e m p l o y e d .  A p p e n d i x II.  under  complex  compounds t h e n  in  'organic'  were d e t e c t e d  i n which  0 . 1 % w/v  and  systems  (water,  15% aqueous  detection  Flavonoids  After  and the  pre-coated  cellulose  nm)  system  commonly, TLC was  either  (366  detailed  methanol, water;  using  Nagel)  is  other  was  them i n of  air  column d u r i n g  dissolved  i n .1-2 ml  filled taken  with to  a filter avoids  1  the  development. of water  and  gently  pipetted  onto  column i n v o l v e d mixtures water,  in  top of  use o f w a t e r ,  increments  of  250 ml o f  Since  each was  acetone.  light,  used.  of  they  taken  pair  polyamide  solvent  were  fractions  residue of  before of  0.5  other  was  from two  of  the only  500 ml  finally  each  washed  m o n i t o r e d by UV  absorbing bands.  collected  except  bands;  to  one b e i n g in  the  in  dryness  when  such  using a variety  in  this  cases  vacuo  Each was  developed  aqueous  in  and  the  s p o t t e d oh a the  system.  organic  After  spraying,  components were c o m b i n e d .  LH20 f r a c t i o n a t i o n ,  of  of  effect,  column was  ml o f m e t h a n o l .  similar  and c h a r a c t e r i s t i c s  majority  1 1 each  continued with  dark  the  taken.  plates;  the  having  Following purified  as  of  water-methano!  found t h a t  flavonoids  appear  Development  had l i t t l e  the  were e v a p o r a t e d  up i n  system,  fractions  was  250 ml were r o u t i n e l y  fractions  All  It  by  removed t h e  p r o c e d u r e would mix m a t e r i a l smaller  followed  Development  The e l u t i o n  under which  Fractions  column.  30%-70% methanol  80% and 100% methanol  with  the  10%.  10% and 20% methanol  flavonoids.  of  the  individual  o f methods  each  compounds were  a c c o r d i n g to  fraction  the  complexity  mixture.  Preparative_TLC  well  Fractions  having  separated  on p o l y a m i d e  banded on p l a t e s  simple mixtures  of  compounds t h a t  in' a p a r t i c u l a r  and d e v e l o p e d  in  that  solvent  system.  flavonoid  bands were d e t e c t e d  u s i n g UV l i g h t ,  polyamide  scraped  compounds e l u t e d  methanol.' dryness, a variety  off  and the  The f l a v o n o i d dissolved of  solutions  in a l i t t l e  solvents  to  test  were t h e n  methanol for  could  system  the  w i t h 80%  evaporated  to  and c h r o m a t o g r a p h e d  purity.  were  Individual marked,  be  in  Partition  chromatography  LH20 f r a c t i o n s exhibiting  similar  chromatography  extract) mixture  was  blender  For  example,  o f water if  would  be b l e n d e d .  compressed a i r was  of  the  The  increasing  to  of  be e l u t e d  7:3,  8:2,  2:8, 5 : 5 ,  0.5  polarity  first  of  followed  acetate).  methylethyl  ketone  10:0.  phase;  : ethyl  organic  compounds were m o n i t o r e d  of  1 g of  The columns were amounts  mixtures  mixtures  caused  (at  other  and f r a c t i o n s  in  then  100% m e t h y l e t h y l  were not m o b i l e  by UV l i g h t  solvent  pressure  onto  acetate  As i n  the  of  petroleum ether  by m o n o g l y c o s i d e s  had been r e a c h e d .  to  Avicel.-  increasing  of  ether.  10 ml  The sequence was  solvent  Diglycosides  of  slight  of  a 60/40  addition  v/w  column.  and f i n a l l y the  weight  used then  with  while  phase.  the  by the  under  amounts  ketone  7:3  the  phase  and p e t r o l e u m  Avicel  mixtures  and d e c r e a s i n g  100% e t h y l  accordingly.  organic  were b e i n g  l o a d e d onto  columns.  stationary  acetate  the  mobile  f1avonoid-mixture , dried  continued with methylethyl ratios  cellulose  proportion of  using solvent  6:4,  the  to  partition  100 t i m e s  Columns were packed  and the  acetate  ratios  ethyl  o f compounds  100-150 ml o f w a t e r - s a t u r a t e d  carefully  developed  in  initial  20 g o f A v i c e l  and perhaps  ethyl  or a p p r o x i m a t e l y  applied  by  formed the  formed t h e  blended with the  water  Avicel,  g,  phase was  the  the  layer  of water-saturated  The water  of  microcrystal1ine  organic  (20-50  complex m i x t u r e s  were s e p a r a t e d  a d s o r b e d onto A v i c e l ,  Avicel  then  mobility  on A v i c e l  A water-saturated water,  containing  in  ketone. aglycones  approximately until  50%  columns, taken  Polyamide  column  chromatography  A column o f appropriate TLC.  larger  amounts  SC6  system  advantage  when aqueous  procedures. to  solvent  The major  especially  Polyamid  eluted  c o u l d be used i n s t e a d over  the  solvent  of mixtures  A major  (Macherey-Nagel)  can  use o f  systems  TLC i s  are  be h a n d l e d  disadvantage  is  of  any  preparative  speed,  needed.  using  that  with  Furthermore,  column  resolution  is  inferior  TLC. The SC6 column was  particularly  useful  acylated  from n o n - a c y l a t e d  flavonoids.  appeared  as  Rf  spots  with  low  systems.  Acidification  increased  mobility.  placed  solvent.  solvent  acylated  was  acidified  to  readily 2.  of  eluted  the  5% a c e t i c eluted  Flavonoid  in  organic significantly  encountered, with  an  non-acylated acid  in  was  appropriate compounds  whereupon  a pure  it  the  state.  identification  simple observations  considerable flavonoids.  generalizations under  UV l i g h t  shows  the  colour  removal  was  of  compounds  and  resulted  separation  chromatography  Initially, yielded  On t h e  and methods  layer  lupine  solvent  When such a m i x t u r e  compound.was  Materials Thin  the  the  Acylated  both aqueous  on an SC6 column and i n i t i a l l y  neutral the  of  in  in  Mabry  et^ aj_.  based on the before  structural  reactions  and ammonia  information  of  vapours.  of  Rf  regarding ( 1970)  appearance  and a f t e r inferences  and c o l o u r the  identity  described of  reactions  structural  flavonoids  fuming w i t h ammonia. that  can  be drawn from  compounds on p o l y a m i d e w i t h  of  on  paper  Table  III  the  boronate  spray  25  TABLE  III.  Colour 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 and r e s u l t a n t s t r u c t u r a l inferences.  under  UV  light  C o l o u r on pi a t e b e f o r e s p r a y i ng  Colour a f t e r s p r a y i n g with boronate  Colour a f t e r Structural boronate spray and ammonia fumes  purple  v a n ous  various  most f l a v o n o l s w i t h s u b s t i t u t e d 3-posit i o n , most f l a v o n e s  dull  various  various  f l a v o n o l s with 3-pos i t i on  blue  blue  blue  flavones stituted  purple  green  green  f l a v o n e s and f l a v onols with free 4'OH but no f r e e 3'-0H  purple  lemon y e l l o w  lemon  yellow  yellow  inference  free  with sub5-position  flavones with free 5,7,3' ,4'-(0H) 4  purpl e  orange/yellow  orange/yellow  f l a v o n e s with free 5,3' , 4 ' - ( 0 H ) 7-position substiuted 3  purple  orange  orange  f l a v o n o l s with free 5,7,3' ,4'-(0H) 4  purple  green  brown  f l a v o n e with substitution  4'-0H  purple  green  yel1ow/brown  flavone with substitution f r e e 3'-0H  4'-0H but  As t h e  number o f  increasingly  identified  dominant  as  chromotography  of  Identical  and c o l o u r  systems  Rf's  was  compounds i n c r e a s e d , TLC became  an i d e n t i f i c a t i o n  tool  unknown compounds w i t h the  required  reactions  before  two  in  by means  known  of  co -  flavonoids.  a minimum o f  four  solvent  compounds were c o n s i d e r e d to  be  identical UV s p e c t o p h o t o m e t r y UV s p e c t r a  were o b t a i n e d  for  all  purified  flavonoids.  Compounds were d i s s o l v e d  in  methanol  orded a f t e r  of  sodium m e t h o x i d e , aluminum  the  hydrochloric of  Mabry  addition  acid  et_ aj_.  and sodium a c e t a t e / b o r i c  ( 1970).  Peak maxima a r e  and s p e c t r a l  listed  A Pye in  acid  Unicam SP1800  Appendix  shifts  after  rec-  chloride/ the  instrument  method  was  used.  III.  Acid_h^dr olysis ,  Acid  hydrolysis  amount o f water  6 drops of  pipette.  hours.  p e r f o r m e d on a l l  compound was  and 5 or  Pasteur two  pure  was  After  into  was  first  hour  replace  that  point  72°C.)  When h y d r o l y s i s  the  lost  heated  added to of  t u b e were e v a p o r a t e d  mixture  of  ethyl  aqueous  phase was  redissolved cellulose tose,  in  and t h e  plate  was  in  to  was  dryness  and w a t e r . to  xylose  developed twice  has  taken  the  TFA was a  boiling of  up i n  1:1  sugar  and rhamnose was 'sugar'  for  contents a  the residue  onto  a standard mixture  in  (90°C)  separation,  spotted  of  from a  bath  (TFA  and t h e  T h i s was  1 ml  5 drops o f  and t h e n  dryness  a second spot o f  (TFA)  completed the  After  A small  with  a water  by e v a p o r a t i o n .  evaporated  arabinose,  tube  acid  a further  1 drop o f w a t e r .  plate;  glucose,  acetate  a test  trif1uroacetic  The tube the  put  glycosides.  an MN300  of  also  galacapplied  solvent  system was  (ethyl  then  acid for  acetate,  sprayed  pyridine,  with  a 1:1  in water-saturated five  ethyl  minutes  acetate  methanol organic  for  colour  occasions,  sugars,  resistant  compounds w e r e ,  sometimes y i e l d e d (Chopin  C-glycoflavones Alkaline  reactions  (see  products that  each  were s t r i k i n g l y  (110°C)  Concurrently,  dryness,  as  hydrolysis overnight  UV s p e c t r a Prolonged  the  redissolved  & Bouillant,  failed  acid  in  using  the  to y i e l d  treatment.  and NMR s t u d i e s treatment  a second g l y c o s i d e 1975)  by t h e  with  any Thes  also  strong  Wesley-Moser  characteristic  of  2).  on t h e  familiar acylating  UV s p e c t r u m  similar  colour  flavonoids  such as  orientin  moiety  of  glycoside remains there  (Harborne  Their  However,  from the  inferred  discounted since  gave  identical  resembled.  addition  moiety  known  were a l s o  different  was  parent  to w e l l  that  7-0-glucoside.  glycosylating  7-0-glucoside  the  phthalic  an oven  known a g l y c o n e s  compounds were d e t e c t e d  and l u t e o l i n  flavonoid  when,  after  Figure  on s p r a y i n g  hydrolysis  the  in  and  plate  hydrolysis  Several  is  even  acid  C-glycoflavones.  rearrangement  p-anisidine  development. to  The  system.  or  indicated,  10:3.2:2).  and p l a c e d  evaporated  aglycones  of  of  and c h r o m a t o g r a p h e d a g a i n s t solvent  vitexin  mixture  n-butanol  phase was  On numerous  acid  water;  the  in  and  was  all  cases  their  vitexin  and  luteolin  subsequently  confirmed,  2 drops o f  ammonia w a t e r ,  was  produced.  The i d e n t i t y  is  Acylation  no a d d i t i o n a l  & Williams,  of  by a r o m a t i c  absorbance  1975).  It  is  Rf  Acylation  1 or  unknown.  acid  corresponding  common compounds.  of o r i e n t i n , and t h i s  to  UV s p e c t r a  the the acids  peak on  therefore  29 probable Nuclear  that  the  Magnetic  flavonoids Resonance  Where q u a n t i t i e s obtained  (Pierce  (stoppered)  for  evaporated vacuum  to  the  an hour  dryness,  pump.  chloroform,  to  or  first  mixture  after  Mabry  NMR s p e c t r a  addition  was  the  then  filtered an  and  water  I'Tri-Sil' it  mixture  was  pump and t h e n in  at  then a  deuterated  an NMR tube  internal  ejt aj_.  of  leaving  dissolved  into  were  Trimethylsilyl  The r e a c t i o n  at  acid.  (NMR)  mg),  flavonoid  two.  t e t r a m e t h y l s i 1ane added as interpreted  by the  dry  The r e s i d u e the  (5-10  aliphatic  EM 390 i n s t r u m e n t .  were s y n t h e s i z e d  Chemical)  a simple  spectroscopy  permitted  using a Varian  derivatives  carry  and  standard.  Spectra  were  ( 1970).  Results Fifty-six lupine  taxa.  T h e r e was  glycosylation common.  compounds their  present;  However,  glycosylating attached  flavonoids  to  moiety;  isolated  found i n  is the  is  a great  genus  shows  of  in  the  position  remarkable only  throughout  the  study.  shown i n  a  the  whole.  Table  IV  American  and t y p e  B - r i n g was  the  amongst t h e  as  variety  from 73 North  g l u c o s e was  a summary o f genus  identified  methylation  flavonoids  distribution  following  the  were  also  conservatism  sugar  that  A list  and a f u l l  taxa  is  given  in  types  of  flavonoids  of  was of  in  the  found  the  discussion  Chapter that  4; were  the  of  30  1)  Flavones  OR  apigenin  R=Rj=H  acacetin  R=CH ,  luteolin  R=H =  on f o u r  flavones:  A fifth  flavone,  one g l y c o s i d e i)  compounds i s o l a t e d  apigenin, acacetin, d i o s m e t i n , was  R = 0H 1  from L u p i n u s  luteolin  present  in  and  small  3  were  based  chrysoeriol.  amounts  and  as  only.  C - G l y c o f l avones C-Glycoflavones  Two o f  these,  taxa  studied.  were a l s o  were the  orientin  C-glucosylapigenin,  very  and v i t e x i n  respectively), were t h e y  dominant  in  Less  the  were t h e  Table  XI).  above  compounds, i s o o r i e n t i n  addition  to  acid  lemon y e l l o w  simple  present.  on s p r a y i n g w i t h Following  frequent  these  were sometimes  hydrolysis  d e n o t i n g the  product  hydrolysis  indicated  3'-0H.  was  only  The o t h e r  were p r e s e n t  the  o f most  type of  colour  the  the  respectively.  In  in  0-glucosides  of  of  solvents.  had changed  colour  them  colouration  aqueous  hydroxylation  glucose moiety  (see  of  reaction  and t h e  they  present  0-glucosides  Rf  73  and on  isomers  gave a green  and had a h i g h  luteolin  lupines  6-C-glucosyl  these  63 o f found,  flavonoid  C-glycoflavones,  the  in  frequently  and i s o v i t e x i n ,  orientin  that  and 8-  most  were t h e  encountered  (8-C-glucosyl1uteolin  profiles  One o f  boronate  hydrolysis  the  most common f l a v o n o i d s  Not o n l y  numerous o c c a s i o n s  to  =  R = CH.  di o s m e t i n  of  R^OH  V  R H , R^ CH  chr,ysoeriol  The m a j o r i t y  Rj=H  3  to  pattern.  change  had been  This  after attached  C-glycoflavones  were  31  less  complex,  glucose. parent these ii)  In  having  the  consequence, they  C-glycoside.  Methylated  seen to  at  known as  dominate  those  iii)  eight  Flavone This  the  of  of  glucosides  were the  turned  cleaved  was  of  vitexin,  eight  seen  and  The  only  taxa. in  bond  the in  less  former  The  trace  was  latter  amounts  in  four  Hydrolysis colour to  (.within  quantities  Some t a x a  4'-0-glucosides to  were seen  which  from f i v e under  taxa  UV l i g h t  change. hydrolyse  It  was  fully,  the  7-0accumulate  Furthermore,  and a p p e a r e d before parent  found t h a t  and  as after  aglycone  with  7-0-glucosides  4 -0-glucosides 1  and 5 - 0 - g l u c o s i d e s  5 minutes).  7-0-  of to  in  characteristically  ammonia v a p o u r s .  with a c i d y i e l d e d  30 m i n u t e s  C-glycof1avones  A p i g e n i n and l u t e o l i n  found.  were i s o l a t e d  2 hours  second to  most common; s m a l l e r  f l u o r e s c i n g spots  within  immediately  to  ( 8 - C - g l u c o s y l a c a c e t i n ) and  of  profiles.  were a l s o  a concommitant up to  sugar-sugar  some t a x a .  cytisoside  brown when s u b j e c t e d  blue  in  compounds was  and l u t e o l i n  spraying.  took  seen  profiles  lupine  5-0-glucosides bright  UV s p e c t r a  O-glucosides  domination  apigenin  C-bonded  species.  class  diglucosides  4'-position  was  (8-C-glucosyldiosmetin) of  the  the  determined.  the  orientin, is  of  to  C - g l y c o f 1 avones  Methylation  flavonoid  not  attached  had i d e n t i c a l  The p o s i t i o n  compounds was  frequently  second sugar  hydrolysed  were almost  32  iv)  Methylated  flavone  Acacetin luteolin) several v)  0-glucosides  (4 -methylapigenin)  o c c u r r e d as  7-0-glucosides  that  the  was  outstanding  the  presence  as  two  different  these  (called  acyl  twice  that  the  of  immobile,  staying  systems.  On a l l  I)  close  in  is  parent  glucosides .  was  undertaken  Other  Acyl  compound which  to  UV s p e c t r a  the but  sugar.  II  and a l s o ,  of  the  quantities  of  all  the..freed  in  rather  solvent  that  the  II  acylating yielded  perhaps  of  quantities  analysing  It  compounds  determination  the  the  The  component.  acyl  acylating  to  present.  hydrolysis  insufficient  difficulty  always  compounds are  and t h e  of  were i d e n t i c a l  identifiable  also  approximately  neutral  indicates  A more c o m p l e t e  the  was  were always  acylI  glycosides  because o f  in  were  The f i r s t  solvents  Alkaline  no o t h e r  suggested that  charged  small  compound.  UV s p e c t r a  derivatives  unknowns  organic  the  acetate  not  in  Most compounds  A p p e n d i x V)  derivatives.  occasions  glucoside  therefore  map ( s e e  origin  attached  flavonoid  flavones.  the  identical  moiety  Rf's  lupine  to  possession  of  of  acylated  acy,lated  parent  parent  are  structures of  hydrolysate  moiety.  flavonoids  One i s o f l a v o n e compound was as  of  has  corresponding  2)  features  dominated a two-dimensional  present  for  and 7 - 0 - d i g l u c o s i d e s  Acylation  profiles  is  (3'-methyl-  taxa.  One o f  the  and c h r y s o e r i o l  1  was  genistein  an a g l y c o n e  and a l s o  isolated (an  isomer  from s e v e r a l of  glycosylated  taxa.  apigenin)-. at  the  It  This was  7-position.  present It  33 occurred mostly profiles  of  four  as  Lupinus  glucosides  after  kaempferol  species  but  flavonoid  on s p r a y i n g  a few  but  hours.  was  hydrochloric hydrolysis  further  yielded  i n major of  the  t y p e was  slowly  d i d dominate  the  substantiated  flavonols  same c o l o u r  inferred  but  structure  of  type of  aglycone  3-0-  isolated.  that  was  not  colouration  s p e c t r u m was reaction  1964). that  produced with  continued  not  by  Acid  glucoside was  in a  and a n a l y s e d  by a c o l o u r  A flavanone the  were  a red  isolated  (Venkaturaman,  reaction.  Both  encountered  g l u c o s e and an a g l y c o n e  show t h e  were p r e s e n t  amounts.  a p p e a r e d as  A flavanone  acid/magnesium  the  but  and q u e r c e t i n  The compound was  UV s p e c t r o p h o t o m e t r y . and t h i s  never  and 7 - 0 - g l u c o s i d e s  One unknown visible  constituent  taxa.  The f l a v o n o l s few  a minor  to  was  determined.  TABLE  IV.  The f l a v o n o i d s i s o l a t e d from of North American Lupinus  seventy-three  a pi gen i n  1 u t e o1 i n  ap.  lu.  7-0-glucoside  ap . 7 - 0 - g l u .  acyl  11  lu.  7-0-glucoside 7-0-glu.  acyl  I  acyl  11  ap.  7-0-diglucoside  1u . 7 - 0 - g l u .  ap .  5-0-glu.  lu.  7-0-diglucoside  1 u.  5-0-glu . 4'-0-glu .  ap . 4 - 0 - g l u . 1  ap.  4'-0-glu.  acyl  I  1 u.  ap . 4 ' - 0 - g l u .  acyl  11  1 u . 4'-0-glu . acyl  vitexin  11  orientin  vitexin  acyl  I  vitexin  acyl  vitexin  X'-O-glu.  II  i sov i t e x i n isovitexin  acyl  orientin  acyl  I  orientin  acyl  orientin  X'-O-glu.  orientin  3 -0-g1u.  II  1  isoorientin  I  isoorientin  acyl  I  acacetin c h r y s o e r i ol  ac . 7-0-glu .  chry.  7-0-glu.  chry.  7-0-glu . a c y l  chry.  7-0-glu.  I  chry.  7-0-diglu.  11  diosmetin  ac.  7-0-glu . a c y l  ac.  7-0-diglu.  I  c y t i sos i d e cytisoside  acyl  c y t i s o s i de a c y l  8-C-glu.  genistein  kaempferol  gen.  7-0-glu.  kaem.  3-0-glu .  gen.  7-0-glu.  acyl  I  kaem.  7-0-glu.  gen . 7 - 0 - g l u .  acyl  11 quercetin  flavanone  Hi  flavanone  Mid  flavanone  Lo  Rf Rf  quer.  3-0-glu.  quer.  7-0-glu.  Rf unknown  acyl  'green '  I II  35  III.  INFRA-SPECIFIC VARIATION  IN  LUPINE  FLAVONOIDS  Introduction In  the  specific of  days  variation  a plant  ical  early  was  species  actly  in  (Erdtman,  of  controlled  features  were l e s s ological to  to  as  if  affected features  proven  used as to  ological wood,  be as or  for  chemicals  (Harrison, is  for  reliable  as  to  type,  than  is  view  gross this  like  judgements, as  such led  characters  are  characters  that  thereby  This  Currently,  infraspecifical1y character  etc.  than  ex-  enzymatical1y  genes  fundamental  taxonomic  over  formulae  number o f  factors  are  Chem-  be d e s c r i b e d v e r y  closer  1964).  gone; they  a basis  1975).  be e l u c i d a t e d  hair  sample  any  morph-  reverence any  other,  must  be  type  of  similarly  that  used  morph(Hey-  1963).  amongst  to  trinsic plus  shape,  one  have a d v a n t a g e s  a finite  by e x t r i n s i c  (1975) plant  notes  noting factors  extrinsic  genetic such as factors  infraspecific  that  populations  amount o f m o r p h o l o g i c a l tion  (Harborne,  could  could  infra-  only  and c o n f i g u r a t i o n a l  steps  leaf  cytological  Harborne tion  they  conceptually  regard  phytochemicals  and,  since  biosynthetic  some w o r k e r s  be a n a l y s e d  Furthermore,  making p h y t o c h e m i c a l s general  considered; often  structural  1963).  systematics,  were c o n s i d e r e d to  characters  terms  biochemical  rarely  would  characteristics  morphological  of  the is  variation  not  diurnality,  chemical  soil  flavonoid  correlated  within  differences,  such as  amount o f  Fluck  a species. (1963)  ontogeny  and  and c l i m a t e  variation.  In  with  varia-  the In  cites  addiin-  seasonality as  responsible  consequence,  there  is  a continuous  range  constant  in  chemical  races  constant  in  in  production of  their  their  of  Tissut glycosides Cory!us Betula  in  species  of  pigmentation  (1969)  the  leaves  populations worldwide, absent  of  for  populations  Finally,  Aye  (1969)  glucoside or  in  is  the  and from a l l discovered  c o n f i n e d to  ( 1972 )  10 s i t e s  by t r i e i n  in  of  7-0-glucoside flavonoids  can  be a s s e s s e d o n l y  studies  have been e v a l u a t e d .  developmental  flavonoids changes,  populations  of  after  in  as  the  identical  was  strangely  6 North South  America.  and  their  '. flavonoids  luteolin and  is  7-0absent  south. in  infraspecific  end, this  a number o f  geographic  isoflavones  t a x o n o m i c markers  similar  To t h i s  Francis  found  Car ex c u r t a  were i n v a r i a b l e  Lupinus  the  of  1 of in  grown  of  be c h e m i c a l l y  that  northern  plants  roots  levels  populations,  in  1959).  6 - h y d r o x y l u t e o l i n , which  populations  The u s e f u l n e s s  in  and  Aesculus,  Increase  known  Moore ejt aj_.  1975).  flavonol  genera,  C. m i c r o g l o c h i n and C. m a c ! o v i ana  replaced  describes  tree  decreased  variable  Quercus, T i l i a  waterlogging  27 European  are  (Harborne,  1899; A l s t o n ,  PI a n't a go mari t i ma to  from 4 o f  _C. m a g e l 1 a n l c a  other  of  l o n g been  caused  and s t e m s .  except  in  has  showed t h a t  American  whereas  three  leaves  are  distinct  species  found d i f f e r e n c e s old  that  compounds and  constituents  (Overton,  T r i f o l j um s u b t e r r a n e u m in  i n which  showed no d i f f e r e n c e s .  cold temperatures  & Devitt  minor  (1972)  Fra x i n u s ,  anthocyanin  those  species  Most commonly, p l a n t  young v e r s u s  while  to  between  p r o d u c t i o n o f major  & Egger of  and  flavonoids  exist.  their  variation  lupines  and p h e n o t y p i c  chapter tested  for  variation  and  37  the  effects  chemical  of  drying  analysis.  and s t o r i n g  of  plant  material  prior  to  38 The e f f e c t Lupinus  of  coast  age on t h e  flavonoid  content  of a  species  Lupinus studies  plant  arboreus  because  of  its  and c o n s e q u e n t  preliminary arboreus  step,  was  compounds  Sims,  ease  of  in  for  on t h e  study  (Nicholls  of  this  similar  Columbia  As a  necessary  the  flavonoids  & Bohm, 1978)  comparative  and  British  cultivation.  a detailed  use  selected  occurrence  undertaken  for  was  to  I.  of  yield  pure  TLC.  Materials_and_methods Seeds shrub  were c o l l e c t e d  growing  Columbia.  w i l d on t h e  A voucher  herbarium.' found t h a t  in  campus o f  specimen  Germination rapid  September  was  is  the  University  deposited  started  germination  1976 from one  in  in  early  mature  of  the  UBC  April.  c o u l d be e f f e c t e d  British  by  It  was  pricking 2  or  c h i p p i n g away a s m a l l  The seeds  were then  d i s h were t h e y The d i s h e s  area  placed  of  on wet  a b s o r b e d water  were kept  Emergence  of  the  seedlings  were r e a d y  at  for  occurred  potting soil  in  two  three  days  solution the This  roots  and t h e n ,  obtained  Rhi zo bi um and the  I. the  paper  3-4  two  in in  infection  resultant  in  within and i n days  4"  the  development  petri  the  and  hours.  dark.  the  a week.  plastic  irrigated  plants of  a  mm ).  a few  that  with  fine  and  daily the  kept for  aqueous  had been c u t  collected seedling of  of  pots  They were w a t e r e d  days,  arboreus  0.2  a 50/50 m i x t u r e  from b l e n d i n g n o d u l e s  o f mature  broughtabout  weeks.  for  in  potting within  were p l a n t e d  for  filter  and s w e l l e d  sand and s t e r i l i z e d a greenhouse  (perhaps  room t e m p e r a t u r e  radicle  The s e e d l i n g s  testa  root  from  locally. roots  with  nodules.,  39 After  two-weeks a l l  cold-frames  where t h e y  Two p l a n t s 6,  the  Each  of  a polyamide  plate  in  the  solvent  organic  solvent.  those  of  and  Figure flavonoids  of  3 shows of  in  ages  was  1,  at  drying, by  2,  3,  a methanolic the  directions; in  corner first  the  comparing  reaction^  from a b u l k  with  sample  of  & Bohm ( 1 9 7 8 ) .  two-dimensional  arboreus  is  the  A total  of  distribution  in  in  Table  V.  orientin  dominated the  acyl  I).  Traces  immobile  and v i t e x i n  flavonoids  compounds was  in  all  in  its  also  individuals  were some t h a t  member o f  identical  (probably  the  pairs  flavonoid  of  acyl  charged) noted.  analysed;  were p r e s e n t of  plants  content.  six-  profiles,  luteolin,  and a m o b i l e  and 3 - 0 - d i g l u c o s i d e , v i t e x i n of  Vitexin,  found were a p i g e n i n ,  compound ( o r i e n t i n  Each  and t h e i r  shown  map o f  individual.  1  ounds were p r e s e n t  amounts.  two  and c o l o u r  4 -0-methylorientin  and a p a i r  variable  to  spotted  and, a f t e r  Nicholls  constituents  monoglucoside  orientin  in  (4'-0-methylvitexin)  7-0-glucosides,  I  was  required.  each o f  subjected  were i d e n t i f i e d  a typical  a Lupinus  various  w h i l e minor  orientin  was  as  into  Discussion  and c y t i s o s i d e  time  system,  behaviour  described  end o f  extract  compounds were i d e n t i f i e d  plants  acyl  green  outdoor  and r e - p o t t e d  the  known Compounds'.;i s o l a t e d  arboreu»s  teen  plant  Flavonoids  chromatographic  Results  at  and d e v e l o p e d  their  L.  were w a t e r e d  The c r u d e  aqueous  were t r a n s f e r r e d  were h a r v e s t e d  12 and 24 m o n t h s .  extraction.  plants  their  acylated quercetin I,  3-0-  cytisoside acylated Twelve  the in  comp-  only trace  harvested  at  each  FIGURE  3.  Two-dimensional Lupinus  TLC map o f  arboreus  at  the  flavonoids  from  6 months.  aqueous s o l vent di r e c t i on  <f  a b c d e f g h i j k 1 m  = = = = = = = = = = = = =  apigenin apigenin 7-0-glucoside vitexin v i t e x i n acyl I v i t e x i n acyl II cytisoside c y t i s o s i d e acyl I luteolin l u t e o l i n 7-0-glucoside orientin o r i e n t i n acyl I o r i ent i n a c y l 11 4 -0-methylorientin 1  organi c  solvent » direction  41  TABLE V.  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 m a t u r i t y of Lupinus arboreus.  in various  stages of  OJ  T3 •i—  OJ  •r-  (S>  o o  1—i  3  1—1  I—  cn  i i  r—  >>  o  ro  c  r—  c• OJ CD  •r-  Plant  age  CL  •r— C  •j—•  OJ  X  CJ1  •,CL  ca  ca  1 month  0  0  2 months  0  0  3 months  0  0  6 months  0  0  1 year  0  0  2 years  0  0  • denotes o denotes + denotes  i —  »—1  .  OJ  £=  >>  o  ro  c  OJ  T3  •t— IS)  o  </)  X  X  OJ  OJ  •r—  4->  +J  >  >  O  +  +  +  +  +  +  +  +  +  +  +  +  •r-  • • • •  a major a minor a trace  •i—  1—l  >>  >.  • • • • •  OJ  •r-  w  o to  •r-  4->  o  < /) o to  •i—  1—1  i  1—  r—  1  o  O  (0  £ +->  c 1—  1—  o  OJ  +->  +J  +->  o  i—  •  >>  +  3  0  0  0  0  +  0  0  +  0  0  0  0  0  0  +  +  +  constituent constituent constituent  C  •—i  sz  o <u  4->  i—i  OJ  •1—  CD  0}  -a  •I—  't—  1—  ,—  TJ  c  o o  o O rei ca  ,—  • r-  +J  >  >>  l—i  to o o  'i—•  -o  -o  o  •o  OJ  OJ  >>  +J  c  CU  "r-  to  • • • • •  C OJ  •1—  to o o 3  i—  3  '  i—  CD  •r-  CD  1  T3  1  Jo  o  O  oo  CO  ca  1— >1  c  +->  •r—  •r—  OJ  +->  +J  E  4-> OJ  OJ  I  O  CJ  1  OJ  s-  S-  3  3  >>  c  0)  sSo •. o  o  -  0  +  0  0  +  0  0  +  +  0  +  0  0  +  0  0  +  0  1  c  1  OJ  cr  +  + +  +  42  It  i s obvious  flavonoid  content  that there i s l i t t l e o f I.  arboreus  d u r i n g development.  t r a c e compounds t h a t a r e v a r i a b l e trends, that i s , their correlated to  presence  flavonoids It during  of t h i s  should  is intermittent  be  s p e c i e s a t any noted  consequently,  one  would expect  No  winter  frost  damage o r t h e o n s e t  on t h e  experimental  material J_.  i s obvious.  arboreus,  not  harvest time  of seasonal  took fluctuat-  In summary, i t can  For  seen  chemotaxonomic  o f damaged p l a n t be c o n c l u d e d  that  grown u n d e r s t a n d a r d i z e d c o n d i t i o n s , shows in flavonoid  out.  following  o f s e n e s c e n c e t h a t was  o f a v o i d i n g use  major o n t o g e n e t i c v a r i a t i o n  the  was  be r u l e d  changes i n l e a v e s  p l a n t s i n the w i n t e r .  study, the a d v i s a b i l i t y  and  winter collections  possibility  chemical  ontogenetic  assessment of  i o n s o c c u r r i n g d u r i n g t h e w i n t e r months c a n n o t Indeed,  Those  time d u r i n g i t s development.  a l s o t h a t every  the  major  Therefore, i t is possible  accurate q u a l i t a t i v e  s p r i n g , summer o r f a l l .  p l a c e and  show no a p p a r e n t  to i n c r e a s i n g m a t u r i t y .  obtain a reasonably  change i n t h e  content.  no  43 The e f f e c t  of  drying  and s t o r a g e  on t h e  flavonoid  content  of  Lupinus Materials  and methods  Lupinus  arboreus  was  also  and p e t i o l e s  (600  mature  g r o w i n g on t h e  ial  shrub  was  g fresh  used f o r  weight) campus  t h o r o u g h l y mixed and t h e n  of  UBC.  divided  The m i x i n g a v o i d e d  particular  sample came from a s p e c i f i c  low,  sun-side e t c . ) .  immediately flavonoids  u s i n g the  were i s o l a t e d ,  second  p o r t i o n was  jected  to  was  the  air-dried  for  Results  and  between the  Table  three  aglycones  in  are  plant extracted  Chapter  II.  The  The  being  The t h i r d  portion  a paper  extraction  sub-  bag  at  and  no s i g n i f i c a n t  differences  material.  only  is  a slight  the of  dried  Therefore, work  leaves.  glycosidases cleavage.  significantly  compounds are  there  does a p p e a r  some s u g a r  flavonoid  VI  plant  causing  for  to  of  release  plants.  prior  portions  a minor  alter  the  one week b e f o r e  in  equal  any  and i d e n t i f i e d .  stored  mater-  discussion  variation.that  to  that  p o r t i o n was  procedure.  two y e a r s  plant three  of  in  single  identification.  As shown i n  of  for  a week and t h e n for  into  described  purified  air-dried  The  part  Leaves  from a  possibility  The f i r s t  same e x t r a c t i o n  room t e m p e r a t u r e flavonoid  the  procedure  study.  were p i c k e d  portions.  (high,  this  the  the  i n Lupinus  perfectly  was  of  dried is  stable.  in  probably  d u r i n g the  level  use o f  increase  This  This  The  not  drying  equally  amount  results  fresh valid  in  from  period  sufficiently  glycosides or  the  the  plant since  great  dried  material the  TABLE  VI.  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 i n Lupi nus samples f o l l o w i n g d r y i n g and s t o r a g e .  cu -o  -a  •r-  •1—  CO  o 3  1—I  l—l  1  >>  ac  ac  de  c  *i— to  •1—  •1—  •1—  c  X cu  X cu  Treatment of plant material fresh dried  for  dried  and s t o r e d  for  two  1 week  years  •r—  cu co •r-  OfO  Q. ra  'r—  + o o  •r-  •I—  >  >  >  o  «  o  o  • o  • d e n o t e s a major o d e n o t e s a minor + denotes a t r a c e  o  o «  « o  o  (  ,  i —  >> o ra  +J c cu  +->  c:  CO  4-> >> o  o  o o o  constituent constituent constituent  o o  o cu 4-> =5 i —  i —  o cu •f->  3 i—  5o  o  o  +  o o  »  o  £  cu  o ">> +-> cu  £  1  cu  o  s-  -  •I—  s-  +  +  cu •p-  4->  u  >> CJ ra  £  c  o  >> o  «  1—1  o  •r-  -t->  • o  1  I—1  </>  •r—  +J >> O  1—1  CO  •1—  o to  X CU +J  3  i —  o ra  co O CO  •1—  O  >>  o ra  '1—  <L> CO  1—  >>  1—1  o  r~  i —  de  CO  1—1  de  o  i—i  c  CO O  1—1  , —  e  arboreus  o  o  1  +  •  o  o  +  »  o  o  +  45  I n d e e d , Bohm ( u n p u b l . ) f o u n d t h a t w i11i a ms i i (Saxifragaceae) collected  l e a v e s o f Con i mi t e l l a  and d r i e d  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 and  o v e r 80 y e a r s  i n normal  amounts  no e x c e s s o f a g l y c o n e s t o s u g g e s t m a j o r g l y c o s i d i c  down t o have o c c u r r e d . found t h a t  30 m i l l i o n  flavonoid  identical profile  pared d i r e c t l y  two y e a r s , l i e s  at the o u t s e t .  i n the fact  that a l l t o be  Consequently, the  o f t h e two y e a r o l d l e a f l e t s  to i t s o r i g i n a l  present  a r b o r e u s were known  condition;  can be com-  something  n o t be done i n t h e c a s e o f t h e Coni mi t e l l a  studies.  (1977)  ash s e d i m e n t s ,  The r e l e v a n c e o f t h i s  p o r t i o n s o f t h e l e a v e s o f I.  chemically  could  y e a r s o l d , from v o l c a n i c  contained glycosides.  study, spanning merely three  & Gianassi  p r e s e r v e d l e a v e s o f t h e ext.i n e t ..jgenus '-ZeT.ko va  (Ulmaceae), still  Moreover, N i k l a s  break-  that  and Z e l k o v a  46  Geographical Lupinus  variation  i n the  t a x a . Experiment  Two  B o t h t a x a , I.  predominantly  c o a s t a l and  Columbia to C a l i f o r n i a . restricted  to the  member o f t h e an  I: A preliminary  annual,  altitudes  arboreus are The  I.  and  well  perennial  immediate coast  i n l a n d on  b i c o1o r , a r e  s h r u b J..  and  and  may  sandy, open  be  (approximately  0.05  r a n g e s o f t h e two  I).  Ten  evaporated i n 0.5  ml  discussion  1)  arboreus  the  ten  their spot  removed  collections (see  Figure 4  and  i_n v a c u o . and  plates.  organic  The  spotted  and  the  e x t r a c t s were  at the  corners  Two-dimensional  s o l v e n t s was  The  carried  of  develop-, out  and  compared.  R e s u l t s and  Table  species  g) was  methanol, f i l t e r e d  methanol  p o l y a m i d e TLC  profiles  Lupinus  i n 80%  to dryness  ment u s i n g aqueous and the  low  s a m p l e s o f each s p e c i e s were t a k e n .  were g r o u n d up  cm  bi c o l o r ,  areas.  throughout the  X 10  Lupinus found at  specimens t h a t r e p r e s e n t e d  10  arboreus  methods  leaf material  redissolved  British  i s o f t e n a dominant  from herbarium  Appendix  infraspecific  found from s o u t h e r n  i s more w i d e s p r e a d  Dried  extract  study  v e g e t a t i o n o f mature dunes.  Materials_and  leaves  of  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  variation.  is  f l a v o n o i d content  V I I shows t h e  populations  relative s i z e and  distribution  sampled.  concentrations colour intensity  o f f l a v o n o i d s among  A s u b j e c t i v e assessment was  made by o b s e r v a t i o n s  following spraying.  of of  It i s  47  FIGURE 4 .  Map t o show t h e l o c a t i o n s  o f the Lupinus  J_. b i c o 1 o r ( x ) c o l l e c t i o n s specific  flavonoid  monitored  variation.  arboreus  for infra-  (•)  VII.  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 from t e n p o p u l a t i o n s .  among i n d i v i d u a l s  of  Lupinus  arboreus  c  1—1  i—i  i—i  •  1—1  1—1 1—*  1—  1—  ,—  =3  1—• CD  c  'r—  SZ CU  1  o 1  1  i o _i  •r—  c •r-  •r-  CU  CU  OJ  +J  +J  >  >  X  X  .,— iSi  o  tsi •r+->  >> CJ rc  .  >> o to  .  3  r—  c •i—  ,—  CD I  o  CU  -t->  +J  >  0  0  •  0  0  +  0  1  o  3  3  o ra  i— CD 1  o  -  >>  1  3  c •i— +->  cu  tz  •I—  •—  >> o ro c •f—  4o  1—  OJ  £  c  i~  o  0  0  +  1  o 1  0  •  0  +  +  0  •  0  0  0  BC  +  0  •  +  0  0  +  +  0  +  •  0  0  +  R o b e r t s , WA  +  •  •  +  0  0  0  0  +  •  0  0  •  •  +  0  0  0  +  0  •  +  0  0  0  •  +  0  0  •  +  +  •  0  0  OR  R i v e r , OR  Crescent  City,  Westport, Sur  CA  CA River,  CA  0  or  +  Newport,  +  +  •  0  0  +  0  0  +  •  0  0  +  0  0  +  0  •  0  0  +  0  •  0  0  •  • •  +  0. 0  0  0  • • •  •  •  0  •  0  r—  o  •  I s . , BC  •l—  •  3  •p-  c cu  cu  3 i—  'r~  ap  3  CD 1  1  o  +J >> CJ  >> o  1  o 1  >> O  +-> •1—  i— CD  3  i—i  ap  X  cu  i—  •  i—i  0  BC  Monroe, WA  Little  CO  . i— 3  i—i  +  Pender  Victoria,  Pistol  >>  o  ro  0  Vancouver,  Point  o ro  • 3  o  +-> c cu  0  P o p u l a t i on  South  ap  cn  UBC,  >>  cn  •  >>  or  1— >1 o as  • •  •r—  r—  s-  i— CD  IS)  >> S-  CD  1 .  o 1  •  3 i—  CD  ••—  CO  -o  c  o  •r-  1  +J  CO  O  Scu  • scu  3 cr  3 cr  1  1 .  o 1  CU  o  ch  TABLE  •  >>  So  +  0  0  0  + 4^ OO  clear  that  one  California,  is  discussion ten  specimen, that strikingly  of  this  populations  result  present  in  all  major  and two  flavonoids  components  in  two  plants  amounts  to  of  one  seem to  The  be more  dominated  thetic  capabilities  Hence,  we see  ination  plant  of  of  is  however,  one  were  cytisoside  from b e i n g  from major  ranging  is. possible too  low  hand,  major  in .  quantities  individual.  Six  from minor that to  possible  7-0-glucoside other  flavonoids  components  especially  from L i t t l e  which  the  be det-< in  the  were  absent  4'-methylorientin  and may i n d i c a t e  its  possesses not  Sur' R i v e r  collections.  by l u t e o l i n ,  This  in  it  intermittent  other  side.  is  in  the  real  differences.  from the  synthesis  This  other  varied  concentrations  On t h e  'In-dj. vd dual  different  idual  in  of  A  Nine  compounds,  ranged  but  and l u t e o l i n  plant.  three  differences  absence,  Nine  and o r i e n t i n  being;minor  qualitative  present  apigenin  Vitexin  quantities  by TLC m e t h o d s .  qualitative  is  trace  nine.  Quantitatively,  derivatives to  other  similar.  and two  plant.  7-0-glucoside  complete are  from j u s t does  to  showed  compounds  case  some p l a n t s  very  plants  throughout;  orientin  Apigenin  compounds  ected  in  one  River,  separately.  were a p p a r e n t .  acylated  others.  but  all  Sur  from t h e  were q u a l i t a t i v e l y  some d i f f e r e n c e s the  different follows  compounds were common t o were  from L i t t l e  in  1  a typical the  plant  flavonoid  L.  set  arboreus  3 - O ^ J i i e t h y l at i o n 1  area  nothing  profile  and 4 ' - 0 - g l u c o -  Geographically  tree-lupine reveals  conspicuously  a different  other  4 -0-gl u c o s y l a t f on , C-glycof1 avones.  The  7-0-glucoside  quite  seen  is  this  of  biosyn-  plants. and no indiv-  and m a c r o s c o p i c exam-  unusual.  However,  50  microscopic on the  upper  I.  describe faces;  study  all  taxon. would 2)  as  possessing adaxially  specimens  they  state  their  that  plants  of  of  Orientin  except  for  were the  no q u a l i t a t i v e  sides  profile  shown  description.  p u b e s c e n c e on  obtained  another  from t h i s  or  of  from p l a n t s  bicolor.  VIII.  plant  d o m i n a t e d the orientin  assessment  Eighteen  t w e l v e were p r e s e n t  in  com-  every  profiles  and l u t e o l i n  plant  of  all  7-0-  flavonoids. were p r e s e n t  of  in  L.  as  the  major -components  b i c o1o r , but in  their  different  and l u t e o l i n  Similarly, contain  c o n c l u s i o n , there differences  in  the the is  the  showed and  distribution.  plants.  both compounds a r e  that  some minor  showed no v a r i a t i o n  apigenin  in j . . b i c o 1 o r .  i nterpopul ational  Table  were i n t e r m i t t e n t  absence  In  and an a p p r o x i m a t e  in  i n which  differences  repertoires  undramatic.  and L.  such  sur-  speculation.  which  one  consequence s i n c e  glucoside  of  this  leaf  some i n t r o g r e s s i o n w i t h  These d i s c o n t i n u i t t e s > h o w e v e r ,  little  ( 1966 )  glabrous  fitted  presence  and v i t e x i n  major  constituents  presence  Dunn & G i l 1 e t t  study  flavonoids is  The compounds t h a t  synthetic  indumentum  bi c o l or  investigated.  trace  the  support t h i s  concentrations  glucoside  the  flavonoid  pounds were i d e n t i f i e d  the  in  may i n d i c a t e  The a t y p i c a l certainly  leaflets.  considerable  arboreus  The d i s t r i b u t i o n of  of  the  surface  Lupinus  presence  of  other  upper  the  surface  Furthermore, the  reveals  For  in  the  example,  aglycones present  absence  of  as  flavonoids  of  is  the  of  glyco-  apigenin  7-0-diglucoside  no s i g n i f i c a n t  bio-  is  7-0equally  qualitative L..  arboreus  oo  r—  o  oo 0J  O  fD  t«  O O  fD 3 3  o  OJ  3  O  3 CU  i. fD ca o  PJ  •  o  —1  O O •  3"  to c+ CU  O O •  o  c~>  3" CU to riCU  O  —'.  r+ <<  3;' O  to  _Ja  <  o  »  —1  -o c  CO 1—  <<  OJ r+ —i. O 3  OJ  <-+  o  -o  <  00  o  fD -s  -s  1.  CU CO  3  o o  OJ  D>  —'  CO fD  1—1  —••  —i  o  to •  m <  i—i i—i i—i •  i  te  CA  i«  BC  o  ,  CA  CA  o  oo  03 O  o  3  3>  —.  3" fD  -a a . 0  -••  • a to c c+  +  o  +  +  +  —• -i  apigenin  01 ->.  O r+ 3 -"• to O  o  o  ap . 7-0-glu .  +  +  ap.  c+ cr sz  7-0-diglu.  O -b  vitexin o  o  o  o  o  o  o  o  +  vitexin  acyl  I  o  vitexin  acyl  o  vitexin  X'-O-glu  II  i sovi t exin  +  isovit.  acyl  Cu < O 3 O i, CL to  I  1uteolin  ro 3  lu.  7-0-glu.  1 u  7-0-diglu .  to  entin o  o  o  o  o  o  o  o  o  o  o  o  or  entin  acyl  o  o  o  o  o  o  o  o  orientin  acyl  X'-O-glu  +  +  +  0  +  +  +  +  +  o  orientin  +  +  +  +  +  +  +  +  +  +  isoorientin  +  I  +  isoor.  acyl  II  3 CL  CL £ CU  I  52 The  l a c k of chemical  reflected The  former  distinctness  i n t h e m o r p h o l o g y o f L. taxon  has  of lupine populations i s  arboreus  but  l a r g e , h e a v i l y scented  not  i n L^. b i c o l o r .  flowers that  o b v i o u s l y a d a p t e d t o o u t c r o s s i n g ; t h e s p e c i e s has when grown i n t h e a b s e n c e o f p o l l i n a t o r s  (Grant,  1971).  unscented  L u p i nus  f l o w e r s and  b i c o l o r on  seed  ( B r a g d 0 , 1955).  consequence, g e n e t i c d i f f e r e n c e s , whether they o l o g y o r c h e m i s t r y , a r e masked by h i g h  low  levels  control  In  morph-  small  self-pollinated.  This  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 discontinuities isolation variants.  has  between them, as Dunn ( 1 9 5 5 ) n o t e d .  l e d to the development o f d i s t i n c t  This v a r i a t i o n  varieties  and  It should  be n o t e d ,  d i v i s i o n s was  Munz ( 1 9 5 9 ) l i s t e d  from the  The  logical  the  an e n v i r o n m e n t a l l y genotypic II the  and  the  differentiation?  I I I were d e s i g n e d  flavonoid variation  in  four  California.  basis for  such  in flavonoids is  have any  variable  from E x p e r i -  geographical  individuals  range o f the s p e c i e s ?  induced  genetic  morphological  questionsv.that a r i s e  variation  p a t t e r n , or are  and  f l a v o n o i d survey.  ment I a r e : 1) does t h e  random t h r o u g h o u t  chemical  quantitative variation  i n Lupinus.  Such  (1944) to r e c o g n i z e  s i x subspecies  h o w e v e r , t h a t no  apparent  Infraspecific shown t o o c c u r  l e d Smith  set  of h e t e r o z y g o s i t y  t h e o t h e r h a n d , has  i s predominately  are  2)  or  eco-~  distributed Is t h i s  variation  phenomenon o r i s i t an e x p r e s s i o n To  answer t h e s e  to monitor, i n a Lupinus  questions,  more f u l l y species.  at  of  Experiments  and a c c u r a t e l y ,  53 Experiment I I : V a r i a t i o n  i n L u p i n u s ' s e r i c e u s Pursh„  L up i n u s s e r i c e u s was  used f o r t h i s  cause of i t s wide d i s t r i b u t i o n variability.  I t grows i n t h e  from southern  British  Hitchcock  C o l u m b i a and  m a j o r i t y of our  vegetative  characters."  considerable  dry, yellow  e t a_l_.. ( 1 9 6 1 ) d e s c r i b e  more t h a n t h e and  and  this  size,  c o l o u r and  banner p e t a l . Hitchcock beloging  the  other  l u p i n e s , i n both be  spreading.  synonyms a r e Fleak  listed  "even  whether morphological  are  distinctive  and  vary  the  •••'*  species 61  I t i s t h u s an  in which to study  to  Flowers  f o r the  .'  floral  sparse  (1971) regarded  L^. s e r i ceus c o m p l e x .  by  Arizona.  varying  I t s p u b e s c e n c e may  ejt aj_. ( 1 9 6 1 ) w h i l e  paralleled  as  areas  amount o f a d a x i a l p u b e s c e n c e on  H e n c e , 23  to the  pine/sagebrush  taxon  be-  morphological  A l b e r t a to n o r t h e r n  a b u n d a n t , s h o r t a p p r e s s e d t o l o n g and in  more d e t a i l e d s t u d y  geographic  by  taxa  ideal  e  as taxon  patterns  flavonoid patterns.  MatflCi^ls.and^M^th^ds A total f r o m 32 during the  of  181  populations the  individuals  i n the western U n i t e d  summers o f  populations.  1978/79.  as  was  i n a p a p e r bag  prepared dried in  shown i n A p p e n d i x IV. and  from each p o p u l a t i o n .  leaflets  readiness  Figure  was  States  collected  and  5 maps t h e  air-dreid.  pressure  from  (or part  usual  liquid  of  each thereofY  A voucher specimen  From each i n d i v i d u a l  e x t r a c t e d i n the  f o r high  Each p l a n t  Canada  localities  From 2 t o 10 p l a n t s were o b t a i n e d  population placed  o f L^. s e r i c e u s were  way  (see  one  gram  Chapter  chromatography.  was  II)  of  54  FIGURE 5.  Map  showing the l o c a t i o n s  sericeus monitored variation.  o f 32 p o p u l a t i o n s  for infraspecific  of  flavonoid  Lupinus  55 High  Pressure_(Performance) The use o f  specimens sons. of  of  Not  HPLC to  Lupinus  only  is  extraction, tive has  injection  measure  of  197 9 ) .  only  in  and a d v a n c e s  High  1iquid  of  of  column or  a pump (see  6)  to  solvent  may be a u t o m a t i c a l l y  solvent  prior  to  entering  are  monitored  leave  the  column.  other  chromatographic  high  resolution;  for  derivatization of  All  to  types  exclusion equipment. fast,  of  compounds.  comparison  gas  iii)  But,  the  in  (1975)  easy  reproducible  can  practical  simply  two of  flow  and q u a n t i f i a b l e  (King-  five short  no r i s k  of  refined  of  the  use  solvent The  programmer  injected  set  into  separation,  detection  the  constit-  system  as  they  advantages  of  HPLC  analysis  of  advantages  iv)  time;  no  requirement  thermal are  in  decompodirect  flavonoids. partition,  be m o d i f i e d terms,  a  involves  quantification;  The l a t t e r  years  materials.  It  After  i)  technique  availability  a solvent  lists  methods:  a compara-  packing m a t e r i a l .  column.  LC ( l i q u i d - s o l i d ,  in  is  rea-  analysis  The  eight  Compounds are  compounds; v)  chromatography)  to  a pre-set  mixed  chromatography  of  obtained.  packing  uniform  a number o f  standardizing  increasing  by a UV a b s o r b a n c e Markham  ii)  sition  fine,  individual  procedures,  six  column  a precisegradient.  uents  over  in  force  a column o f  deliver  last  by  chromatography.  through  to  very  is  of  qualitative  also,  chromatography  liquid  Figure  the  for  the  gradient  from the  instrumentation  method  for  quantities  results  pressure  method  (HPLC)  flavonoids  preferred  solvent  flavonoid  This  was  the  compounds, but and  become w i d e s p r e a d  ston,  a fast  of  Chromatography  investigate  sericeus  .it  complex m i x t u r e s  Liquid  the  to  exchange  be p e r f o r m e d on  use o f  results  ion  is  HPLC to beset  and HPLC  give  with  pit-  56  FIGURE 6.  Diagrammatic  r e p r e s e n t a t i o n o f a high  chromatograph  pressure  system.  SOLVENT  SOLVENT  A  B  SOLVENT PROGRAMMER  PUMP  SAMPLE INJECTION  COLUMN  UV DETECTOR  CHART RECORDER OUT  liquid  57 falls.  Speed o f  chromatography.  separation  affects  the  Resolution  is  affected  solvent/packing/compound rectly  to  study.  problems o f  are  can  much more  cause (or  choice  of  ients  each o t h e r .  wavelength  of  any  any  flow-rate lead  and  di-  quantitative  (microcrystal1ine use,  HPLC columns  Interactions  the  that  packing material  a gradual  and b r o a d e r  sample  Finally,  different  in  p a c k i n g and compound m i x t u r e s  of  and c o n s e q u e n t l y also  depends  injections,  on  solvent  problems w i t h  peak  by  deterioration  extinction  may show s i m i l a r  concentrations  to  Avicel  Reproducibility  and column w a s h i n g s .  detector  in  become l o w e r  Compounds h a v i n g  vital  expensive!).  result  standardized cycles  exist.  their  (and  decomposition of  solvents  peaks  merge i n t o  use o f  or  by t h e  and d i s c a r d e d a f t e r  b i n d i n g between  thereof)  resolution; to  permanent  column o f  as  These e f f e c t s  reproducibility,  be c l e a n e d  irreversible  part  poor  interactions.  Whereas a p a r t i t i o n  cellulose)  also  resolution  the  grad-  quantification  coefficients  heights  tend  even  at  the  though  differ.  U ? ! r ? _ ° f _ f l ^ v o n o i ds Normal-phase been  used by  (adsorption)  Kingston  amide columns e l u t e d to  separate  mixtures  However,  most o f  from t h e  use o f  flavones.  with water/methanol  gradients  that  bile  in  to  (Manley  & Shubiak,  (Ward & P e l t e r , the  successful  reverse-phase  phase means  decreasing  separate  anthocyanins  chalcone  phase;  (1979)  c h r o m a t o g r a p h y on s i l i c a  the  1974 ;  separations  partition  stationary  phase  of  is  less  used  1975).  flavonoids  eluted  poly-  flavanone/  ejt aj_. ,  polar  has  have been  chromatography.  c o n s e q u e n c e , compounds a r e  polarity.  Also,  1975 ) and  Collet  gel  than  in order  come Reverset h e moof  58 Non-polar inert are  stationary  support,  usually  esterified  phenyl, with  alkylamine  the  nolic  with  first  silica  are  bonded c h e m i c a l l y  gel.  functional or  two  phases  The s i l o n y l  groups such as  alkylnitrile  most w i d e l y  (Johnson  used f o r  groups  an  (=Si-0H)  octyl , octadecyl ,  & Stevenson,  the  separation  1978)  of  phe-  compounds. Several  separation ified  to  oats  (Strack  et  glycoflavones  (1978),  and t o b a c c o  HPLC o f  Lupinus  sericeus  each  glycosides  by  of  in  sample was  c h r o m a t o g r a p h y was  Court  each  into  mm ID  30 m i n .  set  to  continued  ( 1977) a methto  & Brederode  pure MeOH.  sericeus One m i c r o -  HPLC s y s t e m .  Partisil  A gradient  The  instrument 10 ODS  of  (5%  methanol/water  (KH^PO^) was  employed.  10% MeOH f o r  10 m i n . ,  gradient for  I.  900 s e r i e s  deliver  non-linearity)  The 90% MeOH was  column wash w i t h  the  potassium phosphate  programmer was (80%  by Niemann  of methanol.  column o f  C-^g o c t a d e c y l s i 1 ane , Whatman L t d . ) .  a concave  a]_.  been used  1 g sample o f  p e r f o r m e d on a T r a c o r  w i t h 0.033M m o n o b a s i c  were  Lastly,  has  and  (1977).  1 ml a l i q u o t s  injected  w i t h a 25 cm X 4.6  The s o l v e n t  buffer  1978)  gradients et  of  flavonoids  extracts  litre  & Krause,  ( 1978).  acid-  flavonoids  by B e c k e r  by West ejt aj_.  phenolics  were d i s s o l v e d  fitted  g l y c o f l avones  flavonoid  gradients  the  Water/acetonitri1e  and t h e i r  leaflets  resolved (Strack  with a phosphate  resolve  of  buckwheat  1979).  isoflavones  The p h e n o l i c  Methanol/water  successfully  isomeric  gradient  HPLC.  1976 ),  al_. ,  separate  anol/water  acid  & Nagel ,  and i s o m e r i c  systems .have been employed f o r  reverse-phase  5% a c e t i c  (Wulf  used to  solvent  in  wine  then  to  to  5 min.  The system was  90% MeOH before  lasting  a 10 m i n .  re-equilibrated  with  10% MeOH f o r 5 min.  before i n j e c t i o n  t i m e p e r s a m p l e was 1 h o u r .  The  o f t h e next sample.  procedure  p e r a t u r e and a t a f l o w r a t e o f 1 m l / m i n . psi  was m a i n t a i n e d d e p e n d i n g  tion  involved  A typical  Total  was r u n a t room tem-  A p r e s s u r e o f 800-1400  upon t h e s o l v e n t .  a 254 nm UV d e t e c t o r c o n n e c t e d  Compound d e t e c -  to a chart recorder.  c h r o m a t o g r a m i s shown i n F i g u r e 7.  B?§ul t s _ a ^ d _ a ^ a j ^ s i s Fifteen  flavonoid  pounds by c o m p a r i s o n known f l a v o n o i d s .  of their  cation each  peak.  extiction  sericeus  p e a k s were n o t e d  o f each  retention  collected  but remain  to specific  previously  from a  f r o m Lumby, BC.  unidentified.  compound was a c h i e v e d by m e a s u r i n g  Quantifi-  since a l l the  c o e f f i c i e n t s , a t 254 nm, o f t h e f l a v o n o i d s o f L^. s e r identical  (Jurd,  Of t h e 17 peaks o b s e r v e d , constituents.  1964).  10 were c o n s i d e r e d m a j o r  f r o m c o - d o m i n a n c e i n many p r o f i l e s 4 populations.  pared  t o a b s e n c e f r o m 63 o t h e r s and  b u t , when p r e s e n t , were u s u a l l y  o n l y (Appendix  IV).  i n trace  The 181 c h r o m a t o g r a m s , when com-  showed no o b v i o u s  groupings  Q u a n t i t a t i v e l y , e v e r y compound v a r i e d it  varied  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  distribution,  visually,  o r minor  N i n e o f t h e s e 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  o v e r 9 0 % o f i n d i v i d u a l s , b u t one compound, i s o o r i e n t i n ,  quantities  Two  the height of  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  iceus are almost  their  com- •  times w i t h those o f  These had been i s o l a t e d  b u l k sample o f Lupinus additional  p e a k s were i d e n t i f i e d  or d i s c o n t i n u i t i e s .  continuously.  However,  was o b v i o u s t h a t some compounds v a r i e d more t h a n 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 ranged  (peak  2 ) , a major  f r o m a s i t u a t i o n where i t  component t h r o u h o u t ,  co-dominated  60  FIGURE 7.  HPLC o f  a typical  Lupi nus s e r i c e u s  individual  l i 2  I  1  i I I i  . 6  1 = orientin 3'-0-glu. 2=orientin 3 = isoorientin 4 = o r i e n t i n acyl I 5 = vitexin 6 = v i t e x i n acyl I 7 = l u t e o l i n 7-0-glu. 8 = apigenin 7-0-glu. 9 = luteolin 10= v i t e x i n a c y l II 11= unknown b i o s i d e 12= o r i e n t i n 4 ' - 0 - g 1 u . 13= o r i e n t i n a c y l II 14= a p i g e n i n 15 = i s o v i t e x i n  the  profile  quantities as  with as  to  mentioned,  by v i s u a l  other  compounds to  where i t  dwarf  the  the  isoorientin  assessment,  fore of  analysis  d e c i d e d to  analysis.  available  analyses  fully  (1975)  data  of  analysis  lines  of  the in  spanning t r e e s ,  etc.  there  ures.  They  is  avoided Since  excercise earlier  generation go on to  the  of  suggest  in  present  workers  that data  it  was  Principal  components a n a l y s i s  available  through  •Model ted  II)  u s i n g MIDAS.  by the  Statistical  Michigan System  (Fox  (MTS) .  & Guire,  more  various  Limnanthaceae. unsatisfactory  a multivariate between  Similarly,  much o f in  three  ordination  the  divergent  Clifford  & Stephen  d e n d r o g r a m s , minimum loss  of  this  information  d e c i d e d to  (PCA)  was  is  take  Research 1976)  of  advice the  chosen s i n c e  Laboratory  and run  one and not  the  analysis  a package  under  loss  or more d i m e n s i o n s .  UBC c o m p u t i n g f a c i l i t y This  fig-  information  a phytochemical  and use a m u l t i v a r i a t e  the  to  compared  considerable  s t u d y was  statistics  there-  such s i m p l i f i e d t w o - d i m e n s i o n a l  by e x a m i n i n g the the  was  t a x o n o m i c method  rather  production of was  It  analysis  from t h e  family.  be made.  programmes were  data  relationships  Also,  from HPLC made an  (1976)  generated  large  Nevertheless,  desirable.  clustering  dendrograms whereas  that  the  data  Parker  flavonoid  such  flavonoids.  could  classificatory  methods.  note  during  of  in  variable.  divisions  highly  from s i m p l e  e x p r e s s e d the  evolutionary son  the  cluster  two-dimensional  extremely  such o b j e c t i v e  Many c h o i c e s  He found t h a t  other  use a c o m p u t e r - a i d e d n u m e r i c a l  complex m u l t i v a r i a t e  more  of  of  and r a n g e d  numerical  was  of  no c l e a r - c u t  The a c c u m u l a t i o n objective  peaks  was  of  (an  it  Amdhal  programmes  of the  the  of  the  data. was 470V/6 distribu  University  Michigan  an  of  Terminal  62 Principal  components a n a l y s i s (PCA)  Principal plant  components a n a l y s i s has an i m p o r t a n t  taxonomy ( C l i f f o r d  to t h e study  1971)  & Stephenson, 1975); recent  of infraspecific  phyl 1 a ( C o l d r a k e ,  place i n applications  v a r i a t i o n include Acacia  1 9 7 1 ) , E u c a l y p t u s carnal d u l ens i s ( B u r l e y  and t h e L i m n a n t h a c e a e  (Parker,  1976).  among t h e e n t i t i e s  being  the  o f a problem  dimensionality  the  similarity  i.e. operational  two  Consider  far apart  that  tioships  summa-  more e f f i c i e n t l y  x plants, taxa, etc.  (called  In o r d e r  Points  but, only f o r  a t t r i b u t e may be s h a r e d  between OTU's f o r t h r e e  of points.  OTU's  Each OTU can be p l o t t e d a c c u r a t e l y i n  A third  are f a r apart.  than  & S n e a t h , 1963)  on t h e o r d i n a t i o n a r e q u i t e d i s s i m i l a r ,  a x i s must be c o n s t r u c t e d cloud  by c a l c u l a t i n g axes t h a t  f o r each p a i r o f a t t r i b u t e s ( F i g u r e 8 ) .  t h o s e two a t t r i b u t e s . taxa  of groupings  t e r m s , PCA r e d u c e s  taxonomic u n i t s , a f t e r Sokal  fory attributes.  dimensions  In g e n e r a l  r e l a t i o n s among e n t i t i e s  raw a t t r i b u t e s .  scored  studied.  e_t a l . ,  I t i s an o r d i n a t i o n  method t h a t makes no a s s u m p t i o n s a b o u t t h e e x i s t e n c e  rize  harpo-  t o see a c c u r a t e l y  attributes a third  by t h e two  the r e l a orthogonal  and so o n , r e s u l t i n g i n a y - d i m e n s i o n a l  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 . a c c o u n t s f o r t h e most v a r i a t i o n ) w o u l d l o g i c a l l y b e the  best  character  is,  of course,  on w h i c h t o base an i n i t i a l  equivalent  to choosing  have seen i n L u p i n u s , i s o l a t e d subdivide  division.  a key c h a r a c t e r .  characters  This As we  do n o t s a t i s f a c t o r i l y  t h e genus.  Principal  components a n a l y s i s p l o t s t h e OTU's i n y dimen-  sions  and t h e n c h o o s e s a new a x i s , i n m u l t i d i m e n s i o n a l  along  which there  i s greatest  variance  Csee  Figure  space,  9 ) . The new  FIGURE 8.  The r e l a t i o n s h i p to  two  attributes  (A  «  data  -t->  o|  between  OTU's compared w i t h  respect  &; B) .  centroid  , •  CU i £ ro S-  range  of  attribute  A  ^ _  A  FIGURE  9.  Ordination of  the  Fig.8  OTU's w i t h  1st  and 2nd  component axes .  Component axes proportion by the  of  are the  original  uncorrelated variance  attributes  and a c c o u n t  for  a  among OTU's than  was  explained  separately.  greater  axis but  (or  component)  has  acters  contributions contribute  others.  Such  related  of  component spread.  is In  orthogonal  the  sphere equal  from a l l  the  small  first  calculated a stepwise  axes  are  of  each the  (1971)  Results  and d i s c u s s i o n o f  for  72% o f  counts  for  12%.  in  Table  this  of  variance  From the  table  coefficient  that  the  centroid  or  negative  reference orientin  to  the  is  data  centroid.)  to  No.3); the  if  be a  for  For more  an  detailed  Blackith  &  individuals Figure  the  II  10.  It  to  ac-  that  the  axis Its  are  I  is  high  left  of  compound.  (The  posi-  for  spatial  is  rich  ac-  characters  No.2).  far  except  Axis  from Lupi nus  second a x i s  be seen  individuals  meaningless  top of  the  flavonoid  can  of  flavonoids  component a x i s  (variable  levels  sign  (variable  been o r d i n a t e d  higher  the  it  negative  tive  would  HPLC d a t a  while  of  orientin  have  occur  account  sericeus  The f i r s t  d o m i n a t e d by one compound,  data  etc.,  progressively  (1964),  PCA on t h e  10.  total  means  the  greatest  fifth,  would  variance. Seal  cor-  a second  shows  ordination  PCA on L u p i n u s  The e i g e n v e c t o r s IX.  situation  PCA see  than  (1967).  Figure  the  for  component would  p r o d u c e d by  shown i n  account  overall  and M o r r i s o n  counts  to  fourth,  Multidimensional  explanations  The o r d i n a t i o n  that  char-  Following  again  which  some  summarizing  orthogonally  extracted  share  as  characters.  . third,  character  components  variance,  Reyment  shown  Usually,  certain  groups o f  An e x c e p t i o n  (100/y%)  is  to  single  component and i t s  and c o n s e q u e n t l y ,  sericeus  any  attributes.  fashion  were random.  mathematical  c o r r e s p o n d to  more s t r o n g l y  of  variance. data  not  components may be i n t e r p r e t e d  patterns  extraction  less  does  giving  dominated  in is  this  by  iso-  compound have  interesting  to  FIGURE  10.  Ordination  of  181 i n d i v i d u a l s  of  Lupinus  sericeus  by PCA on HPLC d a t a .  Component Component  I II  accounts accounts  for for  72% t o t a l 12% t o t a l  1  1  •  variance variance  •  • •• • • • ••• •  • •  ••  • • • • •• * •. S ! . * -4  ON  6 7  TABLE I X .  Table of eigenvectors for  the f i r s t  three  o f t h e 17 f l a v o n o i d  component axes o f PCA.  <PRINC0M VAR=1-17 CASES=1-181 OPTION=UNSCALED MAX=3> P R I N C I P A L COMPONENTS CASES=CASE/f : 1 - 18 1 TEST  STATISTIC  SPHERICITY  7516.G  DF  SIGNIF  N=  181 OUT  152  COMPONENT % VARIANCE  (1) 7877.3 71.79  (2) 132 1.4 83.84  (3) 1124.5 94.09  SPHERICITY DF SIGNIF  4496.5 135 0.  3513.2 119 0.  1559.1 104 0.  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V1 1 V12 V13 V14 V15 V16 V17  .23858 -.99086 .12088 .90960 .54576 - . 44475 -. 12235 -.41529 -.10792 - . 2284 1 .15624 .24344 -.61093 .20666 .33415 -.45330 .13489  -1 -- .25553 .10748 .90839 .18869 -3 .24231 -2 -2 .13746 -1 .60957 -3 .42099 - 1 •- .99948 -1 .4066 1 -1 . 16776 -1 •- .25529 -2 .15100 -2 -.12117 .23834 -1 -3 .14478 -1 .45901  -1 -2 -2 -3 -2 -1 -2 -1 -2 -1 -1 -1  .95647 .54483 .22244 .114 19 .39062 .38776 .21094 . 11980 .21 188 -- . 13342 . 14057 .58985 .38988 .27373 .7087 1 .55544 .37289  -1  - 1 -1 -1 - 1 -1 -1 - 1 -1 -1 -1 -1 -1 -1  OF  181  variables  68  note t h a t the  PCA  data  (as  present point  objectively extracted being  of highest  dominates the  those i n which o r i e n t i n  of the  (top  total  spread of An  variance  obvious  of the  to the  axis  feature  of  Figure with  10  the  i n d i v i d u a l s of j..  pattern  dient, that population  i s , any  (far left) i n more  accounts  initial  the  for  12%  real  situation.  lack of d i s c r e t e  vary  assessment).  continuously  orientin  plants.  Figure  The  name ' c l i n e ' trend  or s e r i e s of p o p u l a t i o n s in j..  10  i t would  multivariate analysis (canonical  To  gradual  change i n latitude.  for a character  of a s p e c i e s .  the  distribution;  i n space a p p e a r i n g  sericeus  in  accumulation.  r e l a t e d t o some a s p e c t o f  variational  apparent v a r i a t i o n trend  similar  vertical  visual  sericeus  to  I , the  of the  i s the  the  content  of v a r i a t i o n i s e v i d e n t .  mostly northern  (1938) coined  from,  shows a  orientin  i n d i v i d u a l s of southern  l e v e l s is apparently  Huxley  occur  numbers were i n s e r t e d o n t o  a definite  are  high  in axis  exaggeration  agreeing  therefore  II only  compared t o 72%  o r d i n a t i o n are  right  orientin  the  Since  f l a v o n o i d , o r more s p e c i f i c a l l y ,  seen t h a t  left  isoorientin  an  However, i f p l a n t be  10  constituents  be  (again  It appears that their  may  other  and  right).  points  groupings  Figure  compounds  o r i m p o r t a n c e ) as  from t h o s e i n d i v i d u a l s h a v i n g  which t o t a l l y  quantities  same two  variability  author did s u b j e c t i v e l y .  spread  the  To  gra-  in a  investigate  populations  v a r i a t e s a n a l y s i s ) was  another under-  taken . Canonical  variates analysis  Unlike it  then  PCA,  CVA  requires  i n v e s t i g a t e s the  (CVA) t h a t the  data  be  i n s e t s or  r e l a t i o n s h i p s between t h e  groups.  groups; (See  69  Seal, gins  1964  for a f u l l  with  already  fication  i n 32  and  Lupinus  11  axis  II accounts  uous i n t h e is  shows t h e  Axis  shown i n t h e  Figure  CVA  deviation  The  ulations  11-32  tight  The  from the  continuous  present  population  total  of  of each  variance  is less contin-  A more u s e f u l  centroids  and  v a r i a t i o n pattern  plants  each a x i s t o g i v e  populations  centroids  of the  individual  The  have +/-1  diagram standard  some i n d i c a t i o n o f  ordination.  have r e l a t i v e l y  g r o u p shows l i t t l e  form t h e  internal  and  d i s c o n t i n u i t i e s shown by v a r i a t i o n we  i n Lupinus s e r i c e u s .  low  saw  CVA  overlap  with  12.  Pop-  g r o u p i s unwar-  appear q u i t e d i f f e r e n t  from t h e  ecotypic  infra-  external v a r i a t i o n  t h a t a s u b d i v i s i o n of the  s u g g e s t s more o f an  Populations  m i d d l e group i n F i g u r e  have much h i g h e r  rented.  CVA  populations  populations  I t i s c l e a r t h a t the  g r o u p i n g and  which  with, so much o v e r l a p  of the  32  data  a p p a r e n t from t h i s  variability. 7-10  f o r 42%  an  variation.  Three groups are  populations  the  tax-  data  w o u l d seem  Lupinus s e r i c e u s  are  classi-  distribution.  o r d i n a t i o n of p o p u l a t i o n s .  p l o t t e d along  populational  on  a  be-  used i n p l a n t  CVA  o r d i n a t i o n of the  I accounts  i n which the  form a v e r y  properly  CVA  However, s i n c e o u r  groupings,  o r d i n a t i o n of  infrapopulational  1-6  CVA  points  f o r 12%.  PCA  12  been so w i d e l y  o r a more d i s c o n t i n u o u s  sericeus.  population.  not  Since  method t o d e t e r m i n e i f t h e  d i s c u s s i o n of  Figure  c l u s t e r s , i t i s not  populational  analytical  in a c l i n a l  Results  has  explanation.)  & Stephenson, 1975).  natural  appropriate are  defined  s t r a t e g y and  onomy ( C l i f f o r d are  mathematical  PCA.  The  result  type of v a r i a t i o n to  Furthermore, the  p o s i t i o n s of  be the  FIGURE  Axis Axis  11.  Ordination  of  32 p o p u l a t i o n s  of  Lupinus  I a c c o u n t s f o r 42% t o t a l variance II a c c o u n t s f o r 12% t o t a l variance  sericeus  by  CVA on HPLC  II 16  27  13.. 14 5  2 1  data.  *  11  10  12  32 29  20 18  30  2 8  3119 23 26  25  21  22  24  o  FIGURE  12.  Ordination centroids t i onal  of are  32 p o p u l a t i o n s drawn w i t h  vari a bi1i ty.  +/-1  of  Lupinus  standard  sericeus deviation  from CVA. to  show  Population  infrapopula-  72  populations  i n t h e o r d i n a t i o n can be p a r t l y  explained  on a geo-  graphicalbasis. Populations north  o f t h e Grand  sericeus genetic the  in this  south  o f Great  Canyon, A r i z o n a .  area  Salt  The d i s t r i b u t i o n  i s almost continuous  exchange i s very  high.  L a k e , Utah and  area  Lupinus  and t h e p o t e n t i a l f o r  Such a s i t u a t i o n  southerly lupines.  Populations  could  result in  and a j o i n i n g v a l l e y s .  7-10 a r e i n t h e Snake  These p o p u l a t i o n s  s o u t h e r l y g r o u p by G r e a t  Salt  which l i t t l e  arated  from many o f t h e n o r t h e r l y p o p u l a t i o n s  areas  Salt  gene f l o w w o u l d be e x p e c t e d .  o f t h e Sawtooth  closely  grouped.  the  almost continuous  These m o s t l y tracts  M o n t a n a , Idaho and A l b e r t a . t h a t were c o l l e c t e d  represent  o f L.  Divide.  Less s i m i l a r  In t h e  Nos. 1 1 - 1 8 , 32  collections  from  a r e p o p u l a t i o n s 19-31  f r o m t h e more i s o l a t e d  of j..  montane  s e r i c e u s i n the^ g r a s s l a n d s o f  C a s c a d e s and Rocky M o u n t a i n s where t h e r e the d i s t r i b u t i o n  Desert  by t h e h i g h  Range and t h e C o n t i n e n t a l  from  They a r e s e p -  n o r 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 are  River  are separated  Lake and G r e a t  across  in  of  lack 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 that i s apparent i n  these  the  1-6 o c c u r  sericeus.  sites  i n the eastern  i s f a r less continuity  T h i s may have r e s u l t e d i n a  l a c k o f gene f l o w and t h e d e v e l o p m e n t o f d i f f e r e n c e s between populations. sericeus  individuals  flavonoids; be  In summary, we see t h a t PCA r e v e a l e d varied continuously  at the populational l e v e l ,  i n more o f a d i s c o n t i n u o u s  ecotypic  in their  that  Lupinus  accumulation  of  CVA shows t h e s p e c i e s t o  p a t t e r n t h a t may be r e g a r d e d  as  i n nature.  In b o t h m u t i v a r i a t e a n a l y s e s chararacters  (PCA and CVA) a l l 17 f l a v o n o i d  were, used. 0 n e compound h o w e v e r , o r i e n t i n ,  dominated  73  the analyses of o r i e n t i n Bearing  so much t h a t t h e f i r s t accumulation  r a t h e r than c l a r i f y  in single characters  measures  populations.  (1944) t h a t  " t o o much  may s e r v e  to confuse  t h e whole p i c t u r e o f v a r i a t i o n " ,  i t was de-  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  lations The  i n t h e i n d i v i d u a l s and  i n mind t h e s t a t e m e n t o f S t e b b i n s  e m p h a s i s on- g r a d i e n t s  cided  axes were b a s i c a l l y  i n terms o f o r i e n t i n  analysis of orientin  accumulation  accumulation  o f j..  s e r i ceus  popu-  only.  i n Lupinus  sericeus  populations One-way a n a l y s i s o f v a r i a n c e Test  ( S t e e l & T o r r i e , 1960) was used t o compare o r i e n t i n  lation the  means f o r each p o p u l a t i o n .  a n a l y s i s of variance.  a significant orientin  significantly icantly  different  (statistically)  orientin  shows  different  from t h e s o u t h e r n  f r o m each o t h e r .  that  f r o m each  other  s e x t e t but not s i g n i f -  Population  between t h e two g r o u p s .  16 forms a b r i d g e  Geographically  this  i s f a r removed from Nos. 1-6; t h e i n t e r m e d i a t e  level  may s i m p l y  be a c h a n c e o c c u r e n c e .  the a n a l y s i s of populational  I t should  flavonoid characters  were  d i f f e r e n c e s , No.16 was  be  involved  ordinated  away f r o m Nos. 1-6. The  is  means f o r  p o p u l a t i o n , e x c e p t f o r No.16 i s  remembered t h a t when t h e o t h e r  wel1  i n d i c a t e s that there i s  As i n t h e CVA, T a b l e XI  Every other  different  population  The F - v a l u e  1-6 a r e n o t s t a t i s t i c a l l y  a t t h e 1% l e v e l .  accumu-  T a b l e X shows t h e r e s u l t s o f  d i f f e r e n c e between t h e p o p u l a t i o n  accumulation.  populations  in  and Duncan's M u l t i p l e Range  overall  p i c t u r e of o r i e n t i n  accumulation  i n J_. s e r i c e u s  one o f a s h a r p d i s c o n t i n u i t y between t h e p o p u l a t i o n s  south  of  74  TABLE X.  Analysis of variance Lupinus  Source o f variation  among populations  within populations  sericeus  for orientin  accumulation  populations  df  Sum o f s q u a r e s  ~,  980062.08  31614.96  149  415493 .07  2788. 54  Total  ** d e n o t e s s i g n i f i c a n c e  in  1395555 .15  a t t h e 1% l e v e l  Mean s q u a r e  F  11.34**  75  TABLE X I .  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 Multiple (Shown  Range  test  f o r 32 p o p u l a t i o n s  a l s o are the i s o o r i e n t i n  Population number 4 5 2 1 3 6 16 17 14 11 31 15 30 29 19 10 13 8 32 28 24 18 9 7 27 20 21 12 23 22 26 25  D i f f e r e n c e s between populations joined by t h e same l i n e are not s i g n i f i c a n t a t t h e 1% 1 e v e l .  by  mean peak heightof orientin 266 259 231 209 208 201 148 109 106 104 96 95 91 83 83 81 79 76 76 72 69 57 55 43 43 41 38 35 31 29 26 19  Duncan's o f L_. s e r i c e u s .  accumulation  means.)  mean peak heightof isoorientin 0 0 0 10 0 2 88 52 45 32 74 55 77 72 57 48 55 19 54 36 53 21 19 22 52 63 43 43 59 25 21 24  76  Great  Salt  Lake and  suggestion  I.  i n the  far less  lower  same a r e a  apparent  ssp. huffman1i  has  part of the  At t h e  very  l e a s t , there i s a  Fleak  (1971) notes  heavy a n t h o c y a n i n  stems.  ranging  these  pigment-  has  s o u t h e r l y l u p i n e s as  Stebbins  (1944)  led  L_  f r o m n o r t h - c e n t r a l Utah s o u t h  Plateau of A r i z o n a .  that  It is a character that  i n n o r t h e r l y p o p u l a t i o n s .and  & Dunn (.1971) t o d e l i m i t  Kaibab  north.  of e c o t y p i c d i f f e r e n t i a t i o n .  sericeus in this  ation is  those  Fleak  sericeus  to  the  states:  " 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 m o d e r a t e one o r a c o n s t a n t l e v e l i n o t h e r s , t h e n the d i f f e r e n t l e v e l s of 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 t h e c l i n e s may cons 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 . " It  is clear  uous c l i n e  that orientin  accumulation  parallels  of anthocyanin  pigmentation  o f I.  s u b s p e c i f i c r a n k i n g o f I.  supports  the  in this  area.  isoorientin. formed  Although  to the  n o r t h , where F l e a k  between t h e evident.  no  support  (1971) r e c o g n i z e s  from t h e i r  n a t i v e a r e a s , we  much o f t h i s be t h a t t h e plasticity  individuals  variation  in this  have no  that Hitchcock  of  southerly populations  discontinuity  the and  and  of can  here. study  were  evidence  i n f l a v o n o i d content  d i f f e r e n c e s are  devoid  per-  t h r e e more s u b s p e c i e s  f o r taxa d e l i m i t a t i o n  Since a l l the  and  be  U n f o r t u n a t e l y , i n the  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 lend  p o p u l a t i o n means o f  a n a l y s i s o f v a r i a n c e c o u l d not  i s again  thus i t  s e r i ceus s s p . h u f f m a n i i  XI a l s o shows t h e  discontinuity north  discontin-  s e r i ceus;  ( 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  compound) t h e those  Table  the  collected  to determine  how  is genotypic.  It  s i m p l y examples of the  e_t a l _ . ( 1 9 6 1 ) n o t e d  phenotypic  t o be so common  may  i n J_. tion  sericeus . in  this  c a r r i ed  out.  In  order  species,  to  another  establish analysis  the  basis  for  (Experiment  the  III.)  van was  78 Experiment  I I I : The  determination  nature of o r i e n t i n The cations  question  plants  the  of genetic  (1922).  species He  was  environmental and  a uniform analysed healthy  followed  by  environment. by  HPLC.  The  mature p l a n t s  stage.  The  seedlings  difficulty necessitated  earlier i n the  s t u d y i t . was  decided  seedlings  for this  formed on  some s e e d l i n g s  identical  to t h o s e a t t a i n e d  A total  by  i n t o the o f 0.05 was  study.  collec-  range  growth o f t h e  of  plants  were t h e n e x t r a c t e d  their  growing  differ-  the  natural  s e r i ceus  a n a l y s i s at the  by  The  this  and  fresh  the with  injected.  The  material  In t h e  p r o c e e d and  mature  in  and to seed-  The  was and  little mature  I.  per-  qualitatively  plants.)  then  from extracted  young t i s s u e s were r e a d i l y and  way.  butanol  extractions  were  e x t r a c t s were i n j e c t e d  For  dissolved  c a s e o f 0.25  plants  sericeus  a n a l y s i s was  were w e i g h e d and  methanolic  was  use  However,  were s u c c e s s f u l l y r a i s e d  leaves  HPLC i n a s t a n d a r d i z e d plant  there  r e s u l t s were  method; h e a t i n g  unnecessary.  g of  were a t t e m p t e d .  (Two-dimensional  p l a c i n g them i n m e t h a n o l .  considered  their  III involved  found t h a t  to  of twenty s e e d l i n g  populations.  extracted  by  f l a v o n o i d content of seedlings  o f J_. a r b o r e u s , i t was  six  differences  Many i n d i v i d u a l s were n o t e d t o d a m p - o f f even  i n an  difference  modifiby  i n growing j..  t h o u g h numerous g r o w i n g c o n d i t i o n s since  induced  re-assessing  and  phenotypic  answered  from t h r o u g h o u t t h e germination  or  sericeus  posed and  In a s i m i l a r manner, E x p e r i m e n t  species  genotypic  environmentally first  excluded  o f J.. s e r i ceus seed  ling  or  under u n i f o r m c o n d i t i o n s  ences. tion  v a r i a t i o n i n Lupinus  of a p l a n t  Turesson  of the  example, i f the i n 1 ml  extract  m e t h a n o l , 10  g of p l a n t m a t e r i a l  i n 1 ml  ul  79  m e t h a n o l , 2 u l o f e x t r a c t was i n j e c t e d . of  plant material  used made t h e d e t e c t i o n  The  u s u a l l y d o m i n a n t peaks o f o r i e n t i n ,  and  isoorientin  small  poorly  The v e r y  were p r e s e n t  resolved  peaks.  small  of flavonoids  orientin 3  1  amount difficult.  -0-glucoside  b u t l e s s e r compounds a p p e a r e d as Consequently, only  the o r i e n t i n  peak was m e a s u r e d and t h e r e s u l t s a r e shown i n T a b l e X I I . From t h e t a b l e for  i t can be seen t h a t  a l l the seedlings  insignificant  t h e o r i e n t i n peak  r a n g e between 42-66.  F-value, there  As shown by t h e  i s no e v i d e n c e o f p o p u l a t i o n a l  ferences,  clinal  variation  i n the accumulation o f o r i e n t i n i n Lupinus  is  a reflection  or e c o t y p i c .  a p p e a r t o have s i m i l a r 1ation  This  of f1avonoi ds.  dif-  r e s u l t suggests that the  o f some e n v i r o n m e n t a l  some a s p e c t o f l a t i t u d e .  heights  response that  sericeus i s r e l a t e d to  G e n e t i c a l l y , a l l t h e J_. s e r i c e u s  c a p a c i t i e s f o r the production  plants  and accumu-  TABLE  HPLC peak  XII .  sericeus  heights  of o r i e n t i n  i n d i v i d u a l s grown  from 20  from seed  Lupinus in  uniform  conditions.  Population p l a n t No.  Orientin peak h e i g h t  Seed s o u r c e location  and  1.1 1.2  Marysvilie, Utah  50 46  2.1 2.2 2.3 2.4  Joe's Valley Utah  47 66 46 53  3.1 3.2 3.3  Tony G r o v e , L o g a n , Utah  57 42 53  4.1 4.2  Shoshone, Idaho  45 48  5.1 5.2 5.3 5.4  Livingstone, Montana  58 42 49 48  6.1 6.2 6. 3 6.4 6.5  Spokane, Washington  47 66 56 58 42  One-way  d.f  Source of variation Between . within  populations  total  squares  Mean  squares  126.23  25.25  14  874.72  62 .48  19  1000 .95  multiple Population  No s i g n i f i c a n t  Sums o f  5  populat ions  Duncan's  variance  a n a l y s i s of  range  test.  means  difference  between  4 46  1 48  5 49  population  3 51  2 53  means.  F 0.40  6 54  81  IV. I N T E R S P E C I F I C FLAVONOID VARIATION  Materials  IN  LUPINUS  and methods  Small  amounts o f d r i e d  leaflet  material  (0.05-0.1  g) were  removed f r o m h e r b a r i u m s h e e t s 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 of Lupinus.  A p p r o x i m a t e l y 60 t a x a  ( d e p e n d i n g upon t h e t a x o n o m i c s p e c i e s and v a r i e t i e s . s p e c i m e n was unit"  referred  (OTU) a f t e r  and  s y s t e m ) and 13 a r e c o n s i d e r e d  t o as a numbered  Sokal & Sneath  a t 40°C.  separate species  F o r c o n v e n i e n c e , and t o a v o i d  i n 80% m e t h a n o l , f i l t e r e d i n vacuo  are considered  "operational  (1963).  and t h e e x t r a c t  The r e s i d u e was  Each  bias,  sample was  solvent paring  system. their  ammonia  redissolved  Individual  i n 0.5 ml  f l a v o n o i d s were i d e n t i f i e d  R f ' s and c o l o u r r e a c t i o n s  s p o t s were n o t i c e d  cient  manner  amounts o f p l a n t m a t e r i a l and  Two-dimensional V) from w h i c h a t o t a l Several  soon  methanol TLC  i n the  by com-  b o r o n a t e s p r a y and  flavonoids.  Whenever  new  were i s o l a t e d  and  (see Chapter I I ) i f s u f f i -  were  available.  analysis TLC o f each OTU o f 56 d i f f e r e n t  produced  t h e maps i n t o  73 maps ( A p p e n d i x  flavonoids  d a t a t r e a t m e n t s were a t t e m p t e d .  was made t o s o r t problems  (with  o r s u s p e c t e d t h e compounds  i n t h e normal  Data g e n e r a t i o n  ground  system, and, a f t e r thorough d r y i n g , i n the o r g a n i c  v a p o u r s ) w i t h t h o s e o f known  characterized  each  evaporated to dryness  These were d e v e l o p e d i n two d i m e n s i o n s : f i r s t  aqueous s o l v e n t  sub-  taxonomic  t h e n s p o t t e d a t t h e c o r n e r s o f 15 X 15 cm p o l y a m i d e  plates.  taxa  were  Initially,  groups o f s i m i l a r  recognized.  an a t t e m p t  profiles  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  but  Cflavonoids)  82  were s u f f i c i e n t l y  "important"  obvious groupings  d i d appear s i n c e  pounds; c y t i s o s i d e o c c u r r e d glucosides quently  glucoside  that  as  many as 2 8 g r o u p s  amount o f i n f r a g r o u p i t was d e c i d e d  be s p l i t  which  ranking  a c a c e t i n 7-0t h e g r o u p be  of acacetin  7-0-gluco-  s o r t i n g o f maps i n t o obtained  low r e p r o d u c i b i l i t y .  Thus,  g r o u p s and  t h a t was t o l e r a t e d .  Therefore,  t o a n a l y s e t h e d a t a as o b j e c t i v e l y as p o s s i b l e  again  principal  numerical  classification  components a n a l y s i s was c h o s e n .  t h e a n a l y s i s would d e l i m i t t h e groups t h a t  method; once I t was hoped I had p r e v i o u s l y  recognized  by m a p - s o r t i n g , and p o s s i b l y , e x p o s e o t h e r s  had  to see.  that I  Set_I A data m a t r i x  5 s,  5-0-  different profiles,  i n t o as few as e i g h t  variability  a computer-aided  Data  com-  (many o f them s i n g l e t a x a ) d e p e n d i n g on t h e  using  fa i l e d  Some  infre-  Moreover, the grouping  s o r t i n g t h e maps had v e r y could  7-0-glucoside  Or s h o u l d  ignoring the d i s t r i b u t i o n  73 p r o f i l e s  that  thus  character?  g r o u p s an u n s a t i s f a c t o r y method.  the  and f l a v o n e  o t h e r w i s e had v e r y  Such p r o b l e m s made t h e s u b j e c t i v e  by v i s u a l l y  taxa  unusual  T h e r e w e r e , h o w e v e r , many  be lumped t o g e t h e r  as an i m p o r t a n t  upthus  side?  i n just eight  species.  i n a few t a x a such t a x a  split  they accumulated  s e e n compounds s u c h as a c a c e t i n  occurred Should  in five  upon w h i c h t o base d i v i s i o n s .  (73 OTUs X 56 f l a v o n o i d s )  r  l ' s and O's was c o n s t r u c t e d  was  an a s s e s s m e n t o f t h e r e l a t i v e  within  any one OTU.  c o n s i s t i n g o f 9.'s,  (see Table X I I I ) . concentration  o f each  T h u s , a 9: d e n o t e d a f l a v o n o i d  m a j o r q u a n t i t i e s ; 5, a m i n o r c o n s t i t u e n t ; 1 d e n o t e d present i n trace  q u a n t i t i e s and 0 meant t h a t  Each  value compound  present i n a flavonoid  t h e compound was  TABLE X I I I .  Data S e t I .  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  Fl a v o n o l d s Nos. compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56  orientin vitexin o r i e n t i n acyl I v i t e x i n acyl I o r i e n t i n acyl I I v i t e x i n acyl I I l u t e o l i n 7-0-glucoslde apigenin 7-0-glucoslde l u . 7-0-glu. acyl I unknown ' g r e e n ' l u . 7-0-glu. acyl I I ap. 7-0-glu. a c y l I I luteolin apigenin tsoorlentln tsovltexin pink flavanone (high Rf) pink flavanone (mid Rf) pink flavanone (low Rf) cytisoside c y t i s o s i d e acyl I cytisoside acyl I I 4'-0-methylortent1n chrysoertol acacetin chry. 7-0-glu. chry. 7-0-glu. acyl I chry. 7-0-glu. acyl I I ac. 7-0-glu. ac. 7-0-glu. acyl I l u . 7-0-dlglu. ap. 7 - 0 - d i g l u . chry. 7-0-dlglu. ac. 7-0-dlglu. kaempferol kaem. 3 - 0 - g l u . kaem. 7 - 0 - g l u . quercetln quer. 3-0-glu. quer. 7-0-glu. Isoorlentfn acyl I genlstein genlstln gen. 7-0-glu. a c y l I gen. 7-0-glu. a c y l II o r i e n t i n X'-O-glu. v i t e x i n X'-O-glu. o r i e n t i n 3'-0-glu. l u . 4'-0-glu. l u . 4'-0-glu. a c y l II ap. 4 ' - 0 - g l u . ap. 4 ' - 0 - g l u . a c y l I ap. 4 ' - 0 - g l u . a c y l II Isovltextn acyl I l u . 5-0-glu. ap. 5 - 0 - g l u .  t n 73 L u p i n u s  taxa,  (9=maJor c o n s t i t u e n t ,  5=minor,  l'trace.)  Taxa (OTU's) 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 77 Nos. 12345678901234 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3456 789012 3456 789012 3 999999999999999959999999959199999999999 999995 1 1 1 5 9 9 9 9 9 9 5 9 9 5 9 9 911 9 1 5 5 9 5 9 5 9 5 9 5 5 5 9 5 5 5 9 9 5 5 9 5 1 5 5 1 5 5 555 595955 15555 111555555555555 555 55555555555 5 5555555515 5 5 9 9 5 9 9 5 5 5 5 15555 111 55555 5 5 515551515 5 5 5555555 15 555 5511 1 15555 111 55555 5 5 1 9 5 1 9 5 5 5 5 5 5 1 5 5 5 5 5 1 5 9 5 5 5 5 5 55 5555551555 55155 15 59 5 55 5 5 11519555555 515551595555 51 51 5555551551 5555551551 1 55 15 59 1 5 5 599 55151151 15 59599995 55559 5955555 555 5511115 55 555555 155555555511 559 111 1 1559 55115 1 55 1555 5 5 55 55 5 1 555 15 5 955 55 5 9 9 5 5 5 5 5 5 5 5 55 5555555 55 1 5 5 11 555 5155 5555 15 555 55 555 55 1 191991 11195 5 111 191951 1 15 111 1 1 151 1 999959 5 55 5 995555 15111 5 5  5  1  5 5 5 999 555 555 555 5 1 1 1  1 1  1  1  1 1  1 5 55 1  1111  1  11 1115 1 11 1 15111  11  11  1  1  115 1  11 111  5 1  555  5 1  55 5 11  5 5 5 555 55 55 55  115 111  55 95  1  1 55 55 5 55 5 5  11 1 1 11 1 1  55555 55555  5 5  5 51 1  1 1 11 11  11151 11151  5 55 55 1  55 155 5 5  111 11 55 5 55 5 5 55 55 55 5 9999 5 555  5 55 55 5 5 555  111111 1 1 515 5 5 555  absent. major,  Although s u b j e c t i v e , minor,  identical example,  the  L.  sellulus  dominated  they  hand,  profile  of L^.  in  widely  of  var.  quantities (No.  minor or  of  63)  had  concentrations.  7-0-glucoside as  compound as  some t a x a  1o b b i i  such c o n c e n t r a t i o n s  perennis  each  since  different  sellulus  by l u t e o l i n  small  with only  rating  deemed n e c e s s a r y  ssp.  were i n  presence  other  was  compounds but  a profile atives;  etc.,  the  (OTU No.  53)  and a c y l a t e d  to  almost  For had deriv-  obscure  C-glycoflavones.  On the  had a C-gl y c o f l avone-domi nated  trace  amounts  of  flavone  7-0-glu-  co s i d es . The c r i t e r i a  for  delimiting  the  each compound s h o u l d be e x p l a i n e d . seen  that  tuents. before large in  the  majority  This  means  spraying.  until  amounts  after  that  Major  and i n t e n s e l y  trace  of  the  From the  are  plainly  constituents  were t h o s e they  that then  as  it  be  consti-  under  that  for  can  minor  visible  were t h o s e  spots.  score  matrix  compounds o c c u r  they  coloured  spraying;  concentration  UV  light  appeared  Compounds c o n s i d e r e d to  were not appeared  visible as  under  rather  UV  dull,  as be  light  diffuse  spots. Results  of_PCA_on_Data_Set_ I  The o r d i n a t i o n s Figures ations  13 and 14. of  account  The e i g e n v e c t o r s  seen  axes that  variables  are axis (1  I  of  for  the  in  & 7).  the  the  Table  ordinates  Set  are  shown  component axes  total  variance for  From t h i s  1 is  the  table  on t h e  orientin;  in  in  represent-  three  OTU's m a i n l y  No.  I  two-dimensional  coefficients  XIV.  the  Variable  are  first  54% o f  containing  shown  PCA on Data  These f i g u r e s  combinations  cumulatively  three  p r o d u c e d by  which  the  data.  first it  can  basis  variable  of  be two  No. 7  FIGURE 13.  Ordination  o f 73 l u p i n e t a x a  from  PCA on Data  Set I .  66 35  61  58  34  63  57  31  Components I/11  60  55  32 20 23  19 11 27  24  1  3 0 2  2  18 45  3 9  10 37  47  9 21 46  33  44 8  53  4  65  59  2  41  51  29 36  42  54 62  38  64  25  68  26 48  67 69  71  43 28 /15 . s  70 16  axis A  x  y  56, /  14 17  4  3  axis II 10%  I 36%  86  FIGURE  14.  Ordination  of  73 l u p i n e  Components  taxa  from PCA on Data  11/ 111  ,4fr i  J3  /« L 4 2 _ 41  73 18  ^  13 14  A  16. 70.  ~" ~ - - - 56.  axis  II-  28  43  38 „ , 51 26 29 25  72  57  837  a ^ ° 3 2 7 .35 5 ^ 6 60 5 8  3  4 6  44., * 59q 24  K  36  O  4 5  J  '54~ • '52  10%  47 ^8  55  64  \  50  \  \ i  67 6  9  49  53  63 \  V_62  axis  7%  III  \ 61\  — —J  Set  I  87 TABLE  XIV.  T a b l e o f e i g e n v e c t o r s o f t h e 56 f l a v o n o i d v a r i a b l e s f o r t h e f i r s t t h r e e component axes o f PCA on Data Set I. <PRINCOM VAR=1-56 CASES=1-73 OPTION=UNSCALED MAX=3> P R I N C I P A L COMPONENTS CASES=CASE#:1-73 TEST  STATISTIC  SPHERICITY  10808.  DF 1595  SIGNIF O.  COMPONENT % VARIANCE  (1) 54.824 36.20  (2) 15.34 1 46.32  SPHERICITY DF SIGNIF  9317.0 1539 0.  8872.3 1484 0.  - .41739 - .2801 1 -.26804 -.18390 -.28064 -.26440 .33874 .22168 .75379 -.15355 .69381 .31920 .28418 .22414 -.10373 -.94921 .13563 -.92028 -.77377 -.50930 -.15673 - . 4 0 2 21 -.69334 .14088 .66533 .16232 .70220 .89427 .94983 .27660 .22606 -.93599 .20373 .39786 .86177 .13836 .10360 .17997 .50844 .11463 -.13497 .11575 . 13261 -.31056 .11091 -.33244 -.26615 - . 144 10 .11722 .14910 .98737 . 1837 1 .13054 -.10702 -.73820 - .73820  .37867 .84 120 .38193 .33931 .88388 .13198 .2304 7 . 134 18 .27102 .54786 .97713 .12873 . 13676 .13254 .18508 . 1337 1 . 144 15 .27190 -.34469 -.55009 - . 177 15 -.38928 -.71074 .53087 .44571 .10545 .54546 .52130 .42876 -.12548 .38738 .14664 -.16787 .66016 .28485 .30423 .61552 .13896 .89844 .28452 .24405 .64764 .11641 . 19675 .38121 .37991 .21068 .28678 .31882 -.30043 -.88075 .31857 -.2751 1 . 15740 -.20254 -.20254  1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56  V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 1V1 1 V12 V13 V14 V15 V16 V17 V18 V19 V20 V2 1 V22 V23 V24 V25 V26 V27 V28 V29 V30 V31 V32 V33 V34 V35 V36 V37 V38 V39 V40 V4 1 V42 V43 V44 V45 V46 V4 7 V48 V49 V50 V51 V52 V53 V54 V55 V56  -1 -1 -1 -1  -1 -1 -2 -2 -1 -1 -1 -2 -1 -1 -1 -1 -1 -1 -3 -1 -2 -1 -1 - 1 -1 -1 -1 -3 -1 -1 - 1 -1 -1 -1 -1 -1 -1 -2 -2  N= 73 OUT OF 73  (3) 11.249 53.75 8520.1 1430 0. -1 - 1  .44374 .42551 .23421 . 1221 1 - 1 .18307 .10830 - .48522 .87906 - 1 .66963 - 1 .37763 -2 .10132 -1 .11502 -.22019 -.10653 -.78851 .15205 - 1•.27428 -2 .25431 -3 .20531 .47818 .18798 .36289 - 1 -.17457 - 1 .10742 -1 .36931 .46185 -1 .29599 -1 .30274 -1 .30664 -2 .56225 - 1 -.37890 -1 . 12148 -1 .20446 -3 .13880 - 1 -.15848 -1 .66157 -2 .53200 -1 --.14064 -2 .55538 -1 .10442 - 1- .24 144 -1 --.32429 -.51324 -1 --.37922 -1 •-.12968 -2 - . 2 6 0 4 0 - 1 .51248 -1 - .26443 -2 .66923 -2 -.33308 -2 .75608 -2 -.28801 -3 -.32492 -1 .36619 -1 - . 1 0 9 7 5 - 1 -.10975  - 1 -1 -1 -1 -1 -1 - 1 -1 -1 -1 -2 - 1 - 1 -2 -2 -1 - 1 -1 -1 -1  -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -3 -1 -2 -2 -2 - 1 -1 -1 -1 -1 -2 -1 -1  88  is  luteolin  butes  OTUs  taining  noted  from  taxa  glucoside  it  7-0-glucoside.  to  that  component  of  positive  of  the  axis  ative, and  eriol  flavones rection  67-69  and  axes  the  I  but  14  alone  luteolin  by  & 22,  they  d i s t r i -  luteolin  It  should  common  in  7-0-  be  Lupinus  dominate  to  as by  of  the  so  first  and  appears Nos.  to 47,  in  for  a  accounts  for  36%  abundant  the  negative  a  an  acylated  OTU N o s .  26-29  groups  and most  combined but  axes  43  and  and  compounds,  but  12-17,  56  II  and 65  44  of  Nos.  (the  isod i -  groupings III  (Fig.  & 66)  49,  are 14).  that  Group  C  is  5 0 , 5 3 ,  61-64,  55. clearly  total  of  weighting effect  even  though  18% o f  groupings  compounds,  the  chryso-  negative  More  40-42,  obscures  dominating  deriv-  (acacetin ,  positive  composed 54  end  C-glycof1 avones  respectively.  Character  offset  the  1  52,  account  and  C-glycoflavones  4 -0-methylflavones .  be  three  but  variables  (OTU N o s .  and  by  A.  involving  taxa  3'-  & 4)  variables  centroid,  five  3  Group  genistin)  ordination of  dominated  (containing  7-0-glucoside.  universally  that  cytisoside  ubiquitous  to  high  1  almost  help  extremely  I  7-0-glucoside-con-  4 -methylated  group  shows  the  and  axis  direction.  find  which  This  and  levels  only  dominated  could  20  data  possibly  Figure  are  7-0-glucosides)  B consists high  right  to  luteolin  orientin  (variables  dominated  the  diffuse  Axis  is  from  low  similarly  in  referred  their  from  produce  two  is  OTUs  genistein  apparent Group  be  III  and  more  II  (variables  Axis  to  direction  groups  will  low  compounds  respectively).  70)  and  component  analysis.  axis  the  left  surprising  the  in  having  a  two  be  Component  found  in  these not  orientin  those  levels  should  are  high  Therefore,  on  ease  the  data  certain with  variance.  since  orientin and  the  it  is  and scaling axes  which  the  by ordin-  89  at i o n s  could  be r e l a t e d  back  to  the  original  data  might  well  decrease. Data_Set_II Data set  I  to  Set  same t i m e ,  data  avoids  obviously  a transformation  scores  the  (0/1's).  subjectivity  total  three  variance.  Data  Set data  causing first  I,  set.  data  The change of  Data  Set  this  be a t t r i b u t e d  to  As e x p e c t e d ,  component of  of  the  accounted  figure, the  loss  overall  OTUs i n  axes. the  from  to I  purely  but  at  taxa  the  such  identical.  axes  The drop i n  separation  the  II  component  much c r o w d i n g  three  slight  can  of  1 oses: consi derabl e i n f o r m a t i o n ;  PCA_on_Data_Set  The f i r s t  No.  simply  OTU 53 and 63 now become a l m o s t  Results_of  raw  was  presence/absence  qualitative  as  II  all  The o n l y  Group A t a x a  for  4 4 % . o f the  compared to of  that  variability  point  ordinations noteworthy and the  from  in  s p r e a d was  isolation  the  small  involving points  . . t  the  were a of  taxon  55.  Data_Set_111 Each OTU was  scored  for  its  o n o i d : p r o d u c t i on and m o d i f i c a t i o n a simplified chemical shared For  affinities  by two  example,  onoids :  version  taxa  of  that  biosynthetic  capabilities.  prepared  were based on the instead  consider  three  of  the  repertoire  by  Levy  hypothetical  is  which  biosynthetic  compounds i n  plants  flav-  method  ( 1977 ) . i n  number o f  number o f  This  of  and t h e i r  steps  common. flav-  90  luteolin  A  B  +  + +  luteolin  7-0-glucoside  +  luteolin  4 -0-glucoside  +  1  from A by the  a basic,  statistical  dissimilar  from A.  mediate  the  sized  in  the  of  4 -0-glucoside, 1  quently,  B can  A.  argument  flavonoids bilities  does  surety  to  share  example, cepted found  types  is  hiosynthetic i n Lupinus. as  etc.  for  (Sutter  sylation fore, Oata  and t h a t  at  III  C  terminal  steps  in  relationships As an e x a m p l e ,  7-0-glucosylation  the  intersynthe-  from A i s  makes  no  pathway.  C's  luteolin ConseC is  biosynthesis  biosynthetic  the  from  of-  capa-  was  biosynthetically compounds known  biosynthesis,  15 shows  the  for  currently  ac-  between the  types  all  7-0-glucosides  since is, of  flavone the  the  as  o f compounds  7-0-glucosylation  same enzyme i s  apigenin,  requires  treated  11  groups o f  1 973 ; S u t t e r  position  consequently  is,  Figure  one compound t y p e  another  B lacks  have  from A than  we know the  compounds, t h a t  & Grisebach,  must  however,  a biosynthetic  equally  a necessary  difference  were condensed i n t o  s u c h compounds a r e Set  B,  be  Using  4'-0-glucoside.  n o n - s p e c i f i c , that  the  only  Species  5-0-glucosylation.  substrate  sible  of  identical  considered  its  in  is  glucosides,  assume t h a t  make l u t e o l i n  The 56 f l a v o n o i d s different  luteolin  would  be c o n s i d e r e d more d i s s i m i l a r  with  to  its  an end p o i n t  o f one compound.  method, they  therefore,  luteolin.  absence  since  production of  aglycone;  accumulation  This  However,  +  +  Both B and C d i f f e r unweighted  C  luteolin,  another  acacetin,  enzyme;  flavonoid  a 73 X 11 m a t r i x  of  step  respon-  e_t al_. , 1 9 7 2 ) .  a different  were  0-Glucothere-  type.  presence/  FIGURE 1 5 .  Biosynthetic  interrelationships  ( U n d e r l i n e d compound-types are t h o s e v a r i a b l e numbers are a l s o shown.)  of  used i n  3.  isoflavones  io  '  8 4'-0-glucosides  4'-0-methyl flavones  3'-0-methyl flavones  n  7-0-glucosides -irr a  (Diagram  adapted  from H a h l b r o c k  III,  6-C-glycoflavones  flavones  5-0-glucosides  Set  8-C-glycoflavones  ^f1 avanones  f1 a v o n o l s  Data  flavonoids.  4'-0-methyl C-glycoflavones  3' - 0 - g l u c o s i d e s  1.  lupine  & Grisebach,  1975)  4  92 TABLE  XV.  Data  Set  III:  the  distribution  1  2  of  3  4  CO  «/)  C o  c o  4-  <*-  o u  o u  o>  en  Qi  > ta  </>  <u c o  OTU No.  Lupinus  taxon  1 elatus 2 barbiger 3 sericeus ser. 4 ser. huffmanii 5 ser. egglestonianus 6 ser. flexuosus 7 bi c o l o r 8 polycarpus 9 neomexicanus 10 lemmonii 11 texensis 12 d e n s i f l o r u s scopulorum 13 dens . a u r e u s 14 ruber 15 arboreus 16 hori z o n t a l i s 17 flavoculatus 18 benthami i 19 c o n c i nnus 20 sparsiflorus 21 arizonicus 22 stoloniferus 23 wyethii 24 hillii 25 holosericeus 26 parv i f l o r u s 27 caudatus X h i l l i i 28 nevadensis 29 1 e u c o p h y l 1 us 30 alpestri s 31 greenei 32 psuedoparvif1orus 33 1utescens 34 sulphureus 35 palmeri 36 argenteus a r g . 37 arg. rubricaulis 38 caudatus 39 arbustus 40 grayii 41 albifrons 42 chamissonis 43 humi c o l a 44 arcticus arct. 45 arct. canadensis 46 arct. subalpinus 47 latifolius 48 . 1i t t o r a l i s 49. a.ridus 50 confertus 51 mi niraus 52 lyallii 53 sel1ulus 54 kuschel 55 diffusus 56 u n c i a 1 is 57 cusi cki i 58 caespitosus 59 lepidus 60 cul bersoni i 61 nootkatensis 62 burkei 63 perennis 64 polyphyllus 65 excubitus 66 longifolius 67 a l b i ca u l i s 68 andersonii 69 formosus 70 ki ngi i 71 p u s i l l u s rubens 72 pusi11 us p u s i l 1 us 73 hirsuttssimus  CO I—  > to  O c o  O  re u «*-  r— 4ts>  >  11 f 1 a v o n o i d - t y p e s  >> 1  oo  OI  > to >. • i  ca  1 0  5  > ra c — Mo u  >>  r— cn t >>  •*-> O)  E  =3  i —  +J  in 1 1  73 L u p i n u s  taxa  93  absence  scores  for  flavonoid  The o r d i n a t i o n s Figures 59% o f  16 and 1 7 . the  From the effects in  total  table of  five  such a way  flavonols, at  the  The f i r s t  can  to  flavone  positive  of  X,V)..'  the  eigenvectors that  axis  flavonoids.  4 -0-glucosides 1  at  the  the  axis  to  negative  Set  III  component axes  show a g r a d a t i o n  end o f  8-C-glycoflavones  three  be seen  types  as  (Table  p r o d u c e d by PCA on Data  variance;  it  types  I  shown  accounted  appear  mainly  are  in  Table  separates  The OTUs are from t h o s e  in  for XV;I .  the  distributed  that  synthesize  and B - r i n g m e t h y l a t e d  flavones  those  mostly  end.  that Axis  synthesize II  spreads  taxa  that  0  contain  3 - 0-gl ucos i des  extent,  isoflavones,  toward  1  the  negative  glucosides of are  axis  while  found at  containing  up o f  the  end o f  the  those  that  are  axis. are  contain  apparent  Taxa r i c h  24-28 which  from F i g s .  are  In  peculiar  addition,  by 8 - C - g l y c o f l a v o n e s  A. is  in  Fig.  17, taxa  Group B i s  also  readily  less  clearcut  than  in  Nos.  to  5-0-  regions  ordinations  Taxa  profiles  1  their  Group E i s  be a d i s c r e t e  arising  of  contain chemical  and m e t h y l a t e d  Fig.  made  production  group E t a x a  in  Nos.  3 -0-glucoside,  71 and 72 a p p e a r  recognizable  17.  by h a v i n g  in  all  octet  but,  .  C-glycoflavones  and i s o f l a v o n e s .  Group A c o n t i n u e s  atives  flavone  positive  16 and  orientin  8-C-glycoflavones. characterized  the  ...  7-0-glucosides,  in  methylated  characterized  7-0-glucosides  5-0-glucosides .  a lesser  and f l a v o n e  found i n  8- and 6 - C - g l y c o f l a v o n e s ,  OTU N o s .  a n d , to  end.  Group D and a r e  flavone  flavone  6-C-glycoflavones  negative  groupings  2-6 c o m p r i s e  simple  C - g l y c o f l avones  and 4 ' - 0 - g l u c o s i d e s  III,  More  of  deriv-  close  to  Group  16 a l t h o u g h  from Data  Set  .  it I.  FIGURE  16.  Ordination  of  73 l u p i n e  Components  axis  II  taxa  from PCA on Data  Set  II  I/11  17%  28 26 25  ,15  27  46$%  65 42 - - '  C66 71 72 73  59V  33^20  j27°31  \2\ \ \ \ \  \  63 6247 61  19 52 53  axis 67 6968 64  49  I 30%  50  FIGURE  17.  Ordination  of  73 l u p i n e  Components  taxa  from  PCA on Data  Set  III  11 /111  '26 / 28/  < 27 x  /V.  C2  ' / 4  24 /  s 25/ N/  5  3  6  42 34  T33]  -3i, 31 I S  5(M  19  66  37  49 54 55  48  40 41  36  axis  4645 44 65  2 2°73  52 53 64  57I860 58 71 72  70 cit  \ 11  axis III 11%  'A  II  17%  TABLE X V I . T a b l e o f e i g e n v e c t o r s variables  o f t h e 11 f l a v o n o i d - t y p e  f o r the f i r s t  PCA on Data  three  Set I I I .  <PPINC0M VAR=1-11 C A S E S M - 7 3 MAX = 3> P R I N C I P A L COMPONENTS CASES=CASE#:1-73 TEST  STATISTIC  DF  SIGNIF  INDEPENDENCE EOUICORRELATION  236.05 299.4 1  55 54  0. 0.  N= 73  COMPONENT % VARIANCE  3.2577 29.62  (D  (2) 2.0101 47 . 8 9  (3) 1.2701 59.44  INDEPENDENCE DF SIGNIF  141.64 54  87.087 44 .0001  61 . 8 8 9 35 .0034  1 . V1  2 . V2  3 . V3 4 . V4 5 . V5 6 . V6 7 . V7 8 . V8 9 . V9 10 . V10 11 . V.1 1  .oooo  . 15589 -.43605 .40326 .49684 -.42036 -.14801 -.18164 - .4434 1 - . 15543 .27097 .29159 - .4 1740 - . 9 1 6 9 6 -1 .24830 .39544 .14013 - . 7 5 4 7 5 -1 - . 5 2 2 4 6 .38985 .61103 .41442 .70227  .78507 -1 •- .11481 -1 •- . 1 6 4 5 6 .20653 - .37627 - .22484 .69058 .39942 .15109 -1 •- . 2 3 9 3 5 -1 .47217  component axes  from  Group near  C is the  also  periphery  Group C2. it  is  diffuse of  Finally,  therefore  the  taxon  classed  Half  the  taxa  remainder  are  centrally  (Group  have r a t h e r  F)  substitutions. and 17 s i n c e flavonoids  OTUs 67-69 form a c l o s e  ordinations No.  as  (albeit are  compounds.  Their  55 s t a n d s  have now been  typical  in  lupine  in  each  The s i m p l e s t  all  tend  Nos.  to  flavonoid  0-G1ucosylation occur  in  at  the  7-position  plus  flavones.  remaining taxa  are  accumulates table  of  the  flavone  3 '-0-methylated groups  is  shown i n  of  v e r y common  simple  8-C-glyco-  flavone shared  7-0by  8-C-glycoflavones.  and  8-C-glyco-  Nos.  19 and 4 8 ,  which  and No.  which  7-0-glucosides .  Table  7,21,30,32,  both 8- and 6 - C - g l y c o -  7-0-glucosides  flavone  types  16  Between-group  and 59 and N o s .  7-0-glucosides  4'-methylated  only  Figs.  both 8- and 6 - C - g l y c o -  35 and 39 s y n t h e s i z e  produce  of  in  be d o m i n a t e d by  having  OTUs 1 , 8 , 2 2 , 5 1 , 5 4 ,  The t h r e e  identical  of  taxa  w i t h no u n u s u a l  groups  types  The  These  compliments are  1 0 , 2 0 , 3 3 and 34 a c c u m u l a t e  flavones. flavones  two  quantities  OTUs 9 , 1 1 , 2 3 , 2 9 , 3 6 - 3 8 and 4 3 - 4 6 , Taxa  discrete  one or  as  ordinations  groups.  concentrations).  and many have s m a l l e r  glucosides.  both  ordinations.  group c o n t a i n  based on j u s t profiles  six  flavonoids  tight  different  in  to  group.  in  all  grouping  be r e f e r r e d  alone  placed  clumped i n  members o f  and w i l l  a monospecific  They a p p e a r  differences  flavones  but  XVII.  73  A summary  98 TABLE  XVII.  Group  Summary  of  ponents  analysis  OTU's  Lupinus  groupings  on Data  Sets  Characteristic cytisoside;  from I  principal.'com-  and  III.  flavonoids  12-17,56,70  B  40-42,65,66  Cl(i)  47,61-64  genistein  •  Cl(ii)  49-54,57-60  variable;  group based on m o r p h o l o g y  C2  67-69  flavone  D  2-6  orientin  E  24-28  flavone  F  1,7-11,18-23 29-39,43-46 48,73  mostly simple f l a v o n o i d s ; i n t e r n a l g r o u p i n g s to be based on m o r p h o l o g y (see d i s c u s s i o n ) .  55  s i n g l e taxon with p r o f i l e of simple and f1 a v o n o l s .  acacetin  of  and c h r y s o e r i o l  major  (see  outliers  remarks  A  as  possible  and  constituents.  71 & 72  7-0-glucosides  discussion).  4'-0-glucosides 3'-0-glucosides,  6-C-glycoflavones  major  amounts  5-0-glucosides  u n u s u a l l y complex compounds; flavones  99  Discussion GROUP A OTU 12  L.  densiflorus  Benth.  13  L.  densiflorus  var.  14  L.  ruber  15  L.  arboreus  16  L . horizontal is  Heller  17  1.  flavoculatus  Heller  56  L.  uncialis  70  L.  kingii  group, taxa  factors  linking  of  annual  else  the  are  the  L_.  erected  that  study.  arboreus  derivative peculiar  Seven o f is  of  to  the  this eight  a perennial.  but  are  the  subgeneric  had c o n n a t e  which  there  any  Their  morphological  has  subgenus  six  of  it  in  the  its  solitary,  seven  piatycarps  into  the  taxa  has  axillary to  was  in  Group A  connate  In  flowers.  include  addition,  PIatycarpus  It  own s u b g e n e r i c  PIatycarpus subgroup.  group  cotyledons.  unciali s likewise  placed  a monospecific  racemose  the  obvious,  first  to  it  compound i s  possession  eight?  Lupinus  as  while  by t h e i r  4'-methylated  in  (1873)  Lupine! 1 us,  the  nowhere  lupines  but Watson  as  including  characterized  This  taxa  belong.  extended  group a r e  cytisoside.  (1873)  accomodate  the  this  affinities  to  it  in  annual ^1 u p i n e s ,  Watson  would  densiflorus  Wats.  occurring  chemical  Munz  Wats.  known as  are  (Kel.l.)  Sims.  C-glycoflavones  vitexin  aureus  Heller  The t a x a of  var.  a  group  cotyledons  group, Smith  (.1946)  I. u n c i a l i s ,  treating  Smith  divided  (.1944)  M i c r o c a r p i . a n d /Pusi11i•based  on  100  keel in  ciliation  the  talis L.  Micro car pi) . were  placed  ki ngi i  of  in  the  close  f 1 avonoid  is  it  sandy tion  others  and t .  h o r i zon^ and  The n a t u r a l n e s s but of  the  of  basic  this  seed-  Group A s u g g e s t s  reflected  the  platycarps  of  are  in  divide  their  similar  to  cotyledons,  Group A.  other  (Smith,  lupines  1944)  similarities  con-  between  blinkers  from p e r e n n i a l  of  and  have had s i m i l a r  the  I.  Morpho-  shrubby  can we remove  annual  small  lupines  with  taxa  United  that  and  see  scented  and p o s s e s s a d i l a t e  coastal  meadows.  are  may r e a c h  (an  striking  a height  It  length, is  feature  in  small  of  the  while  aureus  is  4 dm, w i t h are  Smith's  bright  found i n is  excep-  The f l o w e r s  imperfection  14-17 mm i n keel.  typically  1959).  dry,  An o b v i o u s  dens i f 1 o r u s v a r .  2 dm (Munz,  s u b v e r t i c i l 1 ate  coloured  Most o c c u p y  States.  Lupinus  that  characteristics),  variously  indumentum.  Some v a r i e t i e s  lupine  A very  to  more p r o b l e m -  exist?  adding another  verticillate  petiolate  flavonoid  platycarps,  much more r o b u s t .  racemes  group i s  & Dunn (1964)  unexpected  are  this  member o f  close  southwestern  appressed  to  I. 1 o n g i f o l i us  by Nowacki  I. dens i f ! o r u s .  Microcarpt  is  present  r_. f l a v o c u l a t u s  annuals  taxon w i t h  and commonly a v i l l o u s  an e r e c t , its  seven  placed  similarities  habitats is  ruber  debatable,  and t h i s  such as  immediately  other  Most  is  an a t y p i c a l  been  and the  us to  flowers  has  Given  L^. a r b o r e u s  any  perennial  studies  clusions.  lead  by the  while  P u si 1 1 i .  of j . . arboreus  California  alkaloid  the  PIatycarpus  obviously  logically, coastal  Mi c r o c a r p i  (both  profiles.  A shrubby  arboreus  if  the  relationship,  The a f f i n i t y atic.  flowers  Lupinus: dens i f l o r us , J..  morphology shared  a very  verticillate  became members o f  subdivision ling  and s t r o n g l y  yellow,  mesic  stout  to  woody  dry  stem-base to  refer  that to  it  Lupinus nial,  but,  pubescent  led as  two  a perennial  arboreus,  like stems,  inflorescence  I.  in  taxa  morphology. mise the  that  striking  lations  section the  Group A t a x a .  ated In  other  present taxon  complex, short  habit  while  the  different Figures  possible  very  ciliate  derived  to  are  cytisoside  quantities  other  lupines.  these to  seeds  as  ssp.  Group A.  between  the  specu-  rubens  (Rydb.)  members o f  rather  different  the from  and have p r o f i l e s  the to  pre-  ancestors,  also  and  and  that  so,  it  do a p p e a r  flavonols unlike  this  platycarps, make i t  domin-  chrysoeriol.  profiles  notes  Evert  two' t a x a  seedling  affinity.  chemical  with  in  evolu-  interesting  luteolin  Dunn (1959)  features  the  numerous  parallel  C-glycoflavones  overall  study.  close  of  scented  and  are  similarities  p u s i 1 1 u s Pursh  apigenin,  appressed  similarities  from p e r e n n i a l  can l e a d  respectively)  of  of  peren  H u t c h i n s o n ' s (1959)  evolutionary  and unusual  from a l l  with  has  heavily  difference  but were c h e m i c a l l y  14 and 17 t h a t  cidentally)  is  platycarps  sharing  peduncles  is  result  and m o r p h o l o g i c a l  making t h e i r in  be the  we a g r e e  Both l a c k  small  yellow,  keel  have such a b a s i c  by 7 - 0 - g l u c o s i d e s  addition,  were  may s i m p l y  pusi11us  PIatycarpus  the  too  aureus  71 and 72 ( L .  Dunn and s s p .  bright  it  densi f1orus v a r .  chemical  OTU N o s .  aureus,  and 1 .  if  genus)  undoubtedly a  The  annual  concerning  are  is  subverticil1 ate.  that  and the  w i t h the  to  However,-  the  tree-lupine  but  hand,  var.  Furthermore,  arboreus  two  other  that  scattered  and s u r p r i s i n g tion  on t h e  flowers  (unfamiliar  lupine.  j . . dens i f 1 o r u s  and 14-17 mm l o n g .  between  taxonomtsts  any  species  have  such  unmistakably can  be seen  (possibly  in  coin-  102  GR0UP_B OTU 40  L.  grayi  CWats.)  41  L.  a l b i frons  42  I. cha m i s s o n i s  65  JL.  66  j . . 1 ongi f o l i us  perennial  of  species  four  taxa  ially  pubescent  _L. a r b o r e u s considered  in  (Wats.)  Abrams  characterized  of  the  taxa  close  c h a m i s s o n i s and I. e x c u b i t u s  As m e n t i o n e d , the  flavonoids  different  from the  supported  by K i n g h o r n e t  and J_.  chamissonis  trasts  dramatically  these  sympatric  had but with  species  The m o r p h o l o g i c a l be i l l u s t r a t e d Watson  (1873)  I. a n d e r s o n i i dissimilar aration. Smith  Jepson  (1938)  ( 1980)  Nowacki  of  as  who  described  the  it  two  as  all  in  of  taxa  of  the  have abax-  alkaloid  were seen  (1964)  studies.  to  be  is  quite  further  arboreus  This  con-  assertion  that  similar. taxa  their as  specific tend  to  d e s c r i b e d J..  a l b i frons  a variety  I. g r a y i  of  placed  I. a l b i f r o n s ,  common.  I. g r a y i to  are  grouped w i t h  found t h a t _L.  among the  a -Little  he r a i s e d  (.1944)  dissimilarity  & Dunn's  similarity  ( 1936)  treated  and t h i s  All  & Dunn (.1964)  of  basis  were c h e m i c a l l y  originally  flavonoids  relatives  one a l k a l o i d  by o u t l i n i n g  before  Nowacki  I. a r b o r e u s  Group B t a x a aj_'.  Sericei  on t h e  of  Smith  1 o n g i f o l i u s was  However,  major  7-0-glucosides.  section  Lupinus  Arborei .  production of  California.  large  banners.  latter  by t h e  and c h r y s o e r i o l  coastal  into  the  the  Lindl .  Jon.es  acacetin  first  ex  Eschsch.  group i s  quantities  J_.  Benth.  excubitus  This  Wats.  of  Group B can  taxonomic  histories.  a variety rank  of  in  1876.  The  support  their  sep-  var.  medi u s ,  before  Munz  which  (1959)  placed  the  Lupinus as  taxon  I. e x c u b i tiis  1ongi f o l i us has  a variety  raised  into  to  of  had a . s i m i l a r  I. c h a m i s s o n i s  specific  var  rank  m e d i u s (Jeps.) history,  by Watson  by Abrams  first  (1876)  Munz.  being  before  viewed  being  (1904).  GR0UP_C1 Group CI perennial 64, J_.  is  a rather  lupines  perennis,  native  diffuse to  aggregation  western  North  of  taxa.  America  All  except  which  extends  from e a s t e r n  Canada to  The i n t e r r e l a t i o n s h i p s  between  the  c o n f u s i n g and  tinuous.  be d i v i d e d  Group CI  identifiable for  reasons  1'ose  units. of  chemical  Cl(l)  can  The two  clarity,  but,  g r o u p , the  and C l ( i i )  taxa  into  are two  s u b g r o u p s are since  possible  will  taxa  they  are  for  No.  Georgia. discon-  morphologically treated  separately  do form a : s i n g l e , _ a l b e i t  cross-relationships  between  be d i s c u s s e d .  GR0UP_C1(|) OTU 61  J_.  nootkatensis  62  I. b u r k e i  63  I. p e r e n n i s  64  I. p o l y p h y l 1 us L i n d l .  47  L. 1 at i f o l tus These  or  its  workers  Don.n.'.e.x  Wats.  five  L.  Agardh  taxa  7-0-glucoside, as c l o s e l y  considerable  Sims.  all  accumulate  genistin.  interrelated  changes  in  rank  the  They  isoflavone  have  and t h e  genistein  been r e g a r d e d  taxa  have  and name, t y p i f y i n g  and/  by many  undergone the  nomenclat-  104  ural  and t a x o n o m i c  problems  Lupinus  burkei  was  by H i t c h c o c k  ejt al_.  (1961)  the  name a l t o g e t h e r ,  ssp.  polyphyl1 us.  tained close  the  in  the  differ  by  keel  burkei  having  reduced  with  section  of  C187 3)  I. p e r e n n i s .  able  since  suggested been L.  but  derived  arct icus,  Lati folius (1955)  complex.  and l i s t  eight  versies  evidence  the  present  (.1955)  Smith  mainits  ( 1944), the  who  two  and f l o w e r  o f which  size,  L.  of  of  1 ati f o l i u s , as  I. p e r e n n i s  The above  being  has it  further  as  rank  unten-  Dunn  (1965)  may  have  arboreus  part  of  and  the  Phillips  recently a  by  subspecies  J_.  which  been  separate  taxonomic  similarities are  a  I. n o o t k a t e n s i s  who r e c o g n i z e  that  as  species  specimens.  L . 1ati f o l i u s ,  morphological  study.  in  it  transfer  he views  subspecies.  similarities  a separate  considered  traits  & Dunn (1977)  flavonoid  as  found t h i s  a subspecies  species  Cl(i)»  have  indicating  did  with  I. p o l y p h y l 1 us  Morphologically,  treated  Meanwhile,  by  Group  dispensed  however,  i n _L. b u r k e i )  pol y p h y l 1 us ,  latter  Kenny  (1955)  taxon w h i l e  had examined so few  treated  of  was  morphological  to  this  I. p o l y p h y l 1 u s  a synonym o f  (1966),  (present  Phillips  from 1 .  reduced  of  Dunn (1965)  the  as  of  smaller.  Phillips that  it  Phillips  Polyphyl1i.  ciliation the  a variety  IL. pol y p h y l 1 us as  Lupinus . n o o t k a t e n s i s Watson  to  whereas  treating  status  relationship it  Lupinus.  Dunn & G i l l e t t  specific  placed  in  between  supported  controthe by  taxa the  105 GRQUP_Cl(ii) OTU 49  I. a r i d u s  50  _L.  confertus  52  L.  lyallii  53  J_.  s e l 1 ul us  54  L_.  kuschei  Kel 1 .  Gray ssp.  comprises  from t h o s e  and by e x a m i n a t i o n (Appendix ations  V)  is,  it  in  of  Groups  and  Except caespitose (1972). but  seven  subspecies  Cl(ii)  14  Set  of  or  seen  that  their  in  the  rather  o f _L.  been  ex W a t s . )  Cox  complex  Kartesz  perennial  that  into the  that  the  & Kartesz,  caespitose  high  this  perennial and Cox variable  belong  and held  similarly with  to  subspecies.  variability  complex then  taxa.  similarly  four  recognition  lupines  of  the  (1955)  ordin-  Otherwise,  sets  them to  maps  common  (1951)  for  just  1980)  chemically  the  taxa.  Detling  of  in  their  b e l o n g to  by  deserved  of  dissimilar  Phillips  lupines  decided  placement  of::the  considered all  caespitose  variable  quite  two-dimensional  published  1 epidus.  are  because  several  treated  have  epithets.so  Dunn ( i n  species  (Gray  morphologically  They  data  appear  (1951)  1 e p i d u s - c a e s pi t o s us  taxa.  are  raw  lupines  (1972),'however,  many s p e c i f i c  that  k u s c h e i , t h e s e ;.taxa  Detling  the  taxa  isoflavones  80 names  group  Cox  the  been  group o f  grouped a l l  1 ob b i i  l i k e l i h o o d , merely  for  Over  var.  Group C l ( i ) .  of  of  can  all  accumulation C1 ( i )  s e l 1 ul us  Eastw.  Cl(ii)  Group different  Dougl .  in'the reinstated  30  lists  valid 11  16 s u b s p e c i e s  and  varieties.  I  The most  central  (Jigs.  13 &114)  taxa  in  the  were Nos.  ordinations  49 and  5.0.  arising  Their  from  Data  positioning  so  106  close of the  to  the  the  p o l y p h y l 1 us group i s  isoflavone  genistein.  C-glycoflavones  Detling  (1951)  that  treated as  ( 1955)  I. a r i dus  It  is  in  the  tribe  Group C I . to  the  dominate  separate  interesting  to  into  taxa  53,  and i n  this  this  ex Greene  but  (1951)  Detling  with  52)  has var.  and P h i l l i p ' s the  central  caespitose otherwise  Tobbi i  (see  group C t a x a  case  of  Group  L..  confertus  (Nos.  of  I. 1 e p i d u s  make  Fig.:  appears:to  As we get  thus  to  to  b e l o n g to  L.  1epidus  the  I. caes p i t o s u s  59),  the  taxon  var.  into  and  lupines  L.  culbersonii  that  also  of  close  considered  repertoire  supporting  taxa  it  the  lyallii as  further  from another  but  is  and j . . l y a l l i i .  that  One o f  which  Gray  Detling's  profile  all  some t i m e .  on m o r p h o l o g i c a l  quite  I. 1 o b b i i  1 obbi i  complete  1e p i d u s .  1 epi dus  rest  no i s o f l a v o n e s  group.  have been i n c l u d e d at  L_.  OTU 5 1 , L_. m i n i m u s ,  other  same m o r p h o l o g i c a l  (OTU No.  lupines  by a number o f  Phillips  C h e m i c a l l y , J_.  8-C-gl y c o f 1 a vone , 7-0-gl u c o f l avone shared  49 & 5 0 ,  be c h e m i c a l l y  progressively  produces  similar  is  d e s c r i b e d as  16)  Cl(i).  while  up the  1obbi i  lyallii.  of  1e p i d u s s s p .  same b i o s y n t h e t i c  taxon  very  L.  and:-Phi H i ps:.(1955 ) ssp.  presence  p r o d u c e none  profiles  var.  we e n c o u n t e r  This  chemically  caespitose  the  treatments.  I. minimus i s  pitose  exactly  the  1835 Agardh p l a c e d  t a x o n was  I. 1 e p i d u s  lupine.  The s i m p l e  in  I. s e l 1 u l u s  Originally,  s e l l ul us  they  synonomy w i t h  that  similar.  L.  owing to  P o l y p h y l 1 i , members o f which  CICi)  COTU No.  the  subspecies  note  Taxon No.  conspecific  Otherwise,  j . . a r d i us and  respectively) placed  clearly  of  are  thought  these the  other  Lupi nus list  of  grounds w i l l  is caes-  cusikii,  perennial be added  to  Group C I ( i i ) . Cox cited  (19.73)  their  found many isozyme d i f f e r e n c e s  geographic  isolation  as  in  a contributing  this  g r o u p and  factor  in  107  their  interspecific  elevations  differentiation  from the  Sierra  Nevada  to  since  they  the  Rocky  grow  at  high  Mountains  of  Canada . Taxon No. the  Cl(i)  Alaska  I. k u s c h e i , has  and C l ( i i )  and n o r t h e r n  species  to  BC,  similar  plant  with  to  the  a woody  The  flowers  but  rather  the  taxon  It  occur  from p a s t  is  0-glucosides  of  in  than  flavonoids,  to  L.  fail  substantiate  affinities  have s u g g e s t e d u s i n g m o r p h o l o g i c a l and f l a v o n o i d s  ally  it  more c l o s e l y  appears  being  a.low  leaves  banners  the  are  arise.  glabrous  Chemically,  suggested  Lupinus  C-glycoflavones,  overall,  this  6-C-glycoflavones  convincing.  only  kuschei , but, the  complex,  makes  the that  with  and i s  affinity  quite  of j_. kuschei  Dunn & G i l l e t t  the  or  a r c t i c u s has  flavonoids  characters.  Yukon,  between  long p e t i o l e d  of  both  the  suggest  many 1 e p i d u s - 1 i ke t a x a . The l a c k  to  in  morphology  and t h e  C-glycoflavones  less  similar  (1966)  overall  racemes  simple.  the  lupine  introgression  from which  dense  than  L .' s e r i c e u s  much s i m p l e r  caudex  quite  Its  flavonoids  an endemic  1epi d u s - c a e s pi t o s u s  in  longer  is  similar  Dunn & G i l l e t t  and I. s e r i c e u s .  quite  to  taxa.  have r e s u l t e d  _L. a r c t i cus  with  54,  (1966)  The growth  taxa  of  the  habit  1epi dus  group. GR0L)P_C2 OTU 67  I. a l b i c a u l i s  Dougl.  68  I. a n d e r s o n i i  Wats.  69"  I. formosus This  trio  ex Hook.  Greene of  taxa  differ  from t h e  rest  of  Group C by  its  accumulation J_.  of  andersonii  logical  flavone  as  being  characters  4 -0-glucosides. "close"  and  of  species.  one o r d i n a t i o n  Dunn ( p e r s o n a l  these  taxa  evidence  and t h o s e  for  this  descriptions are  almost  (not  its  OTU No.  of  rest  in  this  of  it  Principal  components a n a l y s i s  Set as  C but  III  it  in is  the  a monospecific  is  native  Dunn (1971) America.  to  Long-distance  f e d on t h e  shrubby,  coastal  Although  the  sites  flavonoids  as  Phillips  considers  a species  ( 1955)  L_. I.  close  of  Set  I  placed  from t h e It  is  has seed  this  of  species  in  to Data  classed  this  in  area.  It  study leaves  South  which  northern  not  of  simple  by m i g r a t i n g are  close  chemically  origins  by b i r d s  Florida  frequented  taxon  and has  its  it  analysis  both  is  taxon.  therefore  is  States  lupines the  ordinations  any o t h e r  di f f u s u s  taxon  into  are  the  with  D.  United  dispersal  of  The  be a synonym o f  from any o t h e r  simple-leaved  that  between  literature.  ( 1955 )  it  in  arising  this  have i n t r o d u c e d _L. di f f usus mesic  distinct,  and j _ . 1 at i f o 1 i u s .  it  Group  different  that  the  to  treat  on Data  Lupi nus  southeastern  suggests  study)  ally  to  group.  in  di f f u s u s  ordinations  and m o r p h o l o g i c a l l y It  to  found c l o s e  related  Nutt.  Lupinus  difficult  chemically  a 1 b i c a u 1 i s in  nootkatensis  di f f u s u s  a closely  relationship  Phillips  (1966)  such t h a t  Group  is  while  regards  Group C, and m o r p h o l o g i c a l  appears  and I.  also,  Dunn & G i l l e t t  The p l a c e m e n t  group i s  the  examined  55 L u p i n u s  constituting  a close  relationship  m o r p h o l o g y to J..  communication)  reflects  of j . . perennis  a 1b i c a u1i s , in  of  identical;  r i v u1 a r i s  as  Although t h i s  (Fig.14)  describes  to j . . a 1 b i c a u 1 i s i n many morpo-  I. a 1 b i c a u 1 i s and j . . formosus complex  Munz (1959)  1  have  Brazil occurs birds.  unusual,  they  may in  •109  are  numerous and v a r i e d ,  species.  Therefore,  observations,  creating  owing to  I. di f f u s us i s  a profile  unlike  both m o r p h o l o g i c a l treated  the  and  western  chemical  as a s i n g l e - t a x o n  group.  GR0UP_D OTU 2  L^. b a r b i ger  Wats.  3  L.  Pursh  4  j . . sericeus  ssp.  huf fmani i  (C.P.Sm.)  5  j . . sericeus  ssp.  ser.  eggl e s t o n i anus  6  L..  ssp.  ser . var.  sericeus  sericeus All  five  taxa  ssp.  in  sericeus  var.  this  the  sericeus  systematic  pi ex c o n s i s t s leaves  of  at  or  near  Lupinus  sericeus  species  and i s  of  its  ianus  has  thought  group. all  Variety  to  flowers dense  h u f fmani, i. i s  s e r i ceus side  has  be d e r i v e d  (Dunn  & Gi 1 1 e t t ,  arate  species  flowers  while  and s p r e a d i n g  Utah  as  part  detailed com?:  cauline  banners  are  pubescent.  racemes  racemes.  Fl ea k , 1971) .  from s o u t h e r n  calyces;  southern  anthocyanin  from i n t r o g r e s s i o n  19:66 ;  numerous  c o n s i d e r e d the  branches  a  both  F l e a k , 'the  abaxially  dense  of  treated  undertook  with  gibbous  ex Aga r dh ) C . P . Sm .  amounts  was  who  perennials  (.9-11 mm) and l a x  small  C.P.Sm.  A c c o r d i n g to  sometimes  (.11-13 mm) and few  small  (1971)  m i d - p o i n t and are  ssp.  & Dunn  (Lindl.  barbiger  d i s t i n g u i s h e d by heavy  stems.  flowers has  the  and u n s p u r r e d but  reflexed  Lupinus  17 t a x a ,  Fleak  f 1 exuos us  complex by F l e a k  analysis.of  sericeus  group p r o d u c e l a r g e  8- and 6 - C - g l y c o f 1 a v o n e s . of  var.  sub-  pigmentation of  var.  large f l exuos us  Variety retrorse  eggl e s t o n hairs,  from _L. 1 e u c o p h y l 1 us Lupinus  and n o r t h e r n  barbi ger,  Arizona,  has  a sepwhite  110  flowers  but  similarity Fleak's  is of  otherwise the  very  similar  flavonoidrprofi1es  conclusions  that  these  to of  lupines  I. s e r i c e u s . these  are  taxa  The  would  closely  close  support  related.  GR0UP_E  OTU 24  J_.  hillii  25  J_.  holosericeus  26  j . . p a r v i f 1 orus  27  L.  28  Greene  hi 1 I i i  X L.  . nevadens i s These  flavone  five  Nutt. Nutt.  ex Hook.  caudatus  Kell.  lupines  8-C-glycoflavones.  of  the  J..  nevadens j s were  j^.  s u l phureus  L.  argenteus.  of  complex  [personal it  as  L^. h i l l i i . of  gression  from L.  placed  have  their  they  (.Abrams,  in  the  the  In  1944)  however,  that  with  are  after  Dunn n o t e s  the  of  that  whereas  by  present the  the  small  hilli i .  The f l a v o n o i d  supports  its  the  as  parvi f1orus placed.  Prof.  flowers  identified  ciliation  indicate of  affinity  The  D.B.Dunn  had  and wing  morphological  to  communication)  are  profile  and  caudatus ,  I.  author  keel  with  treatment  now r e f e r r e d  Dunn ( p e r s o n a l  determined  typical  L. h o i o s e r i ceus  and I. h o l o s e r i c e u s  27 was  of  taxa  Smith's  published a treatment  OTU No.  the  perennial  Cal c a r i t i  taxa  production  accumulate  smal1 -f1owered  h i 11 i i  I. c a u d a t u s  certainly  in  interrelationships.  communication)  typical  hybrid  are  Recently  of  peculiar  California  i n which JL. origin  & Arn.  Otherwise,  and many o f  and Harmon (1980)  hybrid  are  All  labyrinthine lupines  & Gray  Heller  5-0-glucosides.  typical  ex T o r r .  the to  is  intro-  putative I. hi. 1 I i i .  Ill The o n l y Its  affinity  controversial to  Smith's  dividing  this  ication)  places  than  pa r v i f 1 o r u s  the  pubescent Flower  rather  is  of  chemical  of  is  group o f  group o f  in  taxa,  as  L_.  s u l ph'ureus  in  by J_.  communrather  have  nevadensis.  (10-12  flavonoids  makes  sub-  complex  Both complexes  shared  the  parvi f l o r u s  j . . nevadensis .  Dunn ( p e r s o n a l  I. s u l p h u r e u s  5-0-glucosides the  Group E i s  u n q u e s t i o n e d but  the  not  to  mm) but  in  lupines.  a character  (8-10  flavone ally  large  more s i m i l a r  _L. hoi os e r i ceus uction  Calcariti  nevadensis  banners,  size  placement  mm) t h a n  show,  the  I. n e v a d e n s i s a  to  prod^'  distinct  complex.  GR0UP_F We now t u r n profiles. addition  They a l l  results i n t h e i r  difficult,  members o f to  compound t y p e . possible  are  accumulate  that  have l e s s  outstanding  8-C-glycoflavones  and some i n  To d e l i m i t but  other  extracted  reviewed.  e x p l a i n i n g the  for  approached  between  some a r e  Group F,  (Group  by j u s t the  one  to  all  is  the similar  flavonoid  discussion  or  of  point  be c l o s e l y  chemical  in:all  taxa  clearly  from a d i f f e r e n t  group and t h e i r  group  these  • .: J  7-0-  and c o n g e s t e d  affinities  differ  flavone  this  groups w i t h i n  c o n s i d e r e d , by morphol o g i s t s from t h e  simple  placed  a problem s i n c e  groups t h a t  is  and/or  compounds i n  chemical  Therefore,  affinities taxa,  unusual  being c e n t r a l l y  a group i s  members o f  Sets of  many t a x a  The l a c k . o f  ordinations. not  the  produce 6 - C - g l y c o f l a v o n e s  glucosides. F)  to  of  view.  allied  affinities  112  GR0UP_F1 OTU 23  L.  wyethi i  43  JL.  humi c o l a  44  J..  a r c t i c u s Wats.  45  L^. a r c t i cus  46  L . ar c t i c u s ssp. These  found i n  but  their  ssp.  of  phy 11 u s .  Phillips (1961) I.  and  as  Lupinus of_L.  also  of  is  a relictual  to  the  that  Dunn  )  & Robinson  (1966)  o f J_.  are  refected  of  into  it  Group  Cl(i)  them.  Lupinus  the  their  I.  synonomy  but as  a  ( 1944)  polywith viewed by  Hitchcock  et  al .  synonym;of  taxonomies.  considered  Such  of  humi c o l a was  their  although  between  As a  1 a t i f o l i us by Smith  treated  in  taxa  Lupinus  arcti cus.  similarity  present.  a subspecies  in  Dunn profiles  distinguishes  as  rank  )  flavonoid  w i t h the  t e t o n e n s i s was  also  .  recognition,  specific  element  is  I  I. wyeth i i  Cox & Dunn ( 196 9)  morphological  similarity  it  placed  specific  of  R  simplest  p r u n o p h y l 1 us .  humi c o l a v a r . by  S  clearly  treated  Dunn & G i 1 1 e t t  wyeth i i  .  a synonym o f J..  (1955)  wyethi i , a taxon  P  allied  isoflavones  as worthy  .  8 - C - g l y c o f 1 avones  closely  I. pol y p h y l 1 us v a r .  C  (Piper  have the  only  Phillips  (  subalpinus  treated  Phillips  a r c t i cus  canadensis  have been lack  ssp.  lupines  study;  a r c t i c u s was whereas  Nels.  five  the  group t h e y  Wats .  they  to  be a  suggest  variety that  a situation Group F l  flavonoid  taxa,  it  attests a  profiles'.  113 GR0UP_F2 OTU 10  L.  1emmonii  30  L.  X alpestris  31  L . greenei  32  L.  arbustus  33  L.  lutescens  34  L.  sulphureus  35  1.  palmeri  36  1.  argenteus  ssp.  argenteus  37  1.  arqenteus  ssp.  rubricaulis  38  JL.  caudatus  39  L . arbustus This  Dougl.  simple  the  Dougl.  Lindl.  ssp.  pseudoparviflorus  (Rydb.)  Dunn  ex Hook.  Pursh (Greene)  Hess  & Dunn  Kel1. Dougl.  ex Hook.  lupines  genus.  in  may w e l l  Chemically,  which  accumulate  addition  to  but  almost strongly  of the as  are  lupines  7-0-glucosides .  affinties  treated  the  is  ex  be t h e the  most t a n g l e d  simplest  only  forest  members a r e  C-glycoflavones.  8-C-glycoflavones,  other  one two  is  chemical chemical  possible,  6-C-glycoplus  however,  type without  Nos.  to  including  types;, consequently,  they  one.  rather of  of  It  of  Taxa  OTUs 3 0 , 3 1 , 3 2 , 3 5 and 39 have 8 - C - g l y c o f l a v o n e s  nomenclature  All  caudatus)  Wats.  the  flavone  discuss  X  C.P.Sm.  and 3 8 , t a x a  flavones.  (argenteus  Nels.  1 0 , 3 3 and 34 have  are  Nels.  group o f  synonomy i n 36,37  C.P.Sm.  smal1-f1owered  Group E.  The banner  perennial petal  is  glabrous  i n JL.  s u l p h u r e u s and J..  spurred  i n J..  caudatus  pubescent  to  f o r .JL.' a r g e n t e u s  often  adaxially  has  that  pubescent argenteus.  and g i b b o u s  entum may be a p p r e s s e d which  taxa  sericeus  resemble i n many The  in o t h e r s .  throughout  glabrous  calyx Indum-  except  leaves.  Smith F2 i n t o into  the  and merge  taxonomic  tions  of  one  paper,  and t a x o n o m i c  a continuous  the  other  end i s  of  (1980)  L.  is  the  the  24 synonyms  of  I. a r g e n t e u s . than  of  taxa  of  little  group  have been  respect  split  intro-  for  their  of  synonyms  parents. control  hybridization. may be i n v o l v e d  they  are  have  identical  in  This through  L_.  charac-  pubescence; L^.  arg-  the  varieties more  may have genome  another  origin  :  caudatus  I.  no fewer  are  '  than of  compli-  resulted interaction  lupine  (19.66) o f JL.  being  suggest X al p e s t r i s  flavonoids.  treated to  the  is  Dunn & Harmon  are  hybrid  of  extremes  evidences  Dunn & G i l l e t t  L. a r b u s t u s  related  in  o f which  this  complexes  calyx-bearing  included  six  that  being c l o s e l y  and heavy  list  two  was  one extreme  gibbous  c o u l d be because  arbustus  two  ( 1980)  genetic  Lupinus  At  fact  & Kartesz  of  to  in  are  com-  observa-  two  between  from a breakdown  seen  they  the  hybrid  the  the  that  Field  a putative  flavonoids  it  and c a u d a t u s  revealed  of  Also  L. X a l p e s t r i s ,  alternatively,  argenteus  similarities.  that  two.  of  in  their  scores  Kartesz  "The  the  glabrate,  those  as  natives  unfortunately  a spurred calyx  X alpestris,  and j . . a r g e n t e u s .  involved  the  that  variation.  with  The e x i s t e n c e between  or  time  with  treated  extent  range  gradation  cated  that  descriptions  j^. caudatus  other  enteus.  Californian  complexes  noting  such a g r e a t  teristic at  each  & Dunn (1970)  in  to  Since  different into  the  ranking!  Hess plexes  grouped a l l  Calcari t i .  a number o f  gress  vary  (1944)  by Dunn (.1957) who viewed  argenteus  and J..  caudatus.  synonym  of  I. a r b u s t u s  ssp.  arbustus  var.  Dunn was  L.  1utescens.  This  taxon  now t r e a t e d  is  montanus  it One  (Howell)  by Dunn  (per-  sonal  communication)  greenei  is  Peebles j..  (Dunn,  suggest  1 emmon i i  (1960);  they  (1939)  argenteus/caudatus  this  group o f  find  lupines  is  All as  complex o f  robust  habit  an e x t r e m e l y and t h e  profile.  than wooly  pubescent  p r o d u c i n g a. f l o w e r does,  however,  the  since  indumentum. is  similar remain  Kearney  Its  reflexed to J_.  a very  &  between  many  others)  extremely  Arizona.  vari-  Clearly and t h e r e  ex L i n d l .  it,  Morphologically and  flav-  is  taxonomic subgroups.  Dougl.  f o r e g o i n g taxa  banner  the  northern  delimiting  Group F2 melee  the  (and  of  Lupinus  1  for  range  its  a m o r p h o l o g i s t s nightmare  foundation  Lupinus  similarity  taxa  members o f  a place  chemical  although  L. a r b u s t u s .  three  I. 1 e u c o p h y l 1 us  in  morphological  a strong  Taxon No.29 i s  simple  It  with  indicate  1 emmoni i .  by Smith  chemical  the  sulphureus.  communication)  alliance  also  able  little  of  o f J_.  r e s e m b l e j . . pa 1 mer i , a c c o r d i n g to  and J..  were t r e a t e d  ally  be p a r t  personal  a closer  greatly  greenei  a close  c o n s i d e r e d to  L. c a u d a t u s onoids  as  it it  flowers above  too, has is are  the  has  It  may  a  very  a much more  characterized small  (9-11 mm)  mid-point  sulphureus  and L..  distinctive  taxon.  by  thereby  arbustus.  GROUP_ F3 OTU 7  L.  bi c o l o r  Lindl.  8  1.  polycarpus  9  1.  texensis  Hook.  10  L.  benthanii  Heller  19  L.  concinnus  20  L.  sparsiflorus  21  L.  arizonicus  22  L.  stoloniferus  73  L.  hirsutissimus  1  1.  elatus  9  1.  neomexicanus  48  L.  1i t t o r a l i s Dougl .  Greene  Argardh Benth.  (Wats.)  Wats.  L. Benth.  Johnston  The t h i r t e e n morphologically  Greene  taxa  that  diverse.  B-ring methylated  flavone  comprise  7-0-glucosides .  Perhaps  i n which  to  place  lupines  of.doubtful  The  group  F3 t a x a  would  such a g r o u p !  show while  flavonoid others  and s i m i l a r For about  such to  as  of  the  unlike  is  and ^L. desert  the  for any  little  reported.  spars i f 1 o r u s of  a page  be prime  has  but that  is  Lupi nus  have  should  and  affin-  candidates  hirsutissimus,  lupine  in  a profile  there  and  I n q u i rendae  relationships  other  are  others  Species  e x a m p l e , J..  species,  regions  the  certainly  I. neomexi canus  many o t h e r  lupines  I. a r i z o n i c u s  who e r e c t e d  Some t a x a ,  completeness,  these  species  ( 1835)  profiles  chemically  profiles,  from Agardh  for  F3 a r e  Some have s i m p l e  be t a k e n  ities.  Group  the  the  study,  that  is  simpl  similarity  known or  ends  inferred  cone i nnus ,  three  very  southwestern  similar  United  annual  States.  117  Christian this  & Dunn (1970)  complex and d i s c o v e r e d  species.  species  c o m p l e x ; the  would t e n d t o  an e x t r e m e l y  i c a n u s . the Since  their  infer  any  texensis  simple  out  flavonoid  and i s  are  as  so s i m p l e  between  one o f  taxa  the  it  these  taxa.  two  lupines  just  w i t h a h a p l o i d chromosome number o f  in  comprise the  Lupinus to  texensis  taxa.  unwarranted Moreover, in  18 (compared  three  I. neomex-  argenteus  seems  the  should  profiles  similar  within  between  separation.  group and some o f  affinities  stands  barriers  of these  their  profile  chemistries  real  each  differing  support  arcticus  c r o s s i n g experiments  sterility  They c o n c l u d e d t h a t  a separate  has  undertook  North to  the  to  I. America typical  24). Lupinus coastal Smith  bicolor  annuals  (19-6)  Chemically,  which  I.  into  Smith  (1944)  placed  is  Few workers  have t r e a t e d  remain  of  taxa  Lupinus  el at us  (Smith,  1944).  variety  of  L.  unknown is  Jepson  British  Columbia.  Micranthi  be c l o s e l y  7-0-glucosides  I. s t o l o n i f e r u s , available.  into  the  absent  atypical  and Dunn  related. and 8-C-  a taxon  Lupinus  section  Sparsiflori  and _L . hi rs ut i s s i mu§ , s y n t h e s i z e s  compound i s  Lupi nus hi r s u t i ss imus  is  to  section  information  pi u s " a p i g e n i n , l u t e o l i n  The l a s t  his  flavone  similar  little  s pars i f 1 o r u s  flavones, ides.  is  both smal 1 - f 1 owered  from C a l i f o r n i a  both a c c u m u l a t e  there  are  c o n s i d e r them to  chemically  b e n t h a m i i , which with  occur  also  they  glycoflavones; about  that  polycarpus  p l a c e d both t a x a  C1944)  & Gillett  and J_.  and c h r y s o e r i o l  8-C-glyco-  7-0-glucos-  from L^. s pars i f 1 orus . in  j _ . benthami i  its or  lack  of  C-glycof1 avones.  L_. hi r s u t i ss i n n i s ;  they  relationships. a perennial ( 1936)  a 1bicau1is  that  resembles  c o n s i d e r e d J_.  and Smith  (1944),  elatus  I. a n d e r s o n i i to  a variety  be a of  ' ,  118  j..  formosus  ically, though  before  I. el a t u s its  he r e - e l e v a t e d is  quite  morphological  it  to  different  specific  from the  similarities  status.  Chem-  group C2 t a x a  do s u g g e s t  a  al-  possible  affinity. Lastly, placed  with  1 i t t o r a l i s should L_. a r b o r e u s  and L..  Dunn & G i l l e t t  ( 1966)  consider  closest  ally.  The l a t t e r  flavone  of  the  it  J..  acacetin  7-0-glucoside  I. c h a m i s s o n i s which  acacetin.  As a summary o f t h e Table  XVIII  L^. a r b o r e u s the  while This  be  lupine  should  Smith his  to  be  1  I. 1i 11 o r a 1i s  sea-shore  also  ( 1944)  Ar bo r e i .  4 -0-methylated  compound i s  I. a l b i f r o n s , o t h e r  I. 1i t t o r a l i s amy w e l l  cussed,  r i v u 1 a r i s in  produces  (cytisoside)  of  and  also  be d i s c u s s e d .  its C-glycoaccumulates present  lupines  to  related.  groupings that  be r e f e r r e d  to.  have been  dis-  in  TABLE  Group  XVIII  Summary t a b l e o f the groups o f morphological evidence.  Lupinus  Characteristic' flavonoids (if any)  taxa  d e n s i f l o r u s var dens i f1 or us var ruber h o r i z o n t a l is flavoculatus unci a l i s ki ngi i  Lupinus  taxa  delimited  by f l a v o n o i d  data  and  Remarks W a t s o n ' s (1873) s e c t i o n P I a t y c a r p u s . Annual t a x a w i t h s e s s i l e , c o n n a t e cotyledons .  dens . aureus c y t i so s i de  arboreus  I. a r b o r e u s i s an e x c e p t i o n . It i s a shrubby p e r e n n i a l with p e t i o l a t e cotyledons. C o n s i d e r e d u n r e l a t e d , by o t h e r workers. C y t i s o s i d e a c c u m u l a t i o n and 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 to J.. d e n s i f l o r u s v a r . aureus suggest p o s s i b l e link. Group A o u t l i e r s : p u s i 1 1 u s s s p . rubens and p u s i 1 1 u s . P l a t y c a r p s but w i t h o u t c y t i sosi de.  grayii a l b i frons chamissonis excubitus 1ongi f o l i us  Cl(i)  nootkatensis burkei perennis polyphyllus 1 a t i f o l i us  major amounts of a c a c e t i n and c h r y s o e r i o l 7-0-glucosides  genistein  C a l i f o r n i a n c o a s t a l p e r e n n i a l s . Nowacki & Dunn (1965) c o n s i d e r them to be r e l a t e d ( e x c e p t j . . g r a y i i ) . to each o t h e r , and to I. a r b o r e u s ( c h e m i c a l l y distinct) J.. g r a y i i c o n s i d e r e d m o r p h o l o g i c a l l y s i m i l a r t o I. a l b i f r o n s by Munz ( 1 9 5 9 ) ; chemistry supports this c o n t e n t i o n . M o r p h o l o g i c a l l y s i m i l a r to each o t h e r and to t h e C2 and F l t a x a . Genistein accumulation is d i s t i n g u i s h i n g feature 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 o f each o t h e r i n the p a s t .  Cl(ii)  C2  a n ' dus confertus 1ya 11 i i sel1ulus 1 epi dus caespitosus minimus cus i cki i culbersonii kuschei  g e n i s t e i n in some, o t h e r s more s i m p l e Group d i s t i n g u i s h e d by morphology.  a 1b i c a u1 i s andersonii formos u s  f1 avone 4 ' •0glucosides  Some a r e 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 tinct. A l l are p e r e n n i a l c a e s p i t o s e lupines. D e t l i n g (1951) & P h i l l i p s (1955) t r e a t e d them a l l as s s p . o f L. 1epidus . Cox (1972 ) r e g a r d s them as having s p e c i f i c s t a t u s , j . . kuschei is an e x c e p t i o n and has not been c o n s i d e r e d p a r t o f the c a e s p i t o s e g r o u p . However, 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 and c h e m i c a l a f f i n i t i e s ( t o some) s u g g e s t a p o s s i b l e relationship. 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 o t h e r and to C l ( i ) t a x a . Flavone 4 - 0 - g l u c o s i d e s are p e c u l i a r to C2 t a x a 1  barbiger sericeus sericeus sericeus sericeus  E  var. var-. var. ssp.  Major amounts o f sericeus both 8-C- and 6-Cflexuosus gl y c o f l avones p l u s e g g l e s t o n i a n u s 3 ' - 0 - g l u c o s i d e of huffmanii orientin  hill i i parvi f1orus holosericeus h i11i i X caudatus nevadensis  di f f u s u s  f l a v o n e 5-0glucosides  A l l are members o f the s e r i c e u s complex of Fleak (1971). Chemically uniform q u a l i t a t i v e l y but h i g h 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 taxon. 5-0-glucosides d i s t i n g u i s h t h i s group. 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 o f S m i t h ' s (1938-1952) section C a l c a r i t i . E x c e p t f o r L. n e v a d e n s i s a l l t a x a have e x t r e m e l y s m a l l f1owers . Monospecific group. Complex p r o f i l e o f s i m p l e compounds; u n l i k e any o t h e r lupine in this study. Morphologically d i s t i n c t ; a simple-1eaved taxon of s.e U.S. P u t a t i v e a n c e s t o r s i n S. A m e r i c a (Dunn, 1 9 7 1 ) .  Fl  wyethi i h u mi c o1 a a r c t i cus s s p . a r c t i cus s s p . a r c t i c u s ssp.  F2  1emmoni i X alpestris greenei 1utescens arbustus pseudoparviflorus suphureus pa 1 mer i argenteus s s p . argenteus arg. ssp. r u b r i c a u 1 i s caudatus  F3  bi co 1 o r polycarpus texensis bent hami i concinnus s pa rs i f 1 o r u s arizonicus s t o l o n i ferus elatus h i. r s u t i s s i m u s 1i t t o r a l i s neomexi canus  a r c t i cus canadensis subalpinus  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 ) taxa. Their l a c k o f g e n i s t e i n and f l a v o n e 7 - 0 - g l u c o s i d e s makes t h i s group d i s t i n c t from C1 ( i ) . A l l members o f 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 morpho l o g y and p a r t l y by c h e m i c a l simplicity.  varied simple  but  profiles  v a r i e d but mostly simple profi1es  j . . 1 e u c o p h y l 1 us not c o n s i d e r e d a p a r t o f the C a l c a r i t i but f l o r a l characteri s t i c s a l l y i t with lupines in t h i s group.  A s s o r t m e n t o f a n n u a l s 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 u s i n g e i t h e r c h e m i c a l or m o r p h o l o g i c a l c h a r a c t e r s ; a l t h o u g h some have been a l l i e d w i t h members o f o t h e r groups (see d i s c u s s i o n ) .  122  •V. OVERVIEW  Flavonoids Cain (or  as  taxonomic  & Harrison  descriptive  haviour for  which  a particular  Davis ture  & Heywood whose  sessed. but,  taxonomist  (1963)  than  describe  "good"  wide v a r i a t i o n intrinsic  structed  natural  quirement acters ments  is  discussed  1)  characters  Intraspecific  (.Hitchcock and has  in  et  led  al_. ,  of  in  of  size,  more s i m p l y  any  useful  in  fea-  as  those  samples  being:  4)  Davis  1)  readily  consistent, existing  that  in  classification. be i n c l u d e d samples.  with  not  are  & Heywood they  subject  b e i n g c o n s i d e r e d ; 2) not  as-  some c h a r a c t e r s  "bad" characters;  and  to  not  high  susceptible is,  they  a previously  con-  One a d d i t i o n a l  here:  5)  that  Each  of  these  the  to  agree  re-  char-  require-  reference  to m o r p h o l o g i c a l  of  lupine  morphology i s  1955 ;  Dunn & G i l l e t t ,  and  Lupinus.  1961; P h i l l i p s , to  Lupinus hair  as  "good"  variation  taxonomists  The v a r i a b i l i t y range  turn  interpretation."  taxa.  characters  feel,  be-  organism  delimiting  be homologous between  flavonoid  the  I  whole  or  classification  in  system o f  should,  from the  group b e i n g a s s e s s e d ,  the  structure  c o m p a r i s o n or  v a r i a b i 1 i t y ; . 3)  of  form,  attribute  theoretically  modification;  with-correlations  the  "any  otherwise  characters within  to  as  be m e a s u r e d , c o u n t e d or  between  genetic  environmental  is  others  distinguish  characters  view c h a r a c t e r s  Any c h a r a c t e r  d e p e n d i n g on t h e  Lupinus  separates  p u r p o s e such as (1963)  in  define  referring  e x p r e s s i o n can  more u s e f u l  in  (1958)  phrase)  the  characters  describe species  type,  over is  flower  1500 s e p a r a t e  further colour,  abundant 1966) taxa.  illustrated etc.  that  by  appears  123  in  descriptions  however,  are  minor  or  variation  has  little  characters  are  species ified  by any  acters  for  example,  (1963) etic  that  rooted cannot  simple  seed c o l o u r  stable. good as  is  are  to  differences  repertoire  of  capabilities. variability Rollins  in  of  Lupinus  (1958),  a taxonomic  in  characters  determining  treatment  in  constitution  the  genetic  be o b l i t e r a t e d  peas.  to  They c o n c l u d e t h a t  that  simply "a  of a  greatly  other  words,  based  what  charnot  & Heywood  by s i m p l e  gen-  genetically  taxonomic c h a r a c t e r  no m a t t e r  mod-  inheritance,,  Davis vary  Dithyrea  characters  be based s h o u l d  However,  are  consistency,  In  or  system o f  characters that  easily  factor.  two-factorial  upon  of  only  between  its  qualitative  we s h o u l d r e l y  in  Where  restricted  biosynthetic  1  although  commonly s e e n .  usually  the  taxon s  segregating  by a one- or  flavonoids,  infraspecifica11y  t a x o n o m i c deMsTons- are  distinguish  as  the  genetic  mechanisms and t h o s e  remain only  they  on which  be g o v e r n e d  they  Moreover,  were u s e f u l  so d e e p l y that  exist,  attention.  stated  Lupine  invariable  and s u b s t i t u t i o n  (Cruciferae), that  floras.  constituents  in  The i n t r i n s i c  had v e r y  which  in  compounds.  no v a r i a t i o n  glycosylation 2)  of  differences  trace  represent  species  comparatively  quantitative qualitative  of  its  but is  genetic  bas i s . " Smith. ( 19 38-52) entities  based on m o r p h o l o g i c a l  C1961) view as useless  for  behave  ple,  as  flower  on t h e  taxa. basis  as  to  that  render  They add t h a t of  characters  s i m p l e Mendel i a n . t r a i t s colour.  Lupinus , taxonomic  characters  varying .so-greatly  delimiting  been d e s c r i b e d to  d i s t i n g u i s h e d , : in  Flavonoid  in  the  that  have  been  genera,  Lupinus  et  al.  characters  many s p e c i e s  in other  genetics  Hi t c h c o c k  is  for as  have shown examyet  124  unknown  but  their  requirements 3)  of  not  for  per  been  phenotypic orientin  4) tent,  reflecting  This  arity  taxonomy acter that  that  use o f  the  then  that with  character  a rather  characters pre-set accepted  of  system  the the  In  is  pre-set  case  quantitative  is  a  exception  of  iso-  are  "good"  be c o n s i s -  unaffected  boundaries  disagreed with  various  and u s e f u l  (as  any  previously  established  in  they  taxonomies  to  boundaries).  particular  a former If  data  be even  the  char-  strengthens judge-  past  fit  there  Lupinus  circul-  taxonomies  dishonesty  do not  this that  were t r e a t e d  should  to a  This  Since  flavonoid  & Heywood,  and t h e r e b y  lead  groupings.  any  new d a t a  to  by c o m p a r i n g them to  classification  consequence,  If  "bad".  valuable.  because  constructed  admit,  contrary  considered  was  (Davis  weighting.  a p p r o a c h and can  taxonomic  that  authors  a way  is  must  question  the  character  ignored simply of  as  in  a_ p o s t e r i o r i  usefulness  subgeneric  averted.  characters  a group i n  blinkered are  the  L_^_ s e r i c e u s  classification  character  taxonomy t h e n  ment o f  considered  statement* l e a d s ,  subdivides  agrees  is  a natural  and a measure  character  the  genus  In  that  sericeus  (with of  the  genotypic.  Lupinus  flavonoids  in  not  factors.  A character  1963).  the  we do see  or  our  m o r p h o l o g y has  r- •• s uggesteci  Qualitatively  production)  the  that  satisfy  variability.  lupine  phenotypic study  seems to  intrinsic of  compounds i n  response.  without  as  present  certain  by e n v i r o m e n t a l  low  The-'Variation  identified  in  of  constancy  modification  se.  f l a v o n o i d s , the  variation  is  characters  Enviromental  been t e s t e d has  infraspecific  as  is  if  into no  the widely  difficulty agreed  was  or  equally  valid  if  they  cut  across  The f a c t  that  all  the  125  profiles the  of  same f l a v o n o i d s  and a l s o markers  to in  5}  related  the  group such as  does  that  samples  a p r o b l e m at  of  are  compared.  plants the  the of  is  of  biochemical  Lupinus  are  had  integrity  "good"  luteolin  taxonomic  has  lupines  document i n s t a n c e s  where  in  two  state of  & Levy  genotypes  lupines the  that  the  genetic the  ( 1978)  give  less  one must  been q u e s t i o n e d  and C r a w f o r d  various  closely  biosynthesize  a measure  has  import-  however,  been made t h r o u g h o u t it  a  Chemically  7-0-glucoside  both  but  (1974)  extreme  is,  We may f u r t h e r m o r e  systematics  e_t aj_.  of  there  level.  compound r e f l e c t s  an a s s u m p t i o n t h a t  by B r e d e r o d e  group's  homology i s  morphological  same way.  the  It  that  Platycarpus  When d e a l i n g w i t h  such as  h o m o l o g o u s , we assume t h a t  pos e s s i o n  to  flavonoids  character  example, that  compound i n  section  genus.  question  for  the  lend weight  credibility  group o f  assume, is  the  The  ance when  of  a natural  rise  identity.  history recently who to  the  same  f1avonoi d . An example o f gives  false  by the  evidence  of  where  compared t o  lysine  from p y r u v a t e  latter  make  lysine  (Luckner,  1972);  there  are  pathways  cannot  character. onoid  In  lysine,  obviously  and from  closely  the  L-aspartate  related  species,  homologous and  production of The f o r m e r  steps via  taxonomic  but,  the  since  we can  lysine syn-  -semialdehyde  -ketoglutarate  knowledge o f  negligible,  not  fungi.  being manufactured  Lu p i n u s , o u r is  in  no b i o s y n t h e t i c  be c o n s i d e r e d , f o r  biosynthesis  is  most o t h e r  the  same compound,  a compound i s  similarity  Oomycetes  thesize while  a case  and a c e t y l  in  two  common.  CoA The  different  p u r p o s e s , the  same  genetics  of  they  a group o f  continue  are to  flav-  assume  that  126 each  compound r e p r e s e n t s  genus five  until  proven  requirements  by the cases  flavonoids superior  a homologous c h a r a c t e r  otherwise.  It  of  a taxonomic  of  Lupinus.  is  the  author's  character  They are  to m o r p h o l o g i c a l  throughout view  have been  that  in  terms  the  satisfied  c o m p a r a b l e and i n  characters  the  of  some  reliab-  ility.  Relationships  in  Lupinus  The g r o u p i n g s 73 t a x a  of  Further  interpretation  sary  an e v o l u t i o n a r y  if  Stages  in  Lupinus  p r o d u c e d by t h e  the  illustrated groups  results of  evolution  fossil  a purely  the  of  of  the  phenetic  flavonoids  is  morphological sequences  record.  of  to  be  are  Some w o r k e r s ,  neces-  attempted.  characters  form i n  of  classification.  groups and c h a r a c t e r s  classification  by d e s c r i b i n g  and the  in  analysis  have  existing  notably  been plant  Harborne  (.1967 , 1 972 , 1 977 ) and Swain  ( 197 5 ) ,  flavonoids  sequence  by d e s c r i b i n g  substit-  advanced.  A c c o r d i n g to  Harborne  ution  into  patterns  an e v o l u t i o n a r y as  primitive  [1975)  a simple  flavonoid  and i s  considered  primitive.  highly  methylated  flavonoids  biosynthetic  steps'is  (.1978)  point  out,  account  for  the  these  are  reduction  f l avones in  the  Gornall in  that  of  a few  are  the  advanced.  processes  in  and the  Compositae.  of  views  evolution  the  arrange  enzymatic  steps  & Bohm  that  do not  leading  chemistry.  presence Cyperaceae  many  numerous  As G o r n a l l  of morphology and/or cite  to  synthesizes  result  over-simplistic  bryophytes  and the  result  An o r g a n i s m t h a t  & Bohm (1978)  both the  bryophytes  the  therefore  secondary s i m p ! i f i c a t i o n examples,  is  or  have a t t e m p t e d  of  to As  C-glyco-  and  aurones  127  The f l a v o n o i d s serve  as  a basis  intergroup of  cannot only  (David is ers  in  in  be d e r i v e d  putative  ancestors  to  of  are  compare t h e  placed  into  sect ion  ically  distinct  platycarps, similarities All  other  Since  relationship  especially between  annuals  in  in  the  the  perennial  They lupines  exhibit but  strong  since  a closely  trend  evolution considLupinus its  be  inter-  some o f Watson  the  (1873)  (with the  that  group  with  L_.  tempts  derived arb-  one  and  we  to  the  numerous m o r p h o l o g i c a l  d e n s i f 1 orus  b e l o n g to W a t s o n ' s profiles  notable  annuals,  tree.lupin  the  chemical  are  related  affinity  evidence  and L_.  their  a  life-cycle  would  platycarps  light.of  study  is  leaves,  that  studied  between t h e  I. a r b o r e u s  Perhaps  through  herb w i t h  The i n t e r e s t i n g  and show u n s p e c i a l i z e d f l a v o n o i d compounds.  the  represent  reflexion  m o r p h o l o g i c a l l y and chem-  lupines  p e r e n n i a l , >provides  a direct  the  it  species  are  phenetic  Dunn .(1971)  with simple  of  the  shrubby a n c e s t o r s .  simple-1eaved;  other  from a common a n c e s t o r .  suggest  American  PIatycarpus  that'they  o r e u s , a woody  of woodiness  The annual  from a l l  type of  position  been  be n o t i c e d  most  and  sequence.  an annual  flavonoids  o f _L. a r b o r e u s ) .  may s p e c u l a t e  can  perennial  species.  have a l s o  I. di f f usus which  also  type  indumentum, banner  The l o s s  a herbaceous  American  exception  case  to  from South  is  lupines  sequence t h a t habit  in  to  since  Furthermore,  an e v o l u t i o n a r y  1963).  the  of  variation  classification  differences  size,  into  perennial  d i f f u s us  South  flower  evolutionary  apparent  esting  represent  be p l a c e d  & Heywood,  to  an e v o l u t i o n a r y  classifications  from a woody  show i n s u f f i c i e n t  and B - r i n g m e t h y l a t i o n .  differences  etc.  Lupinus  differences  morphological  the  for  glycosylation  since  of  Lupi nus  containing resemblances  chemistries  are  var.  no to  aureus. proper  unusual many  so unspec^  ialized,  a dirct  and p e r e n n i a l  evolutionary  lupine  is  relationship  unwarranted  at  between  this  time.  both groups do have such s i m i l a r  chemistries,  initial  and p e r e n n i a l s  ural  division  and p h e n e t i c .  chemical  the  It  annuals  is  hoped t h a t  or m o r p h o l o g i c a l , w i l l  tween s m a l l to  between  numbers  recognition  of  annuals  future  find  any  annual  The f a c t  however, even  makes  more  research,  of  smaller  more n a t u r a l  to  discuss  is  the  unnat-  be  it  some common c h a r a c t e r  and p e r e n n i a l s  that  and t h e r e b y  belead  groups.  Convergence A final ical If  groups  our  gence  point seen  ultimate is  tionary  in  this  goal  is  irrelevant.  study  important.  Cain  parallelism  resulting  we a r e  then  in  Consequently,  convergence  which  it  is  taxa  Our o n l y  between  taxa  sible.  The p r o b a b i l i t y  every  solution  to  simi. l a r m u s t  be l o w e r  thens  similarities  the  argument  is  take  than  place to  in  problem i s types  the  fact  noted  within-group  to  related  (Davis  to  of  that  of  more  occur  be-  forms.  & Heywood,  make c o m p a r i s o n s  characters  character  lupine  as  pos-  for being  them b e i n g  similar  flavonoids  by m o r p h o l o g i s t s members are  or  those  being r e s p o n s i b l e  probability The  is  precisely  detect  evolu-  convergence  distantly  and c y t o l o g i c a l  previously  that  that  chem-  conver-  f o r m i n g an  more l i k e l y  between  convergence  by an assumed common o r i g i n . reflect  convergence  this  chemical, morphological  then  problem o f suggest  the  convergence.  in  many d i f f e r e n t of  of  interested  most d i f f i c u l t  1963).  u s i n g as  will  result  that  classification  (1960)  than  possibility  the  similarity  related  in  the  & Harrison  tween c l o s e l y  groups  are  a phenetic  If  classification  the  more  streng-  129  closely  related  flavonoid eric  evidence  groupings  unrelated  than  tends  that  lupines  members to  of  different  support  were c o n s t r u c t e d  having  similar  the  groups.  notion  are  profiles  that  natural by  In  short,  the  groups  the  subgenand  convergence.  not  130 VI.  At the o u t s e t of t h i s detailed analysis  SUMMARY  s t u d y , f o u r g o a l s were s t a t e d : 1) a  o f t h e f l a v o n o i d s o f some N o r t h A m e r i c a n  i n e s ; 2) a s s e s s m e n t o f l u p i n e f l a v o n o i d s f o r t h e i r  lup-  reliability  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  on  d a t a ; 4)  chemical  other c l a s s i f i c a t i o n s must now  were a n a l y s e d  of  individuals,  for flavonoid  currently  K a r t e s z , 1980).  o r so s p e c i e s had  compounds i s o l a t e d list  content  new  onoids acters.  This  the m a t u r a t i o n  study  their extraction  Few  although  America  of a Lupinus  variation  of the  and  identification,  reliability  fresh  the  the  dried  ulation  reflected  1)  during  geographic  analyses  In t h e c a s e  differences in orientin  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  to the south of the taxon's  char-  amounts o f t h e c o n s t i t u e n t  more common i n f r a s p e c i f i c a l l y .  sericeus quantitative  flav-  p l a n t mat-  d i f f e r e n c e s were s e e n i n t h e s e  i n the r e l a t i v e  complete  lupines.  variation  versus  after  t h a t the  as s p e c i f i c  s p e c i e s ; 2) t h r o u g h o u t  Lupinus  species  (Kartesz &  i n North American  involved monitoring flavonoid  qualitative  f l a v o n o i d s was  third  compounds  represent a reasonably  as t o t h e i r  r a n g e o f t h r e e s p e c i e s ; 3) f r o m erial.  one  different  been a n a l y s e d , i t i s c o n c l u d e d  in this  were a s s e s s e d  56  We  taxa  f l a v o n o i d s were d i s c o v e r e d  o f the f l a v o n o i d s t h a t e x i s t Following  north  and  and  attained.  r e p r e s e n t i n g 73 d i f f e r e n t  r e c o g n i z e d i n North  S i n c e few  classification  observations.  g o a l s have been  This represents approximately  Lupinus  ten  between t h i s  b a s e d on m o r p h o l o g i c a l  decide whether these  Seventy-three  noted.  a comparison  based  distribution.  of  accumr from  Subsequent  the  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 u n d e r u n i f o r m suggested the  that the v a r i a t i o n  r e s u l t of a phenotypic C h a p t e r IV d e t a i l e d  onoids. rather  Initially, uniform  represented  conditions,  i n the mature p l a n t s  response.  an i n t e r s p e c i f i c  the d i s t r i b u t i o n  from s p e c i e s  study  of lupine  o f t h e compounds  Several  were  compounds  i n f r e q u e n t and p r e s u m a b l y r e f l e c t e d o p e r a t i o n o f n o v e l routes.  Utilizing  compounds, s i x s u b g e n e r i c discussing within-group assessed  and  chemical  and p e r s o n a l  affinities,  similarities  observations.  morphology c o r r e l a t e d wel1 , t h e r e b y  rity  of these  o f t h e g r o u p s as n a t u r a l  were  biosyn-  more  unusual  g r o u p s o f l u p i n e s were d e f i n e d .  f o r morphological  literature  the d i s t r i b u t i o n  flav-  appeared  t o s p e c i e s ; some compounds  almost u b i q u i t o u s , f o r example, o r i e n t i n .  thetic  was  After  t h e g r o u p s were  by r e f e r e n c e  to the  In many c a s e s ,  chemistry  strengthening  the i n t e g -  units.  F i n a l l y . w e must a s k t h e q u e s t i o n : a r e f l a v o n o i d s u s e f u l as taxonomic markers i n Lupi nus? On t h e p o s i t i v e  The a n s w e r i s r a t h e r i n c o n c l u s i v e .  s i d e , t h e r e were g r o u p s t h a t were e a s i l y  t e d f r o m t h e 73 t a x a a n a l y s e d .  In s e v e r a l c a s e s  g r o u p s a p p e a r e d t o 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 other  cases,  dissimilar ities. and  logical pus  although  allies  these  (having  groups.  revealed  interesting  s i d e , 30 t a x a e x h i b i t e d v e r y  flavonoid profiles;  to d i s c e r n chemical  l u p i n e s seemed u n w a r r a n t e d a l t h o u g h  discontinuities  the chemical In  were shown t o be m o r p h o l o g i c a l l y  f u r t h e r study  On t h e n a g a t i v e  similar  within  chemical  extrac-  exist.  Apart  similarsimple  subgroups:  some morphoT  from the taxa o f P I a t y c a r -  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  species  chemically further the  the  genus.  r e m a i n s on  r e s e m b l e d many o f t h e artificiality  o f the  R e g r e t t a b l y , the  perennials.  annual/perennial  l a r g e and  the w h o l e , t a x o n o m i c a l l y  level.  However, t h e  generic  g r o u p i n g s t h a t a p p e a r t o be  future research, other  present  using  g r o u p i n g s s u c h as  s h i p s w i t h i n the  genus  This  study  has  division  interesting  confusing  Lupinus.  in  Lupinus  species  several  sub-  I t i s hoped  additional characters, will to i n c r e a s e our  genus  a t the  revealed  natural.  indicates  expose  that yet  knowledge of r e l a t i o n ? / ;  133 REFERENCES Abrams,  L.  1904.  Lupinus  1 o n g i f o l i us .  Flora  of  A b r a m s , L. 1944. 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L.  aminopeptidase polymorphism in G a z . 134: 73-76  H . L . 1964. Multivariate Methuen , London .  Smith,  Cochrane  1958. The g e n e t i c e v a l u a t i o n o f a t a x o n o m i c Di t h y r e a ( C r u c i f e r a e ) . Rhodora 6 0 : 145-152  R o t h m a l e r , W. 1944. Die G l i e d e r u n g der R e p e r t . 5 3 : 137-150  Seal,  tribes  f l o r a of California  Taxonomy.  & P.H.A.Sneath. 1963. Principles Freeman & C o . , San F r a n c i s c o .  of  Numerical  Tax-  S t e b b i n s , G.L. 1944. V a r i a t i o n and E v o l u t i o n Columbia U n i v . P r e s s , New Y o r k .  in  S t e e l , R . G . D . & J . H . T o r r i e . 1960. Principles Statistics. M c G r a w - H i l l , New Y o r k .  and P r o c e d u r e s  Plants. of  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 p r e s s u r e l i q u i d c h r o m a t o g r a p h y a n a l y s i s o f o a t (Avena s a t i va) f l a v o n e d e r i v atives. J . Chrom. 1 7 6 : 27Q-273 S t r a c k , D. & J . K r a u s e . 1978. Reversed-phase n a t u r a l l y o c c u r r i n g mixtures of flavone J . Chrom. 1 5 6 : 359-361  HPLC s e p a r a t i o n derivatives.  of  140  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 o f an enzyme from c e l l s u s p e n s i o n c u l t u r e s o f p a r s l e y c a t a l y s i n g the t r a n s f e r o f D-glucose from UDP-Dg l u c o s e to f l a v o n o i d s . B i o c h e m . B i o p h y s . A c t a 2 5 8 : 71-87 S u t t e r , A. & H . G r i s e b a c h . 1973. UDP-glucose: f l a v o n o l g l u c o s y l t r a n s f e r a s e from c e l l s u s p e n s i o n c u l t u r e s parsley. B i o c h e m . B i o p h y s . A c t a 3 0 9 : 289-295 Swain, T. 1975. E v o l u t i o n o f f l a v o n o i d compounds. J . B . Harborne , T . J . M a b r y & H.Mabry ( e d s . ) , The Chapman & H a l l , New Y o r k . T i s s u t , M. & K . E g g e r . 1972. Les de q u e l q u e s a r b r e s , a u . c o u r c h e m i s t r y 1 1 : 631-634 Tournefort,  J.P.  1719.  In: Flavonoids.  Glycosides flavoniques foliaires du c y c l e v e ' g e t a t i . f . Phyto-  Institutiones  rei  herbariae.  T u r e s s o n , G. 1922. The g e n o t y p i c a l r e s p o n s e o f t h e to h a b i t a t and c l i m a t e . H e r e d i t a s 3: 211-350 Turner, of  3-0of  Paris. plant  species  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 L u p i n u s t e x e n s i s and L_. s u b c a r n o s u s . Madrono 1 4 : 13-16  T u r n e r , B.L. Austin.  1958.  The Legumes o f  V a u g n , P. & D . B . D u n n . 1 9 7 7 . plex. T r a n s . Mo. A c a d .  Texas.  Univ.  Texas  Press,  The Lupi nus 1 a t i f o l i us Agardh S c i . .10: 8 9 1 0 6  com-  r  V e n k a t a r a m a n , K. 1 9 6 2 . Methods f o r d e t e r m i n i n g the s t r u c t u r e s o f f l a v o n o i d compounds. I n : T . A . G e i s s m a n ( e d . ) , t h e Chemi s t r y o f f l a v o n o i d compounds. M a c M i l l a n , New Y o r k . Waller, in Ward,  G.R. & E . N o w a c k i . 1978. Alkaloid plants. P l e n u m , New Y o r k .  biology  and m e t a b o l i s m  R.S. & A . P e l t e r . 1974. The a n a l y s i s o f m i x t u r e s o f 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 r g a n i c compounds u s i n g HPLC. J . Chrom. S c i . 12 : 570  W a t s o n , S. 1873. R e v i s i o n s o f the e x t r a - t r o p i c a l North American s p e c i e s o f t h e genera L u p i n u s , P o t e n t i 1 1 a and O e n o t h e r a . P r o c . Amer. A c a d . 8: 517-618 Watson, West,  S.  1876.  Proc.  Acad.  1 2 : 250  L . G . , P.M.Birac & D.E.Pratt. S e p a r a t i o n o f the i s o m e r i c i s o f l a v o n e s from soybeans by HPLC. J . Chrom. 1 5 0 : 266-268  W i l k i n s o n , J . G . 1854. II. New Y o r k . Wulf,  Amer.  A popular account  of  the  ancient  L.W. & C . W . N a g e l . 1976. A n a l y s i s of phenolic f l a v o n o i d s by HPLC. J . Chrom. 1 1 6 : 271-279  Egyptians.  acids  and  141 APPENDIX Plants  and  Details of collection A.  Sources c o l l e c t i o n s of plant housed i n UBC e x c e p t  m a t e r i a l used where n o t e d .  in  Nicholls  arboreus 7702,  campus,  argenteus  excubitus  Nicholls  San  j..  1 June 1977.  Thunderbird  Grown  Road.  Jones v a r .  from seed  collected  900 g e x t r a c t e d  johhstonii  7 8 1 7 , 15 June 1978.  (fresh  on UBC  weight).  Gabriel  Mtns.,  Nutt.  Los A n g e l e s  ex T o r r .  Cooper  Dougl . ex  Darlington,  Canyon  Co. C a l i f o r n i a .  entrance, 25 g  (dry).  & Gray  7 8 3 2 , 28 June 1978. 2 miles along H w y . l l near E l k o , E l k o Co. Nevada.  1 e u c o p h y l 1 us  s.  C.P.Sm.  Hwy.2,  L a n o i l l e Canyon Road, 15 g ( d r y ) extracted.  Lindl.  N i c h o l l s 7 7 0 8 , 20 June 1977. Lumby, BC. 400 g ( f r e s h ) j..  All  Pursh  holosericeus  Nicholls off L_.  study.  Sims.  N i c h o l l s 7 8 4 0 , 30 June 1978. Hwy.93, 2 m i l e s Butte C o . , Idaho. 15 g . e x t r a c t e d ( d r y ) . I.  the  Bulk samples o f L u p i n u s s p e c i e s used f o 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 a c c u m u l a t i o n o f f l a v o n o i d compounds t h a t were s u b s e q u e n t l y used i n c o m p a r a t i v e T L C .  Lupinus  L_.  I.  Shuswap Road, extracted..  10 m i l e s  east  of  1 i 11 o r a 1 i s Dougl .  N i c h o l l s 7 7 0 1 , 1 June 1977. R o a d s i d e near T h u n d e r b i r d S t a d i u m , UBC campus, V a n c o u v e r , BC. 50 g ( f r e s h ) e x t r a c t e d . I. r u b e r  Heller  N i c h o l l s 7 8 1 0 , 14 June 1978. h m i l e s. 2 9 9 , C a n b y , Modoc Co. C a l i f o r n i a . J_.  s e l 1 ul us  Kell.  var.  1 obbi i  (Gray  P i t t River 20 g ( d r y )  ex W a t s . )  B r i d g e , Hwy. extracted.  Cox  N i c h o l l s 7 8 3 7 , 29 June 1978. E l k Meadows, Hwy.21, 50 m i l e s Lowman, C u s t e r Co. I d a h o . 12 g dry e x t r a c t e d . j..  s e r i ceus  var.  sericeus  n.  Pursh  N i c h o l l s 7 6 1 5 , 14 May 1976. (fresh) extracted.  Trinity  Valley,  Lumby,  BC.  1.1  kg  142 B.  Lupinus  arboreus  s p e c i m e n s , used i n  Experiment  I,Chapt.  III.  N i c h o l l s 7 7 0 3 , 1 June 1977. T h u n d e r b i r d Road, UBC campus, V a n c o u v e r , BC. Franko s . n . , 22 May 1976. S o . Pender I s l a n d , BC. Eastham s . n . , 2 June 1938. Beacon H i l l P a r k , V i c t o r i a , BC. Hebda s . n . , 1 4 June 1975. P o i n t R o b e r t s , Whatcom C o . W a s h i n g t o n . S p e l l e n b u r g s . n . , 17 June 1966. M o n r o e , King Co. W a s h i n g t o n . S t e w a r t 6 1 6 3 , 22 June 1952. Newport, L i n c o l n Co. O r e g o n . K r a j i n a s . n . , 10 June 1953. P i s t o l R i v e r , J o s e p h i n e Co. O r e g o n . F i n c h 3 1 5 , 6 June 1953. C r e s c e n t C i t y , Del N o r t e Co. C a l i f o r n i a G o r n a l l 0 0 9 0 , 10 June 1977. W e s t p o r t , Mendocino Co. C a l i f o r n i a . Frank 9 2 , 8 J u l y 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 c o l or  Lindl.  specimens  used  in  Experiment  I  Ch.  III.  D a v i d s o n s . n . , 23 May 1914. S a v a r y I s l a n d , BC. ( s s p . b i c o1o r ) N i c h o l l s 7 6 5 1 3 , 13 May 1 9 7 6 . Nanoose H i l l , Nanaimo, BC. ( s s p . b i c o1o r ) D a v i d s o n s . n . , 15 May 1913. R i f l e Range, V i c t o r i a , BC. ( s s p . b i c o 1 o r) M c C a l l a 5 3 2 2 , 24 A p r i l 1939. M o s i e r , Wasco Co. O r e g o n . Maze et_ aj_. 765 , 4 A p r i l 1971. S h a s t a C i t y , S h a s t a Co. C a l i f o r nia, (ssp. tridentatus (Eastw.)Dunn) Maze e t aj_. 742 , 4 A p r i l 1971. O a s i s Rd. e x i t ' I 5, S h a s t a C o . California. ( s s p . umbel 1 a t u s ( G r e e n e ) D u n n ) Maze et aj_. 9 1 4 , 13 A p r i l 1971. E l k C r e e k , Glen Co. C a l i f o r n i a . T~ssp. umbel 1 a t u s ) Booras 1, 26 A p r i l 1955. C l e a r l a k e P a r k , Lake Co. C a l i f o r n i a . Dunn 9 3 8 , 29 March 1 9 4 1 . Pomona, Los A n g e l e s Co. C a l i f o r n i a , ( s s p . mi c r o p h y l 1 us ( W a t s . )Dunn Dunn 1278, 10 June 1 9 4 1 . H a r p e r ' s R a n c h , San Diego Co. C a l i f o r nia, ( s s p . m a r g i n a t u s Dunn)  143  D.  Lupinus  sericeus  collections  f o r Experiment  II,  Ch.III.  Population number 1  N i c h o l l s 7 8 2 5 , 22 June 1978. M i l e Jacob Lake, Coconino Co. A r i z o n a .  5 9 3 , Hwy.89  5 miles  2  N i c h o l l s 7827, D i x i e l a n d Nat.  3  Nicholls turnoff,  4  N i c h o l l s 7 8 2 9 , 27 June 1 9 7 8 . P i u t e Co. Utah.  5  N i c h o l l s 7 8 3 0 , 27 June 1 9 7 8 . 3 m i l e p a s t J o e ' s V a l l e y R e s e v o i r , Emery C o . U t a h .  6  N i c h o l l s 7 8 3 1 , 27 June 1978. R e c r e a t i o n S c h o f i e l d R e s e v o i r , Carbon Co. U t a h .  7  N i c h o l l s 7 8 3 5 , 29 June Boise Co. Idaho.  8  N i c h o l l s 7836 , 29 June 1978'. Lowman, B o i s e Co. I d a h o .  9  N i c h o l l s 7 8 4 1 , 1 m i l e w. c a m p g r o u n d , P e b b l e Lava Hot S p r i n g s , Bannock C o . I d a h o .  10  N i c h o l l s 7 8 5 1 , 5 J u l y 1 9 7 8 . Banks o f H e n r y ' s n. A s h t o n , Fremont C o . I d a h o .  11  N i c h o l l s 7853 , 6 J u l y 1 9 7 8 . M i l e 2 8 , Hwy.19.1, West Y e l l o w s t o n e , G a l l a t i n C o . M o n t a n a .  20 m i l e s  n.  12  N i c h o l l s 7854, 6 J u l y 1 9 7 8 . \ m i l e L i v i n g s t o n e , Park Co. M o n t a n a .  I90/89N,  e.  13  N i c h o l l s 7 8 5 5 , 7 J u l y 1 9 7 8 . Hwy.89, 15 m i l e s S u l p h u r S p r i n g s , Meagher C o . M o n t a n a .  14  N i c h o l l s 7858, 7 J u l y Cascade C o . M o n t a n a .  1978.  Hwy.89,  15  N i c h o l l s 7859, 7 J u l y Teton Co. Montana.  1978.  Hwy.287,  16  Nicholls Browning  24 June 1978. North o f E s c a l e n t e F o r . G a r f i e l d Co. U t a h .  7828, 26 June 1 9 7 8 . S e v i e r Co. Utah.  1978.  Hwy.24,  1 mile  Mountain  Hwy.21,  s.  Little  Creek  Marysvale,  campgroud sw. area,  27 m i l e s  Mountain  on Loa Rd.  s.  road w.  s.  w. s i d e o f n.  Boise,  View c a m p g r o u n d , Creek  Fork,  juction  5 miles 9 miles  7 8 6 0 , 7 J u l y 1978. Hwy.89, m i l e and C h o t e a u , T e t o n C o . M o n t a n a .  88,  Road,  n.  2 miles  White  n. Monach, s.  Choteau,  between  144  D.  (contd . )  P o p u l a t i on number Hwy.2,  8 miles  e.  17  N i c h o l l s 7 8 6 1 , 8 J u l y 1978 P a r k , F l a t h e a d Co. Montana  18  N i c h o l l s 7862, 8 J u l y campground. 10 m i l e s  19  N i c h o l l s 7 8 6 3 , 8 J u l y 1978. Hwy.2, M o y i e r M o y i e r R i v e r b r i d g e , Boundary C o . I d a h o .  20  N i c h o l l s 7 8 6 7 , 8 J u l y 1978. Coeur d ' A l e n e L a k e , K o o t e n a i  Hwy.95a, 2 m i l e s Co. I d a h o .  21  N i c h o l l s 7872, Washington.  9 July  1978.  Central  22  N i c h o l l s 7873, 9 J u l y P e r c e Co. I d a h o .  1978.  Hwy.95,  23  Packer N i c h o l l s 7 8 7 4 , 9 J u l y 1978. New Meadows, Adams Co. Idaho  24  N i c h o l l s 7 8 7 7 , 10 J u l y 1978. Powder, Union Co. O r e g o n .  Hwy.237,  25  N i c h o l l s 7 8 7 9 , 10 J u l y 1978. Yakima Co. W a s h i n g t o n .  Hwy.12,  26  Hwy.821, m i l e N i c h o l l s 7880, 10 J u l y 1978. o f Yakima R i v e r , Yakima C o . Washi ngton .  27  N i c h o l 1 s 7881 , 11 J u l y 1978 Okanogan C o . W a s h i n g t o n .  Hwy.97,  28  N i c h o l 1 s 7882 , 11 J u l y 1978 Douglas C o . W a s h i n g t o n .  Hwy.151,  29  N i c h o l l s 7905 F r u i t v a l e , BC  2 July  1979.  Route  30  Nicholls  5 July  1979  Trinity  Valley,  31  N i c h o l l s 7910 P a r k . Route 6  2 J u l y 1979 Alberta .  1 km n.  Waterton  32  N i c h o l l s 7914, 3 J u l y Nanto.n, A l b e r t a .  7916,  West  Glacier  1978. Hwy.2, 2 m i l e s w. Paul s. L i b b y , L i n c o l n Co. Montana  1979.  Route  River  Ferry, s.  3,  2,  Rd  s.on  w e.  Garfield  Lewiston,  John's  Bunyan  side Co.  Nez  Cabin S t a t e  4 miles 6 miles  n. nw.  10,  1 mile  s.  7 miles between  Park,  North Yakima,  flood  plain  Brewster, e.  Orondo,  Creston Lumby,  BC  Lake  Nat  Between P a r k l a n d  and  and  145  E.  Lupinus  col 1ections  Chapter  VI.  OTU or No . 1  used f o r  interspecific  variation  taxon  Lupinus  elatus  Johnston  N i c h o l l s 7 8 1 8 , 15 June 1978. Cooper C a n y o n , o f f G a b r i e l M o u n t a i n s , Los A n g e l e s Co. C a l i f o r n i a . 2  I. b a r b i ger  4  L_.  sericeus  5  var.  j . . s e r i ceus  huf fmani i  L_.  I.  ssp.  var.  var.  _L.  bi'color  Nicholls 8  L.  9  10  f 1 exuosus  7 6 1 3 5 , 13 May  12  79-448.  I. 1emmoni i  C . P . Sm.  Route  1976.  1 mile  s.  Little  Creek  3,  mile  3 5 , near  ex Agardh) 3,  Johnstone  Wardner  C.P.Sm. Creek  camp-  Nanoose  Hill,  Nanaimo,  BC.  Nanoose H i l l ,  Nanaimo,  BC.  New M e x i c o .  1978. Hwy.89, m i l e 5 7 3 , 5% L a k e , C o c o n i n o Co. A r i z o n a .  miles  Hook.  Benson 9 3 2 8 , 24 A p r i l [Logan),  1966.  L.  var.  den s i f l o r us B e n t h .  Eastham  & Dunn  Greene  Yatskievych  texensis  Anarchist,  Greene  I. neomexicanus  J_.  Mount Fleak  (Lindl.  7 6 5 1 3 , 13 May 1976.  N i c h o l l s 7 8 2 4 , 22 June e. J e t . 6 7 . . near J a c o b 11  Plateau, Co. Utah  Lindl .  p o l y e a r pus  Nicholls  Aquarius Garfield  C.P.Sm.  Route  N i c h o l l s 7 9 0 2 , 1 J u l y 1979. g r o u n d , B r i d e s i l i e , BC. 7  1964.  Hwy.24,  egglestonianus  7907, 2 J u l y ' l 9 7 9 .  sericeus  Posy L a k e , Escalente,  (C.P.Sm.)  7 8 2 8 , 26 June 1978. S e v i e r Co. U t a h .  sericeus  San  sericeus  6 4 5 - 3 7 , 23 May  Nicholls BC. 6  Pursh  Krajina & Fraser Osoyo.os, BC.  Nicholls turnoff,  Hwy.2,  Wats.  Holmgren 2 4 6 1 , 14 August 1965. D i x i e l a n d N a t . F o r . 15 m i l e s n. 3  study  6 8 6 9 , 11 June 1 9 3 9 .  Brownwood,  Brown Co.  Texas  dens i f T o r u s Beacon  Hill,  Victoria,  BC.  146 13  J..  dens i f 1 orus  var.  aureus  (Kell.)  Bohm & Banek 1 4 1 3 , 9 May 1980. Mendocino Co. C a l i f o r n i a . 14  j . . ruber  L_.arboreus  L_.  horizontal is  L.  L_.  f1avoculatus  L_.  bent hami i  L_.  23  Kern  Co.  27, Test  Site,  Nye  New Cayama, San  Luis  Obispo  1962.  Temescal  Canyon,  Benth.  '(Wats .)Wats .  I. s t o l o n i f e r u s  1935.  Egglestone (Logan).  8 9 0 4 , 13 September  L.  Wats .  wyethii  & Fraser  hill i i  Graham Mtn.s.  Graham C o .  L. 1972.  645-21 , 22 May 1964.  Athol,  Bonner  Olalla,  Co.  Idaho  B.C.  Greene  H i g g i n s 9072, 9 August 1 9 7 3 . U t a h . (Logan) 25  Plot  & M u h l i c k 2 2 1 5 3 , 16 March Co. C a l i f o r n i a .  I. a r i z o n i c u s  I..  Desert,  007 ,  Krajina 24  Mojave  1965.  12 May 1962.  M a g u i r e 10152 , 20 March A r i z o n a . (Logan) 22  UBC campus,  Heller  s pa rs i f 1 or u s  Foust 21  Canby,  cone i nnus A g a r d h  Hitchcock Riverside 20  Bridge,  Heller  B r e e d l o v e 2774, Co . Cal i f o r m ' a . 19  River  T h u n d e r b i r d Rd.  1974.  B e a t t y & C a r l 3 3 8 2 , 9 June Co. Nevada. (Logan) 18  Pitt  Heller  Holmgren 7 6 5 9 , 7 A p r i l C a l i f o r n i a . (Logan ) . 17  76.16,  Sims.  N i c h o l l s 7 7 0 2 , 1 June 1977. V a n c o u v e r , BC. 16  Hwy.101, m i l e  Heller  N i c h o l l s 7 8 1 0 , 13 June 1978. Modoc Co. C a l i f o r n i a . 15  Munz  I. h o i o s e r i ceus  Nutt.  ex T o r r .  N i c h o l l s 7 8 3 2 , 28 June 1978. Rd. E l k o , E l k o Co . Nevada .  Pine V a l l e y ,  Washington  Co.,  Lanoille  Canyon  & Gray 2 miles  along  147  26  j . . parvi f l orus  Nutt.  Morgan 7 , 17 August Co. I d a h o . (Logan) 27  I. c a u d a t u s Nicholls Coconino  28  Kell.  7 8 2 2 , 22 June Co. A r i z o n a .  J _ . nevadens i s  30  L.  31  7708,  33  2399,  35  Dougl.  Eastham  8 May  s.n.,  L_. . a r g e n t e u s  L.  argenteus  caudatus  Canyon,  Humboldt  Co.  Shuswap  Rd.  10 m i l e s  e.  Lumby  Red B u t t e  Canyon, S a l t  Lake  City  Panaca,  Lincoln  Co.  Nevada.  Rydb. Riggins,  Idaho  Co.  Idaho  (Logan)  1976.  ex  East  banks  of  Lake  Okanagan,  Hook.  1940.  Kaleden,  BC.  Wats.  Welsh 1 7 5 4 9 , (Logan) 38  Grand  Lindl.  1944.  10 J u l y  J _ . s u l phureus  L..  ex  1909.  var.  1948.  var.  Sierra  argenteus  N i c h o l l s 7 9 1 2 , 30 June bridge, Alberta. 37  Rim,  C.P.Sm.  Gould 4 7 1 5 , 1 June A r i zona . 36  South  Sage Hen H i l l s ,  28 June 1 9 4 0 .  7622,  I. p a l m e r i  Freemont  Nels.  7 July  I. 1 u t e s c e n s Nicholls BC.  34  Dougl.  j . . pseudoparvi f 1 orus Davis  Resevoir,  Nels.  Galway 2 1 2 5 9 , (Logan) 32  1978.  1978.  1 9 0 5 , 21 J u l y (Logan)  _L . g r e e n e i  Park  Greene  20 June 1 9 7 7 .  X alpestri s  Smith Utah.  Island  Heller  j . . 1 e u c o p h y l 1 us Nicholls BC.  1963.  & Arn.  X I. h i l l i i  Tiehm 4 2 1 0 , 19 May N e v a d a . (Logan) 29  ex Hook.  1979.  1978.  Gila  Co.  Pursh  rubricaulis  13 J u l y  Ancha M t n s .  Route  5,  (Greene)  Monroe M t n .  30 m i l e s  s.  leth-  H e s s & Dunn Sevier  Co.  Utah.  Kel1.  Nicholls 7843, Colorado.  3 July  1978.  Hwy.  1 - 7 0 , near  Idaho  Springs  148  39  j..  arbustus  Grierson 40  j..  Dougl . ex L i n d l .  & J a c k s o n s . n . , 16 May  grayi i  L.. a l b i f r o n s B e n t h . ex  Seed  I. a r c t i c u s Wats. Taylor  45  47  1963.  Costa  Co.  1945.  H a l f Moon  Canyon,  Cassiar,  BC.  JL. a r c t i c u s s s p . c a n a d e n s i s (C.P.Sm.) Dunn 5 0 5 1 , 30 J u l y  1938.  P a r a d i s e , Mt. R a n i e r , Wash.  L_. a r c t i cus s s p . s u b a l p i n u s  (Piper  & R o b b i n s ) Dunn  Brayshaw  1954.  Mt. C h e l a n , BC  s . n . , 21 September  I. 1 a t i f o l i us A g a r d h Sisters,  D e s c h u t e s Co.  Oregon.  L. 1 i t t o r a l i s Dougl . N i c h o l l s 7 7 0 1 , 1 June 1977. campus, V a n c o u v e r , BC.  49  San  s s p . a r c t i cus  M a r t i n 4 8 3 0 , 21 June 1939. (Logan) 48  A n t i o c , Contra  11916, 3 J u l y (Logan)  375, 18 June 1956.  McCalla 46  Empire Union High S c h o o l ,  L.. humi c o l a N e l s . Hitchcock & Muhlick F e r g u s Co. M o n t a n a .  44  s o u r c e : Rancho S a n t a Anna Gard  I. c h a m i s s o n i s E s c h s . B r e e d l o v e 4 2 8 1 , 29 March C a l i f o r n i a.  43  Valley  Lindl.  M c C a l l a 6050 , 9. May 1940. Diego Co. C a l i f o r n i a . 42  Wenachee  (Wats.)Wats.  Grown from seed a t UBC. 41  1948.  J_. a r i d u s D o u g l . ex  Near T h u n d e r b i r d  Stadium,  UBC  Lindl.  H i t c h c o c k 4 9 7 7 , 28 June 1939. F l o o d p l a i n o f Roger R i v e r , F a r e w e l l Bend F o r e s t camp, J a c k s o n Co. Oregon (.Logan). 50  J.. c o n f e r t u s  Ke 11 .  H e l l e r 1 5 1 6 0 , 14 June 1938. C a l i f o r n i a . (Logan) 51  L. minimus  Feather  River  hwy.  Gray's  Dougl.  C o n s t a n c e 1873, 1 June 1937. Anatone B u t t e , Blue A s o t i n Co. W a s h i n g t o n . ( L o g a n ) 52  Flat  Mtns.  L.. 1 ya 11 i i G r a y Taylor  1330 , 14 J u l y  195.1.  Cathedral  Lakes, Ashnola,  BC.  149  53  JL.  s el 1 ul us  Nicholls 54  _L.  55  J_.  J_.  & Gillett  L_.  di f f usus  58  59  1384, 25 A p r i l  6  0  k-  1967.  7 3 3 2 , 30 June  Atlin,  1968.  62  1953.  Grant  11292 ,  1e p i d u s D o u g l .  ex  1953.  63  1954.  L_.  1 2 8 , 5 September BC.  1 952.  64  65  7904, 2 J u l y  J_.  Grant  Co.  Co. O r e g o n .  Vermillion (Logan)  Thornhill  Mtn.  Valley,  Prince  43,  1979.  Route  3 near  Nancy  Green  17 A p r i l  s.n.,  excubitus  1962.  Laxahaw,  South  Carolina.  Lindl .  12 August  1951.  Burnaby,  BC.  Jones  N i c h o l l s 7 8 1 7 , 15 June 1 9 7 8 . Hwy.2, Cooper C a n y o n , G a b r i e l M t n s , Los A n g e l e s Co. C a l i f o r n i a . 66  Lake,  L.  _L . pol y p h y l 1 us Davis  Baker,  Wats .  perennis  McGaha  Co.  Greene  Eastham Rupert,  Nicholls BC.  Humboldt  Co. Oregon.  Grant  Sims.  burkei  Co.  Lindl.  7 3 1 8 , 28 June  culbersonii  17 June  j . . n o o t k a t e n s i s Donn. ex  J_.  BC  Chesterfield  Winnemucha,  E v e r e t t & Johnson 7 4 1 9 , 18 J u l y 1935. S i e r r a N e v a d a , F r e s n o Co. C a l i f o r n i a . 61  Oregon.  Nutt.  & Taylor  Cronquist  Klamath,  Wats.  I. c a e s p i t o s u s  J_.  n.  Cox  Wats .  cu s i c k i i  Gillett Oregon.  Hwy.58,  ex W a t s . )  Nutt.  uncial is  Cronquist  1978.  (Gray  2 5 1 4 4 , 9 June 1960.  G e n t r y 1 6 0 0 , 21 June Nevada. (Logan) 57  1 obbi i  Eastw.  Leonard & Radford South C a r o l i n a . 56  var.  7 8 0 2 , 13 June  kuschei  Calder  Kell.  j _ . 1 o n g i f o l i us  San  (..Wats . )Abrams  N i c h o l l s 7920 , 9. A u g u s t 1 9 7 9 . Grown from seed at UBC. c o l l e c t e d at Rancho Santa Anna B o t . G a r d . 16 June 1978.  Seed  150  67  J.. a 1 b i c a u 1 i s D o u g l . ex  Hook.  H i t c h c o c k 2 4 3 9 6 , 22 May 1966. A r e a , Multnomah Co. O r e g o n . 68  L_. a n d e r s o n i i  J_. formosus  J_. k i n g i i  72  73  Pursh  ssp.  9 0 4 3 , 16 May  J.. p u s i 11 us s s p . Boivin  Lake,  Mt.  Lassen  Vol-  Lockwood, M o n t e r e y  Co.  Cali-  Wats.  J_. p u s i 11 us Cronquist  Summit  1960.  Holmgren 7 3 9 6 , 25 August New M e x i c o . (Logan) 71  Conservancy  Greene  B a c i q u l u p i 7 3 8 5 , 9 May f o r n i a . (Logan) 70  Nature  Wats.  M c C a l l a 6 3 1 2 , 26 June 1940. canic Park, C a l i f o r n i a . 69  Camassia  I. hi r s u t i ssimus  rubens  (Rydb.)Dunn  1961.  Hite,  p u s i 11 us  9 9 3 1 , 27 J u l y  Manzano M t n s .  1973.  1952.  Garfield  Bernalillo  Co.  Co.  Utah.  Pursh Agassiz  Coulee,  Empress,  Alberta.  Benth.  H i t c h c o c k 5978 , 9 A p r i l Cal i f o r n i a . (Logan)  1940'.  Cuyamara  Lake,  San  Diego  Co.  151  APPENDIX  II.  S o l v e n t systems and media used i n the t h i n - l a y e r c h r o m a t o g r a p h y o f f l a v o n o i d s from L u p i n u s . R a t i o s a r e by v o l u m e .  Polyamide  DC6.6  a)  water  70: n-butanol  b)  water  55: methylethyl  c)  benzene 5 5 : m e t h y l e t h y l  d)  dichioroethane  50: methylethyl  ketone  2 5 : methanol  2 1 : water  4  e)  dichloroethane  50: m e t h y l e t h y l  ketone  2 0 : methanol  2 0 : water  5  acetic  Silica a)  acid  15: acetone ketone  10: dioxane  20:  ketone  5.  isoporponol 20: a c e t i c  2 2 : methanol  2 0 : water  acid 5  3.  5.  gel  n-butyl water  acetate  60: m e t h y l e t h y l  ketone  25: a c e t i c  acid  12:  3.  Cel1ulose a)  water  90: a c e t i c  acid 10.  b)  water  85: a c e t i c  acid  c)  ethyl  acetate  15.  10: p y r i d i n e  3.2:  water  2,(for  sugars  only).  APPENDIX UV S p e c t r a l a b s o r p t i o n i s o l a t e d from L u p i n u s  maxima  (nm)  III. of  some o f  the  flavonoids  apigenin  apigenin  MeOH: 268 334 NaOMe: 275 326 390 A l C I - : 276 300 348 382 A l C I , / H C l : 275 302 378 NaOAc : 275 302 378 N a O A c / H B 0 : 268 336  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 N a O A c / H B 0 : 268 335  apigenin  vitexin  3  3  4'-0-glucoside  7-0-glucoside  3  3  MeOH: 268 300s 329 NaOMe: 275 295s 260 A l C I - : 277 295s 340 384 A l C I , / H C l : 277 292s 336 379 Na OAc: 276 356 N a O A c / H B 0 : 269 333  MeOH: 270 302s 339 NaOMe: 280 331 397 A l C I - : 279 306 350 386 A l C I , / H C l : 279 304 344 384 NaOAc: 278 304 381 N a O A c / H B 0 : 270 342  i s ovi t ex i n  acacetin  MeOH: 269 303 338 NaOMe: 279 330 396 A 1 C 1 , : 279 306 349 385 A l C I ~ / H C l : 279 305 344 382 NaOAc : 278 304 386 N a O A c / H B 0 : 270 344  MeOH: 269 327 NaOMe: 276 364 A l C I - : 276 300 338 382 A l C I ; / H C l : 276 296 334 378 Na OAc: 276 359 N a O A c / H B 0 : 69 329  acacetin  cytisoside  3  3  3  3  7-0-glucoside  3  3  3  3  MeOH: 268 325 NaOMe: 287 359 A 1 C 1 - : 277 302 347 386 A l C I ^ / H C l : 277 299 339 382 NaOAc: 268 324 N a O A c / H B 0 : 268 326  MeOH: 272 342 NaOMe: 278 396 A l C I , : 266 273 300 358 390 A l C I , / H C l 266 273 300 352 380 NaOAc : 277 370 N a O A c / H B 0 : 272 348  luteolin  luteolin  MeOH: 254 268 351 NaOMe: 268 329s 405 Al CI : 274 302s 328 426. A l C I ^ / H C l : 274 298s 354 388 NaOAc : 269 325s 382 N a O A c / H B 0 : 259 302s 374 425  MeOH: 256 269s 350 NaOMe: 265 300s 398 A l C I - : 275 302s 330 429 A l C I ; / H C l : 275 295s 358 390 NaOAc: 260 365s 405 N a 0 A c / H , B 0 , : 260 372  3  3  3  3  3  3  7-0-glucoside  153  luteolin  4 -0-glucoside  orientin  1  MeOH: 243 272 335 NaOMe: 270 295s 376 A l C I : 278 298s 352 386 A l C I ^ / H C l : 278 290s 344 382 NaOAc : 278 320s 370 N a O A c / H B 0 : 272 344  MeOH: 258 271 297s 351 NaOMe: 272 336s 408 A l C I • 276 302s 334 428 A 1 C U / H C 1 : 268 277 298 358 386 NaOAc: 279 330 388 N a O A c / H . B 0 : 266 378 434s  isoorientin  orientin  MeOH: 258 271 353 NaOMe: 272 336s 409 A l C I _ : 276 302s 332 429 A l C I | / H C 1 : 278 298 357 384 NaOAc: 276 328 392 N a O A c / H B 0 : 266 377 430s  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 N a O A c / H B 0 : 276 338  c h r y s o e r i ol  chrysoeriol  MeOH: 244 270 347 NaOMe: 264 274s 329s 405 A 1 C U : 264 274 295 388 A l C I | / H C 1 : 275 292 350 384 NaOAc: 271 320 391 N a O A c / H B 0 : 268 349  MeOH: 254 269 346 NaOMe: 254 270 350 400 A l C I o : 264 275 300 386 A l C I ^ / H C l : 264 272 296 350 380 NaOAc: 267 385 N a O A c / H B 0 : 268 347  4'-0-methylorientin  genistein  MeOH: 254 269 290s 343 NaOMe: 274 302s 390 A 1 C U : 270 294s 366 386 A l C I f / H C I : 266 350 378 NaOAc: 281 324 372 N a O A c / H B 0 : 254 272 346  MeOH: 261 330s NaOMe: 276 330s A 1 C U : 272 305s 372 A l C I f / H C I : 272 305s 372 NaOAc: 271 336s N a O A c / H B 0 : 262 336s  genistein  unknown  3  3  3  3  3  3  3  3'-0-glucoside  3  3  3  3  3  3  3  3  7-0-glucoside  7-0-glucoside  3  'pink'  (flavanone)  MeOH: 263 326s NaOMe: 272 360 A l C I _ : 274 302s 372 A l C I ^ / H C l : 274 302s 370 NaOAC : 263 327s N a O A c / H B 0 : 263 327s  MeOH: 287 326s NaOMe: 246 329 A 1 C U : 276 312 367 A l C I , / H C l : 274 308 366 NaOAC: 278 330 N a O A c / H B 0 : 286 330s  kaempferol  quercetin  3  3  3  7-0-glucoside  MeOH: 268 273 283 315 364 NaOMe: 274 283s 4 2 4 d e c . A 1 C U : 269 305s 356 420 A l C I ; / H C l : 269 304s 350 420 NaOAC: 275 283 380 415s N a O A c / H B 0 : 273 283 318s 366 3  s denotes dec  3  3  3-0-glucoside  MeOH: 257 269s 302s 365 NaOMe: 273 330 310 A 1 C U : 275 306s 330s 440 A l C I ~ / H C l : 268 298s 366 405 NaOAc: 274 324 376 N a O A c / H B 0 : 262 294s 370 3  3  shoulder  denotes  decomposition  of  peak  after  3 minutes.  154  APPENDIX  Raw HPLC d a t a : of  peak h e i g h t s  of  IV  17 f l a v o n o i d s  from 181 i n d i v i d u a l s  Lupinus s e r i c e u s .  (For  e x p l a n a t i o n of  P o p u l a t i on No .  compound Nos. see t e x t  Plant code No.  or  Fig.  Compound Nos t—1  CM  ro "3-  LO  to  00  cn  O  >—i  6  x—1CM »—1 I—1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  251 252 253 254 255 257 258 259  24156 0 20 25 13 11 34 196 0 25 21 12 15 22252 0 0 19 10 1 1 9 26 13100 80 16 12 23240 0 0 23 14 15 34360 0 32 32 14 15 22142 0 22 27 14 19 24228 0 14 17 10 12  10 11 10 9 1 1 13 16 10  9 10 9 9 1 1 10 13 9  12 11 8 0 0 13 10  271 272 273 274 275 276 277 278 279 27 10  25260 15194 28102 16172 34228 763 12 36220 51224 4 1 182 46420  0 0 0 0 0 0 O 0 0 0  18 18 0 20 14 16 23 28 23 16  25 20 13 21 12 14 37 16 16 25 14 13 25 1 1 12 22 15 13 29 15 20 31 2 1 28 23 14 14 17 10 13  10 1 1 12 1 1 9 9 12 18 1 1 8  10 10 12 13 8 9 1 1 16 1 1 8  0 12 0 0 12 15 0 21 0 9  0 0 0 0 0 0 0  28 1 282 28 3 284 285 '286 287 288 289  28 176 33176 23268 28196 26204 40252 34248 23200 31 158  0 0 O 0 0 0 0 0 0  2 1 27 18 30 16 29 27 25 21  26 24 21 28 18 29 29 26 24  1 1 10 9 12 7 13 12 1 1 10  1 1 10 9 1 1 6 12 1 1 1 1 9  13 13 13 15 9 0 15 12 12  0 0 0 0 0 0 0 0 0  29 1 292 293 294 295  19 160 37332 39346 21 188 34304  18 19 1 t 10 9 1 1 0 21 22 10 1 1 9 9 0 27 33 19 16 13 14 0 22 25 13 12 10 9 0 19 22 13 1 1 10 9  301 302 303 305 304 306  42368 59392 20170 24 160 64308 20156  0 28 30 15 0 0 26 15 0 15 25 12 0 23 31 20 0 31 34 16 0 16 20 1 1  31 1 312 313 314 315 316 351 352 353 354 355 356 357 .358  7)  13 14 1 1 15 8 17 14 12 12  13 15 13 15 8 15 15 14 13  o  o 0 0  10 0 1 1 0 0 12 15  0 0 0  LO  LO  I-H  r*H  I—1  t  8 9 7 7 9 9 9 0  0  0  0 0 0 0 0  0 0 0 0 0  0 8 7 6  0 0 0 0 0 0 0 0  7 0 0 9 0 10 0 10 0 0 7 0 0 10 0 12 0 9 0 7  0  0 0 0 0 0  0 0  0 0  0  0 0  0 0  0 0 0  0 0 0  o 0 0 0 0 0 0  CO I—1  10 9 8 10 6 10 10 9 8  9 0 0 9 0 13 0 9 0 0  o o 0 0  o 0 0 0 0 0 0 0 0  o 0  o 0 0  o o o o o  0 0  o  0 0 0  0 0 0 0  16 12 15 1 1 14 1 1 22 18 15 9 12 9  12 15 12 16 16 0 15 20 8 14 1 1 1 1  6  0 16 o 10 o 0 0 12 7 0 0 8  0  0  0 0 0 0 0  0 0  7 12 10 O 19 0 36300 0 29 26 16 19232 0 25 26 22 33108 0 22 25 18 23208 0 21 23 12 53350 0 28 32 21  6 15 18 14 14 15  6 12 14 1 1 12 37  0 16 20 17 13 19  0 0 0 0 0 0  0 0 0 0 0 0  O 10 11 10 10 11  0 0 0  61 38 24 15 77 38 63 91 13102 47 31 31 14 16 18  9 10 9 10 8 0 8 8 8 8 9 0 1 1 10 10 14 1 1 13 17 0 9 10 10 9 0 0 8 8 7 8 7 0  0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0  0 0 0 0 0 00 10 0 11 0 0 0 o 0 0 0  27 0 34 12 12 0 19 9 29 10 27 0 57 23 23 20 0 22 37 12 23 14 26 14 13 10 22 9 14 14 16 8  6 13 15 1 1 12 14  8 8 8 0 14 8 10 9 0 7 9 0 8 7  0  0 8 8 9 O 9 9 8 0  0 0 0 0 0 0 0 0 0  O 7  0 0  o 0 0 0 0  o o 0 0  1 1 0 O 0 0 0  o o 0 o o o 0 0  0 0 10 11 9 0  0 0 0 0 0 0  0 0 0 o 0 0 0 00  O 10 IO 0 0 10  o  0 0 0 9 0 9 0 0  0 0 0  oo  0 0 0 0  o 0 0 0  0 c  0' 0 0  o 0 0  o 0  155  36 361 362 363 364 365  13 63 29 64 25 37 36134 44 28 8130  23 33 29 0 31 0  1 1 14 15 14 19 0  12 19 19 23 24 25  41 1 4 12 4 13 4 14 4 15 4 16  1 1. 26 65 15 18 36 71 14 16 52 16 84  17 16 21 0 35 27  10 10 0 14 16 18  7 8 0 15 O 8 14 9 8 7 7 0 17 8 8 6 6 8 19 10 9 8 8 0 22 1 1 10 9 10 0 23 1 1 16 10 14 10  511 512 513 514 515 516  124105 38 87 41 61 4 7 90 44 47 42 94  64 64 42 43 42 35  27 22 39 33 31 26  30 26 22 12 38 25 30 19 28 16 28 8  53 531 532 533 534 535 54 1 542 543 544 545 546  16 66 13100 14 92 14 89 162 10 13 67 9 16 53 22 40 10  32 28 59 22 54 12  50 84 0 32 82 96 27 42 60 38 69 22  21 31 36 22 32 16 16 22 32 17 29 18  30 37 24 19 38 27 28 43 42 25 38 25  9 10 10 8 10 0 7 15 28 1 1 17 15  12 13 10 13 13  7 8 0 7 7 1 1 7 7 1 1 6 7 10 0 8 10 8 7 11 6 5 0 12 O 12 12 16 1 1 10 9 10 12 1 1 14 12 1 1 0  13  55 551 553 554 555 556  14 31 27 46 18 27  33 55 88 53 79 168  24 0 97 65 73 72  15 28 38 78 32 62  22 46 40 79 41 49  10 18 19 18 1 1 12  8 13 17 12 1 1 9  9 9 1 1 1 1 12 12 1 1 10 9 9 0 8  14  58 1 582 '583 584 585 586  83 132 86 22124 7 1 20140110 26100 0 16 88 0 9 52 0  60 42 48 38 48 24  57 17 40 14 52 15 27 13 36 15 33 12  "14 13 12 10 1 1 10  15  59 1 •592 593 594 595 596  12 57 52 24110104 38104 0 10 73 0 35144 7 1 11 8 1 1 0 0  25 40 53 27 33 28  38 12 54 15 28 13 38 14 40 14 51 14  12 10 1 1 9 10 9 0 10 9 1 1 9 12  13 12 13 1 1 12 1 1 0 0 12 12 12 12  0 7 1 1 8 0 9 7 0 10 0 0 9 0 6 0 0 0 9 8 0 8 0 0 10 7 10 9 0 0 10 0 5 8 8 0 1 1  0 0 1 1 0 0 9 0 0 9 0 31 9 0 34 9 0 0 8 0 0 0 0 29 9 0 0 19 0 0 9 0 0 7 0 0 9  131OO110  49200 37 130 21 190 39130 31 138  77 66. 90 94 90  0 59 34 58 45 57  65 41 41 66 51 77  7 9 9 5 0 0  0  16  601" 602 603 604 605 606 61 1 612 614 615 616  31204 8 67 42 60 21 91 13124  79 4 1 42 22 0134 66 32 72 28  621 622 623 624 625 626  7 20 10 10 98 0 5 33 0 13 50 0 39100114 1 1 46 0  8  9  11  12  17  18 29 27 19 52 20  13 10 9 1 1 12 9  10 10 10 9 9 9 8 7 7 13 12 1 1 12 13 10 8 8 10  13 10 18 15 14 9 8 8 7 7 8 8 6  12 10 15 1 1 14 8  13 10 1 1 10 10 9 10 9 13 10 14 9 1 1 53 1 1 9 13 10  1 1 9 14 10 13 8  10 0 0 1 1 12 0  15 0 0 0 0 8  9 13 0 0 10 10  0 0 0 0 0 0  0 10 00 0 8 0 0 0 o 0 9 0 0 9 0 0 O 0  0 0 0 0 0 0  0 0 O 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0  0 0 0 0 o 8  0 0 0 0 0 0  0 0 0 0 0 0  0  10 0 0 0 12 0  52 20 0 20 31 0  0 0 0 0 7 0 10 0 0 9 10  15 8 0 0 9 17 0 0 0 1 1 28 0  O 9 14 10 8 0  0 0 0 0 0 O  12 9 10 10 14 14  1 1 9 10 10 13 12  14 10 13 14 15 0  45 14 22 1 1 73 21 38 16 36 1 1  1 1 8 16 13 9  10 0 14 1 1 8  9 7 15 1 1 8  8 0 0 6 0 0 14 1 1 0 8  16 75 24 24 65 39  10 13 0 14 19 10  10 0 12 0 0 0 13 12 18 17 10 10  1 1 17 14 24 24 0  0 0 0 0 o 0  0 0 0 o o 0  10  0 0 0 0 0 0  0^ 0 0 0 0 o  0 0 0 0 0  8  0 0 12 0  0 0 7 0 0 0 0 6 0 25 8 0 7 0 0 6 6 0 0 0 0 0 6 0 0 0 0 6 0 0 5 0 0 0 4 10 9 0 0 0 7 10 0 36 10 8 0 0 0 0 0 0 10 0 ' 0 9 10 11 0 27 0 7 8 0 0 0 7 11 o 0 0 0 15 0 31 0 0 1 1 o 30 0 0 0 0 0 7 0 7 0 o  12 10 12 12 14 15  17 14 16 16 16 21  0 0 0 0 0 12 0 0 0 8 0  6  9 7  0 8  8  10 9 9 13 14 1 1  0  0 o 0 0  6  0 0 8 8 0 O 0 8 0 7 0 0  9 0 0 o 8 0 24 18 0 0 8 o 0 12 0 0 10 10  O 8 5 0 7 12 8 13 0 0  8 o 0 0 20 8 0 0 14 0 0 11 0 20 8  o  8 7 0 0 0 25 8 0 0 14 4 0 11 8 0 0 0 0 10 15 0 0 8 0  0 13 0 0 O O 0 0 0 0 0 0 0 O 14 0 0 o  b  0 0 0 0 0 0  11  0 9  631 633 634 635 636 671  672  47108108 17 110 85 150 54 0 15104. 89 1 138 0  63 48 27 46 32  16 14 15 0 17  14 12 14 1 13  14 44 68 31 66 14 38 58 19 38  19 12  14 12  14 13 1 110  26 16 25 17 31 27 30 16 21 10  14 14 16 14 10  14 14 17 14 7  '72A 72C 72F 72H 72X  24 83 1 15 128 84  73E  64" 40 47 13 66 24 130 34 87 34 120 26  73 73K 73J 73M 73P 74A" 74H 74K 74N 74U  13 25 14 14 19  55100 35 34. 23 30 30 49 50 0  19 36 37 20 42  30 1 26 23 26 33  50 35 30 64 26  26 33 36 35 25  24 30 16 32 23 26 25  13 12 12 11 12  12 10 1 1 10 12  13 14 17 13 7  0 0 0 0 0 0 0 14 14 0 0 0  10 8 14 14 16 13  9 7 1 12 12 12  9 9 8 7, 0 0 11 1 0 0 13 1 1 0 12 1 1 12 13 1 1 0  32 16 23 12 54 26 29 19 55 23 26 15 25 17 2 1 13 130 27 24 17  1 16 14 1 14  11 1 10 16 13 15 12 11 1 0 14 1 1  118 4 1 32 37 26  77 A 130 44 19 14 22 13 9 77B 96 46 19 15 19 77N 260 67 56 32 28 18 77R 291 10170 29 26 14 77X 192 76 0 17 13 13 17 20 26 26 20 17 22 18 24 15 23 29 27 22 13  9 0 7 0 17 15 13 13 1 110 13 13 14  8 7 14 12 10  10 15 0 0 13  0 11 1 1 0 0 1 120 7 9 0 0 33 9 13 0 16 1 1 0 0 10 17 0 7 0 0 o 0 10 10 7 12 0 38 o 0 0 10  0 30 0 19  0 9  10 10  10 9 12 13 15 13 0 14 17 14 18 13 0 0 7  14 0 29 0 0 0 0 0 0  0 13 19 12 18  0 0 18 0 0  0 9  0 0  o  8 0 8 0 0 0 7 7 0 0 0 0 13 1 1 0 0 20 1 1 0 21 0 14 10 0 24 0 1 110 0 31  8 8 9 9 0 0 10 8 12 0 26 1 1 0 8 1 1 0 19 0 0 10 10 0 0 10 9 1 11 1 0 2 111  0  1 1 8 1 1 0 20 13 1 1 8 12 0 24 18 10 8 10 0 2 1 10  0 0 0  0 0 0 0 23 0 0 23 14 0 19 9 0 18 8 0 18 10  0  79D 79E 79V  38 49 54  "80 80A 80L 80T 80X 80Y  9 39 15 19 19 22 13 1 1 10 10 1 1 0 19 25 32 23 18 12 1 11 11 110 13 7 61 18 21 22 22 14 14 14 12 13 12 14 0 10 8 45 13 15 14 18 1 110 1 1 9 9 9 8 8 8 0 8 62 43 19 18 18 84 42 22 17 18 13 12 10 10 0 0 7 1 1 9 112 9 14 O 15 10 14 10 15 0  8 7 8 8 7 9  10 10 12 9 8 10  10 0 o 11 12 0 0 0 12 0 1 10  16 18 28 25  25 27 39 40 35 39  37 '17 10 1 110 45 18 12 1 11 55 2 1 14 13 12 55 2 1 15 13 0 50 19 14 12 12 50 18 14 13 12  21 63 20 17 80140 30 40 60100  43 30 0 37 71  18 17 38 24 39  0 9 0 6 9 1 10 23 12 1 1 9 24 13 12 1 110 1 1 8 6 10 0 0 32 21 19 17 0 19 0 16 0 0 29 25 18 13 14 13 0 12 9 1 10 0 39 19 16 15 13 0 0 10 12 0 0  C 1A C1B C 1C C 1D  13 91 261 10 18 50 42 82  58 20 25 98 38 38 60 15 30 70 21 6 0  C2A C2B C2C C2D  28  C3A C3B C3C C3D  48 82 28 47  "C4A C4B C4C C4D  36 21 49 55  19 13 28 38 23 49  82 82B 82D 82F 82J  29 27 35 53 43 72  0 0 10 10 10 12 10 O 17 1 110 7 0 1 11 1 9  48 28 28 15 18 0 89 90 18 56 15150100 62 78 20 21 78 90 42 30 21  3e  90 130 82 80  80 18 25 78 25 35 70 33 41 66 42 52  12 12 10 15 13 9 10 10 5 10 10 9  5 0 O O 0 15 0 10 0 5 0 10  0 0 0 0  11  0  10  0  8 0 12 0 10 2 1 10 0  0 0 0 0 0  O 1 10  31 45 50 65 52 66  8'i 8 1C 81L 8 1M 8 1R 8 1S  8 15 15 1 1 14  2 1 9  8 0 8 5 0 0 0 -9 6 0 0 22 13 14 22 0 1 19 1 1o 0 0 9 1 10  13' 12 14 14 12 12 13 1 1 0  7 0 7 O 10 12 10 0 10 12 10 0  0 0 0 0 0  0  1 1 0  o  o o  0 0  0 0 0  0  0  o  o  0  6"  0 0 o12 1 10  0 0  o 12 12 0 1 1 0  10  0 20 . O 0 15 10 0 0 10 0 10 0  0  IO  o  o  0 0 0  0 22 10 0 0 18  0 0 0  0  0  1 10  10  0  0  12  0  0  7 0 16 0 10 10 1 1 1 1 0 0 0 1 113 10 10 12 1 1 13 7 ' 5  0 5 0 0  0  1 10 1 130 0 O  5 7 0  1 10 ' 5 1 19 0 10 10 0 0 0 110  0  O 0 0 10 0 0 10 10 9 11 0 1 10 7 0 0 0 0 9 0  0  68 38 '18 26 15 58 21 15 30 17 78 80 19 38 21 0 98 78 29 4 0  12 10 10 1  0 0 5  0 12 5 13 7 0  0 0  0 0 0  APPENDIX Two- d i m e n s i o n a l that  are  chromatograms  described  Solvents:  in  Chapter  A s c e n d i n g ; the  organic solvent  to r i g h t ) ;  tern  7 0 : n-butanol  Polyamide  1  compounds  (as used  1  present  dotted  the  73 L u p i n u s  taxa  the  system  (benzene  20: water aqueous  15: acetone  55:  3). solvent  10: dioxane  sys5).  DC6.6  orientin vitexin o r i e n t i n acyl I v i t e x i n acyl I o r i e n t i n acyl II v i t e x i n acyl II l u t e o l i n 7-0-glucoside apigenin 7-0-glucoside l u t e o l i n 7-0-glu. acyl I unknown green l u t . 7-0-glu. acyl II ap. 7-0-glu, acyl II 1u t e o1 i n apigenin i s oo r i e nt in i sovi t ex in pink f l a v a n o n e (high Rf) pink f l a v a n o n e (mid Rf) p i n k f l a v a n o n e (low Rf) c y t i sos i de c y t i s o s i d e acyl I c y t i s o s i d e acyl II 4'-0-methylorientin chrysoeriol acacetin c h r y s o e r i o l 7-0-glu. chry. 7-0-glu. acyl I c h r y . 7 - 0 - g l u . a c y l 11  with  methanol  (left  (water  of  IV.  Horizontal  Compound numbering system 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28  (2- D maps)  ketone 2 2 :  methylethyl  Medium:  V.  in  29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56  trace  perimeters.  in  Data  Set  I):  acacetin 7-0-glucoside a c a c e t i n 7 - 0 - g l u c o s i d e 'acyl l u t e o l i n 7-0-diglucoside apigenin 7-0-diglucoside chrysoeriol 7-0-diglucoside acacetin 7-0-diglucoside kaempferol kaempferol 3-0-glucoside kaempferol 7-0-glucoside quercetin quercetin 3-0-glucoside quercetin 7-0-glucoside i soor i enti n acyl I. gen i st ein genistein 7-0-glucoside gen. 7-0-glu. acyl I gen . 7 - 0 - g l u . a c y l 11 o r i e n t i n X'-O-glucoside vitexin X'-O-glucoside orientin 3'-0-glucoside luteolin 4'-0-glucoside 1 u t e o l i n 4 ' - 0 - g l u . a c y l 11 apigenin 4'-0-glucoside apigenin 4'-0-glu. scyl I apigenin 4'-0-g1u . acyl II i s o v i t e x i n acyl I luteolin 5-0-glucoside apigenin 5-0-glucoside quantities  are  shown  as  spots  I  158  2-D map o f  the  flavonoids  of  L.  elatus,  OTU 1  159  2-D map o f  the  flavonoids  of  L.  barbiger,  OTU 2  2-D  map  of  the  flavonoids OTU  of 3  L.  sericeus  var.  sericeus  161  2-D  map  of  the  flavonoids  OTU  of  4  L.  sericeus  ssp.  huffmanii  162  -D  map-of. t h e  flavonoids OTU  of  L.  sericeus  5  -S 8  42;  var.  e g g l e s t o n i anus  163  2-D  map  of  the  flavonoids OTU  of 6  L.  sericeus  var.  flexuosus  164  2-D map o f  •.46; •''  the  flavonoids  of  L.  bicolor,  OTU 7  165  2-D map o f  the  flavonoids  of  L.  polycarpus,  OTU 8  166  2-D map o f  the  flavonoids  of  L.  neomexicanus,  OTU 9  2-D maps o f  the  flavonoids  of  L.  lemmonii,  OTU 10  168  2-D map o f  the  flavonoids  '.47, l  o  of 1 .  texensis,  OTU  11  169  2-D map o f  the  flavonoids OTU  of 12  L.  densiflorus  var.  densiflorus  170  2-D  map  of  the  flavonoids  of  OTU  13  L•  densiflorus  var.  aureus  172  2-D map o f  the  flavonoids  of  L.  arboreus,  OTU 15  173  2-D map o f  the  flavonoids  of  L.  horizontal i s ,  OTU 16  174  2-D map o f  the  flavonoids  of  L.  flavoculatus,  OTU 17  2-D.map o f  the  flavonoids  of  L.  benthami i , OTU 18  177  -D map o f  the  flavonoids  of  L.  spars i f T o r u s ,  OTU 20  2-D map o f  \46 ;  the  flavonoids  of 1 .  arizoni cus,  OTU  21  2-D map o f  the  f1avonoids  o f 1.  s t o l o n i f e r u s , OTU 22  180  2-D map o f  the  flavonoids  of  L. wyethi i ,  OTU 2 3  2-D map o f  V  the  flavonoids  of  L.  h i l l i i , OTU 2 4  182  2-D map o f  the  flavonoids  o f 1.  holosericeus,  OTU 25  183  2-D map o f  the  flavonoids  13)  of  L.  parviflorus,  24)  OTU 26  184  2-D map o f  the  f 1 a v o n o i ds o f  L.  caudatus  X L ; h i 11 i i , OTU 27  185  map o f  the  flavonoids  '••47\ "'46  6  13J  of  L.  nevadensis,  OTU 28  1 8 6  2-D map o f  1 4 6 :  the  flavonoids  of  L.  1eucophylIus,  W  w 14  OTU 29  187  2-D map o f  A6,'  :  the  flavonoids  of  L.X  alpestris,  OTU 30  188  2-D map o f  the  flavonoids  of  L.  greenei,  OTU 31  189  2-D map o f  the  flavonoids  of  L.  pseudoparvi f 1 o r u s ,  OTU 32  map o f  •46  is;'  the  flavonoids  of  L.  1utescens,  OTU 3 3  191  2-D map o f  the  flavonoids  of  L.  sulphureus,  OTU 34  192  -D map o f  -47,  :  ' 46. ; :  the  f 1 a v o n o i ds o f 1 .  paTmeri,  OTU 35  193  2-D map of the flavonoids of L. argenteus var. argenteus OTU 36  •  \ 4  i'5 /'  ;  1 9 4  2-D map o f  the  flavonoids  of 1 . argenteus  OTU 37  var.  rubricaulis  195  2-D map o f  the  flavonoids  o f 1.  caudatus,  OTU 38  196  2-D map o f  •47 •46  :32  the  flavonoids  of  L.  arbustus,  OTU 39  198  Oi  199  200  2-D map o f  ',46  ;  the  flavonoids  of  L.  humocola,  OTU 43  201  2-D  map  of  the  flavonoids  OTU  of  44  L.  arcticus  ssp.  arcticus  202  2-D  map  of  the  flavonoids  OTU  of  45  L.  acticus  ssp.  canadensis,  203  2-D map o f  the  flavonoids  of  OTU 46  L.  arcticus  ssp.  subalpinus,  205  2-D map o f  the  flavonoids  '.34.:'  of  L.  1 i t t o r a 1 i s , OTU 48  207  2-D map o f  the  flavonoids  35^7^)  of  L.  confertus,  > ) \)  14  42  2  OTU 50  25  )  208  2-D map o f  the  flavonoids  of  L.  minimus,  OTU  51  209  2-D map o f  the  flavonoids  of  L.  lyallii,  OTU 52  210  2-D map o f t h e f l a v o n o i d s  o f L. s e l l u l u s  OTU 53  var.  lobbii,  211  2-D-map o f  46 ;  the  flavonoids  of  L.  k u s c h e i , OTU 54  212  2-D map o f  the  flavonoids  of  L.  diffusus,  OTU 55  213  2-D map o f  the  flavonoids  of  L.  uncial i s O T U  56  214  215  2-D map o f  V ' f c 31  •46 )  {  the  flavonoids  of  L.  caespitosus,  OTU 58  216  2-D map o f  (3i;  '-32;  the  flavonoids  of  L.  1epidus,  OTU 59  218  219  2-D  map  of  the  flavonoids  of  L.  burkei,  OTU  62  220  2-D map o f  the  flavonoids  of  L.  perennis,  OTU 63  221  2-D map o f  the  flavonoids  of  L.  p o l y p h y l ! u s , OTU 64  222  2-D map o f  the  flavonoids  of  L.  excubitus,  OTU 65  223  1  2-D map o f  the  flavonoids  of  L.  1ongifolius,  OTU 66  224  2-D map o f  the  flavonoids  of  L.  albicaulis,  OTU 67  225  2-D map o f  the  flavonoids  o f 1.  andersonii,  i \ •31 ••  13)  1?)  OTU 68  226  2-D map o f t h e f l a v o n o i d s  (  30/  • 8 ;  o f L. f o r m o s u s , OTU 69  227  2-D map o f  the  flavonoids  of 1.  kingii,  OTU 70  228  2-D map o f  the  flavonoids  of  \ 31>  / T N 26  L.  pusillus  ssp.  rubens,  OTU 71  229  2-D map o f  the  flavonoids  of  L.  pusillus  OTU 72  3>  35)  lO  24)  ssp.  pusillus,  230  2-D map o f  the  flavonoids  of  L.  hirsutissimus,  OTU 73  

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