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Flavonoids and taxonomy of the Limnanthaceae Parker, William Harrison 1975

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FLAVONOIDS  AND  TAXONOMY  OF  THE  LIMNANTHACEAE  by  WILLIAM HARRISON PARKER "  B.A. , Reed C o l l e g e ,  1968  M . S c , Univ. B r i t i s h Columbia, 1 9 7 2  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of Botany  We accept t h i s t h e s i s as conforming t o t h e r e q u i r e d standard.  THE UNIVERSITY OF BRITISH COLUMBIA August, 1975  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree the Library shal make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of m y Department by his representatives. It is understood that copying or publication of this thesis for financial gain shal not be alowed without m y written permission.  Depa rtment The University of British Cou lmba i 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  ABSTRACT The Limnanthaceae i s a small family of North American annual herbs w i t h uncertain r e l a t i o n s h i p s .  I t i s generally recognized t o consist of two  genera, Limnanthes and Floerkea, together containing 10 species w i t h 13 v a r i e t i e s and subspecies.  To help c l a r i f y r e l a t i o n s h i p s w i t h i n t h i s f a m i l y , a com-  parative f l a v o n o i d i n v e s t i g a t i o n was undertaken.  A l l taxa were compared on  the basis of flavonoids occurring i n a l l t i s s u e s , and most Limnanthes taxa were a d d i t i o n a l l y compared on the basis of flavonoids occurring i n the petals and the u l t r a v i o l e t r e f l e c t a n c e c h a r a c t e r i s t i c s of t h e i r flowers. f l a v o n o l glycosides were encountered.  A t o t a l of hd  Of these, 35 were i d e n t i f i e d as d e r i v a -  t i v e s of s i x f l a v o n o l aglycone types: s y r i n g e t i n , isorhamnetin,  kaempferol,  l a r i c y t r i n (myricetin 3'-methyl e t h e r ) , quereetin and m y r i c e t i n , a l l g l y cosylated w i t h combinations of glucose and rhamnose. The f l a v o n o i d data were analyzed by three numerical taxonomic techniques:  c l u s t e r i n g by the weighted  p a i r group method, P r i n c i p a l Components A n a l y s i s , and Varimax Factor Analysis with rotation.  Duplicate comparisons were made according to type of t i s s u e  analyzed and concentrations of flavonoids considered.  C l a s s i f i c a t i o n s based  on p e t a l flavonoids occurring i n r e l a t i v e l y higher concentrations were found to r e f l e c t most c l e a r l y n a t u r a l r e l a t i o n s h i p s i n Limnanthes.  The u l t r a v i o l e t  r e f l e c t a n c e c h a r a c t e r i s t i c s of flowers were found t o d i s t i n g u i s h one supras p e c i f i c group of Limnanthes taxa from the remainder of the genus.  The adap-  t i v e s i g n i f i c a n c e of u l t r a v i o l e t patterning i s discussed, together with i t s i m p l i c a t i o n s concerning f l a v o n o i d compositions.  The method and v a l i d i t y of  using flavonoids as taxonomic characters i s also discussed.  Flavonoid and  u l t r a v i o l e t r e f l e c t a n c e c h a r a c t e r i s t i c s are integrated with a l l other taxonomic a l l y s i g n i f i c a n t information known f o r the Limnanthaceae, and proposals f o r taxonomic r e v i s i o n are made. A synopsis of the family i s presented which r e cognizes one genus, Floerkea, containing 15 species with 5 .varieties._  - i i i-  TABLE OF CONTENTS  Abstract  i i  L i s t o f Tables  v  List  of Figures  List  o f Appendices  v  i i i  Acknowledgement  i  x  xiii  Introduction Family D e s c r i p t i o n  1  Taxonomic Background A. Below the Family  Level  3 7  B. Family A f f i n i t i e s Previous  Chemical I n v e s t i g a t i o n s  10  Agronomic E v a l u a t i o n o f Limnanthes  11  Thesis Objectives  12  M a t e r i a l s and Methods lh  The P l a n t s and T h e i r Sources . P l a n t Growth  18  Plant Tissue S e l e c t i o n F l o r a l Patterning i n Ultra Violet  20 Light  A. I n t r o d u c t i o n  22  B. Method  2h c  F l a v o n o i d I s o l a t i o n and I d e n t i f i c a t i o n A. I n t r o d u c t i o n  25  B. P l a n t E x t r a c t i o n s  27  C. I d e n t i f i c a t i o n o f t h e F l a y o n o i d s y a r . d o u g l a s i i , OTU 1 i. ii.  Chromatography S p e c t r a l Analyses  o f L. d o u g l a s i i .  28 30  - iv-  TABLE OF CONTENTS —  iii.  Continued  Hydrolyses  i v . Acetate  31  Preparation  32  D. I d e n t i f i c a t i o n o f t h e Remaining Limnanthes F l a v o n o i d s • Expression  of Flavonoids  as Taxonomic C h a r a c t e r s  33 33  Treatment o f t h e F l a v o n o i d Data A.  3h  Introduction  B. Method  '  35  F l o r a l Patterning i n U l t r a Violet Light  39  Results  F l a v o n o i d C h a r a c t e r i z a t i o n and. I d e n t i f i c a t i o n A. Data P r e s e n t a t i o n  kk  B. E x p l a n a t i o n o f t h e Data  51  S i g n i f i c a n c e o f the Flavonoids  o f t h e Limnanthaceae  A. Aglycones B. G l y c o s i d e s The  I  60 '  62  C o n t r i b u t i o n t o V i s i b l e Flower C o l o u r by S y r i n g e t i n  Derivatives  62  E n v i r o n m e n t a l M o d i f i c a t i o n o f F l a v o n o i d Composition  63  Comparative 'Flavonoid Data  65  F l a v o n o i d D i f f e r e n c e s Between P e t a l and.Whole P l a n t M a t e r i a l  68  Numerical Taxonomic R e s u l t s  68  Comparisons, o f OTUs by F l a v o n o i d s A. Occurrence T a b l e s  83  B.  Qh  C l u s t e r Analyses  C. 'Factor Analyses  88  D. G l y c o s y l a t i o n C l a s s e s  89  TABLE OF CONTENTS —  Continued  Discussion Method and V a l i d i t y o f Using F l a v o n o i d s A. F l a v o n o i d s i.  as T a x i m e t r i c  as Taxonomic  Characters  Characters  Introduction  ii.  92  I n c l u s i o n o f N e g a t i v e Matches  iii.  92  Flavonoid Concentration  94  i v . Lack o f V a r i a t i o n among C h a r a c t e r s B. Use o f Dry v e r s u s  Fresh Plant M a t e r i a l  C. P e t a l v e r s u s Whole P l a n t R e s u l t s D. F a c t o r A n a l y s i s v e r s u s  Conventional  96 97  Cluster Analysis  E. F l a v o n o i d D i f f e r e n c e s "between D u p l i c a t e Taxa. Taxonomy o f t h e Limnanthaceae  95  98 99  .  A. I n t r o d u c t i o n  101  B. H y p o t h e t i c a l E v o l u t i o n a r y H i s t o r y o f t h e Limnanthaceae  104  C. Assumptions t h a t 'Led t o t h e H y p o t h e t i c a l H i s t o r y  107  D. Comparison o f t h e Proposed C l a s s i f i c a t i o n t o the' E x i s t i n g C l a s s i f i c a t i o n i.  Above t h e S p e c i e s  ii.  Species  Level'and  E. Summary o f P r o p o s a l s . i, ii. iii.  Level' Below'  f o r R e v i s i o n o f t h e Limnanthaceae  111 116 \^  Introduction  128  Genus L e v e l  129  Section Level  i v . Species F. Synopsis  and V a r i e t a l L e v e l s  o f t h e F a m i l y Limnanthaceae  129 129. 130 .  Conclusions  133  Literature Cited  135  - vi -  LIST OF TABLES  TABLE I —  Present Day C l a s s i f i c a t i o n o f t h e Limnanthaceae  TABLE I I — I d e n t i f i c a t i o n and Sources o f O p e r a t i o n a l Taxonomic U n i t s o f Limnanthes and F l o e r k e a 1  TABLE I I I — I d e n t i f i c a t i o n s o f t h e F l a v o n o i d s o f t h e Limnanthaceae TABLE IV —  Rfs and Colours' o f F l a v o n o i d s o f t h e Limnanthaceae  TABLE V — Products 1.0 N HC1  Obtained upon T o t a l H y d r o l y s i s w i t h  TABLE VI — D e r i v a t i v e s Obtained upon P a r t i a l A c i d H y d r o l y s i s and H y d r o l y s i s w i t h Emulsin TABLE V I I — UV A b s o r p t i o n Maxima o f F l a v o n o i d s o f t h e Limnanthaceae TABLE V I I I — P r o t o n Chemical S h i f t Values o f TMS E t h e r s o f S i x F l a v o n o l y3-D-Rutinosides o f t h e Limnanthaceae TABLE IX —  Whole P l a n t F l a v o n o i d s o f 30 OTUs o f the Limnanthaceae  TABLE X -.- P e t a l F l a v o n o i d s o f 1 8 OTUs o f Limnanthes TABLE XI •— Synopsis o f t h e R e s u l t s o f Four C l u s t e r A n a l y s e s Based on Whole P l a n t F l a v o n o i d s o f 30 OTUs TABLE X I I — Synopsis of'•the R e s u l t s o f Four C l u s t e r A n a l y s e s Based on P e t a l F l a v o n o i d s o f 1 8 OTUs  -  V l l  -  LIST OF FIGURES  FIGURE I —  D i s t r i b u t i o n o f Limnanthes i n Oregon and C a l i f o r n i a  FIGURE I I — The Flowers o f S i x S e l e c t e d Limnanthes t a x a Taken in " V i s i b l e and. U l t r a v i o l e t L i g h t :  FIGURE I I I — Composite Two-Dimensional Map o f 48 F l a v o n o i d G l y c o s i d e s o f t h e Limnanthaceae FIGURE IV — Stepwise E l a b o r a t i o n o f t h e B-Ring o f F l a v o n o l s o f the Limnanthaceae FIGURE V — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. montana. Taken i n U l t r a v i o l e t L i g h t FIGURE VI — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. montana Taken i n V i s i b l e L i g h t FIGURE VIT -- Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by J a c c a r d C o e f f i c i e n t : H i g h C o n c e n t r a t i o n s o f Whole P l a n t 'Flavonoids • 'FIGURE V I I I — Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by Simple Match C o e f f i c i e n t : H i g h C o n c e n t r a t i o n s o f Whole Plant Flavonoids FIGURE IX — Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by J a c c a r d C o e f f i c i e n t : A l l C o n c e n t r a t i o n s o f Whole P l a n t F l a v o n o i d s FIGURE X — Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by Simple Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s o f Whole P l a n t Flavonoids FIGURE XI — Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by J a c c a r d C o e f f i c i e n t : High C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s • FIGURE X I I — Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by Simple Match C o e f f i c i e n t : H i g h O C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s FIGURE X I I I — Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by Jaccard C o e f f i c i e n t : A l l Concentrations of P e t a l Flavonoids FIGURE XIV — Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by Simple Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s FIGURE XV - - P r i n c i p a l Components A n a l y s i s o f 30 OTUs Based on Whole P l a n t 'Flavonoids FIGURE XVI — Varimax F a c t o r A n a l y s i s o f 30 OTUs Based on Whole P l a n t F l a v o n o i d s FIGURE XVII — P r i n c i p a l Components A n a l y s i s o f 18 OTUs Based on P e t a l F l a v o n o i d s  -  VI11  -  LIST OF FIGURES —  Continued  FIGURE XVIII — Varimax F a c t o r A n a l y s i s o f 18 OTUs Based on P e t a l Flavonoids FIGURE XIX —  Hypothetical Phylogenetic  Tree o f t h e Limnanthaceae  FIGURE XX — P r i n c i p a l Components A n a l y s i s o f 9 OTUs o f L. f l o c c o s a Based on Whole P l a n t F l a v o n o i d s  - ix -  LIST OF APPENDICES  APPENDIX I —  Mason's. C l a s s i f i c a t i o n o f Limnanthes R. B r .  APPENDIX I I — Voucher Specimens from seed  139  o f Limnanthes Taxa Grown  APPENDIX I I I — TLC Map o f P e t a l F l a v o n o i d s o f L. d o u g l a s i i v a r . d o u g l a s i i , OTU 1 APPENDIX IV — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. d o u g l a s i i v a r . d o u g l a s i i , OTU 1 1  ikO  l 4 l  142  APPENDIX V — TLC Map o f P e t a l F l a v o n o i d s o f L. d o u g l a s i i v a r . d o u g l a s i i , OTU 2  143  APPENDIX VI — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. d o u g l a s i i v a r . d o u g l a s i i , OTU 2-  ihk  APPENDIX V I I — TLC Map of'- P e t a l F l a v o n o i d s o f L. d o u g l a s i i v a r . n i v e a , OTU 3  1^5  APPENDIX V I I I — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. d o u g l a s i i v a r . n i v e a , OTU 3  146  APPENDIX IX — TLC Map o f P e t a l F l a v o n o i d s o f L. d o u g l a s i i v a r . r o s e a , OTU h  ikj  APPENDIX X — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. d o u g l a s i i v a r . r o s e a , OTU h.  148  APPENDIX XI — TLC Map o f P e t a l F l a v o n o i d s o f L. d o u g l a s i i v a r . s u l p h u r e a , OTU 5  149  APPENDIX XII -- TLC Map o f Whole P l a n t F l a v o n o i d s o f L. d o u g l a s i i v a r . s u l p h u r e a , OTU 5 APPENDIX X I I I —  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. v i n c u l a n s  APPENDIX XIV -- TLC Map o f P e t a l F l a v o n o i d s o f L. b a k e r i , OTU 7 APPENDIX- XV — OTU 7  •  152  153  TLC Map o f P e t a l F l a v o n o i d s o f L. s t r i a t a , OTU 8  APPENDIX XVII — OTU 8 APPENDIX XVTII — OTU 9  OTU'9-'  151  TLC Map .of Whole P l a n t F l a v o n o i d s o f L. b a k e r i ,  APPENDIX XVI —  APPENDIX XIX —  150  154  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. s t r i a t a , 155  TLC Map o f P e t a l F l a v o n o i d s o f L. m a c o u n i i , 156  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. m a c o u n i i 157  -  X  -  LIST OF APPENDICES —  APPENDIX XX —  Continued  TLC Map o f P e t a l F l a v o n o i d s  APPENDIX XXI — OTU 10  o f L. m a c o u n i i , OTU 1<0)  TLC Map o f Whole P l a n t F l a v o n o i d s  APPENDIX XXII — OTU 11  TLC Map o f P e t a l F l a v o n o i d s  o f L. m a c o u n i i ,  o f L. a l b a v a r .  APPENDIX XXIII — TLC .Map o f Whole P l a n t F l a v o n o i d s v a r . a l b a , OTU 11 APPENDIX XXIV — OTU 12  TLC Map o f P e t a l F l a v o n o i d s  o f L. alba.  o f L. a l b a v a r .  •APPENDIX XXV — TLC Map o f Whole P l a n t F l a v o n o i d s v a r . a l b a , OTU 12 APPENDIX XXVI — TLC Map o f P e t a l F l a v o n o i d s v a r . v e r s i c o l o r O T U 13  o f L. a l b a  APPENDIX X X V I I l — TLC Map o f P e t a l F l a v o n o i d s v a r . g r a c i l i s , OTU 'l.te  o f L. a l b a  o f L. g r a c i l i s  APPENDIX XXIX — TLC Map o f Whole P l a n t F l a v o n o i d s L_. g r a c i l i s v a r . g r a c i l i s , OTU ik  of  o f L. g r a c i l i s  APPENDIX XXXI — TLC Map o f Whole P l a n t F l a v o n o i d s L_. g r a c i l i s v a r . g r a c i l i s , OTU 15 APPENDIX XXXII — TLC Map o f P e t a l F l a v o n o i d s v a r . p a r i s h i i , OTU 16  of  o f L. g r a c i l i s  APPENDIX XXXITI — TLC Map o f Whole P l a n t F l a v o n o i d s L_. g r a c i l i s v a r . p a r i s h i i , OTU 16 APPENDIX XXXIV — OTU 17 APPENDIX XXXV — OTU 17  TLC Map o f P e t a l F l a v o n o i d s  of  o f L. montana,  TLC Map o f Whole P l a n t F l a v o n o i d s  APPENDIX XXXVI :— TLC Map o f P e t a l F l a v o n o i d s ssp. b e l l i r i g e r i a n a , OTU 18 1  alba,  o f L. a l b a  APPENDIX XXVII — TLC Map o f Whole P l a n t F l a v o n o i d s v a r . v e r s i c o l o r , OTU 13  APPENDIX XXX — TLC Map o f P e t a l F l a v o n o i d s v a r . g r a c i l i s , OTU 15  alba,  o f L. montana,  o f L. f l o c c o s a  APPENDIX XXXVTI — TLC Map. o f Whole P l a n t F l a v o n o i d s L. f l o c c o s a ' ssp. b e l l i r i g e r i a n a , OTU 18  of  - xi_  LIST OF APPENDICES —  Continued  APPENDIX XXXVIII — TLC Map o f Whole P l a n t F l a v o n o i d s o f L_. f l o c c o s a s s p . b e l l i n g e r i a n a , OTU 19 APPENDIX XXXIX — TLC Map o f P e t a l F l a v o n o i d s o f L. f l o c c o s a ssp. p u m i l a , OTU 20 APPENDIX XL — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. pumila., OTU 20 APPENDIX XLI — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. p u m i l a , OTU 21 APPENDIX XLII — TLC Map o f Whole P l a n t F l a v o n o i d s o f L_. f l o c c o s a s s p . g r a n d i f l o r a , OTU 22 APPENDIX XLI'II. — TLC. Map o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. f l o e cos a,;-OTU 23 APPENDIX XLIV — TLC Map' o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. f l o c c o s a , OTU 2k . APPENDIX XLV — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. f l o c c o s a , OTU 25 APPENDIX XLVI — TLC Map o f Whole P l a n t F l a v o n o i d s o f L. f l o c c o s a ssp. ' c a l i f o r n i c a , OTU 26 APPENDIX XLVII — TLC Map. o f Whole P l a n t -Flavonoids o f F_. p r o s e r p i n a c o i d e s , . OTU 27 • APPENDIX XLVITI — TLC Map o f Whole P l a n t F l a v o n o i d s o f F_. p r o s e r p i n a c o i d e s ^ ©TU 28 APPENDIX XLIX — TLC Map o f Whole P l a n t F l a v o n o i d s o f F_. p r o s e r p i n a c o i d e s , OTU 29 APPENDIX L — TLC Map o f Whole P l a n t F l a v o n o i d s o f F. p r o s e r p i n a c o i d e s , OTU 30 APPENDIX LT — 100. mHz NMR Spectrum o f TMS E t h e r o f Kaempferol 3-0-y3-D-Rutinoside APPENDIX L I T — 100,mHz NMR 3-0-y3-D-Rut i n o s i d e  Spectrum o f TMS E t h e r o f Q u e r c e t i n  APPENDIX H I T r,-, 100. mHz NMR, Spectrum o f - TMS E t h e r o f Isorhamnetin 3-0-/3-D-Rut inos i d e APPENDIX LTV — 100. mHz NMR 3-0-/3-D-Rut i n o s i d e  Spectrum o f TMS E t h e r o f M y r i c e t i n  - xii _  LIST OF APPENDICES —  Continued  APPENDIX LV — 100 mHz NMR Spectrum of TMS Ether of L a r i c y t r i n 3-0-y3-D-Rutinoside .  193  APPENDIX LVI — 100 mHz NMR Spectrum of TMS Ether'of Syringetin 3-0-y3-D-Rutinoside  19^  APPENDIX LVII — Matrix of S i m i l a r i t y Coefficients Calculated for 30 OTUs by Jaccard C o e f f i c i e n t : High Concentrations.  195  APPENDIX LVIIl — Matrix of S i m i l a r i t y Coefficients Calculated f o r 30 OTUs by Simple Match C o e f f i c i e n t : High Concentrations APPENDIX LIX — Matrix of S i m i l a r i t y Coefficients Calculated f o r 30 OTUs by Jaccard.Coefficient: A l l Concentrations APPENDIX LX — Matrix of. S i m i l a r i t y Coefficients Calculated f o r 30 OTUs 'by'"; Simple Match C o e f f i c i e n t : A l l Concentrations  . 196  197  198  APPENDIX LXl — Matrix of S i m i l a r i t y Coefficients Calculated f o r 18 OTUs by Jaccard C o e f f i c i e n t : High Concentrations  199  APPENDIX LXII — Matrix of S i m i l a r i t y Coefficients Calculated f o r 18 OTUs by Simple Match C o e f f i c i e n t : High Concentrations  200  APPENDIX L X I I I — Matrix of S i m i l a r i t y Coefficients Calculated f 6 r r l 8 OTUs by Jaccard C o e f f i c i e n t : A l l Concentrations APPENDIX LXIV —  Matrix of S i m i l a r i t y Coefficients Calculated f o r  18 OTUs by Simple Match Coefficient!:;-All Concentrations APPENDIX. LXV —  201  Coordinates of 30 OTUs Plotted i n Figure XV  APPENDIX LXVI —  Coordinates of 30 OTUs Plotted i n Figure XVI  APPENDIX LXVIT — APPENDIX LXVIIT —  Coordinates of 18 OTUs Plotted i n Figure XVII Coordinates of 18 OTUs Plotted i n Figure XVIII  7  202 203. 20k 205 206  - xiii -  ACKNOWLEDGEMENT  I thank t h e f o l l o w i n g people who a s s i s t e d i n t h e c o m p l e t i o n o f t h i s  thesis:  Dr. B.A. Bohm f o r h i s support and a d v i c e throughout t h e c o u r s e o f t h i s work;  Drs. C.J. Marchant, J.R. Maze, W.B. S c h o f i e l d , I.E.P. T a y l o r and R.L. T a y l o r f o r t h e i r c r i t i c i s m s o f t h e manuscript and h e l p f u l s u g g e s t i o n s f o r its  improvement;  Dr. F;,-.W\ C o l l i n s f o r t e c h n i c a l  advice;  Mr. Steve Borden, I n s t i t u t e o f Animal Resource E c o l o g y , U n i v e r s i t y o f B r i t i s h Columbia, f o r p e r f o r m i n g t h e computer  Dr. Bob Bos.e, Environment Canada, Department d e t e r m i n i n g t h e NMR  analyses;  o f F i s h e r i e s , Vancouver, f o r  spectra;  Mr. G.A. White, U.S.D.A, ,' A g r i c u l t u r a l Research S e r v i c e , B e l t s v i l l e , M a r y l a n d , f o r p r o v i d i n g seed o f Limnanthes;  Dr. R. O r n d u f f , Dept. o f Botany, U n i v e r s i t y o f C a l i f o r n i a , Berkeley), f o r p r o v i d i n g d r i e d m a t e r i a l o f Limnanthes.;  and my w i f e Jane C. P a r k e r f o r h e r c o n t i n u e d support and a s s i s t a n c e .  INTRODUCTION  FAMILY DESCRIPTION The Limnanthaceae i s a s m a l l f a m i l y o f annual herbs c o n t a i n i n g two genera:  F l o e r k e a W i l l d . , which i s monotypic, and Limnanthes R. B r . ,  which c o n t a i n s 18 r e c o g n i z e d t a x a .  Floerkea proserpinacoides W i l l d . i s  w i d e l y d i s t r i b u t e d a c r o s s the U n i t e d S t a t e s and s o u t h e r n Canada.  Limnanthes  i s . r e s t r i c t e d t o s o u t h e r n Oregon, C a l i f o r n i a and t h e s o u t h e r n t i p o f Vancou- • ver Island, B r i t i s h  Columbia.  F i g u r e I i s a map  showing t h e d i s t r i b u t i o n s  o f a l l Limnanthes t a x a except L_. macounii T r e l . which o c c u r s o n l y i n t h e v i c i n i t y o f V i c t o r i a , B.C. • All  o f t h e Limnanthaceae a r e e a r l y s p r i n g a n n u a l s , and most a r e  a s s o c i a t e d w i t h v e r n a l p o o l s or s o i l p o c k e t s kept m o i s t by seepage.  Limnanthes  t a x a v a r y i n t h e i r m o i s t u r e r e q u i r e m e n t s ; some a c t u a l l y grow i n s t a n d i n g water, o t h e r s at t h e edges o f p o o l s or streams, and o t h e r s on moist h i l l s i d e s . With t h e e x c e p t i o n o f L_. montana Jepso'n and L. s t r i a t a . J e p s o n , b o t h o f which f r e q u e n t l y occupy more shaded s t r e a m s i d e h a b i t a t s , Limnanthes grows i n open spots.  Taxa o f Limnanthes o c c u r over a range o f e l e v a t i o n s from n e a r l y sea  l e v e l t o about lB'OO meters Munz & Keck, 1 9 6 3 ) .  [Mason, 1952;. Gentry & M i l l e r , 1 9 6 5 ; A r r o y o ,  F l o e r k e a grows i n t h e shade o f shrubs or t r e e s .  t h e s e p l a n t s are ephemeral;  1973a;  A l l of  t h e y appear e a r l y i n t h e s p r i n g , t h e n f l o w e r  and s e t seed, and a r e soon o v e r t o p p e d by o t h e r p l a n t s and d i e . A l t h o u g h all.members o f t h e Limnanthaceae a r e . s e l f - c o m p a t i b l e ,  their  b r e e d i n g systems v a r y from n e a r l y t o t a l o u t c r o s s i n g t o complete autogamy. O u t c r o s s i n g members a r e adapted t o i n s e c t p o l l i n a t i o n .  These p l a n t s have,  l a r g e numbers o f a t t r a c t i v e , f r a g r a n t f l o w e r s , and t e n d t o produce a c a r p e t  - 2FIGURE I D i s t r i b u t i o n o f Limnanthes in California  and Oregon  L. doug.var.L.  striata  L_. baker i  ^ -- .  L. a l b a v a r . a l b a L. f l o e . ssp.. floccosaL_. v i n c u l a n s - ' " L_. doug. v a r . sulphurea-- " L. doug. v a r . d o u g l a s i i "  L. doug. v a r . n i v e a —  L_. grac . v a r . p a r i s h i i - -  - 3 -  o f blooms over a meadow.  From t h i s c h a r a c t e r  "meadow foam", i s d e r i v e d . species  Additional t r a i t s associated with  a r e p r o t a n d r y and t h e presence o f n e c t a r  filaments.  F. p r o s e r p i n a c o i d e s ,  flowers  outcrossing  glands a t t h e base o f t h e  Autogamous members o f t h e f a m i l y t e n d t o have u n s c e n t e d ,  l e s s , and g e n e r a l l y i n c o n s p i c u o u s f l o w e r s . and  t h e common name f o r Limnanthes,  nectar-  The extreme i s found i n L. macounii  which a r e t o f a l l y s e l f - p o l l i n a t i n g , h a v e - s m a l l  and.reduced numbers o f f l o r a l parts..  These c h a r a c t e r s ,  s m a l l s t a t u r e , make t h e s e p l a n t s v e r y d i f f i c u l t  greenish  coupled with  to find.  TAXONOMIC BACKGROUND Past  taxonomic t r e a t m e n t s o f t h e Limnanthaceae r e c o g n i z e d  f a m i l y comprises a d i s c r e t e group o f c l o s e l y  this  r e l a t e d p l a n t s . . However, t h e s e  treatments have expressed c o n f l i c t i n g views r e g a r d i n g f a m i l i e s and r e l a t i o n s h i p s w i t h i n t h e f a m i l y .  that  a f f i n i t i e s with  others  To some e x t e n t , t h e s e c o n f l i c t s .  remain u n s e t t l e d today.  A.  Below t h e F a m i l y  Level  Robert Brown's o r i g i n a l c i r c u m s c r i p t i o n o f t h e f a m i l y d e l i m i t s two genera, Limnanthes and F l o e r k e a . tioned the v a l i d i t y of this i n t h e genus F l o e r k e a . o f Limnanthes (1952).  This  separation subject  Some.later authors have ques-  and u n i t e d a l l members o f t h e f a m i l y  i s reviewed by Mason i n h i s monograph  A c t u a l l y , the questrop of whether t h e f a m i l y  o f one genus o r two has not y e t been a d e q u a t e l y r e s o l v e d . for this  (Brown, 1833)  i s t h a t Mason excluded F. p r o s e r p i n a c o i d e s  consists-  The p r i m a r y r e a s o n  from h i s treatment o f  Limnanthes. ;•' S i n c e t h e modern c l a s s i f i c a t i o n o f t h e Limnanthaceae i s based on Mason's t r e a t m e n t , t h i s .exclusion has r e s u l t e d i n a de f a c t o s e p a r a t i o n f a m i l y i n t o two genera.  Whether t h i s  of the  s e p a r a t i o n was j u s t i f i e d cannot be  determined without a p p l y i n g t h e same taxonomic c r i t e r i a used by Mason t o a l l  -  h -  members o f t h e f a m i l y s i m u l t a n e o u s l y , A more r e c e n t metric  including Floerkea.  study by O r n d u f f and C r o v e l l o ( 1 9 6 8 ) , based on a t a x i -  a n a l y s i s of morphological  c h a r a c t e r s , concluded that the d i f f e r e n c e s  between Limnanthes and F l o e r k e a a r e p r o b a b l y two genera on e i t h e r p h e n e t i c  or phylogenetic  not g r e a t  enough t o d i s t i n g u i s h  grounds.  However, t h e two  genera have not been f o r m a l l y r e u n i t e d . Mason ( 1 9 5 2 ) d i v i d e d Limnanthes i n t o 8 s p e c i e s and 1 1 v a r i e t i e s t o t a l i n g 1 5 taxa.  H i s c l a s s i f i c a t i o n i s presented  as Appendix I .  taxonomic scheme i s based on t h e r e s u l t s o f a r t i f i c i a l  crosses  Mason's  and m o r p h o l o g i -  c a l and c y t o l o g i c a l a n a l y s e s . The  r e s u l t s o f Mason's c y t o l o g i c a l i n v e s t i g a t i o n show u n i f o r m  pat-  t e r n s o f 5 i d e n t i f i a b l e p a i r s o f chromosomes, i n d i c a t i n g a c l o s e r e l a t i o n s h i p between a l l Limnanthes t a x a tinct  (except  L_. macounii which was thought t o be ex-  a t t h a t time and f o r which Mason had no l i v i n g m a t e r i a l ) .  l a c k o f karyotype v a r i a t i o n suggests e i t h e r a c o m p a r a t i v e l y from a n c e s t r a l s t o c k o f t h e t a x a i n v e s t i g a t e d , o r a s t r o n g  This  recent  complete  divergence  evolutionary  conservancy o f karyotype i n t h e f a m i l y . Mason's c l a s s i f i c a t i o n o f Limnanthes r e l i e s h e a v i l y on t h e b i o l o g i c a l species  concept.  Thus, i n many i n s t a n c e s  f o r m e r l y t r e a t e d as separate  experimentally  interfertile  s p e c i e s , a r e reduced t o v a r i e t y rank.  taxa,  Taxa so  reduced i n c l u d e two o f t h e f o u r v a r i e t i e s o f L. d o u g l a s i i . R . B r . , t h e two v a r i e t i e s o f L. a l b a Benth. , and t h e two v a r i e t i e s o f L_. g r a c i l i s According  Howell.  t o Mason, Limnanthes can be d i v i d e d n a t u r a l l y i n t o two  s e c t i o n s , the Reflexae  and t h e I n f l e x a e  (See Appendix I ) .  Names o f t h e s e two  s e c t i o n s a r e d e r i v e d from t h e p e t a l p o s i t i o n s assumed and m a i n t a i n e d a f t e r pollination.  Although; Mason d i d not d e t e c t o t h e r m o r p h o l o g i c a l  differences  c o i n c i d e n t w i t h t h i s d i v i s i o n , he c o n c l u d e d t h a t t h e two s e c t i o n s two p h y l o g e n e t i c a l l y d i s t i n c t groups s i n c e no s u c c e s s f u l c r o s s e s  represent c o u l d be made  -  5 -  across the s e c t i o n a l b a r r i e r . Ornduff and C r o v e l l o (1968) agreed w i t h the separation of Limnanthes i n t o two s e c t i o n s , although t h e i r presented r e s u l t s only weakly support t h i s separation.'- JJsing a t a x i m e t r i c a n a l y s i s based on 35 morphological characters, a l l recognized taxa of the Limnanthaceae were c l u s t e r e d using the weighted p a i r group method and a v a r i a t i o n of f a c t o r a n a l y s i s .  Two a d d i t i o n a l f a c t o r  analyses were also performed, one on 18 f l o r a l characters, and the other on the remaining 17, or "vegetative" characters. analysis are markedly d i f f e r e n t .  The r e s u l t s of each c l u s t e r i n g  Only the r e s u l t s of the vegetative a n a l y s i s  f o l l o w Mason's s e c t i o n a l alignment, producing a d i s t i n c t c l u s t e r of taxa corresponding t o the s e c t i o n Reflexae and a f a i r l y d i f f u s e grouping corresponding t o the s e c t i o n I n f l e x a e . I t i s notable that i n t h i s a n a l y s i s F. proserpinacoides a l s o grouped w i t h the Reflexae.  The r e s u l t s of the a n a l y s i s u t i -  l i z i n g f l o r a l characters apparently c o n t r a d i c t the presence of a s e c t i o n a l d i v i s i o n since no groups were separated.  According t o the authors, these r e s u l t s  a r e . e a s i l y explained, i f not expected, since i n t h i s Instance a c l a s s i f i c a t i o n based on f l o r a l characters i s i n e f f e c t c l o s e l y l i n k e d t o l e v e l of autogamy. Therefore, they hypothesize that the two sections are not separated by the f l o r a l a n a l y s i s , since p a r a l l e l trends toward autogamy have apparently occurred simultaneously i n both s e c t i o n s . The a n a l y s i s based on the combined data of the f l o r a l and vegetative a n a l y s i s separated three c l u s t e r s of taxa: the f i r s t contains L_. macounii and F. proserpinacoides, the second contains Mason's.three v a r i e t i e s of L. f l o c c o s a Howell, and the t h i r d contains the remaining Limnanthes. taxa.  Again the authors explained that the'observed  phenetic a f f i n i t i e s are c l o s e l y l i n k e d t o l e v e l of autogamy and do not i n v a l i date Mason's s e c t i o n a l d i v i s i o n , which they considered an accurate phylo-• genetic arrangement. In t h e i r 1968 study, Ornduff and.Crovello included two.undescribed  -  6  -  Limnanthes t a x a w i t h a f f i n i t i e s t o members o f t h e R e f l e x a e . subsequently Ornduff lasii  d e s c r i b e d one  (l969 -i-  o f t h e s e as a new  I t i s a narrow endemic growing s y m p a t r i c a l l y w i t h L_. doug-  a  i n a l l but i t s  morphology. Arroyo  ( 1 9 7 3 a ) reexamined L_. f l o c c o s a , e l e v a t i n g Mason's t h r e e  v a r i e t i e s t o the rank o f s u b s p e c i e s and c r e a t i n g two of  has  s p e c i e s , Limnanthes v i n c u l a n s  R. Br. v a r . n i v e a Mason which i t c l o s e l y resembles  leaflet  Ornduff  new  subspecies.  Level  autogamy, as demonstrated by a p h e n e t i c c l u s t e r a n a l y s i s h e a v i l y weighted  with f l o r a l  c h a r a c t e r s , was  the s o l e c r i t e r i o n used t o d i s t i n g u i s h t h e  s u b s p e c i e s , a l t h o u g h g e o g r a p h i c a l and this  separation.  sent stages  e c o l o g i c a l evidence was  five  c i t e d to  support  A r r o y o c o n c l u d e d t h a t the f i v e t a x a o f L_. f l o c c o s a r e p r e -  i n t h e t r e n d toward complete autogamy i n t h e I n f l e x a e and  a n a t u r a l group r e c e n t l y d e r i v e d from t h e predominantly Benth. ( 1 9 7 3 a , 1 9 7 3 b ) . cosa i n t o s u b s p e c i e s  She  d i d not e x p l a i n why  form  o u t c r o s s i n g L. a l b a  she chose t o d i v i d e L_. f l o c -  i n c o n t r a s t t o the v a r i e t a l s u b d i v i s i o n used u n i f o r m l y  by Mason f o r t h e r e s t o f the genus. In  h i s most r e c e n t p u b l i c a t i o n c o n c e r n i n g t h e Limnanthaceae,  C l 9 7 l ) r e p o r t e d t h a t both L. macounii numbers o f n=5,  and F_.. proserpinacoides.' have chromosome  t h e same as the r e s t o f t h e f a m i l y .  Ornduff  sented the r e s u l t s of extensive Limnanthes.interbreeding •his r e s u l t s c o n f i r m e d the work' - of,';' Mas on, demonstrating 1  (1971) a l s o  trials. low  riers  i n Limnanthes-.  interspecific  of s t e r i l i t y b a r r i e r s  tween t a x a does not c o r r e s p o n d w i t h t h e m o p h o l o g i c a l these taxa.  differences  3) The presence  where p r e s e n t , p r o b a b l y has  o f a s t e r i l i t y b a r r i e r between two  barbe-  distinguishing  •2)'. S i n c e t h e c y t o l o g i c a l d i f f e r e n c e s between t a x a a r e o f a  t i v e l y minor n a t u r e , h y b r i d s t e r i l i t y , basis.  macounii).  observations regarding s t e r i l i t y  l ) In g e n e r a l , t h e presence  pre-  In g e n e r a l  c r o s s a b i l i t y and a h i g h degree o f chromosomal homology ( i n c l u d i n g L. However, Ornduff made t h r e e important  Ornduff  rela-  a genetic  t a x a i s most  likely  - 7 i f t h e two  lack d i s t i n c t geographical differences.  i s o f p a r t i c u l a r i n t e r e s t t h a t L. m a c o u n i i ,  In t h i s l a s t r e g a r d , i t  which i s the most d i s j u n c t species,  of Limnanthes, produced the; h i g h e s t number of i n t e r s p e c i f i c h y b r i d s i n c l u d i n g t h e o n l y i n t e r s e c t i o n a l h y b r i d , a s t e r i l e o f f s p r i n g r e s u l t i n g from a c r o s s w i t h L_. montana. T a b l e I shows the modern c l a s s i f i c a t i o n o f the Limnari'thaceae. i s based on Mason's c l a s s i f i c a t i o n o f Limnanthes, and r e c e n t taxonomic a d d i t i o n s p r e v i o u s l y c i t e d . as a d i s t i n c t  It •  i t i n c l u d e s t h e more  In a d d i t i o n , i t l i s t s  Floerkea  genus.  I n summary, r e l a t i o n s h i p s below t h e f a m i l y l e v e l i n t h e Limnanthaceae remain u n c l e a r f o r two  primary  reasons;  l ) i t i s probable  that certain simi-  l a r i t i e s have a r i s e n by convergence; t h e - t r e n d toward autogamy has most occurred often.  independently  i n the f a m i l y at l e a s t t h r e e times  2) t h e b i o l o g i c a l s p e c i e s concept  likely  and p o s s i b l y more  employed by Mason i s p r o b a b l y  of  l i m i t e d v a l u e i n t h i s c o n t e x t , . s i n c e genetic, b a r r i e r s t o i n t e r b r e e d i n g e x i s t between c l o s e l y r e l a t e d sympatric distantly related allopatric  B.  t a x a , but may  not be p r e s e n t between more  taxa.  Family- A f f i n i t i e s In h i s d e s c r i p t i o n o f t h e Limnanthaceae, Robert Brown (1833)  uncertain regarding a f f i n i t i e s with other f a m i l i e s . examinations of F l o e r k e a suggested  He  was  implied that his  an a s s o c i a t i o n w i t h t h e perigynous  families.  However, a f t e r the d i s c o v e r y o f Limnanthes, he d e c i d e d t h a t t h e f a m i l y more p r o p e r l y a l l i e d w i t h the hypogynous f a m i l i e s . authors  Other n i n e t e e n t h  earlier  was  century  g e n e r a l l y agreed w i t h Brown, e i t h e r l i n k i n g t o , or i n c l u d i n g members  of t h e Limnanthaceae i n , t h e Geraniaceae. i n c l u d i n g the f a m i l y i n the  E n g l e r and P r a n t l (1896) d i s a g r e e d  Sapindales.  Most modern a u t h o r s ,  i n c l u d i n g Hutchinson  (1926), C r o n q u i s t  (1968),  - 8TABLE  I  Present Day C l a s s i f i c a t i o n  o f t h e Limnanthaceae  Floerkea W i l l d . l ) F_. p r o s e r p i n a c o i d e s W i l l d .  Limnanthes R. B r .  Section Reflexae 2) L. b a k e r i  J.T. Howell  3) L_. d o u g l a s i i R. B r . v a r . d o u g l a s i i "  var.- n i v e a Mason  "  v a r . r o s e a (Benth.) Mason  "  v a r . s u l p h u r e a Mason  4) L_. macounii  Trel.  5) L_. s t r i a t a Jepson 6) L. v i n c u l a n s . O r n d u f f  Section  Inflexae  7) L_. a l b a Benth. y a r . . a l b a ".  var. versicolor  (Greene) Mason  8) L_. f l o c c o s a Howell s s p . f l o c c o s a .  9)  "  ssp. b e l l i n g e r i a n a  (M.E. Peck) A r r o y o  "  ssp. c a l i f o r n i c a Arroyo  "  ssp. g r a n d i f l o r a Arroyo  "  s s p . p u m i l a (Howell) A r r o y o  L_. g r a c i l i s Howell v a r . g r a c i l i s "  10) L_. montana Jepson  var. p a r i s h i i  (Jepson) Mason  - 9 and T a k h t a j a n  (1969), agree t h a t t h e Limnanthaceae c o n s t i t u t e s a d i s t i n c t  family belonging t o the Geraniales. t i o n t o t h i s viewpoint and t h e G e r a n i a l e s .  (1956) took excep-  Maheshwari and J o h r i  a f t e r comparing t h e Limnanthaceae t o t h e S a p i n d a l e s  T h e i r comparison was based on e m b r y o l o g i c a l and morpho-  l o g i c a l f e a t u r e s o f Limnanthes d i s c o v e r e d by Mason  (1951) and Mathur (1956),  t o g e t h e r w i t h t h e i r own f i n d i n g s on embryo development, f l o r a l morphology, and  seed morphology o f F. p r o s e r p i n a c o i d e s .  Limnanthaceae has few m o r p h o l o g i c a l  The authors  showed t h a t t h e  s i m i l a r i t i e s t o either the Geraniales or  S a p i n d a l e s , w i t h r e g a r d t o embryogenesis, and seed, p o l l e n , and gynoecium morphology.  On t h i s b a s i s , Maheswari and J o h r i concluded  t h e Limnanthaceae t o e i t h e r t h e S a p i n d a l e s  or Geraniales  t h a t assignment o f i s unsatisfactory,  and t h a t p r o b a b l y t h e f a m i l y s h o u l d be r a i s e d t o o r d i n a l rank. t h e authors suggest  Unfortunately,  do not s p e c u l a t e . o n t h e d e r i v a t i o n o f t h e Limnanthaceae, nor do t h e y  w i t h which o r d e r s , i f any, t h e Limnanthaceae shares t h e g r e a t e s t  affinity. Warburg (1938) d i s c u s s e d i n c l u s i o n o f t h e Limnanthaceae i n t h e G e r a n i a l e s i n l i g h t o f i t s chromosome c y t o l o g y . mined karyotype  morphology, and observed  He made chromosome c o u n t s , chromosome behaviour  at meiosis  f o r at l e a s t onfe-'representative o f each f a m i l y i n t h e G e r a n i a l e s Hutchinson  (1926), i..e/, Geraniaceae,  Oxalidaceae,  L i n a c e a e , Balsaminaceae and Z y g o p h y l l a c e a e .  Tropaeolaceae,  The behaviour  deter-  sensu Limnanthaceae,  o f chromosomes  of Limnanthes a t m e i o s i s , t h e i r l a r g e s i z e , and s m a l l number caused Warburg t o propose two a l t e r n a t e hypotheses r e g a r d i n g placement o f t h e Limnanthaceae; e i t h e r t h e f a m i l y i s i m p r o p e r l y p l a c e d i n t h e G e r a n i a l e s and.more l i k e l y i n t o the Sapindales primitive relic  fits  as proposed by E n g l e r a n d P r a n t l ; o r t h e f a m i l y i s a  c l o s e l y r e l a t e d t o a n c e s t r a l t a x a from which members o f t h e  Geraniales are derived.  N e i t h e r o f t h e s e hypotheses has been supported by  more r e c e n t d i s c o v e r i e s , b u t t h e c y t o l o g i c a l o b s e r v a t i o n s  on which they were  - 10 based remain v a l u a b l e . ,  ..  PREVIOUS CHEMICAL INVESTIGATIONS C o n s i d e r i n g the s m a l l s i z e of the Limnanthaceae, a r e l a t i v e l y l a r g e amount o f research' has been d i r e c t e d toward c h a r a c t e r i z i n g t h i s family'.;s cherr.risitry-^been  T h i s i s not  s u r p r i s i n g i n view of the i n t e r e s t i n g r e s u l t s t h a t have,  obtained. Members o f the f a m i l y c o n t a i n g l u c o s i n o l a t e s , a c h a r a c t e r known t o  be  shared by o n l y about a dozen other f a m i l i e s .  cyanate was  i s o l a t e d and  and Lundeen (.1956).  m-Methoxybenzyl i s o t h i o -  c h a r a c t e r i z e d from seed o f L_. d o u g l a s i i by  Actually, this  Ettlinger  i s o t h i o c y a n a t e i s not n a t u r a l l y o c c u r r i n g ;  i n s t e a d , i t i s a d e r i v a t i v e o f the ,glueoside p r e s e n t formed by the enzymic r e l e a s e o f g l u c o s e .  i n the seed, which i s  Hence, the n a t u r a l l y o c c u r r i n g  g l u c o s i d e o f L_. d o u g l a s i i i s m-methoxybenzyl g l u c o s i n o l a t e , g i v e n the name g l u c o l i m n a n t h i n .  T h i s compound possesses an extremely r a r e  trivial  substi-  t u t i o n p a t t e r n not known o u t s i d e the Limnanthaceae.' M i l l e r et a l . (1964) have d e t e c t e d an a d d i t i o n a l u n i d e n t i f i e d g l u c o s i n o l a t e i n L. d o u g l a s i i v a r . n i v e a , and a l s o r e p o r t e d the presence o f g l u c o s i n o l a t e s i n a l l other nanthes t a x a . ( G e n t r y  & M i l l e r , 1965).  The U n i t e d S t a t e s Department o f A g r i c u l t u r e (U.S.D.A.) has e x t e n s i v e r e s e a r c h on the seed o i l chemistry  1965;  Lim-  M i l l e r et a l . , 1964).  conducted  of Limnanthes (Gentry  U n l i k e common v e g e t a b l e  oils  & Miller,  based on 16  or  l 8 - c a r b o n f a t t y acids', the seed o i l o f Limnanthes i s based on 20 and fatty acids.  22-carbon  P o s i t i o n s o f u n s a t u r a t i o n o f t h e f a t t y a c i d s o f Limnanthes are'-  a l s o uncommon.  Four such f a t t y a c i d s comprise about 95%  o i l f r a c t i o n o f Limnanthes;  of t h e s e , t h r e e are unknown from other  w h i l e the" f o u r t h i s e r u c i c a c i d , a l s o p r e s e n t B e f o r e the p r e s e n t  of the t o t a l  i n v e s t i g a t i o n was  i n rape and mustard completed, l i t t l e was  seed  sources,  oils. known o f  - l i the p h e n o l i c substances that  hydrolyzed  kaempferol,  e x t r a c t s o f l e a v e s o f L_. d o u g l a s i i c o n t a i n e d c a f f e i c  i n v e s t i g a t i o n , Parker  AGRONOMIC EVALUATION OF  in later  of  s i n c e the o i l content  implications of  i n the seed o i l of Limnanthes a r e w e l l s u i t e d products.  Because o f t h i s  o f t h e n u t l e t s i s 20-33% (Gentry  Limnanthes as a crop p l a n t .  suitability  T r i a l s were made.in A l a s k a , Oregon, C a l i f o r n i a  of p l a n t i n g , h a r v e s t i n g and  most o f the Limnanthes t a x a  character,  & Miller,1965),  conducted e x t e n s i v e t r i a l s t o determine t h e  and Maryland, a n d . c o n s i s t e d for  r e s u l t s and  chapters.  the p r o d u c t i o n o f h i g h q u a l i t y wax  t h e U.S.D.A. has  The  LIMNANTHES  T h e . f a t t y acids present  and  As  and Bohm (1975) c h a r a c t e r i z e d  f l a v o n o l g l y c o s i d e s from L_. d o u g l a s i i .  t h a t study are p r e s e n t e d  to  acid,  q u e r c e t i n , m y r i c e t i n , l e u c o c y a n i d i n and l e u c o d e l p h i n i d i n .  p a r t o f the p r e s e n t 18  Bate-Smith (1962) r e p o r t e d  o f t h e Limnanthaceae.  calculating  ( H i g g i n s et_ a l . , 1971).  yields  In a d d i t i o n ,  v a r i o u s r e s e a r c h e r s have t r i e d t o determine o p t i m a l c o n d i t i o n s f o r germin a t i o n o f Limnanthes. seed The populations  (Toy & W i l l i n g h a m ,  r e s u l t s of the f i e l d t r i a l s  1966;  1967;  C o l e , 197*0.  i n d i c a t e t h a t most of t h e w i l d  o f Limnanthes produce abundant seed i n t h e range of 1000  h e c t a r e when c u l t i v a t e d .  From an agronomic s t a n d p o i n t L_. a l b a i s c o n s i d e r e d  t h e most d e s i r a b l e s p e c i e s due  t o i t s u p r i g h t form and  both q u a l i t i e s being necessary  f o r mechanical harvesting.  are'marginal  kg/  at p r e s e n t , H i g g i n e t a l . - (1971) concluded  good seed r e t e n t i o n , Although  profits  t h a t L_. a l b a prob-  Yiably c o n t a i n s s u f f i c i e n t g e n e t i c v a r i a b i l i t y such t h a t an a r t i f i c i a l s e l e c t i o n program c o u l d s u b s t a n t i a l l y i n c r e a s e seed y i e l d s . c o n t a i n s 15-25% p r o t e i n , the authors c o u l d be used as s t o c k f o d d e r , a r e not t o o high.'  suggested  S i n c e Limnanthes  seed  t h a t the e x t r a c t e d meal  i f r e s i d u a l l e v e l s of t o x i c g l u c o s i n o l a t e s  -  12  -  THESIS OBJECTIVES The  Limnanthaceae i s p a r t i c u l a r l y w e l l s u i t e d t o the a p p l i c a t i o n  o f c h e m i c a l taxonomic t e c h n i q u e s .  Due  b o t h w i t h i n the f a m i l y and w i t h other o f new  information  information  can be  t o the u n c e r t a i n t y  of r e l a t i o n s h i p s ,  f a m i l i e s , the d i s c o v e r y  and  w i t h taxonomic s i g n i f i c a n c e i s d e s i r a b l e . a p p l i e d , . i t i s probably impossible  f a m i l y r e l a t i o n s h i p s , and t h e r e f o r e , i m p o s s i b l e  application  Unless  to f u l l y  new  elucidate  t o t e s t the v a l i d i t y of  the  c l a s s i f i c a t i o n shown i n T a b l e I. The.chemical c h a r a c t e r s p o t e n t i a l l y valuable have s e r v e d l a r g e and  f a m i l y are an untapped source o f  taxonomic i n f o r m a t i o n .  as u s e f u l taxonomic• c h a r a c t e r s  s m a l l , and  characters  o f the  Secondary c h e m i c a l  f o r many groups of p l a n t s , both  i t i s p o s s i b l e t h a t the s y s t e m a t i c  might be u s e f u l i n the context  constituents  u t i l i s a t i o n of these  of the Limnanthaceae.  The  infor-  mation a v a i l a b l e on seed f a t s suggests t h a t t h e s e c o n s t i t u e n t s possess taxonomic s i g n i f i c a n c e ( M i l l e r . e t a l . , f i c a n c e o f other cannot be  The  p o s s i b l e taxonomic  c l a s s e s o f secondary compounds, i n c l u d i n g the  into this latter  primary i n t e n t i o n s o f the p r e s e n t  sitions;  flavonoids  study a r e :  l ) t o determine 2).to  the  produce  an  c l a s s i f i c a t i o n ( s ) o f t h e f a m i l y based s o l e l y on f l a v o n o i d compo3)  t o compare the c h e m i c a l c l a s s i f i c a t i o n w i t h the  t y p i c treatments;  and  k) t o d i s c u s s any  w i t h i n the f a m i l y or r e g a r d i n g course .of t h i s work.  lh  There are t h r e e value  The  category.  f l a v o n o i d c o m p o s i t i o n of each,member of the Limnanthaceae; artificial  signi-  glucosinolates,  e s t i m a t e d u n t i l t h e y are more c o m p l e t e l y e l u c i d a t e d .  of the Limnanthaceae a l s o f a l l The  196k).  little  of the  e x i s t i n g pheno-  i m p l i c a t i o n s .regarding  a f f i n i t i e s to other  evolution  f a m i l i e s uncovered i n the  Y  secondary o b j e c t i v e s 'o§ t h i s  contribution flavonoid character  m a t i c s o f the Limnanthaceae; . 2)  study:  l ) to assess  taxonomy can make t o the  the  syste-  t o d i s c u s s b r i e f l y the method o f a p p l i c a t i o n  - 13 of flavonoids  as c h a r a c t e r s  nomic s i g n i f i c a n c e o f f l o w e r  i n p l a n t taxonomy; patterning  i n the  and  3) t o a s s e s s the  family.  taxo-  - 14 -  MATERIALS AND METHODS  THE PLANTS AND THEIR SOURCES Whenever p o s s i b l e f r e s h l y grown p l a n t material'was comparisons. of  used f o r f l a v o n o i d  T h i s p r a c t i c e tends t o e l i m i n a t e s e v e r a l p o t e n t i a l  chemical v a r i a t i o n f o r t h e f o l l o w i n g r e a s o n s :  sources  t h e use o f f r e s h m a t e r i a l  e l i m i n a t e s most a m b i g u i t i e s a r i s i n g e i t h e r from compounds b e i n g p r e s e n t i n low c o n c e n t r a t i o n o r from i n d i v i d u a l v a r i a t i o n , s i n c e a l a r g e amount o f p l a n t m a t e r i a l i s u s u a l l y a v a i l a b l e and many p l a n t s can be a n a l y z e d taneously; any  simul-  e n v i r o n m e n t a l l y induced v a r i a t i o n i n f l a v o n o i d c o m p o s i t i o n , i f  e x i s t s , a l s o i s e l i m i n a t e d , s i n c e a l l p l a n t s can be grown under n e a r l y  uniform c o n d i t i o n s ;  and a l t h o u g h f l a v o n o i d breakdown i s a p p a r e n t l y i n s i g n i f i -  cant i n p r e s s e d specimens o f Limnanthes and F l o e r k e a , t h e use o f f r e s h m a t e r i a l for  chemical comparisons e l i m i n a t e s t h i s p o s s i b l e source o f v a r i a t i o n . Approximately  t h r e e q u a r t e r s o f t h e Limnanthes t a x a were grown from  seed p r o v i d e d by G.A. White, U.S.D.A., A g r i c u l t u r a l Research S e r v i c e , B e l t s v i l l e , Maryland.  T h i s seed was a v a i l a b l e as a r e s u l t o f t h e agronomic  e v a l u a t i o n b e i n g conducted  by t h e U.S.D.A.  The t a x a f o r which no seed was  a v a i l a b l e were t h o s e d e s c r i b e d a f t e r t h e U.S.D.A. work was underway, i n c l u d i n g L_. v i n c u l a n s and t h e v a r i o u s s u b s p e c i e s o f L_. f l o c c o s a . of F l o e r k e a was u n o b t a i n a b l e  Unfortunately,  from any source.  S i n c e i t i s n e a r l y e s s e n t i a l t o i n c l u d e a l l members i n a study, herbarium thaceae  seed  comparative  specimens were used f o r a n a l y s i s o f t h o s e t a x a o f t h e Limnan-  f o r which no seed c o u l d be o b t a i n e d .  D r i e d m a t e r i a l o f L_. v i n c u l a n s  and L_. f l o c c o s a was p r o v i d e d by Dr.. R. O r n d u f f , Department o f Botany, U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , w h i l e s e v e r a l c o l l e c t i o n s o f F l o e r k e a were  p r o v i d e d by t h e U n i v e r s i t y o f B r i t i s h Columbia Herbarium. The  p a r t i a l use o f d r i e d m a t e r i a l causes c e r t a i n i n t e r p r e t i v e prob-  lems a r i s i n g from the a n a l y s i s o f n o n - p a r a l l e l m a t e r i a l . p r a c t i c e on the r e s u l t s o f t h i s  effects of t h i s  study -will be d i s c u s s e d i n t h e l a s t  In t h e course of t h i s work, i t was  r e a l i z e d t h a t Dr. W.B.  Department o f Botany, U n i v e r s i t y of B r i t i s h Columbia, had l a t i o n of t h e extremely  The  chapter. Schofield,  d i s c o v e r e d a popu-  r a r e L_. macounii at .William Head, Vancouver I s l a n d  about 25 m i l e s from t h e o n l y p r e v i o u s l y known p o p u l a t i o n at V i c t o r i a , (Mr. A.  Ceska, Department o f B i o l o g y , U n i v e r s i t y o f V i c t o r i a , has  t h a t he and h i s w i f e have d i s c o v e r e d a d d i t i o n a l p o p u l a t i o n s M a t e r i a l was  informed  T a b l e I I i d e n t i f i e s t h e sources  me  s i n c e t h a t time.)  grown from seed taken from t h e S c h o f i e l d c o l l e c t i o n , and  t i o n a l p l a n t s were c o l l e c t e d from t h i s new  B.C.  addi-  population.  o f a l l p l a n t s used i n t h i s  study.  The  c o l l e c t o r s and t h e i r c o l l e c t i o n numbers are g i v e n f o r the herbarium materi;  The  remaining  numbers.  (1971)  p l a n t s are ' I d e n t i f i e d by t h e o r i g i n a l U.S.D.A. p l a n t a c c e s s i o n  These numbers a r e the same as those r e f e r r e d t o by H i g g i n s  i n t h e i r agronomic e v a l u a t i o n Each e n t r y i n T a b l e I I was  s e p a r a t e l y , and number.  The  et a l .  study. t r e a t e d as a d i s t i n c t taxon  and  analyzed  each has been a s s i g n e d an O p e r a t i o n a l Taxonomic U n i t  assignment o f t h e s e numbers r o u g h l y f o l l o w s t h e accepted  f i c a t i o n of t h e f a m i l y , but t h i s assignment was C e r t a i n v a r i e t i e s , subspecies i n Table I I .  classi-  made o n l y t o i d e n t i f y OTUs.  or s p e c i e s a r e r e p r e s e n t e d by more than  one  OTU  and  almost" c e r t a i n l y d i f f e r e n t p o p u l a t i o n s as w e l l .  separately different  (OTU)  These r e p e t i t i v e OTUs r e p r e s e n t d i f f e r e n t The  collections  decision to  c o l l e c t i o n s o f a taxon does not r e f l e c t  any  analyze  preconceived  i taxonomic judgements.  A primary  reason  f o r separateutreatment  i s t h a t some  o f the t a x a were i d e n t i f i e d a c c o r d i n g t o d i f f e r e n t taxonomic c r i t e r i a and • conventions.  'For i n s t a n c e , i t was  c o n s i d e r e d unwise t o group f r e s h m a t e r i a l  - 16 TABLE I I I d e n t i f i c a t i o n and Sources o f O p e r a t i o n a l Taxonomic U n i t s Of Limnanthes and F l o e r k e a  Taxon Name  U.S.D.A. P l a n t A c c e s s i o n No.  1  L. d o u g l a s i i v a r . d o u g l a s i i  2T81T0  2  L. d o u g l a s i i v a r . d o u g l a s i i  283T08  3  L. d o u g l a s i i v a r . n i v e a  283T13  k  L. d o u g l a s i i v a r . r o s e a  283T15  5  L. d o u g l a s i i v a r . s u l p h u r e a  283T18  6  L. v i n c u l a n s  7 8  L. b a k e r i  283T06  L. s t r i a t a  283T2T  9  L. macounii  OTU  C o l l e c t o r and C o l l e c t i o n No.  5699  Rubtzoff  (Victoria)  315048  10  L. macounii ( W i l l i a m  11  L. a l b a v a r . a l b a  283T01  12  L. a l b a v a r . a l b a  B55689  13  L. a l b a v a r . v e r s i c o l o r  283705  14  L. g r a c i l i s  var. g r a c i l i s  283722  15  L. g r a c i l i s  var. g r a c i l i s  283723  16  L. g r a c i l i s , v a r . p a r i s h i i  283724  IT  L. montana  283725  18  L. f l o c c o s a  Schofield  Head)  ssp. b e l l i n g e r i a n a .  283720  19 • L. f l o c c o s a ssp. b e l l i n g e r i a n a  Kalin  ( A r r o y o ) 7031  Kalin  7033  283721  20  L. f l o c c o s a  ssp. pumila  21  L. f l o c c o s a  ssp. p u m i l a  22  L. f l o c c o s a ssp. g r a n d i f l o r a .  Kalin  7028  23  L. f l o c c o s a ssp. f l o c c o s a  Kalin  7026  24.  L. f l o c c o s a ssp. f l o c c o s a -  Kalin  7022  25  L. f l o c c o s a  Kalin  6917  26.  L.' f l o c c o s a s s p . ' c a l i f o r n i c a  2T  F.- p r o s e r p i n a c o i d e s  (Dutchess Co. , N.Y. )  Ahles 76368  28  F. p r o s e r p i n a c o i d e s  ( K e t t l e F a l l s , Wash.)  Beamish  29  F. p r o s e r p i n a c o i d e s  ( M o n t r e a l , Quebec)  Rouleau 4001  30'  F. p r o s e r p i n a c o i d e s  (Shushan, N.Y. .)  Dobbin  ssp. f l o c c o s a  Niehaus  '  371 & O r n d u f f 6885 60352 1758  -  17 -  of L_. f l o c c o s a v a r . b e l l i n g e r i a n a , i d e n t i f i e d on t h e b a s i s o f a t h r e e varietys u b d i v i s i o n o f L_. f l o c c o s a , w i t h p r e s s e d m a t e r i a l o f L_. f l o c c o s a s s p . b e l l i n geriana^ i d e n t i f i e d according t o a f i v e subspecies d i v i s i o n .  T h e r e f o r e , OTUs  18 and 19 were a n a l y z e d s e p a r a t e l y . Three t a x a o b t a i n e d from t h e U.S.D.A. were not i d e n t i f i e d t o Mason's c l a s s i f i c a t i o n .  according  These t a x a a r e : L. d o u g l a s i i . , OTU 1; L_. g r a c i l i s ,  OTU 14; and L_. a l b a , OTU 1 1 .  A f t e r growth and examination,  each o f t h e s e  t a x a was i d e n t i f i e d as t h e r e s p e c t i v e t y p i c a l v a r i e t y , , and t h e y a r e d e s i g n a t e d i n t h i s , f a s h i o n i n T a b l e I I . , I t i s o f i n t e r e s t t h a t t h e seed o f OTU 1 was o r i g i n a l l y from Europe, and p r o b a b l y t h i s taxon  i s descended from t h e p l a n t s  o r i g i n a l l y c o l l e c t e d by David Douglas i n t h e e a r l y 1830s from which Brown d e s c r i b e d t h e f a m i l y (Gentry & M i l l e r , 1 9 6 5 ) . i s o l a t i o n has p r o b a b l y been m a i n t a i n e d  I n t h i s case an a r t i f i c i a l  f o r n e a r l y 150 y e a r s , which a l l o w s an  e s t i m a t i o n o f t h e e f f e c t s o f genetic' d r i f t  and i n b r e e d i n g over t h i s p e r i o d .  F l o e r k e a pros.erpinacoides and L. macounii and two OTUs r e s p e c t i v e l y , as shown i n T a b l e I I .  are subdivided into four Both a r e t o t a l l y  gamous, and p o p u l a t i o n s from d i f f e r e n t l o c a t i o n s a r e e f f e c t i v e l y isolated.  auto-  genetically  F o r t h i s r e a s o n , s e p a r a t e a n a l y s e s o f t h e s e p o p u l a t i o n s may r e v e a l  d i f f e r e n c e s i n f l a v o n o i d composition s e l e c t i o n pressures.  a t t r i b u t a b l e to genetic d r i f t  or d i f f e r e n t  I n t h e c a s e . o f L. m a c o u n i i , t h e two p o p u l a t i o n s a r e  o n l y a few m i l e s a p a r t , but i n t h e c a s e . o f F. p r o s e r p i n a c o i d e s , t h e most w i d e l y d i s t r i b u t e d element o f t h e f a m i l y , t h e p o p u l a t i o n s a r e from t h e extremes o f i t s range. Three d i f f e r e n t c o l l e c t i o n s o f L_. f l o c c o s a s s p . f l o c c o s a were a v a i l a b l e from v a r i o u s l o c a t i o n s .  Each o f t h e s e c o l l e c t i o n s was made by A r r o y o and  was used i n h e r s y s t e m a t i c s t u d i e s .  These c o l l e c t i o n s a r e t r e a t e d . h e r e as  d i s t i n c t OTUs t o assess t h e v a r i a t i o n w i t h i n t h i s taxon and t o t e s t t h e v a l i d i t y - . o f ' A r r o y o ' s r e c e n t taxonomic treatment  o f L. f l o c c o s a  (Arroyo, 1 9 7 3 a ) .  - 18 -  In  t h e course o f growing  L. d o u g l a s i i v a r . s u l p h u r e a , OTU 5, U.S.D.A.  A c c e s s i o n #283718, i t was observed t h a t t h e s e p l a n t s had p e t a l p a t t e r n i n g d i f f e r i n g from Mason's d e s c r i p t i o n o f t h i s v a r i e t y . it  as h a v i n g c o m p l e t e l y y e l l o w p e t a l s , w h i l e t h e U.S.D.A. p l a n t s have a  s m a l l amount o f white a t t h e t i p s .  However, t h e y have a s m a l l e r amount than  t h e p e t a l s o f L. d o u g l a s i i var.. d o u g l a s i i . to  Mason (1952) d e s c r i b e d  Otherwise,' t h e s e p l a n t s c o r r e s p o n d  Mason's d e s c r i p t i o n o f v a r i e t y s u l p h u r e a , i n c l u d i n g l e a f l e t  shape, a c h a r -  a c t e r which d i s t i n g u i s h e s t h i s taxon from o t h e r v a r i e t i e s o f L_. d o u g l a s i i . Mason d e s c r i b e d F^ h y b r i d s o f v a r i e t i e s  s u l p h u r e a a n d . d o u g l a s i i as h a v i n g  p e t a l t i p s w i t h an i n t e r m e d i a t e amount o f w h i t e .  Whether t h e abnormal  c o l o u r i n g o f t h e U.S.D.A. v a r i e t y r e s u l t s from such c r o s s i n g i s not known, but t h e i d e n t i f i c a t i o n o f OTU 5 as L_. d o u g l a s i i v a r . . s u l p h u r e a was accepted f o r t h i s  tentatively  study.  Voucher specimens o f a l l p l a n t s grown f o r t h i s i n v e s t i g a t i o n a r e d e p o s i t e d i n t h e U.B.C. Herbarium. t h e i r c o l l e c t i o n numbers. i n t h e U.B.C. Herbarium.  Appendix I I l i s t s these, vouchers and  The f o u r c o l l e c t i o n s o f F l o e r k e a a r e . a l s o on f i l e • Voucher specimens o f L. f l o c c o s a and. L. v i n c u l a n s ,  t h a t were p r o v i d e d by O r n d u f f , a r e on f i l e a t t h e Herbarium of  of the University  C a l i f o r n i a , Berkeley.  PLAWT GROWTH Most p l a n t s were grown i n a greenhou®^' under n e a r l y u n i f o r m c o n d i tions.  The d i f f e r e n t t a x a were grown i n random groups, two t o f o u r a t a t i m e ,  from t h e end o f 1972 d o u g l a s i i , . OTU 1, of  1973  t o t h e b e g i n n i n g o f 1975-  One taxon,-L.. d o u g l a s i i v a r .  was a l s o grown outdoors on a much l a r g e r s c a l e - i n t h e summer  t o p r o v i d e a q u a n t i t y . o f f r e s h p l a n t m a t e r i a l so t h a t t h e major-  f l a v o n o i d s c o u l d be i s o l a t e d . Mason (1952)'. r e p o r t e d t h a t Limnanthes seed w i l l not germinate, u n l e s s  -  t h e s o i l temperature  19 -  i s kept under 15°.  Preliminary germination t e s t s  con-  f i r m e d t h i s o b s e r v a t i o n and a l s o showed t h a t s t r a t i f i c a t i o n o f t h e seed a t about  5-10° f o r 3-5 days enhanced g e r m i n a t i o n .  T h e r e f o r e , . a l l seed was p l a c e d  i n a r e f r i g e r a t o r f o r a few days b e f o r e p l a n t i n g i n p o t s . p l a c e d i n c o l d frames B r i t i s h Columbia  The pots were  on t o p o f t h e B i o - S c i e n c e s B u i l d i n g , U n i v e r s i t y o f  and kept t h e r e u n t i l g e r m i n a t i o n was complete.  This  practice  was f a i r l y s a t i s f a c t o r y except i n t h e m i d d l e o f w i n t e r o r t h e m i d d l e o f summer,  when temperatures  will  germinate.  i n t h e c o l d frame exceeded t h e range i n which  A f t e r g e r m i n a t i o n , pots o f young Limnanthes t o a greenhouse  and a l l o w e d a s h o r t growth p e r i o d .  Limnanthes  p l a n t s were t r a n s f e r r e d A f t e r development  ofthe  second o r t h i r d permanent l e a f , t h e young s e e d l i n g s were t r a n s p l a n t e d 20 t o a f l a t , . u s i n g a m i x t u r e o f about -  90%. s t e r i l i z e d garden s o i l and 10%. sand.  The  s u r v i v a l r a t e o f t r a n s p l a n t e d s e e d l i n g s w a s - c l o s e t o 100%,, i f they, were not overwatered.  S i n c e t h e m o i s t u r e requirements o f t h e d i f f e r e n t t a x a v a r i e d ,  each taxon was watered an a p p r o p r i a t e amount a r r i v e d a t by t r i a l if  and e r r o r ;  any s i g n s o f damping-ioff appeared, t h e amount o f water.was reduced. P l a n t s used f o r f l a v o n o i d comparisons  under t h e same bank.of  ordinary fluorescent l i g h t s .  a n n u a l s , t a x a o f Limnanthes 1952).  were grown on t h e same bench L i k e many e a r l y  spring  r e q u i r e l o n g d a y l i g h t p e r i o d s t o . f l o w e r (Mason,  I t was found t h a t a l l t a x a r a p i d l y f l o w e r e d u n d e r . l 6 ' h o u r d a y l e n g t h ,  so t h i s l i g h t  d u r a t i o n was used c o n s i s t e n t l y .  A l s o , i t was d i s c o v e r e d e a r l y  i n t h e c o u r s e o f t h i s work t h a t s h o u l d d a y l e n g t h be s h o r t e n e d d r a s t i c a l l y (i.e_. , 16 hour t o an 8 hour d a y l e n g t h ) a f t e r - t h e p l a n t s have.begun f l o w e r i n g , t h e y r a p i d l y cease f l o w e r i n g and d i e .  A complete  g e n e r a t i o n o f Limnanthes,  from seed germination, t o seed r i p e n i n g , o c c u r s i n 3-h months'under t h e ' c o n d i t i o n s o u t l i n e d above. • • The procedure f o r growing L. d o u g l a s i i v a r . d o u g l a s i i outdoors was  - 20 -  s i m i l a r t o t h e above, except t h a t s e e d l i n g s were t r a n s p l a n t e d d i r e c t l y the ground i n s t e a d o f f l a t s . about 50 m l o n g .  into  P l a n t s were spaced every 15 cm i n a s i n g l e row  Seed f o r t h i s p l a n t i n g was germinated i n l a t e June, and  p l a n t s were h a r v e s t e d  i n l a t e September, j u s t p r i o r t o f l o w e r i n g .  Outdoor-  grown p l a n t s produced much c o a r s e r , more l u s h v e g e t a t i o n t h a n t h e c o r r e s p o n d i n g greenhouse grown p l a n t s . shortening  dayleng'th  Whether t h i s d i f f e r e n c e r e s u l t s from t h e n a t u r a l l y  o r some a d d i t i o n a l f a c t o r s a s s o c i a t e d w i t h t h e outdoor  p l a n t i n g i s not known. • There a r e two problems a s s o c i a t e d w i t h t h e growth o f Limnanthes p l a n t s which deserve comment.  Their succulent  vegetation  i s particularly  a t t r a c t i v e t o aphids and w h i t e f l i e s , b o t h common greenhouse p e s t s .  These  i n s e c t s s e l e c t i v e l y i n f e s t Limnanthes p l a n t s , o f t e n o c c u r r i n g i n great?numbers on them w h i l e  adjacent  plants are.unaffected.  Therefore,  i n order t o o b t a i n  s a t i s f a c t o r y y i e l d s , i t was n e c e s s a r y t o spray p l a n t s p e r i o d i c a l l y w i t h insecticide  (.S.C. Johnson & Son, L t d . ) .  I n t e r e s t i n g l y , the f i e l d  "Raid"  grown  p l a n t s . o f L . d o u g l a s i i - were not a t t a c k e d by i n s e c t s . The  second problem a s s o c i a t e d w i t h Limnanthes c u l t u r e regards i t s ;  root c o l l a r . .  In order  remain b u r i e d .  f o r a plant t o reach maturity,  i t s r o o t c o l l a r must  S i n c e t h i s r o o t c o l l a r extends o n l y a s h o r t d i s t a n c e below  the ground (.1-2 cm), care must be t a k e n when w a t e r i n g and weeding not t o d i s t u r b t h e s o i l immediately around t h e p l a n t .  I f t h e r o o t c o l l a r i s uncovered,  the p l a n t w i l l l i v e o n l y a day o r so.  PLANT.. TISSUE. SELECTION The  t y p e o f t i s s u e used f o r f l a v o n o i d a n a l y s i s v a r i e d w i t h t h e source  of p l a n t m a t e r i a l . . S i n c e . o n l y  small.amounts o f p r e s s e d  a b l e , a l l t h e m a t e r i a l a t hand was used.  specimens were a v a i l -  However, for- p l a n t s grown from  seed, c h o i c e s had t o be made r e g a r d i n g .1) whether a l l t i s s u e s should be  - 21  -  i n c l u d e d i n the f l a v o n o i d comparison t e s t s , and s h o u l d be a n a l y z e d  2) whether any  tissues  separately.  A l l members o f the Limnanthaceae have a b a s a l r o s e t t e o f but  f i n e l y d i v i d e d , compound l e a v e s  a r e . l a r g e l y i n d e t e r m i n a t e . a n d may species.  The  a n d . c a u l e s c e n t f l o w e r i n g stems, which  e a s i l y produce over 25 blooms i n most  p l a n t s have s h o r t r o o t c o l l a r s w i t h a d i f f u s e network of  d i s s e c t e d , deeply  penetrating  lection difficult  and.tedious.  the f l a v o n o i d contents  necessary.  For t h i s  experiment t h e r o o t s were e f f i c i e n t l y  showed r e l a t i v e l y weak c o n c e n t r a t i o n s  further'attempts  m a t e r i a l from o t h e r - t a x a .  i n l e a f and  and  soil.  included  present  by  in this  t o comprise the t o t a l f l a v o n o i d composition  composition''  For  this  with pulling  A l t h o u g h t h i s procedure y i e l d s  o n l y small" amounts.af-root t i s s u e , t h e ' f l a v o n o i d s  flavonoid  of any  material given  i s h e r e a f t e r r e f e r r e d t o as the "whole p l a n t  No' r o o t m a t e r i a l was  c o l l e c t e d when L_. d o u g l a s i i v a r . d o u g l a s i i  grown i n the o p e n . f i e l d .  To f a c i l i t a t e . h a r v e s t i n g , t h e s e p l a n t s were  cut o f f at ground l e v e l .  However, f o r comparative a n a l y s i s , t h i s taxon  regrown -under, the same c o n d i t i o n s as the o t h e r The' f l o w e r s having  pre-  were made, t o rec'over q u a n t i t a t i v e , amounts of r o o t  them from the s o i l a f t e r f l o w e r i n g had begun.  t h i s composition  This  stem m a t e r i a l .  However, some r o o t m a t e r i a l was  t a x o n , and  re-  a more s i m p l i f i e d  the p l a n t s used f o r comparative study s i n c e t h e y were h a r v e s t e d  were c o n s i d e r e d  3, were  the'above ground p a r t s t o determine whether r o o t  p a t t e r n of f l a v o n o i d s i n the r o o t s . t h a n r e a s o n , no  col-  E a r l y i n the course of t h i s i n v e s t i g a t i o n ,  covered u s i n g a stream o f water t o wash away the s u r r o u n d i n g liminary, test  finely  r o o t s , a c h a r a c t e r i s t i c which makes r o o t  o f the r o o t s o f L_. d o u g l a s i i v a r . n i v e a , OTU  compared t o the c o n t e n t s . o f r e c o v e r y was  succulent,  was  simply was  taxa.  of many Limnanthes t a x a a r e . q u i t e s t r i k i n g , some t a x a  flowers with.various  shades o f y e l l o w p a t t e r n i n g on o t h e r w i s e w h i t e  - 22 '.to i v o r y c o l o u r e d p e t a l s .  S i n c e t h e r e i s s t r i k i n g v a r i a t i o n between f l o w e r s  o f some t a x a , and p r e l i m i n a r y t e s t s showed h i g h f l a v o n o i d c o n c e n t r a t i o n s i n t h e p e t a l s , t h e d e c i s i o n was made t o a n a l y z e taxon grown from seed.  s e p a r a t e l y t h e p e t a l s o f each  I n t h i s manner t h e f l a v o n o i d compositions  OTUs c o u l d be compared on t h e b a s i s o f p e t a l c o m p o s i t i o n plant composition.  of these  as w e l l as whole  The p e t a l s from 10-20 i n d i v i d u a l s , c o l l e c t e d over a 2-3  week p e r i o d , y i e l d e d q u a n t i t i e s o f f l a v o n o i d s adequate f o r comparative a n a l y s i s .  FLORAL PATTERNING IN ULTRA VIOLET LIGHT A.  Introduction I n s e c t - p o l l i n a t e d , o u t c r o s s i n g p l a n t s f r e q u e n t l y possess  a d a p t a t i o n s , such as n e c t a r glands w i t h t h i s type o f b r e e d i n g  system.  and p a t t e r n e d f l o w e r s t h a t a r e a s s o c i a t e d C o n t r a s t i n g p e t a l c o l o u r i n g i s common  i n such p l a n t s and o f t e n r e s u l t s i n a " b u l l s e y e " e f f e c t . tends t o draw an o b s e r v e r ' s  special  eye toward t h e f l o w e r c e n t e r .  the i n s e c t p o l l i n a t o r i s t h e n e c t a r glands  This type o f p a t t e r n Since the goal o f  r a t h e r than t h e r e p r o d u c t i v e p a r t s  o f a f l o w e r , b u l l s e y e p a t t e r n s have.been i n t e r p r e t e d as a d a p t a t i o n s attract v i s i t i n g termed " n e c t a r  which  i n s e c t s t o t h e c e n t e r o f t h e f l o w e r and.have thus been  guides".  Only by l o o k i n g a t a f l o w e r as a bee sees i t , i s i t p o s s i b l e t o f u l l y a p p r e c i a t e c o l o u r a d a p t a t i o n s that? f a c i l i t a t e p o l l i n a t i o n by t h i s v e c t o r . Although  t h i s cannot be done d i r e c t l y , i t i s p o s s i b l e t o examine f l o w e r s a t  a l l wavelengths t o which bees respond.  Von F r i s c h ' s work on honeybee v i s i o n  (1967) has r e v e a l e d t h a t bees, u n l i k e humans, possess l i g h t r e c e p t o r s s e n s i t i v e t o near u l t r a violet^MY. } l i g h t . 1  parent  o n l y t o UV l i g h t  By photography u s i n g a f i l t e r t r a n s -  and f i l m s e n s i t i v e t o t h i s c o l o u r range, v a r i o u s  i n v e s t i g a t o r s have d i s c o v e r e d t h a t c e r t a i n f l o w e r s have n e c t a r guides are v i s i b l e o n l y i n UV l i g h t .  that  Flowers w i t h such n e c t a r guides have c o n t r a s t i n g  - 23 b r i g h t and dark r e g i o n s , o f UV l i g h t .  c o r r e s p o n d i n g t o areas o f r e f l e c t a n c e and a b s o r p t i o n  To d a t e , two c l a s s e s o f f l a v o n o i d s , f l a v o n o l s a n d . c h a l c o n e s ,  b o t h w i t h a b s o r p t i o n maxima i n t h e near UV range, have been i d e n t i f i e d as t h e pigments r e s p o n s i b l e  f o r t h e UV a b s o r b i n g  portion of the nectar  Rudbeckia and Oenothera v i s i b l e under UV l i g h t  guides o f  (Thompson et_ al_. , 1972;  Dement  & Raven, 19lh). Comparison o f t h e f l o r a l p a t t e r n s bee  o f c l o s e l y r e l a t e d p l a n t s as t h e  sees them may r e v e a l i n f o r m a t i o n w i t h p o t e n t i a l s y s t e m a t i c  importance.  Daumer (1958) has found t h a t f l o w e r s w i t h p a t t e r n i n g v i s i b l e i n UV a r e more a t t r a c t i v e t o p o l l i n a t i n g bees t h a n u n p a t t e r n e d f l o w e r s . led  T h i s phenomenon has  O r n d u f f and Mosquin (1970) t o conclude t h a t p l a n t s which have evolved  system o f n e c t a r  guides v i s i b l e i n UV l i g h t  a  a r e more h i g h l y adapted t o an out-  c r o s s i n g h a b i t than r e l a t e d p l a n t s without such a system. D i s c r i m i n a t i o n by p o l l i n a t i n g agents on t h e b a s i s o f f l o r a l appear- '• ance can p l a y an important r o l e i n t h e d i v e r g e n c e o f p o p u l a t i o n s c r e a t i o n o f new taxonomic e n t i t i e s .  and t h e  Two c l o s e l y r e l a t e d p o p u l a t i o n s  w i t h no  g e n e t i c b a r r i e r s t o c r o s s i n g , d i f f e r i n g o n l y i n t h e i r appearance t o bees, may become r e p r o d u c t i v e l y populations  become i s o l a t e d by p o l l i n a t o r s e l e c t i o n , t h e y may  become d i s t i n c t new s p e c i e s  i s o l a t e d on e t h o l o g i c a l grounds (Mac i o r ,  as a r e s u l t o f g e n e t i c  i n flower  I f two  eventually  d r i f t , and p o s s i b l y d i f f e r e n t i a t e i n t o  i n response t o changing s e l e c t i o n  Differences  1971)•  pressures.  c o l o u r i n g r e v e a l e d by UV photography have.  proven u s e f u l f o r d i f f e r e n t i a t i o n o f c l o s e l y r e l a t e d t a x a .  H o r o v i t z and  Cohen Q-972).have shown t h a t t h e UV r e f l e c t a n c e c h a r a c t e r i s t i c s o f S i n a p i s a l b a and S\ a r v e n s i s h e l p d i s t i n g u i s h t h e s e two c l o s e l y r e l a t e d s p e c i e s . S i m i l a r l y , O r n d u f f and Mosquin (l970),have shown t h a t c l o s e l y r e l a t e d , but geographically tinguished  i s o l a t e d , elements.of t h e Nymphoides i n d i e a complex a r e d i s -  i n t h e same manner.  - 24  -  . S e v e r a l t a x a i n the Limnanthaceae a r e almost t o t a l l y these p l a n t s o f t e n have v i s i b l y p a t t e r n e d bee-pollinated. is  The  outcrossing;  f l o w e r s , n e c t a r g l a n d s , and  are  v a r i e t i e s of L. d o u g l a s i i f o l l o w t h i s p a t t e r n , and i t  i n t e r e s t i n g t h a t t h e most s t r i k i n g d i f f e r e n c e s between these p l a n t s  from v a r i a t i o n i n f l o r a l c o l o u r s and p a t t e r n i n g .  result  S i n c e ,>fTower p e t a l s of  Limnanthes were found t o c o n t a i n h i g h c o n c e n t r a t i o n s  o f f l a v o n o i d s which  might produce UV v i s i b l e n e c t a r g u i d e s , and because i n f o r m a t i o n of p o t e n t i a l systematic  importance might be d i s c o v e r e d , the f l o w e r s o f a l l Limnanthes t a x a  grown from seed were examined by UV photography. d i f f e r e n c e s r e v e a l e d by t h i s t e c h n i q u e  I t was  hoped t h a t any  would complement c h e m i c a l  and would be u s e f u l i n r e e v a l u a t i n g t h e s y s t e m a t i c s  of the  floral  differences  family.  B. Method Flowers o f OTUs grown from seed were photographed on c o l o u r f i r s t w i t h no f i l t e r , only.  and  second w i t h a f i l t e r which t r a n s m i t t e d UV  A l l photographs were t a k e n w i t h an Exa  extension r i n g s adjusted transparency ASA-l60 was  was  35 mm  SLR,  so t h a t the r e s u l t i n g f l o w e r  approximately  used throughout.  life  size.  done i n the l a b , but  l8A  i s transparent  light  on  Preliminary  rephotographed  under t h e same c o n d i t i o n s  source  (366  my*).  o n l y t o l i g h t w i t h wavelengths l e s s t h a n 400  S i n c e a r e g u l a r camera l e n s w i l l t r a n s m i t l i g h t i n p l a c e the frequency  only  satisfactory  each f l o w e r was  (Kodak) 2 i n c h g l a s s f i l t e r  as above, b u t w i t h . a n a d d i t i o n a l hand-held UV filter  colour  conditions.  A f t e r photography' i n n a t u r a l l i g h t ,  18A  present.  showed t h a t ah exposure time o f 1/25.second produced  t h r o u g h a Wjratten No,  and  High Speed Ektachrome (Kodak) f i l m ,  Photography was  p i c t u r e s i n e x i s t i n g d a y l i g h t under t h e s e  light  lens,  image on t h e  b r i g h t sunny days when a s t r o n g n a t u r a l window l i g h t was trials  F 3.5  film,  over 350  nyi, w i t h t h e  range o f l i g h t r e a c h i n g the f i l m i s 350-400 ny*  The ny*. filter  - 25 -  1968).  (Kodak Data Book M-27,  The photographs made by t h i s t e c h n i q u e t h e o -  r e t i c a l l y r e p r e s e n t t h e UV component o f a bee's  vision.  An exposure t i m e o f 30 seconds f o r UV photographs was the  basis of preliminary t r i a l s .  mercial processing.  s e l e c t e d on  The f i l m s were developed by r e g u l a r com-  For p r e s e n t a t i o n i n t h i s t h e s i s , s e l e c t e d t r a n s p a r e n c i e s  were c o p i e d and p r i n t e d i n b l a c k and w h i t e . B e f o r e t h e t e c h n i q u e o f UV f l o w e r photography can produce f a c t o r y r e s u l t s , some t e s t photographs must be made.  satis-  This i s necessary t o  determine exposure and f o c u s i n g d e t a i l s and c h o i c e o f background.  Since the  l i g h t r e f r a c t i o n by an o r d i n a r y camera l e n s i s d i f f e r e n t f o r UV than f o r v i s i ble, l i g h t , t h e l e n s must be r e f o c u s e d b e f o r e a UV exposure i s made. the  UV image i s i n v i s i b l e through t h e camera v i e w f i n d e r .  However,  So, u n l e s s a  flu-  o r e s c e n t v i e w i n g s c r e e n i s a v a i l a b l e , t h e dgree o f l e n s movement must be p r e determined by t r i a l exposures. of  about 3 mm  F o r t h i s work a u n i f o r m d e c r e a s e o f e x t e n s i o n  produced UV exposures t h a t were r e a s o n a b l y w e l l f o c u s e d .  Choice o f f l o w e r background must a l s o be determined by t r i a l error.  and  T h i s i s because t h e UV r e f l e c t a n c e and a b s o r p t i v e c h a r a c t e r s o f t h e  s u b j e c t f l o w e r must be known b e f o r e a c o n t r a s t i n g background can be chosen. For  t h e UV photography o f Limnanthes f l o w e r s , a white background gave t h e best)  r e s u l t s , a l t h o u g h a b l a c k background was  FLAVONOID ISOLATION AND A.  also  tried.  IDENTIFICATION .  Introduction .A comparative s u r v e y o f t h e f l a v o n o i d s o f any group o f p l a n t s con-  s i s t s o f two p a r t s :  l ) i s o l a t i n g and i d e n t i f y i n g a l l t h e f l a v o n o i d s  i n t o t a l , and 2) a s s e s s i n g In  present  which compounds o c c u r i n each t a x o n under study.  p r a c t i c e , however, t h i s t e c h n i q u e i s m o d i f i e d s l i g h t l y ; each.taxon i s ana-  l y z e d i n sequence, r a t h e r t h a n a l l t a x a at.once.  I n c l o s e l y r e l a t e d groups  - 26 of p l a n t s , each w i t h s i m i l a r f l a v o n o i d complements, t h e i s o l a t i o n and i d e n t i f i c a t i o n o f t h e f l a v o n o i d s of. one taxon y i e l d s a s e t o f s t a n d a r d  compounds.  By d i r e c t comparison t o t h e s e s t a n d a r d s , i t i s u s u a l l y p o s s i b l e t o i d e n t i f y most o f t h e compounds p r e s e n t  i n t h e remaining  taxa.  T h e r e f o r e , o n l y a few  a d d i t i o n a l unknown f l a v o n o i d s need he; i s o l a t e d and i d e n t i f i e d from each new taxon.  Once t h e s e compounds have been i d e n t i f i e d , they i n t u r n serve as  standards  f o r comparison. The  taxa.  f a m i l y Limnanthaceae c o n s i s t s o f r e l a t i v e l y c l o s e l y  related  P r e l i m i n a r y o b s e r v a t i o n s i n d i c a t e d t h a t t h e f l a v o n o i d complements o f  t h e s e t a x a were v e r y s i m i l a r .  T h e r e f o r e , L_. d o u g l a s i i v a r . d o u g l a s i i , OTU 1,  w i t h i t s l a r g e y e l l o w and white p a t t e r n e d f l o w e r s was s e l e c t e d f o r i n i t i a l f l a v o n o i d i d e n t i f i c a t i o n s based on t h e amount o f seed a v a i l a b l e and t h e s i z e and.colour  o f i t s f l o w e r s , r a t h e r than t h e uniqueness o f i t s f l a v o n o i d  pattern. P l a n t s o f OTU 1 were grown o u t s i d e t o p r o v i d e enough f r e s h m a t e r i a l f o r f l a v o n o i d i d e n t i f i c a t i o n , and f o u r f l a t s o f p l a n t s were grown i n t h e greenhouse t o p r o v i d e s u f f i c i e n t numbers o f f l o w e r s f o r s e p a r a t e a n a l y s i s o f t h e petals.  S i n c e most o f t h e f l a v o n o i d s p r e s e n t  i n remaining  t a x a were  identi-  f i e d by comparison t o t h e compounds o f OTU 1, l e s s f r e s h m a t e r i a l was r e q u i r e d f o r each taxon grown s u b s e q u e n t l y , o f p l a n t s was u s u a l l y adequate.  and t h e m a t e r i a l from a s i n g l e  However, i n c e r t a i n cases  i t was  flat  necessary  t o regrow a taxon t o i s o l a t e enough o f a p r e v i o u s l y unknown compound(s) f o r identification.• U n f o r t u n a t e l y , a few compounds, always o c c u r r e d i n t r a c e amounts o r were p r e s e n t  i n taxa. f o r which no seed was a v a i l a b l e  (e_.g_. , F l o e r k e a ) .  I d e n t i f i c a t i o n o f t h e s e compounds was i m p o s s i b l e without f o r comparison, and.these, were not a v a i l a b l e . of the f i r s t  reference  standards  However, a f t e r t h e f l a v o n o i d s  s e v e r a l t a x a were c h a r a c t e r i z e d , t h e f l a v o n o i d p a t t e r n s o f t h e  - 27 -  r e m a i n i n g t a x a o f t e n c o u l d be completely  determined on the b a s i s o f two  chromatograms, one o f t h e p e t a l s and one o f whole p l a n t m a t e r i a l . In the c o u r s e o f t h i s work, s l i g h t l y d i f f e r e n t procedures were used t o i s o l a t e f l a v o n o i d s a p p r o p r i a t e t o t h e type and''amount o f m a t e r i a l a b l e and the purpose f o r which the m a t e r i a l was used. cedures w i l l be d e s c r i b e d  B.  Plant  These d i f f e r e n t  pro-  i n the f o l l o w i n g s e c t i o n s .  Extractions F r e s h l e a f - s t e m m a t e r i a l o f f i e l d grown L_. d o u g l a s i i v a r .  OTU  avail-  1, weighing 3.5 kg was r e p e a t e d l y  e x t r a c t was c o n c e n t r a t e d  douglasii,  e x t r a c t e d w i t h hot 90% MeOH.  This  i n vacuo a t 30°, f o l l o w e d by t r i t u r a t i o n w i t h  A n a l y t i c a l F i l t e r A i d t o remove c h l o r o p h y l l s . S.f carbohydrates and o t h e r  Celite  The presence o f l a r g e q u a n t i t i e s  components n e c e s s i t a t e d a step t o remove t h e s e com-  ponents before- f l a v o n o i d s e p a r a t i o n was p o s s i b l e .  T h i s was accomplished by  p a s s i n g t h e e x t r a c t d i s s o l v e d i n water through a s h o r t column o f polyamide (SC-6).  S u f f i c i e n t adsorbent was used- t o h o l d a l l f l a v o n o i d s w h i l e  carbo-  h y d r a t e s and o t h e r non-adsorbing compounds were e l u t e d w i t h water. q u e n t l y , t h e f l a v o n o i d s were removed from the column b y r e p e a t e d 50%, aqueous MeOH u n t i l no t r a c e s c o u l d be d e t e c t e d vonoid  c o n t a i n i n g e l u e n t was t h e n c o n c e n t r a t e d The  Subse-  elution  under UV l i g h t .  with  The f l a -  i n vacuo.  p r e l i m i n a r y , c l e a n i n g o f MeOH e x t r a c t s by means o f a s h o r t  poly-  amide column as d e s c r i b e d above proved i n v a l u a b l e f o r s e p a r a t i o n o f f l a v o n o i d s of Limnanthes, a n d . t h i s  procedure was used as a p r e l i m i n a r y step whenever any  f r e s h m a t e r i a l was e x t r a c t e d .  However, c a u t i o n had t o be e x e r c i s e d  while  e l u t i n g w i t h water, s i n c e h i g h l y g l y c o s y l a t e d f l a v o n o i d s , as a r e - f o u n d i n a l l Limnanthes t a x a , have Rfs approaching 1.0 under t h e s e c i r c u m s t a n c e s . f o r e , t o avoid f l a v o n o i d l o s s or contamination, l i g h t was.required during  elution.  c a r e f u l monitoring  There-  under UV  - 28 -  Approximately  10,000 p e t a l s were c o l l e c t e d from  over a p e r i o d o f f l o w e r i n g  o f 2-3 weeks.  k flats  o f OTU  1  The f l a v o n o i d s were i s o l a t e d from  t h i s m a t e r i a l f o l l o w i n g t h e t e c h n i q u e used f o r l e a f - s t e m m a t e r i a l , t h a t t h e t r i t u r a t i o n s t e p w i t h C e l i t e was e l i m i n a t e d .  except  A l t h o u g h the  presence  o f carbohydrates was not as troublesome i n t h i s and other p e t a l e x t r a c t s , t h e s e e x t r a c t s were e l u t e d from a s h o r t polyamide column w i t h 50% MeOH, l e a v i n g behind carotenoid  pigments which were sometimes p r e s e n t  i n high  con-  centration. Following  t h e work on OTU 1,  t h e i s o l a t i o n procedure was changed  slightly;  r a t h e r t h a n e x t r a c t i o n w i t h hot MeOH, f r e s h p l a n t t i s s u e s were  repeatedly  e x t r a c t e d w i t h MeOH a t room temperature f o r a d u r a t i o n o f 2-5 days.  Hopefully,  t h i s p r e v e n t e d any p o s s i b l e breakdown o f h i g h l y g l y c o s y l a t e d f l a v o -  noids on exposure t o heat. solvent, acceptable  A l t h o u g h l e s s e f f i c i e n t than e x t r a c t i o n w i t h hot  amounts o f f l a v o n o i d s were e x t r a c t e d under the  milder  conditions. The was  simpler  procedure used t o i s o l a t e f l a v o n o i d s than those already  outlined.  from p r e s s e d  plant  material  The d r i e d m a t e r i a l was e x t r a c t e d  w i t h MeOH a t room temperature f o r 2-5 days and t h e n c o n c e n t r a t e d .  No p r e -  l i m i n a r y clean-up steps were r e q u i r e d , a l t h o u g h such steps might have been n e c e s s a r y had l a r g e r amounts o f m a t e r i a l been a v a i l a b l e . a taxon was a n a l y z e d  m a t e r i a l , o n l y about 0.1 g dry weight o f  from p r e s s e d  plant.was a v a i l a b l e .  .  C. I d e n t i f i c a t i o n o f t h e F l a v o n o i d s i.  I n every case where  o f L. d o u g l a s i i v a r . d o u g l a s i i , OTU 1  Chromatography After preliminary  solved  c l e a n i n g , p e t a l and whole p l a n t  e x t r a c t s were d i s -  i n s m a l l volumes o f water and.chromatographed on columns o f SC-6 p o l y -  amide u s i n g a l i n e a r g r a d i e n t  0-50% MeOH i n H O .  This p r a c t i c e  separated  - 29 flavonoids roughly according t o g l y c o s y l a t i o n class — sides, etc.  b i o s i d e s from  trio-  Column f r a c t i o n s , c o n t a i n i n g compounds w i t h a common g l y c o s y l a -  t i o n p a t t e r n , were c o n s o l i d a t e d and c o n c e n t r a t e d in_ vacuo.  These f r a c t i o n s  were taken up i n a s m a l l volume o f Q0% MeOH and s t r e a k e d on TLC p l a t e s (20 x 20 cm) u s i n g DC 6.6 polyamide (MacNarey-Nagel) as t h e adsorbent.  These  p l a t e s were r u n r e p e a t e d l y i n CHCl^MeOH-butanone-HgO (55:22:20:3).  This  o r g a n i c s o l v e n t was developed  flavo-  f o r t h i s purpose and tends t o s e p a r a t e  noids a c c o r d i n g t o t h e i r degree o f s u b s t i t u t i o n on t h e f l a v o n o i d r a t h e r than type o f sugar moiety.  The r e s u l t i n g bands o f pure compounds were  marked under UV l i g h t and s c r a p e d , t o g e t h e r w i t h t h e polyamide, plates. rator.  nucleus,  o f f the glass  The compounds were e l u t e d w i t h 80% aqueous MeOH w i t h a vacuum a s p i The r e s u l t i n g e l u e n t s were c o n c e n t r a t e d , and a g a i n taken up i n s m a l l  volumes o f 80% MeOH. Some groups o f f l a v o n o i d s w i t h d i f f e r e n t g l y c o s y l a t i o n p a t t e r n s , c o u l d not be s e p a r a t e d c o m p l e t e l y by aqueous column chromatography. was  a c h i e v e d u s i n g an aqueous TLC system —  acetone  (8:1:1).  The chloroform-based  Separation  DC 6.6 polyamide w i t h H^O-n-BuOH-  TLC system was then used t o r e s o l v e  t h e s e c l a s s e s o f compounds i n t o i n d i v i d u a l components. The above procedure used t o i s o l a t e pure f l a v o n o i d s was s u c c e s s f u l f o r most types o f f l a v o n o i d s p r e s e n t i n Limnanthes..  However, compounds w i t h  sugars a t t a c h e d a t t h e 3 and 7 p o s i t i o n s o f t h e f l a v o n o i d nucleus were not s e p a r a t e d by TLC i n t h e above o r g a n i c s o l v e n t system. t o s o l v e t h e problem were o n l y p a r t i a l l y s u c c e s s f u l ;  Unfortunately, efforts an a c i d i c system u s i n g  DC 6.6 polyamide and CHCl^-isopropanol-butanone-HOAc ( 1 0 : 3 ^ 3 | , 0 ~ w  as  .developed  which w i l l r e s o l v e t h e 3 , 7 - d i g l y c o s i d e s , but o n l y i f they a r e a p p l i e d t o p l a t e s i n s m a l l amounts.  However, t h i s system made p o s s i b l e t h e i d e n t i -  f i c a t i o n o f t h e s e compounds, even though they were not i s o l a t e d as i n d i v i d u a l pure compounds.  - 30 -  The able.  e f f i c i e n c y o f compound r e c o v e r y  Apparently,  from t h e TLC polyamide was v a r i -  e f f i c i e n c y of e l u t i o n i s inversely correlated with  o f h y d r o x y l a t i o n o f t h e B - r i n g o f t h e . f l a v o n o i d nucleus hydroxyl  groups p r e s e n t  of t h a t compound. hydroxyl  i - i l - > t h e more  on a compound, t h e lower t h e e f f i c i e n c y o f r e c o v e r y  Recovery o f m y r i c e t i n d e r i v a t i v e s w i t h t h e i r t h r e e B - r i n g  groups was p r o b a b l y  w i t h two h y d r o x y l  —  degree  l e s s t h a n 50%, and.the r e c o v e r y  o f compounds  groups' on t h e B - r i n g , such as q u e r c e t i n d e r i v a t i v e s , was  only s l i g h t l y b e t t e r .  Compounds w i t h o n l y one B - r i n g h y d r o x y l group !were  r e c o v e r e d more e f f i c i e n t l y .  Attempts were made t o f i n d a b e t t e r method f o r  . e l u t i n g compounds from TLC polyamide, b u t no b e t t e r e l u t i n g s o l v e n t t h a n 80% MeOH was d i s c o v e r e d .  I t i s a l s o worth n o t i n g t h a t r e c o v e r y o f f l a v o n o i d s  from polyamide columns was by no means complete.  ii.  Spectral  Analyses  Ultra violet isolated i n sufficient by Mabry e t a l .  a b s o r p t i o n s p e c t r a were determined f o r a l l compounds q u a n t i t i e s ( c a . l.mg).  (1970) were f o l l o w e d .  Standard procedures o u t l i n e d -  The t e c h n i q u e  c o n s i s t s o f s p e c t r a l de-  t e r m i n a t i o n i n MeOH, and t h e n i n MeOH w i t h v a r i o u s s h i f t reagents data obtained  are u s e f u l f o r s t r u c t u r a l determination  added.  of flavonoids.  The  UV-spec-  t r a o f Limnanthes compounds were determined on a Pye-Unicam SP 1800 s p e c t r o photometer. N u c l e a r magnetic resonance (NMR) s p e c t r o s c o p y i d e n t i f i c a t i o n o f f l a v o n o i d compounds. t h i s technique  (10-20 mg).  a l s o may be u s e f u l f o r  However, s a t i s f a c t o r y r e s u l t s  r e q u i r e t h e i s o l a t i o n o f comparatively  using  l a r g e amounts o f compound  S i x compounds were i s o l a t e d from f i e l d - g r o w n m a t e r i a l o f OTU  i n quantities ranging  1  from 7-30 mgs, a n d . t h e i r NMR s p e c t r a were determined.  To o b t a i n s a t i s f a c t o r y NMR s p e c t r a , t h e s i x Limnanthes f l a v o n o i d s were c r y s t a l i z e d from 80% MeOH a f t e r i s o l a t i o n by TLC.  T r i m e t h y l s i l y l (TMS)  - 31 e t h e r s o f t h e s i x compounds were prepared u s i n g " T r i s i l K i t s " Chemical  Company).  determined  (Pierce  NMR s p e c t r a o f t h e TMS e t h e r s d i s s o l v e d i n C C l ^ were  on a V a r i a n HA-100 instrument  as i n t e r n a l s t a n d a r d .  (100 mHz) u s i n g t e t r a m e t h y l s i l a n e  TMS e t h e r s were h y d r o l y z e d t o y i e l d t h e o r i g i n a l  f l a v o n o i d s by a d d i t i o n o f aqueous MeOH t o t h e C C l ^ s o l u t i o n s .  The r e s u l t i n g  s o l u t i o n s o f l i b e r a t e d f l a v o n o i d compounds were then c o n c e n t r a t e d i n vacuo.  iii.  Hydrolyses The h y d r o l y s i s o f f l a v o n o i d g l y c o s i d e s was accomplished  d i f f e r e n t procedures.  A l l h y d r o l y s e s were performed  by s e v e r a l  t o determine  either the  number and type o f sugar m o i e t i e s a t t a c h e d t o a f l a v o n o i d n u c l e u s , o r , when a compound was g l y c o s y l a t e d w i t h more.than one sugar, t h e p o s i t i o n and o r d e r o f attachment o f t h e sugars. :  T o t a l h y d r o l y s e s were accomplished  HC1 i n water f o r 30 minutes a t 95-100°.  L i b e r a t e d sugars were i s o l a t e d , neu-  t r a l i z e d and i d e n t i f i e d by TLC f o l l o w i n g a procedure by Mabry et a l . - (1970),  similar t o that described  R e l a t i v e amounts o f t h e i d e n t i f i e d sugars were a l s o  estimated using t h i s technique. co-chromatographyC^^  w i t h 1.0 N  The l i b e r a t e d aglycones were i d e n t i f i e d by .  When a g l y c o s i d e c o n t a i n s more than one s u g a r , . p a r t i a l h y d r o l y s i s may a s s i s t i n i t s - i d e n t i f i c a t i o n by' comparing t h e r e s u l t i n g d e r i v a t i v e s w i t h standard.compounds.  By t h i s process both t h e o r d e r and p o s i t i o n o f sugar  attachment may sometimes be determined.  Limnanthes. compounds were p a r t i a l l y  h y d r o l y z e d b y two methods, depending on t h e mode o f g l y c o s y l a t i o n :  hydrolysis  f o r s h o r t i n t e r v a l s a t 95-100°. w i t h e i t h e r 0.1 N HC1 or 20% (v/v) a c e t i c  acid.  Enzymic h y d r o l y s i s w i t h emulsin proved a u s e f u l t e c h n i q u e f o r t h e c h a r a c t e r i z a t i o n o f many o f - t h e Limnanthes compounds. accomplished  These h y d r o l y s e s were  by m i x i n g a s m a l l amount o f enzymic m i x t u r e  c h e m i c a l Corp.) i n t o . 1 . 0  N a c e t a t e . b u f f e r (pH 5.1)  (Nutritional B i o  containing dissolved  r  - 32 -  compound and a l l o w i n g t h e suspension t o s t a n d o v e r n i g h t a t room temperature (Mabry et a l . , 1970). t h e p-glucosidase  I n t h i s manner g l u c o s e was s e l e c t i v e l y removed by  c o n t a i n e d i n t h e e m u l s i n , when g l u c o s e was t h e o n l y  t u e n t at a g i v e n s i t e .  substi-  (Glucose i s a l s o removed i f i t i s t h e t e r m i n a l sub-  s t i t u e n t o f a g l y c o s i d e moiety; however, enzymic h y d r o l y s e s were not performed on compounds w i t h t h i s mode o f g l y c o s y l a t i o n in t h e present i n v e s t i g a t i o n . )  i v . Acetate Preparation Two p r e v i o u s l y u n r e p o r t e d  compounds, each based on a r a r e  t y p e , were i s o l a t e d i n c o m p a r a t i v e l y douglasii.  l a r g e q u a n t i t i e s from L_. d o u g l a s i i v a r .  To c h a r a c t e r i z e t h e s e compounds more c o m p l e t e l y , t h e i r  d e r i v a t i v e s were p r e p a r e d ,  aglycone  and t h e i r m e l t i n g p o i n t s determined.  acetate  Prior to  d e r i v a t i z a t i o n o f t h e s e two compounds, an a d d i t i o n a l a c e t a t e d e r i v a t i v e o f a t h i r d Limnanthes compound p o s s e s s i n g t h e same g l y c o s y l a t i o n p a t t e r n was p r e pared t o t e s t t h e planned procedure  and t o compare.the m e l t i n g p o i n t o f t h i s  test d e r i v a t i v e with the published value. The j|iKo:'.3iiedure used t o p r e p a r e  a c e t a t e d e r i v a t i v e s was as f o l l o w s :  1 ml o f a c e t i c anhydride was added t o 5 mg of compound i n a 25 ml round bottom flask;  a few drops o f t r i e t h y l a m i n e were added, and t h e s o l u t i o n was  o v e r n i g h t at room temperature;  t h i s s o l u t i o n was then c o n c e n t r a t e d ,  left taken  up i n c h l o r o f o r m and t h e d e r i v a t i v e p r e c i p i t a t e d by t h e a d d i t i o n o f EtOH. The p r e c i p i t a t e d d e r i v a t i v e was removed from s o l u t i o n by f i l t r a t i o n , c r y s t a l i z e d from a m i x t u r e  o f benzene and cyclohexane.  d r i e d and  By f o l l o w i n g t h i s  p r o c e s s , t h e amount o f c r y s t a l i n e d e r i v a t i v e r e c o v e r e d was JO-80% i n each case. These a c e t a t e d e r i v a t i v e s were not s u b s t i t u t e d a t a l l f r e e h y d r o x y l groups of t h e f l a v o n o i d nucleus to  hydrogen bonding.  s i n c e t h e 5-hydroxyl  group remains u n a c e t y l a t e d due  -  33 -  D. I d e n t i f i c a t i o n - o f t h e Remaining Limnanthes F l a v o n o i d s When compounds were encountered which d i d not occur i n L_. d o u g l a s i i , t h e y were i s o l a t e d and i d e n t i f i e d by t h e p r o c e s s e s a l r e a d y d e s c r i b e d , i f t h e y were p r e s e n t i n s u f f i c i e n t  concentration..  i n t r a c e amounts remain u n i d e n t i f i e d .  Compounds  consistently occurring  However, t h e presence o f t h e s e compounds  was s t i l l r e c o r d e d , and they were i n c l u d e d w i t h t h e known compounds f o r analysis. The f l a v o n o i d complement o f each t a x o n , as i t was determined f o r comparative  purposes, was t a k e n t o be a l l t h e f l a v o n o i d s t h a t could, be r e s o l v e d  by two-dimensional  TLC o f whole p l a n t and p e t a l e x t r a c t s .  used was polyamide  DC 6.6, 1 s t dimension  ( 1 6 : 2 : 1 : 1 ) , 2 n d dimension  —  —  H^O-n-BuOH-acetone-HOAc  -  A f t e r d r y i n g thoroughly:;;  maps were sprayed w i t h a boronate r e a g e n t .  i s a m o d i f i c a t i o n o f t h a t d e s c r i b e d by' Somaroo et_ a l . increases the v i s i b i l i t y  system  CHCl^-isopropanol-butanone-HOAc - ( 1 0 : 3 : 3 : 4 ) .  Some p l a t e s were r u n t w i c e i n t h e second dimension. the two-dimensional  The TLC  o f f l a v o n o i d s on polyamide  T h i s reagent  ( 1 9 7 3 ) and g r e a t l y  under UV l i g h t .  p r e p a r e d by d i s s o l v i n g a s m a l l amount o f d i p h e n y l b o r i c a c i d  I t was  ethanolamine  complex ( A l d r i c h ) ifr MeOH, and t h e n d i l u t i n g w i t h water t o 0.1% (w/v).  The  compounds r e s o l v e d by t h i s p r o c e s s were i d e n t i f i e d whenever p o s s i b l e by comp a r i s o n t o s t a n d a r d compounds u s i n g t h e t e c h n i q u e o f co-chromatography, i f necessary.  '  EXPRESSION OF FLAVONOIDS AS TAXONOMIC  CHARACTERS  F l a v o n o i d s a r e . o f t e n used as c h a r a c t e r s i n t a x o n o m i c - s t u d i e s by s i m p l y comparing presence o r absence i n t h e t a x a under study.  In p r a c t i c e ,  however, t h e r e l a t i v e c o n c e n t r a t i o n s o f o c c u r r e n c e a r e . o f t e n a l s o ' c o n s i d e r e d . In t h e p r e s e n t i n v e s t i g a t i o n , t a x a were compared on t h e b a s i s o f 4 6 f l a v o n o i d characters.-  Two d i f f e r e n t l e v e l s o f compound c o n c e n t r a t i o n on  comparative  two-dimensional chromatograms were r e c o g n i z e d  and r e c o r d e d :  r e l a t i v e l y h i g h c o n c e n t r a t i o n and v i s i b l e b e f o r e agent , ( s e v e r a l orders first  present  spraying with boronate r e -  o f magnitude o f c o n c e n t r a t i o n a r e r e p r e s e n t e d  c l a s s i f i c a t i o n ) and 2 ) p r e s e n t  spraying.  l ) present i n  By t h i s process  i n this  i n t r a c e amount and v i s i b l e o n l y  after  each OTU was d e s c r i b e d on t h e b a s i s o f f l a v o n o i d s  i n whole p l a n t m a t e r i a l and a l s o on t h e b a s i s o f p e t a l m a t e r i a l when  i t was a v a i l a b l e . One advantage o f u s i n g a t e c h n i q u e t r a t i o n s o f f l a v o n o i d /occurrences,  on a chromatogram.  Since the r e l a t i v e  o f f l a v o n o i d s seem t o be c o n s i s t e n t f o r a g i v e n t a x o n , a p r o -  cedure which c o n s i d e r s c o n c e n t r a t i o n data p r o b a b l y significant  i n t o account concen-  i s t h a t a b e t t e r a p p r o x i m a t i o n can be made  o f t h e r e s u l t s as they a c t u a l l y occur concentrations  which takes  conserves more t a x o n o m i c a l l y  i n f o r m a t i o n t h a n one t h a t does n o t .  TREATMENT OF THE FLAVONOID DATA A.  Introduction S m a l l groups o f t a x a can be compared onfcthe b a s i s o f t h e i r  by c o n s t r u c t i n g a m a t r i x  o f OTUs v e r s u s  flavonoid characters.  OTUs and/or c h a r a c t e r s can be v i s u a l l y arranged character s i m i l a r i t i e s c l u s t e r together and/or c h a r a c t e r s  c l u s t e r s becomes more d i f f i c u l t  The order o f  so t h a t t a x a w i t h t h e g r e a t e s t  i n the matrix.  i n c r e a s e s , the technique  As t h e number o f OTUs  o f manually r e a r r a n g i n g OTUs i n t o  and.subjective.  T h i s i s because t h e eye can  o n l y c o n s i d e r so much i n f o r m a t i o n a t once, and when c o n f r o n t e d w i t h d a t a , tends t o c o n c e n t r a t e  flavonoids  on a s m a l l p o r t i o n o f t h e d a t a .  excessive  This process  t o r e s u l t s p r e j u d i c e d i n f a v o r o f t h e p o r t i o n o f t h e data c o n s i d e r e d . t h i s reason, phenetic  leads  For  comparisons o f l a r g e numbers o f OTUs on t h e b a s i s o f  many c h a r a c t e r s a r e b e s t made u s i n g t h e t e c h n i q u e s T h e o r e t i c a l l y , the' use o f t h e s e t e c h n i q u e s  of numerical  taxonomy.  e l i m i n a t e s t h e above p r e j u d i c e .  - 35 A most important  t o o l o f t h e n u m e r i c a l taxonomist  i s cluster  analysis,  a t e c h n i q u e r e c e n t l y made p r a c t i c a b l e by t h e development o f t h e computer. There a r e many v a r i a t i o n s o f computerized a l l o f these techniques  c l u s t e r a n a l y s i s , but e s s e n t i a l l y  a r e designed t o c o n s i d e r c h a r a c t e r  between a l l t a x a s i m u l t a n e o u s l y and produce groupings  similarities  or c l u s t e r s o f t a x a  based on r e l a t i v e s i m i l a r i t i e s which p r o v i d e t h e b e s t " f i t " I t was d e s i r a b l e t o use numberical  t o the data.  taxonomic t e c h n i q u e s  o f t h e f l a v o n o i d d a t a o f t h e Limnanthaceae f o r two r e a s o n s :  f o r analysis  l ) a data m a t r i x  c o n t a i n i n g 30 OTUs d e s c r i b e d by 46 c h a r a c t e r s exceeds t h e s i z e f o r which an a c c u r a t e assessment o f s i m i l a r i t y can be made by eye; and 2) c l u s t e r s i s based o n . f l a v o n o i d c h a r a c t e r s a l l o w s a d i r e c t  comparison t o t h e e a r l i e r  n u m e r i c a l taxonomic a n a l y s i s o f t h e Limnanthaceae by Ornduff based on m o r p h o l o g i c a l d a t a  analy-  and C r o v e l l o  (1968).  B, Method To c l u s t e r t a x a by c o n v e n t i o n a l methods, a m a t r i x o f s i m i l a r i t y f i c i e n t s must be c a l c u l a t e d between a l l t a x a .  coef-  S i n c e t h e r e a r e many d i f f e r e n t  ways t o c a l c u l a t e s i m i l a r i t y c o e f f i c i e n t s , . t h e r e s u l t s o f c l u s t e r a n a l y s i s may v a r y w i t h the. type - o f c o e f f i c i e n t used.  Whether or not two t a x a . a r e made  more s i m i l a r due t o the'mutual absence o f a g i v e n f l a v o n o i d c h a r a c t e r t i v e matches' a r e c o n s i d e r e d ) , • i s a q u e s t i o n t h a t has not been answered. • To h e l p answer t h i s q u e s t i o n , c l u s t e r a n a l y s e d  satisfactorily  o f Limnanthes and  F l o e r k e a were performed u s i n g two types o f s i m i l a r i t y c o e f f i c i e n t s : simple matching c o e f f i c i e n t n e g a t i v e matches';  (nega-  l ) the  ( S o k a l & Sneath, 1963, p. 133) which c o n s i d e r s '  and 2) t h e c o e f f i c i e n t o f J a c c a r d ( S o k a l & Sneath, 1963,  p. 133)' which c a l c u l a t e s s i m i l a r i t y o n l y on t h e b a s i s o f mutual F l a y o n o i d d a t a were compiled  occurrence.  i n t o two B a s i c D a t a . M a t r i c e s  listing,  i n one case, a l l OTUs (30) as rows v e r s u s t h e f l a v o n o i d c h a r a c t e r s (46)  found  - 36 -  found i n whole p l a n t m a t e r i a l , and (l8)  i n the o t h e r case t h e OTUs grown f r e s h  versus the f l a v o n o i d c h a r a c t e r s (46)  found  i n the f r e s h p e t a l s .  t h e s e two m a t r i c e s the simple matching and J a c c a r d s i m i l a r i t y were c a l c u l a t e d by computer between a l l t a x a . ranged  coefficients  These c o e f f i c i e n t s were a r (whole p l a n t data) or 18  i n h a l f m a t r i c e s of OTUs, e i t h e r 30 x 30  (petal data).  From  18  x  I n each i n s t a n c e d u p l i c a t e m a t r i c e s were c a l c u l a t e d h a n d l i n g  the data.in.two  ways:  l ) s i m i l a r i t i e s were determined  c o n s i d e r i n g only those  compounds o c c u r r i n g i n r e l a t i v e l y h i g h c o n c e n t r a t i o n s ; were determined  on t h e b a s i s o f any.occurrence  2)  similarities  regardless of concentration.  By t h i s p r o c e s s a t o t a l of e i g h t m a t r i c e s o f s i m i l a r i t y c o e f f i c i e n t s were computed.  From t h i s d a t a , e i g h t d i f f e r e n t  u s i n g the weighted  c l u s t e r a n a l y s e s were computed'  p a i r group c l u s t e r i n g method, and the r e s u l t s were  expressed  i n e i g h t dendrograms (phenograms). In t h e i r t a x i m e t r i c study of the Limnanthaceae, O r n d u f f and C r o v e l l o (.1968) used two  t e c h n i q u e s t o c l u s t e r ' OTUs.  method, t h e weighted  The  a conventional  p a i r group method, t h a t producec&a dendrogram e x p r e s s i n g  o v e r a l l c o p h e n e t i c s i m i l a r i t i e s between OTUs. f a c t o r a n a l y s i s which produced character' space".  f i r s t was  The  second method was  a form  a t h r e e - d i m e n s i o n a l p l o t o f OTUs i n "reduced  D i s t a n c e s between OTUs p l o t t e d by the second t e c h n i q u e  p r o p o r t i o n a l t o major sources o f v a r i a t i o n between t a x a . p u t e r program used by Ornduff and C r o v e l l o was i n v e s t i g a t i o n , two  of  are  A l t h o u g h t h e com-  not a v a i l a b l e f o r t h e p r e s e n t  o t h e r s t a n d a r d types o f f a c t o r a n a l y s i s w e r f e . P f e r  o r m e <  i °n  f l a v o n o i d data. The two Analysis  t e c h n i q u e s , P r i n c i p a l Components A n a l y s i s and Varimax F a c t o r  (Both.programs are from the S c i e n t i f i c S u b r o u t i n e Package of IBM.),  w i l l be b r i e f l y d e s c r i b e d here s i n c e the method d i f f e r e d from s t a n d a r d t e r i n g programs and may  not n e c e s s a r i l y express o v e r a l l c o p h e n e t i c  I n s t e a d t h e s e t e c h n i q u e s a r e based  on key components) of t h e t o t a l  clus-  similarities. variation  - 37 present  between t a x a . The  first  step i n performing  character-by-character t e r i n g of characters computed.  any  f a c t o r a n a l y s i s i s t o set up  c o r r e l a t i o n c o e f f i c i e n t matrix.  a  Then, o v e r a l l  clus-  (not OTUs as by c o n v e n t i o n a l c l u s t e r i n g programs) i s  From t h e n dimensions o f hyperspace, c o r r e s p o n d i n g  t o t h e number  o f c h a r a c t e r s c o n s i d e r e d , t h e r e l a t i v e amount of v a r i a b i l i t y expressed each dimension i s f a c t o r e d . which t o g e t h e r selected.  The  t h r e e ( i n t h i s case) f a c t o r s or v e c t o r s  account f o r t h e g r e a t e s t amount o f v a r i a t i o n between t a x a are.  These f a c t o r s are not c o r r e l a t e d .  each c h a r a c t e r are c a l c u l a t e d ' ^  The  "factor loadings" for  i..e_. , the. r e l a t i v e c o n t r i b u t i o n t h a t each  c h a r a c t e r makes t o t h e v a r i a t i o n expressed vectors.  by  each of the t h r e e s e l e c t e d  Then, t h e c h a r a c t e r complement o f each.OTU i s m u l t i p l i e d by  r e s p e c t i v e f a c t o r l o a d i n g s , producing c o o r d i n a t e s are.used  the  t h r e e c o o r d i n a t e s f o r each OTU.  t o p l o t OTUs i n t h r e e d i m e n s i o n a l  space u s i n g a  type of p l o t t i n g program which p i c t o r i a l i z e s the t h r e e - d i m e n s i o n a l two  by  These standard  plot i n  dimensions. The t y p e o f a n a l y s i s d e s c r i b e d above i s f a c t o r a n a l y s i s by  c i p a l components.  The  Analysis with r o t a t i o n .  o t h e r t e c h n i q u e used i n t h i s study was The  Varimax F a c t o r  p r e l i m i n a r y steps are the same f o r t h i s  A f t e r the f a c t o r s a r e e x t r a c t e d , they are t r a n s f o r m e d  prin-  by r o t a t i o n t o  technique. different  c o o r d i n a t e s which t h e o r e t i c a l l y show i n t e r r e l a t i o n s h i p s between OTUs i n their-s i m p l e s t form.  From these a d j u s t e d f a c t o r s , new  f a c t o r ' l o a d i n g s are c a l c u -  l a t e d , and t h e OTUs a r e p l o t t e d i n three'dimensions  as aboye.  Since p r e l i m i n a r y results, i n d i c a t e d that the c l u s t e r  analyses.were  more t a x o n o m i c a l l y m e a n i n g f u l i f o n l y the' compounds o c c u r r i n g i n r e l a t i v e l y , heavy c o n c e n t r a t i o n were used as c h a r a c t e r s , t h e two  types  of f a c t o r a n a l y s i s  were performed o n l y on the b a s i s o f f l a v o n o i d s v i s i b l e b e f o r e s p r a y i n g chromatograms.  'Eighteen OTUs were a n a l y z e d  on the b a s i s o f 36 v a r i a b l e  the  - 38  f l a v o n o i d s o c c u r r i n g i n the p e t a l s  -  (the r e m a i n i n g 10 f l a v o n o i d s  under these c o n d i t i o n s ) , w h i l e 30 OTUs were a n a l y z e d able flavonoids present  i n whole p l a n t  material.  d i d not  on the b a s i s o f 31  vary vari-  - 39 -  RESULTS  FLORAL PATTERNING IN ULTRA VIOLET LIGHT F i g u r e I I i s a cbmposicb.e p l a t e made up o f s e l e c t e d photograph p a i r s of Limnanthes f l o w e r s taken  in visible  and UV l i g h t .  The t a x a  represented  are t h e v a r i e t i e s o f L_. d o u g l a s i i , OTUs 2,3,4 and 5, L.. m a c o u n i i , and L. g r a c i l i s v a r . g r a c i l i s , OTU 14.  Of t h e s e s i x t a x a , t h e f i r s t  e x h i b i t some form o f UV p a t t e r n i n g w h i l e t h e l a s t the p l a t e t o i l l u s t r a t e the p a t t e r n o f uniform remaining  twelve u n p i c t u r e d  (Figure H E )  ( F i g u r e IIB) shows  r e s u l t r e v e a l e d by t h e f l o w e r photography.  o u t e r p o r t i o n s o f the' p e t a l s o f t h i s v a r i e t y r e f l e c t t o the s t r o n g l y absorbing  effect.  f o r the  taxa.  has w h i t e p e t a l s w i t h r o s e c o l o u r e d v e i n s and a n t h e r s .  trast  five  i s included i n  a b s o r p t i o n observed  The photograph p a i r o f L_. d o u g l a s i i v a r . r o s e a t h e most s t r i k i n g  OTU 10,  Variety rosea  Under UV l i g h t t h e  the l i g h t  i n sharp  i n n e r p e t a l zones, thus p r o d u c i n g  T h i s n e c t a r guide v i s i b l e  o n l y under UV l i g h t  con-  a bullseye  i s analogous t o t h e  familiar^, y e l l o w and w h i t e p a t t e r n o f v a r i e t y d o u g l a s i i ( F i g u r e I I C ) . The similarities  photographs o f L. d o u g l a s i i v a r . n i v e a ( F i g u r e IID) show some t o those  of v a r i e t y rosea.  f l o w e r s under v i s i b l e . l i g h t ,  V a r i e t y n i v e a , which a l s o has w h i t e  a l s o shows t h e b u l l s e y e e f f e c t under UV l i g h t ,  but w i t h l e s s c o n t r a s t than v a r i e t y r o s e a .  A l s o c o n t r i b u t i n g t o t h e UV p a t -  terning effect of v a r i e t y nivea are the l i g h t bases',and t h e l i g h t center. tance  c o l o u r e d a n t h e r s , both  coloured regions at the p e t a l  c o n t r a s t i n g w i t h t h e dark f l o w e r  The p a l e appearance o f t h e p e t a l bases i s due i n p a r t . t o t h e  of the basal h a i r s .  reflec-  A s i m i l a r r e f l e c t a n c e i s a l s o evident f o r v a r i e t y  r o s e a , and somewhat e v i d e n t f o r v a r i e t y s u l p h u r e a  (Figure IIA).  Reflecting  • 40a -  FIGURE I I ~- The 'Flowers o f S i x S e l e c t e d Ultra Violet  4;  nivea,  light.  The lower p i c t u r e s  OTU  10.  are t a k e n through t h e l 8 A  3;  E) L. g r a c i l i s !  filter.  5; B) L. d o u g l a s i i v a r . r o s e a ,  C) L. d o u g l a s i i v a r . d o u g l a s i i , OTU OTU  and  o f t h e photograph p a i r s a r e t a k e n i n  A) L. d o u g l a s i i v a r . s u l p h u r e a , OTU OTU  t a x a Taken i n V i s i b l e  Light.  The upper p i c t u r e s visible  Limnanthes  2;  v a r . g r a c i l i s , OTU  D.), L. d o u g l a s i i v a r . 14;  and F) L. m a c o u n i i ,  -  -  41  h a i r s a t t h e p e t a l hases were o c c a s i o n a l l y observed t a x a as w e l l , i n c l u d i n g L_. a l b a v a r .  I I C ) have some y e l l o w f l o w e r p i g m e n t a t i o n  (Figure HA)  each e x h i b i t s a c e r t a i n amount o f p a t t e r n i n g .  the flower centers.  sulphurea  l e s s UV  d o u g l a s i i (Figure When photographed  s h a r p l y c o n t r a s t i n g zones, but Both have p e t a l s d i v i d e d i n t o  a b s o r p t i o n , w i t h t h e darker  zones toward  The v a r i e t y s u l p h u r e a a l s o e x h i b i t s c e r t a i n f e a t u r e s i n  a d d i t i o n t o the p l a i n p a t t e r n o f v a r i e t y d o u g l a s i i ; ety  and  in visible light.  l i g h t , neither.^'bfi.'ffi'ese v a r i e t i e s has  r e g i o n s of d i f f e r i n g degrees of UV  unpictured  versicolor.  Both L. d o u g l a s i i v a r s . s u l p h u r e a  under UV  f o r other  appear under UV  light  the p e t a l veins of v a r i -  as c o n t r a s t i n g r a d i a l l i n e s , , a b s o r b i n g  l i g h t than the i n n e r zone but more than t h e o u t e r , l i g h t e r p e t a l  parts.. The  appearance under UV  ( F i g u r e ITF) was  surprising.  light  of t h e s m a l l p l a i n f l o w e r s o f L_. macounii  I t s p e t a l bases r e f l e c t t o some extent  i n con-  t r a s t t o the o u t e r p e t a l r e g i o n s , the p e t a l v e i n s , and the h e a v i l y a b s o r b i n g sepals.  Although  t h e r e are.no h a i r s a,t t h e p e t a l bases and no  c o l o u r e d zones at the p e t a l t i p s o f L_. m a c o u n i i ,  lighter  the c e n t r a l zone o f UV  f l e c t a n c e and the c o n t r a s t i n g v e i n s show s i m i l a r i t i e s t o the p a t t e r n s L. d o u g l a s i i v a r s . n i v e a and Flowers a p p e a r i n g UV T i g h t , - are-common and e f f e c t to' a bee  re-  of  rosea.  white i n the v i s i b l e  spectrum, .but which absorb  i n c l u d e t h o s e o f most t a x a of Limnanthes.  The  net  l o o k i n g at t h i s type o f f l o w e r i s a c o l o u r t h a t c o n t r a s t s w i t h  t h e background v e g e t a t i o n .  Presumably, t h i s c o n d i t i o n has  e v o l v e d i n many  l i n e s ' o f p l a n t s r e s u l t i n g from s e l e c t i o n p r e s s u r e s t o i n c r e a s e t h e probab i l i t y , of a v i s i t The  by a p o l l i n a t o r and.thus enhance c r o s s  fertilization.  e l a b o r a t i o n of n e c t a r g u i d e s , which f u n c t i o n t o draw t h e i n s e c t to. t h e  c e n t e r of a f l o w e r , i s a f u r t h e r a d a p t a t i o n t o f a c i l i t a t e c r o s s brought about by.the same s e l e c t i o n  pressures.  pollination  -  The  42--  presence o f U V - v i s i b l e n e c t a r guides  i n the v a r i e t i e s  L_. d o u g l a s i i suggests t h a t these t a x a a r e more h i g h l y e v o l v e d t h a n are o t h e r Limnanthes t a x a .  backwards, i t i s probable unpatterned  The  Extrapolating  t h a t t h e o u t c r o s s i n g Limananthes t a x a w i t h  group,  observed d i f f e r e n c e s i n f l o r a l p a t t e r n i n g o f t h e f o u r v a r i e t i e s  tionship i n t h i s species.  Various  i m p l i c a t i o n r e g a r d i n g d i s t a n c e of  sympatric  t a x a o f Limnanthes have  g e n e t i c b a r r i e r s t o h y b r i d i z a t i o n which were used by Mason guish species.  rela-  evolved  (1952) t o d i s t i n -  S i n c e Mason found t h e f o u r v a r i e t i e s o f L_. d o u g l a s i i e x p e r i -  i n t e r f e r t i l e , he grouped them t o g e t h e r  as v a r i e t i e s o f one  morphic s p e c i e s i n s p i t e o f the f a c t t h a t e a r l i e r authors o f them as d i s t i n c t  species  (Bentham, 1848;  A l t h o u g h Mason s t a t e d t h a t t h e s e  Loudon, 1855;  had  d e s c r i b e d some  Greene, 1891;  r o s e a and n i v e a , i n an a r e a of sympatry.  of the  However, t h e m i x i n g o f  between t h e s e two  v a r i e t i e s appears t o be the extent  the s p e c i e s .  o t h e r v a r i e t i e s p e r s i s t as d i s c r e t e taxonomic  even i n zones o f sympatry.  flowers.  of  varieties,  of intermingling'within  i s a t t r i b u t a b l e to p o l l i n a t o r  r e s u l t i n g from t h e i r d i f f e r e n t l y appearing  fer-  characters  entities,  I t i s p o s s i b l e t h a t t h e g e n e r a l l a c k of  among t h e f o u r v a r i e t i e s  Abrams,  he c i t e d i n support  t h i s statement wlasibthe^intermingling of c h a r a c t e r s between two  The  poly-  f o u r v a r i e t i e s r e a d i l y produce  t i l e n a t u r a l h y b r i d s , t h e o n l y s u b s t a n t i a l evidence  mingling  plain  t h e genus as w e l l .  o f L. d o u g l a s i i suggest an important  1941).  i t s sharply  f l o w e r s most c l o s e l y resemble the a n c e s t r a l form of t h i s  and p r o b a b l y  mentally  outcrossers  Of these v a r i e t i e s , r o s e a , w i t h  c o n t r a s t i n g zones, i s t h e most h i g h l y e v o l v e d o f t h e f o u r .  of  inter-  specificity  This s p e c i f i c i t y  may  c r e a t e an e c o l o g i c a l b a r r i e r t o g e n e t i c exchange, c a u s i n g the i s o l a t i o n  of  these t a x a , even though no b a r r i e r s t o h y b r i d i z a t i o n w i t h a g e n e t i c b a s i s have e v o l v e d .  T h e r e f o r e , the absence of a g e n e t i c b a r r i e r does not  s a r i l y i n d i c a t e that a l l . o f these  f o u r t a x a are v a r i e t i e s o f one  neces-  species.-  - k3  Instead,  some o f them may  nized species  be  -  as d i s t a n t l y r e l a t e d as o t h e r , . i s o l a t e d ,  o f Limnanthes,. - The  presence of c l e a r c u t m o r p h o l o g i c a l  ences between them tends t o support t h i s The  other  patterns  i m p l i c a t i o n s concerning  i n o n l y some t a x a  If this  The  the f o u r v a r i e t i e s  of L_. d o u g l a s i i suggests t h a t t h e s e f i v e t a x a have evolved with s i m i l a r flower patterning.  of  e v o l u t i o n i n ' t h i s genus..  common o c c u r r e n c e o f such p a t t e r n i n g • i n L_. macounii and  ancestor  differ-  possibility.  presence o f U V - v i s i b l e f l o w e r  Limnanthes has  recog-  from a common  i s t r u e , these taxa c o n s t i -  t u t e a n a t u r a l s u p r a s p e c i f i c group d i s t i n g u i s h e d from the r e s t of the genus by t h i s  trait. O r n d u f f ( 1 9 6 9 % O r n d u f f & C r o v e l l o , 1968)  i s a d e r i v e d c o n d i t i o n i n Limnanthes.• p a t t e r n i n L. macounii w i t h no  The  indicated that  presence of a U V - v i s i b l e  obvious s e l e c t i v e advantage t o t h i s  p o l l i n a t i n g species' supports t h i s c o n c l u s i o n . remnant o f an a d a p t a t i o n  has  valuable  autogamy floral  self-  This pattern i s probably a  o n l y t o an o u t c r o s s i n g  plant.  There have been s e v e r a l changes o f Slower morphology accompanying autogamy in- L. m a c o u n i i , such as r e d u c t i o n  o f s i z e and number o f p a r t s .  However, the presence o f U V - v i s i b l e n e c t a r  guides i n t h i s t a x o n suggests t h a t  not  a l l t r a i t s a s s o c i a t e d w i t h the o u t c r o s s i n g h a b i t have been l o s t .  r e t e n t i o n o f such t r a i t s f m a y j , h e e x p l a i n e d l i n k a g e groups caused by  s e l e c t i o n pressures  L i k e L_. m a c o u n i i , some o f the autogamous ( K a l i n , 1 9 7 3 a , i n d e p e n d e n t l y i n the two o f the genus. e x h i b i t UV section;  The  by the p r i o r establishment to preserve  outcrossing.  s u b s p e c i e s o f L. f l o c c o s a are  However, t r e n d s  of  t o autogamy have  totally occurred  s p e c i e s , s i n c e each belongs t o a d i f f e r e n t s e c t i o n  flowers  absorption i/e/j  1973b).  The  o f L_. f l o c c o s a , a member o f t h e  section  Inflexae,  c h a r a c t e r i s t i c s i d e n t i c a l t o o u t c r o s s i n g members of  flowers  photograph o f L. g r a c i l i s  with uniformly (Figure H E ) .  h i g h UV  absorption  Although nectar  as shown by  the  the  guides v i s i b l e i n  UV  - hk -  l i g h t have not e v o l v e d  i n t h i s s e c t i o n , the observed  flower characters  undoubtedly r e p r e s e n t an a d a p t a t i o n t o t h e o u t c r o s s i n g h a b i t . a conservation of flower pigmentation to  t h a t d e s c r i b e d above f o r L.  has  operated  Therefore,  i n L. f l o c c o s a p a r a l l e l  macounii.  R e l a t i v e l y high concentrations  of f l a v o n o i d s w i t h . a b s o r p t i o n maxima  i n t h e near UV were e x t r a c t e d from p e t a l s • o f a l l Limnanthes t a x a . sence suggests .that the a b s o r p t i o n r e c o r d e d by UV from t h e s e compounds.  This  photography r e s u l t s  I f c o r r e c t , t h i s c o n t e n t i o n agrees w i t h t h e  s i o n s o f o t h e r r e c e n t i n v e s t i g a t i o n s designed of U V - v i s i b l e n e c t a r guides  conclu-  absorption exhibited  by f l o w e r s o f Limnanthes t a x a , then t h e s e f l a v o n o i d s are a d a p t a t i o n s S i n c e t h e UV  signifi-  c a n t l y from the o u t c r o s s i n g members, an e v o l u t i o n a r y c o n s e r v a t i o n has working on the f l o w e r f l a v o n o i d s o f a l l t e s t e d members o f t h i s  f l a v o n o i d d i s t r i b u t i o n may  adapta-  t h e s e compounds w i l l tend t o be  con-  i m p l i c a t i o n s r e g a r d i n g the c o n t r i b u t i o n  make t o t h e taxonomy"of t h i s group  These i m p l i c a t i o n s w i l l be d i s c u s s e d f u l l y  FLAVONOID CHARACTERIZATION AND  been  genus.  The' argument t h a t the f l o w e r f l a v o n o i d s o f Limnanthes a r e  s e r v e d by e v o l u t i o n , has profound  which  absorption  c h a r a c t e r i s t i c s o f autogamous members of Limnanthes do not d i f f e r  and.that  nature  Dement & Raven, 197*0.  Assumming t h a t the f l o w e r f l a v o n o i d s do cause t h e observed  c o n f e r a . s e l e c t i v e advantage t o o u t c r o s s i n g p l a n t s .  pre-  directly  t o determine the c h e m i c a l  (Thompson et a l . , 1972;  tions f o r cross f e r t i l i z a t i o n ,  still  of p l a n t s .  i n the f o l l o w i n g chapter.  IDENTIFICATION  A. Data P r e s e n t a t i o n F l a v o n o i d i d e n t i f i c a t i o n s depend on the s y n t h e s i s o f many p i e c e s of information gathered  from a v a r i e t y o f t e c h n i q u e s .  In the f i r s t  p a r t of t h i s  s e c t i o n t h e data gathered f o r Limnanthes and F l o e r k e a . f l a v o n o i d s w i l l be lated.  I n the second p a r t of the s e c t i o n , - an e x p l a n a t i o n w i l l be  tabu-  presented  - 45 of  how  t h e compounds were i d e n t i f i e d . A t o t a l o f 48 f l a v o n o i d g l y c o s i d e s were found  and F l o e r k e a .  Table I I I l i s t s  notations assigned to these f l a v o n o i d s , t h e i r  i d e n t i f i c a t i o n s , and t h e a b b r e v i a t i o n s used i d e n t i f i c a t i o n was  i n t a x a o f Limnanthes  i n following tables.  Where an  not p o s s i b l e , t h e most r e a s o n a b l e guess i s p r o v i d e d .  F l a v o n o i d aglycones were p r e s e n t o c c a s i o n a l l y i n some t a x a , t h o s e a n a l y z e d from p r e s s e d m a t e r i a l . was  However, t h e appearance of  including  aglycones  e r r a t i c , and when p r e s e n t t h e y o c c u r r e d o n l y i n low c o n c e n t r a t i o n .  o b s e r v a t i o n s suggest t h a t t h e appearance o f aglycones was  caused by a  These low  l e v e l o f h y d r o l y t i c a c t i v i t y , e i t h e r i n e x t r a c t s , or i n p r e s s e d m a t e r i a l . For t h i s r e a s o n , i t was  assummed t h a t , f l a v o n o i d aglycones  do not  naturally  occur i n the Limnanthaceae. F i g u r e I I I i s a composite Rfs  (20.x  drawing showing t h e approximate r e l a t i v e  20. cm TLC g l a s s p l a t e s o f Polyamide DC  H^O-n-BuOH-acetone-HOAc - l 6 : 2 : 1 : 1 , 2nd dimension  6.6; —  1st dimension  —  CHCl^-isopropanol-  butanone-HOAc - 10:3:3:4) o f a l l f l a v o n o i d g l y c o s i d e s o c c u r r i n g i n t h e Limnanthaceae.  Although t h i s drawing i s a f u l l s i z e d r e p r o d u c t i o n o f a 20 x  cm two-dimensional  map,  a c t u a l chromatogram.  i t i s not meant t o r e p r e s e n t t h e r e s u l t s o f  20  any  Each of the f l a v o n o i d s i n t h e drawing i s l a b e l e d w i t h  the number or l e t t e r randomly assigned- d u r i n g t h e course o f t h i s work. T a b l e IV l i s t s Rfs o f t h e 48 .flavonoids as t h e y appear i n F i g u r e I I I . These Rfs are approximations  o n l y , s i n c e f l a v o n o i d Rfs on polyamide. are v a r i -  a b l e , b e i n g c o n t r o l l e d by many f a c t o r s ; t h e s e compounds are c o n s i s t e n t .  however, the r e l a t i v e p o s i t i o n s o f  T a b l e IV a l s o l i s t s  colour c h a r a c t e r i s t i c s  of most f l a v o n o i d s as they appear on polyamide chromatograms under UV (366 mji) , w i t h and without NH^ reagent.  vapour, and a l s o a f t e r s p r a y i n g w i t h  light  boronate  C o l o u r d a t a are not l i s t e d f o r compounds t h a t o c c u r r e d o n l y i n  t r a c e amounts, s i n c e no judgement c o u l d be made beyond presence  or absence.  - he TABLE I I I I d e n t i f i c a t i o n s o f t h e F l a v o n o i d s o f t h e Limnanthaceae  Symbol  Compound  H  Unknown kaempferol monoside; perhaps a c y l a t e d  D  P r o b a b l y t h e 7 - 0 - g l u c o s i d e o f kaempferol  E  Probably t h e 7-0-glucoside o f q u e r c e t i n  0  Syringetin-3-0-glucoside  Sg- •3-•0--G  P.  Isorhamnetin-3-0-glucoside  I r - •3-•0--G  Q  Kaempferol-3-0-glucoside•  Kp-•3-•0--G  R  Laricytrin-3-0-glucoside  Lc-•3-•0--G  S  Quercetin-3-0-glucoside  Qu-•3-•0--G  T  Myricetin-3-0-glucoside  My-•3-•0--G  C  Unknown.3-0-monoside o f q u e r c e t i n  A  P r o b a b l y t h e 7 - 0 - r u t i n o s i d e o f kaempferol  B  Probably the 7-0-rutinoside of q u e r c e t i n  19  Unknown 3-0-monoside o r b i o s i d e o f s y r i n g e t i n  20, .  Unknown 3-0-monoside o r b i o s i d e o f i s o r h a m n e t i n  21  Unknown' 3-0-monoside or b i o s i d e o f kaempferol  22.  Unknown 3-0-monoside o r b i o s i d e o f l a r i c y t r i n  23  Unknown 3-0-monoside or b i o s i d e of. q u e r c e t i n  U  Syringetin-3-0-rutinoside  V  Isorhamnetin-3-0-rutinosIde  W  Kaempferol-3-0-rutinoside  Kp-•3-•0--Rut  X  Laricytrin-3-0-rutinoside  Lc-•3-•0--Rut  Y  Quercetin-3-0-rutinoside  Qu-•3-• 0-Rut  Z  Myricetin-3-0-rutinoside  My-•3--0--Rut  I  • Syringetin-3-0-diglucoside  Abbreviation  Sg-•3-•0--Rut ' I r•3- •0--Rut  Sg-•3--0--GG  -  kl  TABLE I I I —  -  Continued  Symbol  Compound  Abbreviation  J  Isorhamnetin-3-0-diglucoside  Ir-3-0-GG  K  Kaempferol-3-0-diglucoside  Kp-3-0-GG  L  Laricytrih-3-0-diglucoside  Lc-3-O-GG  M  Quercetin-3-0-diglucoside •  Qu-3-O-GG  N  Myricetin-3-0-diglucoside  My-3-0-GG  F  A b i o s i d e o f kaempferol; p r o b a b l y  G  A b i o s i d e of quercetin; probably  1  Syringetin-3-0-rhamnosylrutinoside  Sg-3-0-GRR  2  Isorhamnetin-3-0-rhamnosylrutinoside  Ir-3-0-GRR  3  Kaempf e r o l - 3 - 0 - r h a m n o s y l r u t i n o s i d e •'  Kp-3-0-GRR  k  Laricytrin-3-0-rhamnosylrutinoside  Lc-3-O-GRR  5  Quercetin-3-0-rhamnosylrutinoside  Qu-3^0-GRR  6  Myric e t i n - 3 - 0 - r h a m n o s y l r u t  My-3-0-GRR  7 •  S y r i n g e t i n - 3 - 0 - r u t inos i d e , 7 - 0 - g l u c o s i d e  8  Isorhamnetin-3-0-rutinoside,7-0-glucoside  Ir-3-0-Rut,7-  9•  K a e m p f e r o l - 3 - 0 - r u t inos i d e , 7 - 0 - g l u c o s i d e  K p - 3 - 0 - R u t , 7 - -0--G  10 .  Laricytrin-3-0-rutinoside,'7-0-glucoside  L c - 3 - 0 - R u t , 7 - -0--G  11  Quercetin-3-0-rutinoside,7-0-glucoside  Qu-3-0-Rut,7- •0--G  12  Myricetin-3-0-rutinoside,7-0-glucoside  My-3-0-Rut,7- •0--G -  13  Syr i n g e t i n - 3 - O - r u t i n o s i d e , 7-0^-rut inos i d e  S g - 3 - 0 - R u t , 7 - •0--Rut  Ik  Isorhamnetin-3-0-ru'tinoside  I r - 3 - 0 - R u t , 7 - •0--Rut  15 16 1  7  .  k a e m p f e r o l - 3 -•0-glucoside , 7 - 0 --g l u c o s i d e q u e r c e t i n 3 - 0 -• g l u c o s i d e , 7 - 0 - g l u c o s i d e  inos ide  ,7-0-rutinoside  Kaempferol-3-0-rutinoside,7-0-rutinoside  • S g - 3 - 0 - R u t , 7 --0--G  -0--G  K p - 3 - 0 - R u t , 7 - •0--Rut  Laricytrin-3-0-rutinoside,7-0-rutinoside  L c - 3 - 0 - R u t , 7 - •0--Rut  Quercetin-3-0-rutinoside,7-0-rutinoside  Q u - 3 - 0 - R u t , 7 - •0--Rut  .  -  48  -  FIGURE I I I  Composite Two-Dimensional Map^of 48  2  A  Flavonoid  Glycosides- o f t h e Limnanthaceae  ©  ©  '20)  ©  •22 ' ( x s  \  —  R > „ ' '23  •  A  © -°© 1  12 1 —  1 s t dimension ( h o r i z o n t a l ) — . HgO-n-BuOH-acetone-HOAc ( l 6 : 2 : l : l ) 2nd dimension ( v e r t i c a l ) — CHCl^'Vbutanone-isopropanol-HOAc (10:3:3:4)  2 —  S o l i d . c i r c l e s denote i d e n t i f i e d compounds; broken c i r c l e s denote compounds p r e s e n t i n q u a n t i t i e s i n s u f f i c i e n t f o r complete i d e n t i f i c a t i o n .  - 49 TABLE IV  Rfs  1  and C o l o u r s  2  o f F l a v o n o i d s o f t h e Limnanthaceae  Compound  Rf-1  Rf-2  UV  UV/NH^  UV'/Spray-  H  0.08  0.10  P  P  G  D  0.14  0.15  -  Id  E  0.15  0.07 0. 54  0  Sg-3-0-G  0.34  P  Ir-3-0-G  0.24  Q  Kp-3-0-G  0.20  R  Lc-3-O-G  0.24  0.43 0.31 0.18  S  Qu-3-0-G  0.18  T  My-3-0-G  -  3  Id  P  PY  YG  P  G  G  P  P  G  Br  Y  Ro  0.14  Br  Y  Or  0.18  0.06  Br  Y  Ro  C  0.22  0.11  -  -  Id  A  0.18  Y  .' Y  0.06  Y  Y  Or •  0. 54  -  -  Id  0.44  -  -  • Id  21 22  0.31 0.30 0.46 o.4i 0.36 0.41 •  0.34 • 0.23 '  -  -  Id  -  -  Id  23  0.38  0.18  -  -  Id  B  19 20 .  .  G  U  Sg-3-0-Rut  ' 0.54  0. 54  P  PY  YG  V  Ir-3-0-Rut  0.50  0.44  P  G  G  W  Kp-3-0-Rut  0.34 .  P  P  G  X  Lc-3-0-Rut  0.24  Br  Y  Ro  Y  Qu-3-0-Rut .  ' 0.18 •  Br  Y  Or  Z •  My-3-0-Rut  0.43 0.50 . ' 0.46 0.46  0.08  Br  Y  Ro  I  Sg-3-0-GC-  0.55  0.38  P  PY  YG  J  . Ir-3-O-GG  0.31  P  G  G  K  Kp-3-0-GG  0.55 0. 50  0.28  P  P  G  L  Lc-3-0-GG  0.19  -  -  Id  M  Qu-3-0-GG  0.55 0.52  Br  Y  Or  N  My-3-0-GG  0.14 0.'Q6  • -  -  Id.  0.23 •  -  -  Id  F  0.51 0.58 •  G  O.58  0.10  -  -  Id  1  Sg-3-0-GRR  O.69  o.4o  P  PY  YG  2  Ir-3-0-GRR  .0. 70 .  0.31 .  P •  G  G  - 50 TABLE IV —  Continued  Compound  Rf-1  Rf-2  UV  UV/NH^  UV/Spray  3  Kp-3-0-GRR  0.62  0.23  P  P  G  4  Lc-3-O-GRR  0.68  0.18  P  Y  Ro  5  Qu-3-0-GRR  0.63  0.13  P  Y  Or  6  My-3-0-GRR  0.65  0.08  P  Y  Ro  7  Sg-3-0-Rut,7-0-G  0.83  0.38  P  PY  YG  8  Ir-3-0-Rut,7-0-G  0.80  0.32 .  P  Y  G  Kp-3-0-Rut,7-0-G 9 10 . Lc-3-0-Rut,7-0-G 11 Qu-3-0-Rut,7-0-G  0.77  0.24  P  Y  G  0.82  0.14  Br  Y  Rd  0,. 77  ' 0.11  Br  Y  Or  12 . My-3-0-Rut,7-0-G  0.78-  0.03  Br  Y  Rd  13  Sg-3-0-Rut,7-0-Rut  0.90  0.38  P  PY  YG  14  Ir-3-0-Rut,7-0-Rut  0.87  0.31  P  Y  G  15  Kp-3-0-Rut,7-0-Rut  0.84  0.24  P  Y  G  16  Lc - 3-0-Rut., 7- 0-Rut  0.88  0.15  Br  Y  Rd  17  Qu-3-0-Rut,7-0-Rut  0.86  0.10  Br  Y  Or  1 — R f - 1 =. 1 development, Polyamide DC6.6, H 0--n-BuOH-•acetone-HOAc 2  Rf-2  = Xldevelopment, Polyamide (10:3:3:4)  DC6.6, CHCl^-butanone-isopropanol-HOAc  2 — Colour explanation —- P8= purple; yellow; green;  G = green;' Or = oran£;e; I d = limit;.,of  (l6:2:l:l)  Br = brown; Ro = rose;  Y = yellow; Rd. = red;  PY = pale  YG = yellow  detection  - Spray = 0.1% (w/v) d i p h e n y l b o r i c a c i d ethanolamine complex i n HcO & MeOH  -  Table V l i s t s  51 -  t h e products o b t a i n e d upon t o t a l h y d r o l y s i s w i t h HC1,  i n c l u d i n g f l a v o n o i d a g l y c o n e , s u g a r ( s ) , and r e l a t i v e c o n c e n t r a t i o n o f each sugar where a p p r o p r i a t e .  T a b l e VI l i s t s  t h e d e r i v a t i v e s o f p a r t i a l a c i d hy-  d r o l y s i s and t h e d e r i v a t i v e s o f enzymic h y d r o l y s i s w i t h emulsin where t h e s e procedures  (^-glucosidase)  were employed.  T a b l e V I I l i s t s UV a b s o r p t i o n maxima o f t h e f l a v o n o i d g l y c o s i d e s t h a t were i s o l a t e d i n s u f f i c i e n t q u a n t i t i e s f o r d e t e r m i n a t i o n .  T a b l e VII  maxima i n MeOH and a l s o i n MeOH f o l l o w i n g t h e a d d i t i o n o f v a r i o u s  lists  shift  reagents. T a b l e V I I I i s a t a b u l a t i o n o f t h e r e s u l t s o f NMR s p e c t r o s c o p y o f s i x f l a v o n o i d s o f Limnanthes.  I t p r e s e n t s t h e p r o t o n s h i f t s o f TMS.-ethers'.,af s i x  ^ - D - r u t i n o s i d e s r e l a t i v e t o t e t r a m e t h y l s i l a n e . Reproductions of  o f t h e NMR s p e c t r a  t h s e s i x d e r i v a t i v e s a r e p r e s e n t e d as Appendices L I - L V I .  B. E x p l a n a t i o n o f t h e Data The' f l a v o n o i d s o f t h e Limnanthaceae f o l l o w a d e f i n i t e p a t t e r n . A s e r i e s o f s i x f l a v o n o l aglycone types r e p e a t s many t i m e s , each time comp r i s i n g a c l a s s o f compounds p o s s e s s i n g u n i f o r m g l y c o s y l a t i o n . g l y c o s y l a t i o n p a t t e r n s were found in  i n t h e Limnanthaceae.  I n t o t a l 12  A l l o f these  q u a n t i t i e s s u f f i c i e n t f o r h y d r o l y s i s proved t o be based  isolated  on two sugars,  g l u c o s e and rhamnose. Using aqueous s o l v e n t s , f l a v o n o i d s were r e s o l v e d i n t o groups, with uniform g l y c o s y l a t i o n .  each  The a c i d i c c h l o r o f o r m s o l v e n t r e s o l v e d t h e s e  groups i n t o s i x components, each based  on a p a r t i c u l a r aglycone t y p e .  Thus,  the a r r a y o f compounds diagrammed i n F i g u r e I I I i s d i v i d e d a l o n g t h e h o r i zontal axis into glycosylation classes (  ,  U-Z, 1 - 6 ,  s e p a r a t e d by development i n t h e aqueous s o l v e n t .  ... e t c . ) t h a t were  These groups i n t u r n a r e  s e p a r a t e d a l o n g t h e v e r t i c a l a x i s i n t o i n d i v i d u a l compounds based  on each  - 52 TABLE V  Products Obtained upon T o t a l H y d r o l y s i s w i t h 1.0 N HC1  Compound  . Aglycone  Sugar (s ) . Glucose  0  Sg-3-0-G  Syringetin  P  Ir-3-0-G  Isorhamnetin  Q  Kp-3-0-G  Kaempferol  R  Lc-3-O-G  Laricytrin  S  Qu-3-O-G  Quercetin  T  My-3-0-G  Myricetin  U  Sg-3-0-Rut  Syringetin  V  Ir-3-0-Rut  Isorhamnetin  W  Kp-3-0-Rut  Kaempferol  X  Lc-3-0-Rut  Laricytrin  11  Y  Qu£:3-0-Rut .  Quercetin  II  Z  My-3-0-Rut  Myricetin  II  I  Sg-3-0-GG•  Syringetin  K  Kp-3-0-GG  Kaempferol  M  Qu-3-0-GG"  Quercetin  1  Sg-3-0-GRR  Syringetin  2 ,  Ir-3-0-GRR '  Isorhamnetin  3  Kp-3-0-GRR'  Kaempferol  ' "  k  Lc-3-O-GRR  Laricytrin  "  5  Qu-3-O-GRR  Quercetin  6  My-3-0-GRR  Myricetin  7  Sg-3-0r-Rut,7-0-G'  Syringetin  8  Ir-3-0-Rut,7-0-G  Isorhamnetin  9 '  Kp-3-0-Rut,7-0-G  Kaempferol  10  Lc-3-0-Rut,7-0-G  Laricytrin  11  Qu-3-0-Rut,7-0-G  Quercetin'  12  My-3-0-Rut,7-0-G  Myricetin  13  Sg-3-0-Rut,7-0-Rut  Syringetin  Ik  Ir_3_0-Rut,7-0-Rut  Isorhamnetin  15  Kp-3-0-Rut,7-0-Rut  Kaempferol  16  Lc-3-0-Rut,7-0-Rut  Laricytrin  17 •  Qu-S-O-Rut^-O-Rut  Quercetin  G l u c o s e , Rhamnose I! 1!  Glucose ii II  1 G l u c o s e , 2 Rhamnose  2 G l u c o s e , 1 Rhamnose  G.lucose, Rhamnose II  n  ii II  - 53 TABLE V I -  D e r i v a t i v e s Obtained upon P a r t i a l Acid Hydrolysis  and H y d r o l y s i s w i t h Emulsin  Compound  Hydrolysis  1  20% HOAc  Sg-3-0-GRR  Derivatives ,  Sg, Sg-3-O-G, Sg-3-O-Rut  2  Ir-3-0-GRR  3  Kp-3-0-GRR  it  Kp, Kp-3-O-G, Kp-3-O-Rut  k  Lc-3-0-GRR  tt  L c , Lc-3-0-G, Lc-3-O-Rut  5 6  Qu-3-O-GRR  tt  Qu, Qu-3-O-G, Qu-3-O-Rut  My-3-0-GRR.  it  My, My-3-O-G, My-3-0-Rut  TI  I r , I _3_0-G, r  Ir-3-0-Rut  •7 8  Sg-3-0-Rut ,7-0-G  0.1 N HC1  Sg, Sg-7-O-G, G l u c o s e , Rhamnose  Ir-3-0-Rut ,7-0-G  tt  I r , Ir-7-O-G,.Glucose, Rhamnose  9  Kp-3-0-Rut ,7-0-G  tt  Kp, Kp-7-O-G, G l u c o s e , Rhamnose  10 . Lc-3-0-Rut ,7-0-G  tt .  L c , Lc-7-O-G, Glucose, Rhamnose  11  Qu-3-0-Rut ,7-0-G  it  Qu, Qu-7-0-G, Glucose, Rhamnose  12  My-3-0-Rut ,7-0-G  tt  My, Hy-7-0-G, G l u c o s e , Rhamnose  7 8  Sg-3-0-Rut ,7-0-G' Ir-3-0-Rut ,7-0-G  tt  I r - 3-0-Rut, Glucose  9  Kp-3-0-Rut ,7-0-G  tt •  Kp- 3-0-Rut, Glucose  10  Lc-3-0-Rut ,7-0-G  tt.  L c - 3-0-Rut, Glucose.  11  Qu-3-0-Rut ,7-0-G'  tt  Qu- 3-0-Rut, Glucose  12  My-3-0-Rut ,7-0-G  tt  My- 3-0-Rut, Glucose  13  Sg-3-0-Rut ,7-0-Rut'  0.1 N.-HCl  Sg, Sg-7-0-G, Sg-7-0-Rut  Ik  Ir-3-0-Rut ,7-0-Rut  tt..  I r , Ir-7-0-G,.Ir-7-0-Rut  15 16  Kp-3-0-Rut ,7-0-Rut  it  Kp, Kp-7-0-G, Kp-7-0-Rut  Lc-3-O-Rut ,7-0-Rut  tt  L c , Lc-7-O-G, Lc-7-0-Rut  17  Qu-3-0-Rut ,7-0-Rut  tt  Qu, Qu-7-O-G, Qu-7-0-Rut  13 ' Sg-3-0-Rut ,7-0-Rut Ik  Emulsin  Sg- 3-0-Rut, Glucose  . No A l t e r a t i o n  tt  tt  ,7-0-Rut  tt  tt  ,7-0-Rut  it  ti  Qu-3-0-Rut ,7-0-Rut  tt  it  Ir-3-0-Rut ,7-0-Rut  15 ' Kp-3-0-Rut 16 Lc-3-0-Rut 17  Emulsin  a.  TABLE V I I UV*kb:sorp!biph'-Maxiii|, o f ; E l a v o n o i d s  MeOH •M  COMPOUND  .MeOH I  II  Kef  ?•  T T  a0':e  Aica  1  II  272  301s  of-the Limnanthaceae KeOR h A 1 C 1 , & HC1  I  1  -'•  MeOH & NaOAo II  MeOH ftBO TaO.a r M V ? .  v  I  3  • 3  Lc-3-O-G  255,266s ; sos-  36-1  Sg-3-0-G  252,262s , sops  359  266  329  It 27  273  310  306  362  266  322  1*09  273  318 s  1*23  271*  320s .3743,1*08  269  328  1(09  261  306s  383  Qu-3-0-P.ut  258,268s . 299  359  266  322  Ul8 -  276  30os  435  271  299s  366,1(04  271*  328  1(09  261*'  295s  382  Lc-3-0-Rut  255,266s  361  266,286S  329s  1*31  • 27!*  306s  1*39  273  302s  367,407  270  322  1*23  '267  302s  381*  Km-3-0-Rut  266 :  350  266  32!*  1*05  275  306s  35 *,397  275  306s  350,394  275  306s 330  •373s, 1*07  269  203  3oU,Uo6  271*  323  4 09  3 8 0 s . 1*10  271*  307  360,1(07  265  323  427  395-  261  My-3-0-Rut  301 ;  268  327  1*19  271*  309  371,.40 6  272  325  1.11*  3 7 0 s , 1*06  273  310  366,1*05  270  325  1(22  433  ,  262 251,261*  302s  383  306s  361*  355  Ir-3-0-Rut  255,268s ' ;300  356  270  332  1*11  269 ' 293s  255,268s  Sg-3-0-Rut  254,268s \ 300  361  266  330  1*26  272  307 '  Qu-3-O-GG  257,268s ' 298  357  270  322s  1*09  ' 277  302s  i»3l*  271  2Q8s  365,1*06  271  32l(  3I+9  270  322  1*01  275  302s  352,1+00  275  302s  349,396  275  302s 375  38ls,.l*ia  27l*  310s  370,1*0-3  266  322  273  30ks  370,1(07.  270  318s 390  263s  270  301s  368,1*05  271  326  383  262  1(1*1  275  309s  367,1(08  270- 32n  1*12  260  375  267  352  .  266s  308  365  262 "  302s  371*"  269  302s  351  26.IJS  302  365  Km-3-0-GG  268  Sg-3-O-GG  25 »,2,65s  303  362  266,288s  358  1*29  272  310  My-3-0-GRR  255,266s  297  357  266,285S  326s  1*12  271*  310s  1*26  Qu-3-O-GRR  257,267s  305s  356  270  319  hO.P- 271* 306s  1*37  Lc-3-0-GRR  255,266s  300  359  266  324  1H7  271* •30ps  297s  3^9  27 u  324  395  275  305  355,403  276  303  348,399  271*  313  333  255,268s  302s  355  266  326  1*09  270  301s  371 s , 1*09  26o  301s  363.1*01+  273  323  !*0l*  255,26"  Sg-3-0-GRR  254,268s  303  359  266  328  1*18  271*  306s  368s, 1*11  275  30'6s  371,407  . 272  316  388  263s  Lc-3- 0 - R u t , 7-0-.G  258,266  291*  358  269  1*03  273  206s  3 6 2 s , 1*32  273  296s  368s, 1*03  1  . 267  Km-3-0-GRR  1). Expressed 3s  -  -  265  1*30  2°6s 1*03  361  261  366 310s  374  358  S  360  2C2s  373  TABLE V I I I  P r o t o n Chemical S h i f t Values  o f TMS E t h e r s o f  S i x F l a v o n o l J3-D-Rut i n o s ides o f t h e Limnanthaceae  Compound Kp-3-0-Rut  Qu-3-0-Rut  6  8  6.08  5.36  d,J2.5  d,J2.5  •6.08  I _3_0-Rut  6.08  My-3-0-Rut  6.06  r  Carbohydrate  B-Rihg  A-Rin£ 2*  6.32  3'  2H,d,J8.5  .  ' 7-30 d,J2  7.34 q,J2.,9.5  7-45 d,J2  7.27 q.,J2,9  (7.08)  Glc-l-H  5'  Rhm-l-H  Rhm-Me  Other  0.71  3.26-3.83  d,J7  h.l9 d,Jl  5.75  4.20  0.81  3.29-3.80  6.77 d,J9  5.84  4.20  0.72  3.25-3.74  -  5.81  4.22  1.00  3.29-3.85  5-89  4.23  0.81  3.28-3.76  5-93  4.22  0.73  3.25-3.75  5.80  (6.78) 2H,d,j8 .5  (7.68)  '6.36  6.38  6'  6.78  k  d,J9-5  3.84  3H,s^  -  2H,s  Lc-3-0-Rut .  Sg-3-0-Rut  6.09  6.10  6.37  6.41  7.20 • d,J2  (7.12)  2H,s  1 —  6.90 d,J2  -  3.85 3H s  s ?  3  (3.84) 6H,S  5  E x p r e s s e d as ( T i n ppm r e l a t i v e t o t e t r a m e t h y l s i l a r i e ( 0 . 0 0 ) i n C C l ^ . 2 — C o n s i s t e n t value;s f o r H-6 & H-8. 3 — Consistent values f o r glucose-l-H. 4 — C o n s i s t e n t v a l u e s f o r rhamnose-l-H. 5 — 0-methyl groups  - 56 aglycone type • (e_. g_. ,  U,V,W, ... e t c . ) t h a t were r e s o l v e d a f t e r development  i n the organic solvent. Only t h r e e o f the many, g l y c o s y l a t i o n c l a s s e s o c c u r r i n g i n t h e Limnanthaceae are p r e s e n t i n r e l a t i v e l y g r e a t amounts.  The most prominent  group  among t h e s e , one t h a t i s p r e s e n t i n a l l t a x a , consist's^of the s i x compounds d e s i g n a t e d U,V,W,X,Y and.Z.  The i d e n t i f i c a t i o n o f t h e s e s i x f l a v o n o i d s was  c r u c i a l t o t h e e n t i r e i n v e s t i g a t i o n as t h e y were o f t e n o b t a i n e d as h y d r o l y s i s products from o t h e r f l a v o n o i d g l y c o s i d e s , and from t h e s e t h e s i x aglycone types were i d e n t i f i e d . Rfs i n aqueous s o l v e n t s o f t h e compounds U-Z suggested t h a t they were diglycosid'es';'" Upon h y d r o l y s i s each y i e l d e d a d i f f e r e n t a g l y c o n e , and t h e sugars, g l u c o s e and rhamnose ( T a b l e V ) .  Four o f t h e s i x aglycones were kaemp-  f e r o l , q u e r c e t i n , i s o r h a m n e t i n and m y r i c e t i n .  UV and NMR s p e c t r a l d a t a o f  t h e c o r r e s p o n d i n g g l y c o s i d e s (Tables V I I & V I I I ) agreed c o m p l e t e l y w i t h publ i s h e d data f o r 3 - 0 - j J - D - r u t i n o s i d e s . The r e m a i n i n g two r u t i n o s i d e s were new. One  (U) had Rf and UV behaviour s i m i l a r t o i s o r h a m n e t i n 3 - 0 - r u t i n o s i d e (V)  which suggested t h e presence o f an O-methylated  B-ring.  The NMR spectrum o f  the TMS d e r i v a t i v e ; , indeed showed the presence o f two e q u i v a l e n t O-methyl groups, 3.84c*, and. a s i n g l e t a t 7 • 12 S which i n t e g r a t e d f o r two p r o t o n s . T h i s s i n g l e t i n d i c a t e s e q u i v a l e n t protons on the B - r i h g . t h a t t h e B - r i n g i s a syringyl-typevv  These data  indicate  S i g n a l s a t 6.1C& and.6.l4cT showed t h e  expected meta s p l i t t i n g o f the 6-.K and 8-H... The aglycone i s , t h e r e f o r e , m y r i c e t i n 3',5'-dimethyl e t h e r , or s y r i n g e t i n . The behaviour  second new r u t inos idi,e.(x) was s i m i l a r t o r u t i n (Figure I I I , Table V I I ) .  The NMR spectrum  (Y) i n i t s Rf and UV  o f t h e TMS d e r i v a t i v e  showed f o u r aromatic p r o t o n s , as i n the c a s e . o f s y r i n g e t i n r u t i n o s i d e , but o n l y one O-methyl group ( T a b l e VIII':);.  The B - r i n g protons appeared  at 7-20S and 6.90S" and d i s p l a y e d meta c o u p l i n g .  as d o u b l e t s  These r e s u l t s a l l o w t h e  - 57 assignment o f the s t r u c t u r e m y r i c e t i n 3'-methyl ether t o the aglycone, laricytrin The laricytrin  as i f has  r e c e n t l y been named by Tyukavkin et_ al_. (1974).  a c e t a t e d e r i v a t i v e s were prepared 3 - 0 - r u t i n o s i d e and  p o i n t s determined.  q u e r c e t i n 3 - 0 - r u t i n o s i d e , and t h e i r  Il6-ll8°, l a r i c y t r i n  syringetin rutinoside —  6,  of syringetin 3-0-rutinoside, melting  The m e l t i n g p o i n t s o f the t h r e e a c e t a t e d e r i v a t i v e s a r e :  quercetin rutinoside —  The  or  rutinoside —  124-126°,  and  130-132°.  second major group o f g l y c o s i d e s i n v e s t i g a t e d , termed 1,2,3,4,5 and  moved f u r t h e r i n aqueous s o l v e n t s than d i d the r u t i n o s i d e s ( F i g u r e I I I ) ,  suggesting  a-greater  degree o f g l y c o s y l a t i o n .  t o be 3 - 0 - g l y c o s i d e s  (Table V I I ) .  a l r e a d y d e s c r i b e d and, glucose  (Table V ) .  T h e i r UV  T o t a l hydrolyses  i n each case, two  s p e c t r a showed them  gave the .same s i x  e q u i v a l e n t s of rhamnose and  aglycones one  of  P a r t i a l a c i d h y d r o l y s i s o f t h e s e t r i g l y c o s i d e s gave, i n  each case, the c o r r e s p o n d i n g  3 - 0 - r u t i n o s i d e and  3-0-glucoside  (Table V I ) .  The  p o i n t of s u b s t i t u t i o n o f the t e r m i n a l rhamnose has not been determined, but the s i x t r i g l y c o s i d e s are c l e a r l y 3-0-rhamnosyl r u t i n o s i d e s . The  t h i r d major group o f g l y c o s i d e s , c o n s i s t i n g o f the s i x components  7,8,9,10,11 and 12, had h i g h e r Rfs i n aqueous s o l v e n t s than the two groups [ F i g u r e I I I ) .  preceding  A l l attempts t o f r a c t i o n a t e t h e s e g l y c o s i d e s i n t o s i x  components by p r e p a r a t i v e TLC,  as was  r u t i n o s i d e s , were u n s u c c e s s f u l .  done f o r the r u t i n o s i d e s and  However, one  of t h e s e  i s o l a t e d from the r e s t by c r y s t a l i z a t i o n from MeOH, and responded t o a 3 , 7 - d i g l y c o i d e  (Compound 10,' T a b l e V I I ) .  rhamnosyl-  s i x compounds i t s UV  was  spectrum c o r -  Since  separation  c o u l d not be a c h i e v e d , h y d r o l y s i s experiments were performed on a l l s i x g l y cosides simultaneously. the o t h e r groups, two (Table V ) . UV  T o t a l h y d r o l y s i s gave the s i x aglycones  e q u i v a l e n t s o f g l u c o s e and one  present  e q u i v a l e n t o f rhamnose  P a r t i a l a c i d h y d r o l y s i s .yielded f l a v o n o l d e r i v a t i v e s w i t h Rfs  s p e c t r a l c h a r a c t e r i s t i c s t y p i c a l o f 7-0-monosides.  in  Treatment w i t h  and  ^-gluco-  - 58 sidase- (emulsin)  produced t h e s i x 3 - 0 - r u t i n o s i d e s  and g l u c o s e  (Table V I ) .  T h e r e f o r e , these' compounds must be t h e f l a v o n o l 3-0-^-D-rutinoside^7-0-^-Dglucosides. The  18 f l a v o n o l g l y c o s i d e s d e s c r i b e d above a r e u b i q u i t o u s i n t h e  Limnanthaceae, o c c u r r i n g i n h i g h c o n c e n t r a t i o n s tigated.  i n n e a r l y every taxon  inves-  The o t h e r groups o f g l y c o s i d e s encountered i n t h i s work occur  s p o r a d i c a l l y throughout t h e f a m i l y , but i n l e s s e r c o n c e n t r a t i o n s than t h e t h r e e common groups. The  group o f g l y c o s i d e s c o n s i s t i n g o f compounds I,J,K,L,M and N had  Rfs i n aqueous s o l v e n t s i n d i c a t i n g t h a t they were b i o s i d e s .  Rfs i n the  o r g a n i c s o l v e n t and c o l o u r s a f t e r s p r a y i n g i n d i c a t e d t h a t these s i x g l y c o s i d e s were based on t h e s i x aglycones I,K  a l r e a d y encountered.  and M were i s o l a t e d i n q u a n t i t i e s s u f f i c i e n t  analysis.  f o r s p e c t r a l and h y d r o l y t i c  These t h r e e had UV s p e c t r a c o r r e s p o n d i n g  V T l ) , and t o t a l a c i d h y d r o l y s i s gave t h e aglycones and q u e r c e t i n , and t h e sugar g l u c o s e  (Table V ) .  Of t h e s i x , o n l y  t o 3-0-glycosides  (Table  s y r i n g e t i n , kaempferol  This information allows the  conclusion that the three are f l a v o n o l 3-0-digiucosides.  Although  t h e com-  pounds <T,L and.M occur o n l y i n t r a c e amounts, and.were not i s o l a t e d i n q u a n t i t y ^ they a r e almost c e r t a i n l y t h e 3-0-d'iglucoside  analogues o f i s o -  rhamnetin, l a r i c y t r i n a n d . m y r i c e t i n , r e s p e c t i v e l y . The  g l y c o s y l a t i o n s e r i e s c o n t a i n i n g t h e s i x compounds 0,P,Q,R,S and  T had R f s i n aqueous s o l v e n t s c h a r a c t e r i s t i c o f f l a v o n o l monosides ( F i g u r e III).'. T h e i r UV s p e c t r a were those o f f l a v o n o l 3 - 0 - g l y c o s i d e s a c i d h y d r o l y s i s y i e l d e d each o f t h e s i x aglycones T h e r e f o r e , these compounds a r e . f l a v o n o l  .(Table V I I ) . T o t a l  and o n l y glucose- ( T a b l e V ) .  3-0-^-D-glucosides.  A s e r i e s o f f i v e compounds,' 1 3 , l h , 1 5 , 1 6 and 17,  had R f s i n aqueous  s o l v e n t s even h i g h e r than those o f t h e 3,7-branched t r i o s i d e s  (Figure I I I ) ,  s u g g e s t i n g a h i g h e r degree o f g l y c o s y l a t i o n . Attempts t o r e s o l v e t h i s group  - 59 i n t o i t s f i v e components Py p r e p a r a t i v e TLC were u n s u c c e s s f u l . i t was  Therefore,  n e c e s s a r y t o h y d r o l y z e t h e s e compounds as a group. . T o t a l h y d r o l y s i s  y i e l d e d the a g l y c o n e s , s y r i n g e t i n , •isprhamnetin, kaempferol, l a r i c y t r i n q u e r c e t i n , and two  and  s u g a r s , g l u c o s e and rhamnose,. both i n a p p a r e n t l y equal  proportions (Table V).  Attempts  t o h y d r o l y z e t h e s e compounds w i t h emulsin were  u n s u c c e s s f u l , i n d i c a t i n g t h a t g l u c o s e does not occur i n a t e r m i n a l p o s i t i o n on t h e s e compounds. pounds each, one  P a r t i a l a c i d h y d r o l y s i s gave t h r e e s e r i e s o f f i v e com-  s e r i e s c o n s i s t i n g o f t h e f i v e a g l y c o n e s , and t h e o t h e r  two  w i t h Rfs and c o l o u r s c h a r a c t e r i s t i c o f 7-0-monosides and 7 - 0 - b i o s i d e s . f i c i e n t m a t e r i a l and time prevented f u r t h e r experiments  Insuf-  on t h e s e f i v e g l y c o -  s i d e s , but a l l o f the i n f o r m a t i o n g a t h e r e d i s c o n s i s t e n t w i t h t h e  assignments  of f l a v o n o l 3-0-rutinoslde,7-0-rutinosides. A l l o t h e r f l a v o n o i d compounds p r e s e n t i n t h e Limnanthaceae w i t h t h e e x c e p t i o n s o f H,A identification.  and B o c c u r r e d i n c o n c e n t r a t i o n s t o o low f o r i s o l a t i o n Compound H was  found"; o n l y i n F l o e r k e a , of which o n l y s m a l l  amounts o f m a t e r i a l were a v a i l a b l e f o r study.  Its colour characteristics  low Rf i n the o r g a n i c s o l v e n t suggest t h a t t h i s i s a kaempferol (Figure I I I & Table IV).  and  3-0-monoside  I t s extremely low m o b i l i t y i n the aqueous d i r e c t i o n  suggests t h a t t h i s i s an a c y l a t e d d e r i v a t i v e .  Compounds A and B have c o l o u r  c h a r a c t e r i s t i c s and RFs which suggest t h a t they are 7 - 0 - b i o s i d e s o f and q u e r c e t i n .  and  kaempferol  Problems i n o b t a i n i n g s u f f i c i e n t amounts o f t h e s e two  pounds, which w i l l be d i s c u s s e d i n a l a t e r s e c t i o n , have.not  allowed  comcomplete  i d e n t i f i c a t i o n ' , but t h e y a r e p r o b a b l y f l a v o n o l 7 - 0 - r u t i n o s i d e s . I d e n t i f i c a t i o n o f the t h r e e remaining g l y c o s y l a t i o n s e r i e s can o n l y be s p e c u l a t e d upon based on R f s .  Compounds D and.E have Rfs t h a t correspond  t o 7-0-monosides and a r e p r o b a b l y the 7-0-glucosides o f kaempferol cetin.  and  quer-  Compounds F and G a l s o have Rfs i n the o r g a n i c s o l v e n t i n d i c a t i v e  of g l y c o s y l a t i o n at the 7 - p o s i t i o n of the f l a v o n o l nucleus.  T h i s fact-, taken  - 60 t o g e t h e r w i t h t h e i r m o b i l i t y i n t h e aqueous d i r e c t i o n , suggests t h a t t h e y a r e 3,7-dimonosides;  i f t h i s i s t r u e , t h e y a r e p r o b a b l y the  g l u c o s i d e s o f kaempferol a n d - q u e r c e t i n . unknown.  3-0-glucoside,7-0-  Compounds\ 19,20,21,22 and 23  remain  Although c l e a r l y d e r i v a t i v e s o f s y r i n g e t i n , i s o r h a m n e t i n , kaemp-  f e r o l , l a r i c y t r i n and q u e r c e t i n , t h e i r Rfs i n the aqueous s o l v e n t , midway between t h e 3 - 0 - g l u c o s i d e s and 3 - 0 - r u t i n o s i d e s ( F i g u r e I I I ) , makes t h e extent of g l y c o s y l a t i o n unclear.  These compounds a r e e i t h e r 3-0-monosides or b i o s i d e s .  3-0-Dirhamnosides would p r o b a b l y have Rfs s i m i l a r t o t h e s e compounds, but t h e r i n f o r m a t i o n i s r e q u i r e d b e f o r e t h e i r i d e n t i t y can be  SIGNIFICANCE OF THE FLAVONOIDS OF THE A.  fur-  established.  LIMNANTHACEAE  Aglycones Although anthocyanins occur i n t r a c e amounts i n some t a x a , f l a v o n o l s  were t h e major t y p e - o f f l a v o n o i d encountered i n the p r e s e n t  investigation.  S i x f l a v o n o l aglycone types are made and accummulated by every member o f the Limnanthaceae..  These i n c l u d e t h e common f l a v o n o l t y p e s :  kaempferol,  quer-  c e t i n , i s o r h a m n e t i n and m y r i c e t i n , t h e r e l a t i v e l y r a r e s y r i n g e t i n , and the extremely r a r e l a r i c y t r i n , , or •3':-methyl m y r i c e t i n .  These aglycones  differ  o n l y i n t h e i r B - r i n g s , and t o g e t h e r they comprise a s e r i e s o f d e r i v a t i v e s made by s u c c e s s i v e h y d r o x y l a t i o n and m e t h o x y l a t i o n a d d i t i o n s t o t h i s  ring.  T h i s stepwise e l a b o r a t i o n o f t h e B - r i n g , from kaempferol t o s y r i n g e t i n , i s shown i n F i g u r e IV. S y r i n g e t i n has r a r e l y been r e p o r t e d p r e v i o u s l y . b a r k e r & Bohm, 1975)  B e s i d e s Limnanthes  and F l o e r k e a , t h i s aglycone has been r e p o r t e d from  L a t h y r u s (Harborne, 1965), L a r i x ;(Niemann, 1972;  1973; Tyukavkin et a l . , 1974),  Soymida ( P a r k h a s a r a d h i & S i d h u , 1972), and.Philydrum. (Bohm & C o l l i n s , 1975). L a r i c y t r i n - has p r e v i o u s l y been r e p o r t e d o n l y from L a r i x . rence was  s u s p e c t e d by -Niemann(1972, 1973)  and was  I t s occur-  v e r i f i e d by Tyukavkin  and  FIGURE IV Stepwise E l a b o r a t i o n o f t h e B-Ring o f F l a v o n o l s  o f t h e Limnanthaceae  - 62 coworkers (197*0.  However, i t i s p r o b a b l e .that l a r i c y t r i n has a much wider  d i s t r i b u t i o n than t h e s e d a t a i n d i c a t e , s i n c e t h i s compound i s not e a s i l y sepa r a t e d from q u e r c e t i n .  If laricytrin  presence w i l l go) undetected i c a l properties.  i s not s e p a r a t e d from q u e r c e t i n , i t s  s i n c e t h e s e compounds possess  v e r y s i m i l a r phys-  TLC chromatography on polyamide DC 6.6 w i t h o r g a n i c  appears t o be t h e o n l y way p r e s e n t l y known t o separate t h e s e two types.  When t h i s t e c h n i q u e  solvents  aglycone  i s more w i d e l y adopted, seyerrali, a d d i t i o n a l r e -  p o r t s o f l a r i c y t r i n w i l l p r o b a b l y be made.  The r e c e n t d i s c o v e r y o f ' l a r i c y -  t r i n i n Heuchera (C..K. W i l k i n s , p e r s o n a l communication) tends t o support this  observation.  B. G l y c o s i d e s A t o t a l o f twelve of t h i s i n v e s t i g a t i o n .  g l y c o s y l a t i o n p a t t e r n s was>,discovered i n t h e course  Although  monosides do occur i n v a r i o u s members o f t h e  Limnanthac eae, the' m a j o r i t y o f t h e f l a v o n o i d s o f Limnanthes and F l o e r k e a a r e . g l y c o s y l a t e d w i t h two o r more sugars.  A l l of the i d e n t i f i e d f l a v o n o i d  g l y c o s y l a t i o n p a t t e r n s a r e based on d i f f e r e n t combinations o f g l u c o s e and rhamnose. R e l a t i v e l y few t r i o s i d e s and t e t r a s i d e s have been r e p o r t e d , w i t h t h e exception o f 3-0-rutinoside,7-0-glucosides.  To t h e b e s t o f my knowledge,  t h e r e have been no p r e v i o u s r e p o r t s o f 3-0-rhamnosyl r u t i n o s i d e s , nor have. t h e r e been any r e p o r t s o f 3 - 0 - r u t i n o s i d e , 7 - 0 - r u t i n o s i d e s .  THE CONTRIBUTION TO VISIBLE FLOWER COLOUR BY SYRINGETIN DERIVATIVES Harborne (T967) has r e p o r t e d t h a t s y r i n g e t i n a c t s as a y e l l o w pigment i n the flowers o f Lathyrus.  Although  certain.Limnanthes  t a x a have f l o w e r s  t h a t c o n t a i n b r i g h t y e l l o w pigments, i n c l u d i n g two v a r i e t i e s o f L_. d o u g l a s i i , s y r i n g e t i n a p p a r e n t l y does not c o n t r i b u t e t o t h i s phenomenon.  T h i s was  63  -  concluded  since high concentrations  -  o f s y r i n g e t i n g l y c o s i d e s occur  p e t a l s o f most Limnanthes t a x a , t h e m a j o r i t y o f which have w h i t e Furthermore, t h e y e l l o w pigment present  i n Limnanthes f l o w e r s  carotenoid(s) since i t i s r e a d i l y extracted with A l l the white flowered  i s probably  a  forms o f Limnanthes have p e t a l s t h a t a r e t i n g e d  by d r y i n g specimens.  c o s i d e s , as w e l l as other  flowers.  chloroform.  w i t h a p a l e y e l l o w c o l o u r , p a r t i c u l a r l y toward t h e i r b a s e s . accentuated  i n the  I t i s quite probable  This effect i s  that s y r i n g e t i n g l y -  flavonol glycosides, contribute to this  yellowing.  A l l s y r i n g e t i n g l y c o s i d e s i s o l a t e d d u r i n g t h i s i n v e s t i g a t i o n ' share an obvious p r o p e r t y ;  they a r e a l l extremely s e n s i t i v e t o base.  When fumed  w i t h NH^ t h e s e compounds i n s t a n t l y f l u o r e s c e an i n t e n s e p a l e y e l l o w under UV l i g h t .  I n f a c t , o c c a s i o n a l l y s y r i n g e t i n d e r i v a t i v e s were d e t e c t e d  o n l y by t h i s t e c h n i q u e , t o fuming.  colour  being  i n v i s i b l e under UV f o l l o w i n g s p r a y i n g but p r i o r  A f t e r fuming, t h e s e  compounds appear y e l l o w  in visible  light  and remain t h i s way f o r a s h o r t p e r i o d ( n o r m a l l y they appear a v e r y p a l e low when p r e s e n t  yel-  i n h i g h c o n c e n t r a t i o n , and-are i n v i s i b l e when p r e s e n t i n  lower c o n c e n t r a t i o n ) .  This observation indicates that s l i g h t l y basic conditions  i n a f l o w e r might e a s i l y cause s y r i n g e t i n g l y c o s i d e s t o serve as y e l l o w p i g merits i n v i s i b l e  light.  ENVIRONMENTAL MODIFICATION OF FLAVONOID COMPOSITION Taxonomic s t u d i e s u s i n g f l a v o n o i d s as c h a r a c t e r s r o u t i n e l y assume t h a t f l a v o n o i d compositions  do not v a r y w i t h changes i n t h e environment.  ( T h i s statement i s not meant t o i n c l u d e t h e s m a l l amounts o f f l a v o n o i d hydros l y s i s t h a t occur  as p l a n t s age.)  However, t h e presence o f two f l a v o n o i d  compounds o f Limnanthes, A'and B (probable quercetin) apparently  T-O-rutinosides  o f kaempferol and.  depends upon e n v i r o n m e n t a l f a c t o r s .  Germination o f a l l Limnanthes seed drops t o a low l e v e l as s o i l  -  temperatures, i n c r e a s e f r o m ' s p r i n g  64 -  t o summer.  S i n c e seed was germinated o u t -  s i d e , t h i s phenomenon caused an acute problem i n some i n s t a n c e s . attempt, g e r m i n a t i o n  On t h e f i r s t  o f OTUs 12 and 15, L. a l b a v a r . a l b a and L_. g r a c i l i s v a r .  g r a c i l i s , was 1-2%, producing  o n l y two p l a n t s o f each t a x o n .  The-pots con-  t a i n i n g t h e s e p l a n t s were not t r a n s f e r r e d t o t h e greenhouse f o l l o w i n g t h e standard  p r a c t i c e , but were l e f t  No f u r t h e r g e r m i n a t i o n  occurred,  i n c o l d frames pending a d d i t i o n a l  germination.  and e v e n t u a l l y t h e two p l a n t s o f each OTU  were harvested, and t h e f l a v o n o i d compositions o f p e t a l s and whole p l a n t m a t e r i a l were determined. concentration.  Both o f t h e s e t a x a c o n t a i n e d  A and B i n r e a s o n a b l y  high  However, i n s u f f i c i e n t p l a n t m a t e r i a l made it-' i m p o s s i b l e t o  i d e n t i f y these f l a v o n o i d s . An a d d i t i o n a l f l a t of year,  and g e r m i n a t i o n  o f L_. a l b a v a r . a l b a was grown at a c o o l e r time  was much improved.  However, at t h i s l a t e r  date  the p l a n t s were t r a n s f e r r e d t o t h e greenhouse f o l l o w i n g t h e u s u a l p r a c t i c e . Neither the flowers  nor whole p l a n t m a t e r i a l c o n t a i n e d  a l t h o u g h o t h e r w i s e they were c h e m i c a l l y c o l d frames.  compounds A and B,  i d e n t i c a l t o t h e p l a n t s grown i n t h e  The presence o f A and B was not d e t e c t e d  o f t h e Limnanthaceae i n c l u d i n g t h o s e t a x a r e p r e s e n t e d  i n any other members by d r i e d m a t e r i a l .  Presence o f A and B i n p l a n t m a t e r i a l grown i n t h e c o l d frame i s assumed t o be e n v i r o n m e n t a l l y  induced  rather than a genetic p e c u l i a r i t y of  a few p l a n t s , s i n c e the phenomenon was observed f o r two d i f f e r e n t t a x a . n a t u r e o f t h e i n d u c i n g f a c t o r i s not known;  The  however, i t seems u n l i k e l y t h a t  the compounds A and B a r e breakdown p r o d u c t s s i n c e n e i t h e r L_. a l b a nor L_. g r a c i l i s c o n t a i n s any 3 , T - d i r u t i n o s i d e s , presumably t h e o n l y c l a s s o f compounds encountered i n any taxa. o f Limnanthes which c o u l d down t o y i e l d A or-B.  possibly-breab--.  At any r a t e t h e s e two compounds have been e l i m i n a t e d  from t h e comparative a n a l y s i s s i n c e t h e i r presence i s v a r i a b l e and.they a r e , t h e r e f o r e , u n s u i t a b l e f o r taxonomic comparisons w i t h i n t h e Limnanthaceae.  -65-  COMPARATIVE FLAVONOID' DATA . F l a v o n o i d complements o f a l l t a x a were determined from two-dimensional "thin l a y e r chromatograms. two-dimensional map  F i g u r e s V and VI a r e c o l o u r photographs o f an a c t u a l 17,  o f the whole p l a n t f l a v o n o i d s o f L. montana, OTU  a f t e r s p r a y i n g and marking. Exposure d e t a i l s a r e :  F i g u r e V shows t h e chromatogram under UV  2 minutes _at.F 3.5,  and High Speed Ektachrome f i l m . i n d a y l i g h t at 1/25  u s i n g a Wratten 2E f i l t e r  F i g u r e VI shows the  second at F 3.5-  Two  taken  light.  (Kodak)  same chromatogram t a k e n  p o i n t s are noteworthy about  the  ,T  p i c t u r e made w i t h UV l i g h t , l ) , ,The compounds a r e c o r r e c t l y . shown as orescent  spots a f t e r s p r a y i n g , but t h e c o l o u r s are not a c c u r a t e l y  flu-  represented.  A c t u a l d i f f e r e n c e s i n c o l o u r s between compounds are g r e a t e r t h a n t h e y in this picture. eye.  2) The  photographic  f i l m i s more s e n s i t i v e t h a n the human  Compounds which appeared at t h e l i m i t s o f d e t e c t i o n are c l e a r l y  i n t h i s p i c t u r e , and the naked The  appear  visible  c e r t a i n f e a t u r e s o f t h e photograph were i n v i s i b l e t o  eye. p i c t u r e s o f the L_. montana chromatogram e x e m p l i f y  r e s u l t s upon which t h e c o n c l u s i o n s of t h i s t h e s i s o t h e r chromatograms have not been i n c l u d e d ;  the  are based.  experimental Photographs o f  however, t r a c i n g s o f l a b e l e d  and  marked chromatograms'of p e t a l and whole p l a n t m a t e r i a l are i n c l u d e d as appendices  (Appendices I I I - L ) .  pounds v i s i b l e b e f o r e  In b o t h the photographs and the t r a c i n g s , com-  s p r a y i n g are.marked by  s o l i d l i n e s , while  compounds  v i s i b l e o n l y a f t e r s p r a y i n g are marked by broken l i n e s . T a b l e IX l i s t s the f l a v o n o i d s present 30 OTUs i n c l u d e d i n t h i s study.  i n whole p l a n t . m a t e r i a l o f a l l  Compounds p r e s e n t  i n r e l a t i v e l y great  c e n t r a t i o n are i n d i c a t e d by'"Xs", while' compounds t h a t became v i s i b l e a f t e r s p r a y i n g are i n d i c a t e d by " t s " .  The  cononly  compounds are grouped a c c o r d i n g  g l y c o s y l a t i o n type w i t h l i n e s drawn between these groups. d i s t i n g u i s h e d i n t h i s f a s h i o n so t h a t occurrence  The  groups  to  are  of a g l y c o s y l a t i o n c l a s s can  -  66  -  FIGURE V TLC Map of Whole Plant Flavonoids of L. montana Taken i n U l t r a v i o l e t Light  * 4 • FIGURE VT TLC Map of Whole Plant Flavonoids of 1, montana Taken i n V i s i b l e Light  TABLE IX WHOLE PLANT FLAVONOIDS OF 30 OTUS OF THE LIMNANTHACEAE CCMFOUKD:  U  OTU  j-O-Rut V W X  1  3-0- SSI! 2 3 It 5 6  7  3-o- Rut ,7- o-s 8 g 10 11 12  3-o-Ruty-o-Rut 3-0-G '13 I * 15 16 17 0 p Q R 1  s  T  X  t  X  X  X  X  X  X  X  X  X  X  X X  X  doug. doug.  X  X  t  X  X  X X  X  X  X  X  X  X  t  t  X  X  5  dcug. sulph.  X.  X  X  X  X  X X  X  X  X  X  X  X  X  t  t  t  3  doug. nivea  X  X  X  X  X  X X  X  X  X  X  X  X  t  t t  X  X  h  dcav. rosea  X  X  X  X  X  X X  X  X  X  X  X  X  6  vjr.culans  X  X  X  X  X  X X  X  X  X  X  X  t  t  10 1  macounii  X  X  X  X  X  X  X  X  X  t  X  dog;, doug.  X  t  t  X  t  X X  X  X  X  X  X  Q  rcicounii  X  X  X  X . X X  X  X  X  21  floe, pusdla  X  X  X  X  25  floe. floe.  X X  t  X  X  X  X  19  flee, h e l l .  X X  t  t  X  t  X  X  X t  X  X  2k  floe. floe.  X X  t  t  X  X  X X  X  t  X  X  X  striata  2  X  X  X  X  X .X  X t  X  X  X  X  X  X  X  X  X  X  X  t  X  X  t  X  X  X  t  X  X  X  x'  13  alba versic.  X  t  t  X  X  X  X  t  X X  X  X  X  X  X  X  X  X t  X  X. X  X  X  X  t  X  X  X  X  t  X  X  alba alba  X  X  X  X  X  X X  X  12 alba alba  X  t  t  X  X  X X  X  15  grac. grac.  X  X  X  X  X  lit  grac. grac.  X  X  X  X  X  X  X  X  22  floe, grand.  X  X  X  X  X  X  X  X  20  floe, purlla  X  X  X  X  X  X  26  floe, c a l i f .  X  X  X  X  X  X  X  X  23  floe, floe.  X  X  X  X  t  X  X  X  t  -0 Floerkea  X  X  t  X  X  X  X  X  t  X  X  X  t  X  X t  t  X  X  X  X  X  t  t  X  X  X  X  X  t  t  X  X  X  t X  t  X  t  X  X  t  X  X  X  X  X  X X  X t  t  X t  X  t  X  X  X  X  X  t  t  X  X  X  X  X  t  X  t  X  X  X  X  t t  t X  I  X  X t  t  t  t  X  X t  X  X X  t  X t  Floerkea  X  X X  X  X  27  t  B  X  X  X  t  X  A  X  X  X  X  X  t  t  X  28 Flcerkea  t t  X  X  X  X  r> E  t t  X  X  29 Floerkea  X  X  X  t  X  X  X  X  X  X  t  X X  t  3-0-GG K L M  t  X X  11  J  t  X  floe, b o l l ,  t  'I  X X  grac. parish,  X X  c  t X  X  16  X X X  t t  18  17  X X X X  X  19 20 21 22 23 F G  X  X t X t  - .68 be r e a d i l y assessed'"!" or. each. OTU. T a b l e X l i s t s the f l a v o n o i d s present Limnanthes.  The  i n f r e s h p e t a l s o f 18  format i s the same as t h a t d e s c r i b e d  f o r Table  Taxa have been arranged manually i n T a b l e s IX and w i t h the g r e a t e s t  OTUs o f  IX.  X t o p l a c e OTUs  f l a v o n o i d s i m i l a r i t i e s next t o each o t h e r .  Although  the  r e s u l t i n g arrangements i n b o t h t a b l e s are somewhat s u b j e c t i v e , i t i s p o s s i b l e t o see a certain.amount of c l u s t e r i n g between OTUs.  FLAVONOID DIFFERENCES BETWEEN PETAL AND Relative concentrations  WHOLE PLANT MATERIAL  o f c e r t a i n f l a v o n o i d s often"' d i f f e r e d markedly  between p e t a l and whole p l a n t m a t e r i a l .  In the v a r i e t i e s o f L_. d o u g l a s i i ,  L_. f l o c c o s a and-L. g r a c i l i s , the 3 - 0 - r u t i n o s i d e , 7 - 0 - g l u c o s i d e s concentration  i n l e a f m a t e r i a l , but  are n e a r l y absent from the f l o w e r s .  most extreme examples are found i n L_.. d o u g l a s i i v a r s . r o s e a possess no d e t e c t a b l e  occur i n h i g h  and  n i v e a which •  amounts o f t h e s e compounds i n t h e i r p e t a l s .  the 3-0-rhamnosyl r u t i n o s i d e s appear i n h i g h e r  concentrations  whole p l a n t m a t e r i a l , a l t h o u g h the d i f f e r e n c e s are not as A comparison of T a b l e s IX and occur i n p e t a l m a t e r i a l but  not  r e t i c a l l y , the p e t a l f l a v o n o i d s  The  Conversely,  i n p e t a l s than  extreme.  X r e v e a l s t h a t o c c a s i o n a l l y compounds  i n whole p l a n t m a t e r i a l o f some OTUs. should  Theo-  always be a subset o f the whole p l a n t  f l a v o n o i d s s i n c e whole p l a n t m a t e r i a l c o n s i s t e n t l y c o n t a i n e d  flowers.  However,  t h i s s i t u a t i o n has hot been r e a l i z e d , presumably because c e r t a i n p e t a l f l a v o n o i d s are p r e s e n t  i n concentrations  too low  to detect  once the p e t a l s become  a minor component o f the t o t a l t i s s u e a n a l y z e d .  NUMERICAL TAXONOMIC RESULTSThe  data c o n t a i n e d  i n T a b l e s IX and X were a n a l y z e d  several taximetric.techniques  described  i n the p r e v i o u s  by computer u s i n g  chapter.  Figures  PE^AL FLAVONOIDS OF 18 OTUS OF LIMNANTHES  2  z  1  X X  lata  3  COMPOUND: 3-0- Rut 'J V w X Y  2  6  7  t  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  t  X  X  X  X X  X  X  X  X  X  X  X  X  X  X  X  ecu;,  d o-.'.g.  X  X  X  X  X  X  dcug.  sulph.  X  X  X  X  X  X  X x  X  X  X  X  X X  3  cai^.  r.lvea  X  X  X  X  X  X  1*  c^u.;.  rosea  x  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  10  X  1  o 13 lr-  aajcur.il flee, :  :rac.  X  X X  X t  X X  X X  X t  X X  X X  X X  bell.  X  t  t  X  X  X  t  t  X  rarish.  X  X  X  X  X  X  t  X  X  t  t  t  t  X  X  13  3-o- Rut,7- 0-G 8 9 10 11 12  3-0- GRR 3 It •5  X  X X  X X  t  t  X  X  X  X  X  X  X  X  X  X  X  t  X  p  X  3-0 -G Q R  s  T  X  X  X  X  X  X  X  X  t  t  X  X  X  X  X  X  t  t  t  t  t  t  t  t  t  t  t  X  t  X  X  t  t  •  X  t  t  t  t  X  X  X . t  J  3-0 -GG K L M X X  t  X  X  X X  t t  t  X X  -  t  t  X  X  X  t  t  t  X  X  X  t  X  X  t  X  X  X  X  ;i!!'a a l ' . a  X  X  X  X  X  X  t  X  X  t  X  X  X  X  :vac • i':ae .  t  t  X  X  X  X  X  X  t  t  t  t  1U  .;:-ao. ..-'.-ac •  X  X  X  X  X  X  t  X  X  X  X  X  t  t  X  X  X  20  r'.imila  X  X  X  X  X  X  t  t  X  X  t • t  t  X  X  t  X  X  X  X  X  X X  t. t  X  X X  t  X  ;  X t  X  c  X  t aV.-a  G  X  X  11  F  X  alba  17  I  19 20 21 22 23  X  X  X t  0  X  X X  t  X  3-O-Rut7-0-Rut 13 ll+ 15 16 17  t  t X  D  E  A  B  VII t o X a r e t h e ' r e s u l t s o f c l u s t e r a n a l y s i s by the weighted'pair based on whole p l a n t f l a v o n o i d s . of phenetic  group method  The f i g u r e s a r e dendrograms showing degrees  s i m i l a r i t y between a l l 30 OTUs based on h6 f l a v o n o i d s .  VII and V I I I show p h e n e t i c  Figures  s i m i l a r i t i e s based o n l y on compounds o c c u r r i n g  i n h i g h e r c o n c e n t r a t i o n ( t h e Xs i n T a b l e I X ) , w h i l e F i g u r e s IX and X show, a f f i n i t i e s based on f l a v o n o i d s p r e s e n t of Table IX).  The f i r s t  i n a l l concentrations  ( t h e Xs and t s  o f each p a i r o f dendrograms ( F i g u r e s V I I and IX) i s  calculated using the c o e f f i c i e n t  o f J a c c a r d which does not c o n s i d e r  matches, w h i l e t h e second o f each p a i r  negative  ( F i g u r e s V I I I and.X) i s based on  the simple matching c o e f f i c i e n t which does c o n s i d e r n e g a t i v e matches. F i g u r e s XI' t o XIV a r e dendrograms e x p r e s s i n g p h e n e t i c  similarities  between 18 OTUs based on t h e p e t a l f l a v o n o i d data c o n t a i n e d i n T a b l e X.  As  f o r t h e whole p l a n t f l a v o n o i d c l u s t e r a n a l y s e s , two o f t h e dendrograms cons i d e r a l l f l a v o n o i d occurrences s i d e r compounds present  ( F i g u r e s X I I I and XIV) , w h i l e two o n l y con-  i n r e l a t i v e l y h i g h e r c o n c e n t r a t i o n ( F i g u r e XI and X I I ) .  A l s o , two' a r e c a l c u l a t e d u s i n g t h e c o e f f i c i e n t o f J a c c a r d w h i l e two a r e based on t h e simple matching c o e f f i c i e n t The m a t r i c e s  ( F i g u r e s XI and X I I I ) ,  ( F i g u r e s X I I and X I V ) .  o f s i m i l a r i t y c o e f f i c i e n t s used t o produce t h e e i g h t dendrograms  have been attached' as Appendices LVII" t o LXIV. i n t h e same o r d e r as t h e i r c o r r e s p o n d i n g  These m a t r i c e s  a r e arranged  dendrograms.  F i g u r e s XV and XVI a r e r e p r e s e n t a t i o n s o f t h r e e - d i m e n s i o n a l  plots of  30 OTUs u s i n g P r i n c i p a l Components A n a l y s i s and Varimax F a c t o r A n a l y s i s o f 31 whole p l a n t f l a v o n o i d s . dimensional  F i g u r e s XVII and XVIII a r e r e p r e s e n t a t i o n s o f t h r e e -  p l o t s o f 18 OTUs u s i n g 36 p e t a l f l a v o n o i d s .  I n these f o u r f i g u r e s  the p o s i t i o n s o f OTUs i n space a r e r e p r e s e n t e d by t h e l o c a t i o n s o f t h e end s e c t i o n s o f cones.  These cones have been drawn, e i t h e r w i t h p o i n t s downward,  t o i n d i c a t e a p o s i t i o n above t h e p l a n e determined by t h e two h o r i z o n t a l v e c t o r s , o r w i t h p o i n t s upward t o i n d i c a t e a p o s i t i o n below t h i s p l a n e .  When t h e  FIGURE V I I Jaccard  Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by C o e f f i c i e n t : . H i g h Concentrations o f Whole P l a n t  Flavonoids  FIGURE V I I I Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by Simple Match C o e f f i c i e n t : High C o n c e n t r a t i o n s o f Whole P l a n t F l a v o n o i d s  FIGURE IX Jaccard  Ri  Weighted P a i r Group C l u s t e r i n g o f 30 OTUs by C o e f f i c i e n t : M'- C o n c e n t r a t i o n s o f Whole P l a n t F l a v o n o i d s  PJ LU  H-  01  LO  •r-  FIGURE X . Weighted P a i r Group C l u s t e r i n g o f 30 OTUs hy Simple Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s o f Whole P l a n t  Flavonoids  FIGURE XI Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by J a c c a r d C o e f f i c i e n t : High C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s  i  N  HU  H'  Ui  H03  a  ui  in  oi  F I G U R E  Simple  X I I  Weighted P a i r Group C l u s t e r i n g o f 18 OTUs byMatch C o e f f i c i e n t High C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s  r ou  K  CO  O  FIGURE X I I I Jaccard  Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by C o e f f i c i e n t : A l l Concentrations of P e t a l Flavonoids  —3 —] I  3  CD  FIGURE XIV Weighted P a i r Group C l u s t e r i n g o f 18 OTUs by Simple. Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s o f P e t a l F l a v o n o i d s  ...  • FIGURE - XV-  N  ' •  P r i n c i p a l Components A n a l y s i s o f 30 OTUs Based on Whole P l a n t  Flavonoids  FIGURE XVI . Varimax F a c t o r A n a l y s i s o f 30 OTUs Based on Whole P l a n t  Flavonoids  7  7  Co O  FIGURE XVII P r i n c i p a l Components A n a l y s i s Based on P e t a l  o f 18 OTUs  Flavonoids  FIGURE XVIII Varimax F a c t o r A n a l y s i s o f 18 OTUs Based on P e t a l  Flavonoids  - 83 p o s i t i o n o f an OTU.was obscured by t h e presence o f another i n f r o n t o f i t , t h e i r p o s i t i o n s i n t h e drawings were separated  slightly  f o r clarity.-  The  p o s i t i o n o f L_. b a k e r i , OTU 7', has been moved i n somewhat c l o s e r t o t h e other OTUs than was determined by t h e computer a n a l y s i s . of t h e t a x a would be i n c l u d e d i n t h e drawings.  T h i s was done so t h a t a l l  The a c t u a l c o o r d i n a t e s  of the  OTUs determined by computer which were used t o make t h e f o u r drawings a r e p r e s e n t e d i n Appendices LXV-LXVIII.  COMPARISONS OF OTUS BY FLAVONOIDS ' A. Occurrence T a b l e s The m a j o r i t y  o f the flavonoids discovered  i n t h e Limnanthaceae a r e  u n u s u a l , and many have not been p r e v i o u s l y r e p o r t e d . and  X, which l i s t  A p e r u s a l o f T a b l e s IX  f l a v o n o i d o c c u r r e n c e s by OTUs, r e v e a l s t h a t a l l OTUs o f  Limnanthes and F l o e r k e a possess r a r e d e r i v a t i v e s o f s y r i n g e t i n . and l a r i c y t r i n . A l s o , rhamnosyl r u t i n o s i d e s a r e found i n a l l OTUs o f Limnanthes and i n some o f the OTUs o f F l o e r k e a . teristics.'  No other p l a n t s a r e known which share t h e s e c h a r a c -  T h i s i n d i c a t e s two t h i n g s :  l ) t h e OTUs o f F l o e r k e a  c o n s t i t u t e a n a t u r a l g r o u p i n g , and.2) no a f f i n i t i e s t o other i n d i c a t e d on t h e b a s i s o f f l a v o n o i d s i m i l a r i t i e s .  and Limnanthes  families are  However, t h e f l a v o n o i d  compositions o f f a m i l i e s w i t h suspected a f f i n i t i e s t o t h e Limnanthaceae a r e p o o r l y known.  The f u t u r e d i s c o v e r y  o f Limnanthes-type  flavonoids  i n any  o f t h e s e f a m i l i e s would be s i g n i f i c a n t and might h e l p c l a r i f y r e l a t i o n s h i p s above t h e f a m i l y The larities  level.  most s t r i k i n g  f e a t u r e o f T a b l e s IX and X i s t h e o v e r a l l s i m i -  i n f l a v o n o i d c o m p o s i t i o n shown by a l l t h e t a x a i n v e s t i g a t e d .  similarities  a r e not s u r p r i s i n g i n view o f e x i s t i n g i n f o r m a t i o n ,  These  a l l o f which  i n d i c a t e s - t h a t members o f t h e Limnanthaceae a r e c l o s e l y r e l a t e d . The  r e s u l t s p r e s e n t e d i n T a b l e IX i n d i c a t e t h a t F l o e r k e a . i s  distin-  -  81i -  g u i s h e d from Limnanthes by two d i f f e r e n c e s :  T ) t h r e e o f t h e f o u r OTUs o f  F l o e r k e a c o n t a i n compound H ( p o s s i b l y an a c y l a t e d monoside o f kaempferol) , which i s not found of  i n any OTUs o f Limnanthes, and 2) t h e f l a v o n o i d p a t t e r n s  t h e f o u r OTUs o f F l o e r k e a a r e g e n e r a l l y s i m p l e r than t h o s e o f most Limnan-  thes . , However, L_. f l o c c o s a ssp. p u m i l a , OTU 21, has a p a t t e r n o f comparable simplicity..  A l s o t h e r a r e aglycone types are.not p r e s e n t as d e r i v a t i v e s i n  most g l y c o s i d e s e r i e s , o r i f p r e s e n t , occur i n t r a c e amounts. It  i s s i g n i f i c a n t t h a t OTUs o f Limnanthes a r e . s e p a r a t e d i n T a b l e s IX  and X by c e r t a i n d i f f e r e n c e s , c o r r e s p o n d i n g t o t h e s e c t i o n a l d i v i s i o n by Mason.  OTUs d e s c r i b e d as members o f t h e s e c t i o n R e f l e x a e u s u a l l y  proposed possess  more'derivatives of s y t i n g e t i n , isorhamnetin, l a r i c y t r i n and.myricetin i n certain glycosylation series.  This observation i s p a r t i c u l a r l y , t r u e f o r the  p e t a l data. OTUs b e l o n g i n g t o t h e same s p e c i e s have u s u a l l y f a l l e n i n a d j a c e n t rows i n T a b l e s IX and X, a l t h o u g h t h e r e a r e n o t a b l e e x c e p t i o n s : var.  L. d o u g l a s i i  d o u g l a s i i , OTU 1,' from Europe, f a l l s between t h e two OTUs o f L_. m a c o u n i i ; •  Ii.' g r a c i l i s v a r . p a r i s h i i , OTU l 6 , more c l o s e l y resembles  L_. a l b a v a r . v e r s i -  c o l o r and L_. f l o c c o s a than OTUs o f i t s s i b l i n g v a r i e t y ; and s u b s p e c i e s o f L_. f l o c c o s a do not l i e t o g e t h e r , but i n s t e a d form  subgroupings.  B. C l u s t e r . A n a l y s e s The dendrograms a r e c l a s s i f i c a t i o n s o f the'Limnanthaceae based on s i m i l a r i t i e s weight,  i n f l a v o n o i d composition.  purely  A l l f l a v o n o i d c h a r a c t e r s were g i v e n • e q u a l  and c l u s t e r development was not p r e j u d i c e d by p r e v i o u s taxonomic  assessments.  Computation o f t h e dendrograms v a r i e d a c c o r d i n g t o t h e way s i m i -  l a r i t i e s , were determined,  what c o n c e n t r a t i o n s o f f l a v o n o i d s were c o n s i d e r e d ,  and whether whole p l a n t . o r p e t a l m a t e r i a l was a n a l y z e d . grams r e f l e c t  these  differences.  The r e s u l t i n g  dia-  - 85.Although t h e c l a s s i f i c a t i o n s o f OTUs p r e s e n t e d i n t h e dendrograms a r e d i f f e r e n t , a l l e i g h t have c e r t a i n g r o s s f e a t u r e s i n common.  The f e a t u r e s  of t h e s e dendrograms r o u g h l y c o i n c i d e w i t h t h e r e c o g n i z e d taxonomic scheme of  the family.  I n t h r e e o f t h e f o u r dendrograms based  n o i d s , Floerkea. i s s e p a r a t e d from Limnanthes. p u m i l a , OTU 21,  i s also' d i s t i n c t  on whole p l a n t . f l a v o -  However, L_. f l o c c o s a s s p .  from t h e remainder  o f Limnanthes t a x a and  c l u s t e r s w i t h F l o e r k e a i n two o f t h e s e t h r e e dendrograms. • OTUs b e l o n g i n g t o the same s e c t i o n and.species and whole p l a n t ' a n a l y s e s .  g e n e r a l l y h a v e . c l u s t e r e d t o g e t h e r i n both  However, as was observed  petal  f o r t h e manual, a r r a n g e -  ment o f OTUs i n t h e o c c u r r e n c e t a b l e s , c e r t a i n OTUs f a l l out o f p l a c e . T a b l e s XI and X I I a r e attempts i n t h e e i g h t dendrograms^ c l u s t e r a n a l y s e s based  t o summarize t h e i n f o r m a t i o n c o n t a i n e d  T a b l e XI i s a s y n o p s i s o f t h e r e s u l t s o f t h e f o u r  on whole p l a n t f l a v o n o i d s o f 3Q OTUs, and T a b l e XII i s  a s y n o p s i s o f t h e r e s u l t s o f t h e f o u r c l u s t e r a n a l y s e s based n o i d s o f 18 OTUs. with dotted l i n e s  on p e t a l  The OTUs a n a l y z e d from d r i e d m a t e r i a l have been  flavo-  underscored  i n Table XI.  OTUs a r e grouped t o g e t h e r i n t h e l e f t hand columns o f T a b l e s XI and XIr i f t h e y c l u s t e r e d t o g e t h e r i n each o f t h e f o u r a p p r o p r i a t e c l u s t e r ses.  analy-  OTUs t h a t d i d not c l u s t e r c o n s i s t e n t l y w i t h o t h e r t a x a , but had v a r i -  able a f f i n i t i e s  dependent on t h e c o n d i t i o n s o f a n a l y s i s , a r e . l i s t e d i n t h e  r i g h t hand columns o f t h e t a b l e s .  The a f f i n i t i e s o f t h e s e " f l o a t i n g " OTUs  are i n d i c a t e d by d o t t e d l i n e s drawn t o t h e c l u s t e r s a t t h e l e f t o r t o o t h e r f l o a t i n g taxa,.where a p p r o p r i a t e . larity or  I t i s important  t o note t h a t no s e t s i m i -  c o e f f i c i e n t v a l u e was used t o s e p a r a t e f l o a t e r s from c o n s i s t e n t c l u s t e r s ,  t o s e p a r a t e t h e c l u s t e r s themselves.  Therefore, the consistent clusters  shown i n t h e s e t a b l e s a r e somewhat a r b i t r a r y , and they a r e not d i r e c t l y comparable.  • '  In b o t h T a b l e s XI and X I I t h e compositions  of consistent clusters  - 86 TABLE XI  Synopsis  o f t h e R e s u l t s o f Four C l u s t e r  Based on Whole P l a n t . F l a v o n o i d s  Taxa w i t h C o n s i s t e n t Cluster 1 2  o f 30 OTUs  Affinities  1  Taxa w i t h V a r i a b l e A f f i n i t i e s  OTU T  Analyses  OTU bakeri  8 striata 18 : : f l o c . s s p . b e l l i n g 14  grac. var. g r a c i l i s  23.  floe,  9 10  macounii macounii  1  ^  douglasii var. douglasii  —20  floe.  ~l6  grac. var. p a r i s h i i  s s p . pumila  ssp. f l o c c o s a  4 doug. v a r . r o s e a 5 doug. v a r . s u l p h . .6.. v i n c u l a n s . 2 3  8  doug. v a r . doug doug. v a r . n i v e a  15 22. 26  grac. v a r . g r a c i l i s floe. var. grandiflora floe. var. californics  11 12  alba var. alba var. montana  IT  9  27 F l o e r k e a 30 . F l o e r k e a  10.  28 F l o e r k e a .22 . F l o e r k e a  —  —  13  alba var. versicolor floe.  1  __ f  ssp b e l l i n g e r i a n a  ^2.4.  f l o c c o s a ssp. f l o c c o s a  ^2^  f l o c c o s a ssp. f l o c c o s a  — 21.  f l o c c o s a ssp. p u m i l a  1 —  The numbers o f OTUs a n a l y z e d with dotted l i n e s .  2 —  V a r i a b l e a f f i n i t i e s a r e . i n d i c a t e d w i t h broken l i n e s drawn between columns and OTUs where a p p r o p r i a t e .  from d r i e d m a t e r i a l a r e u n d e r s c o r e d  -  87 -  TABLE X I I  Synopsis  o f t h e R e s u l t s o f Four C l u s t e r  Analyses  Based on P e t a l F l a v o n o i d s o f 18 OTUs  Taxa w i t h C o n s i s t e n t A f f i n i t i e s  Taxa w i t h V a r i a b l e A f f i n i t i e s  C l u s t e r OTU  OTU  . .  11  alba var. alba  12  alba var. alba  14  grac. v a r . g r a c i l i s  20  floe  13  alba var. versicolor  15  grac. v a r . g r a c i l i s  17  -T.or.tar.a  s s p . pumila-  .  ,vl8  '  1  s. 3  doug. v a r . n i v e a  k  doug. v a r . r o s e a  v  1  \  floe,  l  ssp. b e l l i n g e r i a n a  douglasii var. douglasii  \  ^16  gracilis  — "10  macounii  var.- p a r i s h i i  2• doug. v a r . d o u g l a s i i 5  9  doug. v a r . s u l p h u r e a macounii  7 • bakeri 8  1  striata  -— V a r i a b l e a f f i n i t i e s a r e i n d i c a t e d w i t h broken l i n e s drawn between columns and OTUs where a p p r o p r i a t e .  -•88 a p p r o x i m a t e l y follow-the, r e c o g n i z e d  f a m i l y taxonomy:  Floerkea  i s distinct;  OTUs o f t h e I n f l e x a e a r e not grouped w i t h OTUs o f t h e R e f l e x a e ,  w i t h t h e ex-  c e p t i o n t h a t L. s t r i a t a and L_. f l o c c o s a ssp. b e l l i n g e r i a n a , OTU 1 8 , together species  group  i n t h e whole p l a n t f l a v o n o i d a n a l y s e s ; . and u s u a l l y OTUs o f t h e same fall  i n t o t h e same group.  But t h e r e a r e numerous exceptions  to this  OTUs o f L_. f l o c c o s a t e n d t o . group w i t h OTUs o f L . g r a c i l i s ,  l a s t observation.  rather than together,  and t h e converse i s a l s o t r u e .  Limnanthes a l b a v a r .  v e r s i c o l o r , OTU 13, groups w i t h L_. montana and L_. g r a c i l i s r a t h e r t h a n i t s sibling  varieties. There a r e e i g h t f l o a t i n g OTUs i n T a b l e XI and f o u r f l o a t i n g  Table XII.  I t i s notable  OTUs i n  t h a t L_. d o u g l a s i i v a r . d o u g l a s i i , OTU 1, and  L_. g r a c i l i s v a r . p a r i s h i i , OTU l 6 , which have v a r i a b l e a f f i n i t i e s  on t h e b a s i s  o f e i t h e r whole p l a n t o r p e t a l f l a v o n o i d s , were a l s o s i n g l e d out as anomalies in  the preceding  s e c t i o n a f t e r v i s u a l arrangement of.OTUs i n T a b l e s IX-and.X.  C. Factor. A n a l y s e s , The  arrangement o f OTUs i n F i g u r e s  XV and XVI, t h e drawings o f t h r e e -  d i m e n s i o n a l p l o t s based on whole p l a n t f l a v o n o i d s , g e n e r a l l y agrees w i t h t h e r e s u l t s o f c l u s t e r a n a l y s i s . • However, t h e f a c t o r a n a l y s e s better representation  provide  a much  o f t h e magnitude and d i r e c t i o n o f v a r i a t i o n between OTUs,  and between c l u s t e r s o f OTUs.  The s t r i k i n g f e a t u r e i n t h e s e diagrams i s t h a t  the Limnanthaceae i s u n e x p e c t e d l y d i v i d e d i n t o t h r e e d i s t i n c t p a r t s : OTUs 27-30, L. b a k e r i , OTU 7, and t h e r e m a i n i n g Limnanthes t a x a .  Floerkea,  Three sub-  groups o f t a x a can be d i s t i n g u i s h e d w i t h i n t h e main c l u s t e r o f Limnanthes OTUs. These subgroups do not f o l l o w t h e r e c o g n i z e d  taxonomy o f t h e genus, b u t gener-  a l l y c o r r e l a t e w i t h t h e - t y p e o f b r e e d i n g system; outcrossers fall  evolved  form a group a t one extreme, while.many o f t h e autogamous t a x a  i n t o t h e group a t t h e o t h e r The  t h e most h i g h l y  features  o f Figures  extreme. XV and XVI d i f f e r by t h e r o t a t i o n o f t h e a r r a y  - 89 o f OTUs i n space.  The  d i f f e r e n c e s between t h e s e f i g u r e s i s comparable t o  d i f f e r e n c e t h a t would be different  detected  i f the  a r r a y o f OTUs were viewed from  XVII and  o f p e t a l flavonoids.A g a i n , L_. b a k e r i  As  XVIII p i c t o r i a l i z e the r e s u l t s o f the- f a c t o r a n a l y s i s above, t h e two  i s sharply  separated  diagrams d i f f e r m a i n l y i n  unavailable  for this analysis).  r e m a i n i n g OTUs of.Limnanthes form a l o n g c o n t i n u o u s g r o u p i n g . s e v e r a l important  features  shows a g r e a t e r  of t h i s g r o u p i n g :  a l l t h e v a r i e t i e s o f L_. d o u g l a s i i (OTUs 1-5)  belonging  c l u s t e r near L.  L_. g r a c i l i s v a r .  by v a r i e t y p a r i s h i i , OTU The  and  14,  extreme,  OTUs;  L_. macounii- (OTUs 9 and below i t ; and  and  3)  10)  the OTUs  cluster linked  to a l e s s e r  extent,  n o h - o v e r l a p p i n g groups.  dimensions, s i n c e the  shown by t h e  t o t h e p l a n e formed by the two  section  to a  smaller  t o become apparent, i t i s n e c e s s a r y  figures i n a l l three  L_. s t r i a t a depend on the a f f i n i t i e s  show the  a f f i n i t i e s t o L_. s t r i a t a and,  For.these features  t o v i s u a l i z e ' t h e s e two  at one  l6.  a subgroup of s e c t i o n R e f l e x a e t o be  L. g r a c i l i s .  The  are  shown by t h e o t h e r  s t r i a t a , but  g r a c i l i s , OTU  However, t h e s e groups are j o i n e d by  pendicular  striata lies  drawings of f a c t o r a n a l y s e s of p e t a l f l a v o n o i d s  I n f l e x a e . and  extent,  There  t o the 'section'.Inflexae a r e a l l d i s t r i b u t e d i n a l o n g  t o L_. s t r i a t a , by  D.  l ) L.  a f f i n i t y , t o L_. b a k e r i t h a n any  form- a r e a s o n a b l y t i g h t  perspective.  from the remainder o f t h e OTUs of Lim-  nanthes ( f l o w e r m a t e r i a l o f F l o e r k e a was  2)  two  points.  Figures  and  the  links  to  dimension of v a r i a t i o n p e r -  horizontal vectors.  G l y c o s y l a t i o n Classes. Differences  more t a x o n o m i c a l l y  i n g l y c o s y l a t i o n p a t t e r n between t a x a  probably  s i g n i f i c a n t t h a n simple d i f f e r e n c e s i n a g l y c o n e types  a s i n g l e g l y c o s y l a t i o n c l a s s , p r o v i d i n g that the missing  sent  are  as d e r i v a t i v e s o f o t h e r  classes.  This  aglycones are  within  pre-  i s because the p r e s e n c e or.i.ab-  - 90 sence o f t h e s y n t h e t i c machinery n e c e s s a r y t o a t t a c h a new l o c a t i o n may r e f l e c t g r e a t e r  an a d d i t i o n a l sugar a t  d i f f e r e n c e s between t a x a t h a n does t h e  presence o r absence o f aglycone types w i t h i n a g l y c o s y l a t i o n s e r i e s . a l l OTUs o f t h e Limnanthaceae c o n t a i n  Since  t h e s i x f l a v o n o l aglycone t y p e s ,  ences i n g l y c o s y l a t i o n between t h e s e t a x a may deserve s p e c i a l  differ-  consideration.  Compounds l i s t e d i n T a b l e s IX and X have been grouped so t h a t  pre-  sence o r absence o f each g l y c o s y l a t i o n t y p e i s e a s i l y a s s e s s e d v i s u a l l y f o r each taxon.  I n t h i s • f a s h i o n OTUs a r e compared on t h e b a s i s o f 12  or t h e number o f g l y c o s y l a t i o n c l a s s e s .  characters,  Although there are d i f f e r e n c e s  OTUs a c c o r d i n g t o g l y c o s y l a t i o n , t h e s e d i f f e r e n c e s  between  a r e s u r p r i s i n g l y few,-and  t h e r e i s l i t t l e v a r i a t i o n between taxa.- I n f a c t , an attempt was made t o c l u s t e r OTUs u s i n g was u n s u c c e s s f u l ,  t h e 12 g l y c o s y l a t i o n c l a s s e s as c h a r a c t e r s .  This  attempt  due t o l a c k o f v a r i a t i o n .  C e r t a i n OTUs i n both T a b l e s IX and X a r e s e t a p a r t . o n t h e b a s i s o f p o s s e s s i n g an i n f r e q u e n t l y o c c u r r i n g  g l y c o s y l a t i o n type.  c a t e d , t h r e e o f t h e f o u r OTUs o f F l o e r k e a  fall  As p r e v i o u s l y  indi-  i n t o t h i s c a t e g o r y , s i n c e they  each possess compound H. Limnanthes s t r i a t a , L. b a k e r i , and L_. d o u g l a s i i v a r . . n i v e a a r e a l l l i n k e d by t h e common o c c u r r e n c e o f two g l y c o s y l a t i o n t y p e s , c o n s i s t i n g o f F and  G, and 19,20,-21,22 and 23.  vars. douglasii  Limnanthes s t r i a t a , L. b a k e r i , L.. d o u g l a s i i  (OTU 2, but not l ) , sulphurea- and r o s e a , and L. v i n c u l a n s  and  L. macounii .(OTU 10', but not 9) a l l • c o n t a i n . at l e a s t one o f t h e 3 , 7 - d i r u t i n o sides. The  g l y c o s y l a t i o n c l a s s c o n s i s t i n g o f t h e s i n g l e compound C i s found  o n l y i n whole p l a n t m a t e r i a l L. g r a c i l i s v a r . var.  o f t h r e e OTUs o f t h e s e c t i o n I n f l e x a e ;  p a r i s h i i , OTU 16,  a l b a , OTU 11,  but not 12.  L . / a l b a v a r . v e r s i c o l o r , OTU 13,  The o n l y o t h e r r e s t r i c t e d g l y c o s i d e  .i.e.. , and L. a l b a class,  composed o f compounds D and E , l i n k s L. m a c o u n i i , OTU 10, w i t h L. a l b a V a r .  - 91 -  a l b a , OTUs 11 and 12, L. f l o c c o s a s s p . g r a n d i f l o r a and.L_. g r a c i l i s v a r . g r a c i l i s , OTU 14. , U n f o r t u n a t e l y , most compounds b e l o n g i n g t o t h e g l y c o s y l a t i o n w i t h r e s t r i c t e d d i s t r i b u t i o n s o c c u r r e d i n .trace amounts. i n t r a c e amounts i n t r o d u c e s absence, a problem t h a t  a problem o f a c c u r a t e l y  Consistent  classes presence  d e t e r m i n i n g presence o r  i s d i r e c t l y r e l a t e d t o the i n a b i l i t y t o completely  i d e n t i f y t h e s e compounds.  Furthermore, compounds c o n s i s t e n t l y a p p e a r i n g i n  t r a c e amounts may be o v e r l o o k e d i n some t a x a , i f t h e y occur i n c o n c e n t r a t i o n s t o o low f o r d e t e c t i o n .  - 92 -  DISCUSSION  METHOD AND  VALIDITY OF USING FLAVONOIDS AS  A. F l a v o n o i d s i.  as T a x i m e t r i c  TAXONOMIC CHARACTERS  Characters  Introduction Both the a p p l i c a t i o n o f t a x i m e t r i c techniques  f l a v o n o i d s as c h a r a c t e r s a r e r e l a t i v e l y new these two  techniques  associated with t h i s process.  q u e s t i o n s , t h e f l a v o n o i d data gat ered i n t h i s  u s i n g a v a r i e t y o f methods t o a s s e s s  ii.  t o p l a n t taxonomy.  i s more r e c e n t and has l e f t u n r e s o l v e d  and p h i l o s o p h i c a l q u e s t i o n s these  and t h e u t i l i z a t i o n  of  The mating of  certain technical To h e l p r e s o l v e  i n v e s t i g a t i o n -were;; a n a l y s e d  s i m i l a r i t i e s between t a x a .  I n c l u s i o n o f N e g a t i v e Matches The  q u e s t i o n o f whether t h e mutual absence of a compound c o n t r i b u t e s  t o s i m i l a r i t y between t a x a i s one t h a t has flavonoid taximetrics. authors  not been d i s c u s s e d i n r e f e r e n c e t o  However, t h i s q u e s t i o n has been t r e a t e d by  i n r e l a t i o n t o o t h e r types  of characters.  Davis  various  and Heywood  (1963)  say t h a t mutual absence does not c o n s t i t u t e s i m i l a r i t y , w h i l e S o k a l and  (1963) say t h a t i t does i n some c a s e s .  The  negative  organisms possess an i n f i n i t e number o f m u t u a l l y  argument says t h a t a l l  absent c h a r a c t e r s , and  s i m i l a r i t i e s based on such absences are i r r e l e v a n t and r e f l e c t no relationships.  The  Sneath  p o s i t i v e argument says t h a t , as l o n g as one  that  taxonomic  of t h e  taxa  under c o n s i d e r a t i o n e x h i b i t s a p a r t i c u l a r c h a r a c t e r , t h e other t a x a , not  pos-  s e s s i n g t h i s c h a r a c t e r , a r e made more s i m i l a r by the common absence. In-at l e a s t . t w o  i n s t a n c e s , the c o n s i d e r a t i o n of n e g a t i v e matches i n  comparisons, based on f l a v o n o i d c h a r a c t e r s may  result  i n t h e misplacement  of  - 93 -  emphasis, and thus l e a d t o unwarranted taxonomic c o n c l u s i o n s .  I f the  absence  of a f l a v o n o i d compound always r e s u l t s i n t h e presence o f a n o t h e r , or t h e  con-  v e r s e i s t r u e , t h e n the, -presence or absence o f t h e s e  be  A  considered  independently  mutual o c c u r r e n c e  i n taxonomic comparisons.  of such compounds s h o u l d not be  compounds should not S i m i l a r i t i e s b a s e d on  i n c r e a s e d f u r t h e r by  the  the  absence o f t h e a l t e r n a t e compounds, s i n c e t h i s p r a c t i c e would i n t r o d u c e r e dundant i n f o r m a t i o n i n t o t h e comparison.  I n s t e a d , presence or absence o f  both compounds s h o u l d be t r e a t e d as a s i n g l e c h a r a c t e r s t a t e . t h a t two  f l a v o n o i d s might occur  It i s possible  i n t h i s l i n k e d , e i t h e r - o r f a s h i o n , i f they  p r o v i d e an i d e n t i c a l f u n c t i o n i n d i f f e r e n t p l a n t s .  each  However, whether t h i s  s i t u a t i o n a c t u a l l y e x i s t s f o r f l a v o n o i d s cannot- be determined on an a p r i o r i b a s i s , s i n c e the f u n c t i o n s o f most o f t h e s e Unnatural occurs  emphasis i s a l s o p l a c e d on mutual absence, i f a compound  i n none o f the t a x a b e i n g  Heywood, and content  compounds are unknown.  compared.  i t i s nearly axiomatic,  T h i s i s t h e o b j e c t i o n o f Davis  s i n c e t h e r e i s no taxonomic  information  i n such c h a r a c t e r s , j u s t as t h e r e i s none f o r c h a r a c t e r s t h a t  c o n s i s t e n t l y p r e s e n t w i t h i n a group o f t a x a . n o i d does occur-.in at l e a s t one  are  However, p r o v i d i n g t h a t a f l a v o -  of t h e t a x a under c o n s i d e r a t i o n , and t h a t  t a x a are o f p a r a l l e l rank and a r e b e i n g  and  compared s i m u l t a n e o u s l y ,  the  t h e mutual  absence o f t h a t f l a v o n o i d does i n d i c a t e a s i m i l a r i t y w i t h p o s s i b l e taxonomic significance. S i n c e r e s o l u t i o n o f t h e above arguments i s not p o s s i b l e without mental evidence,  t.axa;-were£clustered u s i n g b o t h J a c c a r d ' s  f i c i e n t , which does not c o e f f i c i e n t , which does.  similarity  c o n s i d e r n e g a t i v e matches, and the simple I t was  suspected  experi-  coef-  matching  t h a t the r e s u l t s o f t h e  simple  matching c o e f f i c i e n t might possess t h e g r e a t e r taxonomic s i g n i f i c a n c e f o r reasons:  l ) c e r t a i n OTUs of the Limnanthaceae such as L. b a k e r i  n e a r l y every  two  contain  f l a v o n o i d encountered,«which e l i m i n a t e s the p o s s i b i l i t y of any  of  . - h9  t h e s e compounds o c c u r r i n g on an e i t h e r - o r b a s i s ; much reduced f l a v o n o i d complements t h a t are v e r y between t h e s e t a x a are h i g h e r  and  2) c e r t a i n OTUs have  similar;  thus,  i f c a l c u l a t e d considering negative  I n t e r e s t i n g l y , the dendrogram p a i r s , d i f f e r i n g  one  dendrograms, one.from the  matches.  i n t y p e of  c o e f f i c i e n t , are q u i t e s i m i l a r i n each o f t h e f o u r cases. s e l e c t two  similarities  similarity  It i s possible to  f o u r based on whole p l a n t m a t e r i a l  from the f o u r based on p e t a l m a t e r i a l , which b e s t  agree w i t h the  taxonomic scheme o f the Limnanthaceae.  For whole p l a n t m a t e r i a l the  i s F i g u r e VII c a l c u l a t e d by the J a c c a r d  c o e f f i c i e n t , and  i s F i g u r e XII  c a l c u l a t e d w i t h the  vations  i n d i c a t e , t h a t the r e s u l t s o b t a i n e d  roughly  comparable, and  iii.  Flavonoid  using  i n t h i s c a s e , the c h o i c e  accepted choice  f o r p e t a l the  simple matching c o e f f i c i e n t .  and  choice  These obser-  either coefficient o f which t o use  are  is arbitrary.  Concentration  In comparisons o f t a x a based on f l a v o n o i d c o m p o s i t i o n s , i t i s n a t u r a l t o make.comparisons u s i n g as many compounds as p o s s i b l e t o c o n t r i b u t e maximum i n f o r m a t i o n information,  input t o the p r o c e s s .  the  However, at the r i s k of l o s i n g some  perhaps some compounds are b e s t  excluded from c o n s i d e r a t i o n i f  t h e y occur i n amounts which make d e t e c t i o n and/or i d e n t i f i c a t i o n uncertain.,-•the' r e a s o n b e i n g  t h a t the p a r t i a l l o s s o f i n f o r m a t i o n  i n c l u s i o n of misleading Various  the  information.  flavono'ids o c c u r r i n g , i n the Limnanthaceae c o n s i s t e n t l y were  found i n t r a c e amounts. by the use  or i n c o r r e c t  i s preferable to  A l t h o u g h d e t e c t i o n o f t h e s e compounds was  o f a s e n s i t i v e spray r e a g e n t , the d e t e r m i n a t i o n  v i s i b l e by t h i s spray i s d i f f i c u l t .  enhanced  of compounds made  Because of t h i s d i f f i c u l t y and  because  o f the p o s s i b i l i t y t h a t 'compounds c o n s i s t e n t l y o c c u r r i n g  i n t r a c e amounts  might be  analyzed  ways:  o v e r l o o k e d i n some t a x a , the f l a v o n o i d data was  l ) considering  a l l o c c u r r e n c e s i n a taxon r e g a r d l e s s  of  in  two  concentration,  - 95 and,-2) c o n s i d e r i n g o n l y the compounds o c c u r r i n g i n s u f f i c i e n t t o he v i s i b l e under UV l i g h t b e f o r e The  concentration  spraying.  r e s u l t s o f c l u s t e r a n a l y s i s are v a r i a b l e depending on which con-  c e n t r a t i o n of f l a v o n o i d s was  used.  Dendrograms based on f l a v o n o i d s p r e s e n t  i n h i g h e r c o n c e n t r a t i o n more c l o s e l y approximate the accepted the f a m i l y .  taxonomy o f  P r o v i d i n g t h a t t h i s taxonomy i s b a s i c a l l y c o r r e c t , t h i s  result  i n d i c a t e s t h a t the data based on h i g h e r c o n c e n t r a t i o n s have g r e a t e r taxonomic s i g n i f i c a n c e than do the data based on a l l c o n c e n t r a t i o n s . no reason t o suspect less significant;  However, t h e r e i s .  t h a t compounds o c c u r r i n g i n t r a c e amounts are i n h e r e n t l y  T h e r e f o r e , the r e s u l t s themselves p r o b a b l y  t h i s phenomenon, presumably because i n c o r r e c t or incomplete i n t r o d u c e d i n t o the a n a l y s i s by i n c l u d i n g the trace-amount  account  for  information  was  data.  S i n c e t h e f l a v o n o i d d a t a based on h i g h e r c o n c e n t r a t i o n s have g r e a t e r taxonomic s i g n i f i c a n c e t o the Limnanthaceae, the f a c t o r a n a l y s e s were performed o n l y on t h e s e data.  The  amount of time i n v o l v e d i n the computation o f f a c t o r  a n a l y s e s a l s o i n f l u e n c e d the d e c i s i o n t o omit f u r t h e r a n a l y s e s i n c l u d i n g trace-amount  data.  i v . Lack o f V a r i a t i o n among CharactersEach c l u s t e r a n a l y s i s by the weighted p a i r group method was 46 f l a v o n o i d c h a r a c t e r s . v a r i a b l e c h a r a c t e r s was  based  on  However, i n each o f t h e s e a n a l y s e s , the number of l e s s than 46 depending upon t h e c o n d i t i o n s o f a n a l y s i s .  T h e r e f o r e , a c e r t a i n number o f nonvarying  c h a r a c t e r s ( e i t h e r p o s i t i v e or nega-  t i v e ) were i n c l u d e d i n each a n a l y s i s which i n c r e a s e d s i m i l a r i t i e s between t a x a . Although  the r e s u l t i n g i n c r e a s e s are u n i f o r m and the c l u s t e r composition  not v a r y , t h i s procedure i s not c o m p l e t e l y of view.  The  a c c e p t a b l e from a s y s t e m a t i c  j u s t i f i c a t i o n f o r t h i s procedure i s t h a t t h e disadvantage  l i z e d by u n i f o r m l y i n c r e a s i n g s i m i l a r i t y c o e f f i c i e n t s was  outweighed by  does point reathe  - 96 convenience p r o v i d e d t o t h e computer  operator.  I n v a r i a n t c h a r a c t e r s cannot be used i n f a c t o r a n a l y s e s .  Therefore,  t h e whole p l a n t f a c t o r a n a l y s e s were performed on 31 v a r i a b l e f l a v o n o i d s , and the p e t a l f a c t o r a n a l y s e s  on 36 v a r i a b l e compounds.  B. Use o f Dry v e r s u s F r e s h P l a n t M a t e r i a l Whenever p o s s i b l e OTUs o f t h e Limnanthaceae were a n a l y z e d u s i n g f r e s h material.  U n f o r t u n a t e l y , such m a t e r i a l was not a v a i l a b l e f o r a l l t a x a , and  the remainder were n e c e s s a r i l y a n a l y z e d  from d r i e d m a t e r i a l .  i n t r o d u c e d some problems o f i n t e r p r e t i n g comparative r e s u l t s . v o l v e d breakdown i n d r i e d m a t e r i a l .  The f i r s t i n -  Fortunately, the f l a v o n o l glycosides pre-  sent i n Limnanthes and F l o e r k e a a r e c o m p a r a t i v e l y e f f e c t s were p r o b a b l y  T h i s procedure  s t a b l e t y p e s , so breakdown  negligible.  A more s e r i o u s problem a r i s i n g from t h e l a c k o f f r e s h m a t e r i a l was the i n a b i l i t y t o complete the s t u d i e s o f p e t a l f l a v o n o i d s and UV f l o w e r photography.  However, t h e 18 OTUs grown from seed a r e a good r e p r e s e n t a t i o n o f t h e  s p e c i e s and s e c t i o n s o f Limnanthes. been d e s i r a b l e t o b a l a n c e  F r e s h m a t e r i a l o f Floerkea. would have  the i n v e s t i g a t i o n s .  The t h i r d problem r e l a t e d t o l a c k o f f r e s h m a t e r i a l was t h e p o s s i b i l i t y t h a t i n d i v i d u a l s o f a g i v e n t a x o n may v a r y i n t h e i r f l a v o n o i d The  compositions.  f l a v o n o i d p a t t e r n s l i s t e d f o r OTUs grown f r e s h r e p r e s e n t t h e compounds  present  i n many p l a n t s , whereas t h e f l a v o n o i d s determined from d r i e d m a t e r i a l  represent the composition  o f o n l y one o r a few a v a i l a b l e p l a n t s .  I f indi-  v i d u a l v a r i a t i o n i s e x t e n s i v e , r e s u l t s based on s i n g l e p l a n t s c o u l d be t a x o nomically  misleading.  I t i s not p o s s i b l e oh t h e . b a s i s o f t h e a v a i l a b l e d a t a t o c o m p l e t e l y e v a l u a t e t h e e f f e c t s o f comparing i n d i v i d u a l s w i t h groups i n some i n s t a n c e s . . However, no discrepancies''-;' were noted  f o r t h e OTUs a n a l y z e d  from a s i n g l e , o r  few,  dried plants.  Apparently  the e f f e c t s are m i n i m a l , s i n c e t h e s e OTUs  c l u s t e r c o n s i s t e n t l y i n roughly  the same f r e q u e n c i e s  (Table XI) and a c c o r d i n g t o t h e a c c e p t e d  C. P e t a l versus  Whole P l a n t  as the o t h e r OTUs  taxonomic p a t t e r n .  Results  In an e a r l i e r chapter  the  suggestion  was  made t h a t f l a v o n o i d s  are  d i r e c t l y r e s p o n s i b l e f o r b o t h the n e c t a r guides o f L_. d o u g l a s i i . made v i s i b l e under UV  light  and the u n i f o r m l y  of o t h e r  species.  high absorption  T h i s assumption i m p l i e s t h a t f l o w e r  useful function i n outcrossing plants; linators. w i l l be other  c h a r a c t e r i s t i c s o f the  Therefore,  f l a v o n o i d s serve  i-e_. , a t t r a c t i n g and  e v e n t u a l l y , l i n k a g e groups d e t e r m i n i n g  o u t c r o s s i n g t r a i t s may  be  c e r t a i n t r a i t s associated with  formed.  a  guiding p o l -  i n o u t c r o s s i n g t a x a o f Limnanthes t h e s e f l o w e r  s e l e c t e d f o r , and  flowers  pigments  these  and  I f such l i n k a g e groups' are formed,  outcrossing w i l l l i k e l y persist  f o r a time  a f t e r the o u t c r o s s i n g h a b i t has been l o s t . The w i t h UV  presence i n the  absorption  flowers  o f autogamous Limnanthes t a x a o f pigments  c h a r a c t e r i s t i c s comparable t o those observed i n the  c r o s s i n g t a x a i n d i c a t e s t h a t the f l o w e r This . conservation  f l a v o n o i d s are conserved i n the genus.  o f the p e t a l f l a v o n o i d s has  a p p l i e s d i r e c t l y t o the r e s u l t s of t h i s  taxonomic s i g n i f i c a n c e which  study.  I f the p e t a l f l a v o n o i d s have been s e l e c t i v e l y c o n s e r v e d by it  out-  evolution,  i s p r o b a b l e t h a t t h e s e compounds w i l l be b e t t e r i n d i c a t o r s of taxonomic  r e l a t i o n s h i p s i n Limnanthes t h a n w i l l the whole p l a n t f l a v o n o i d s u n l e s s compounds a l s o have been conserved f o r some r e a s o n . sested that flavonoids help discourage t a t i o n unpalatable. t i o n i n the context C l u s t e r and  herbivores  However, t h e r e i s l i t t l e  Stebbins  and.insects  these  (1974) has  by making vege-  evidence t o support  this  sugges-  of Limnanthes. f a c t o r analyses  sug-  based on the p e t a l data more c l o s e l y  - 98 agree w i t h many p o i n t s o f t h e e x i s t i n g taxonomy o f Limnanthes t h a n do t h e a n a l y s e s based on t h e whole p l a n t f l a v o n o i d s .  Providing that the existing  c l a s s i f i c a t i o n i s b a s i c a l l y sound, t h i s o b s e r v a t i o n supports t h e h y p o t h e s i s t h a t t h e p e t a l f l a v o n o i d s a r e indeed b e t t e r taxonomic i n d i c a t o r s i n t h i s genus.  D. F a c t o r A n a l y s i s versus - C o n v e n t i o n a l C l u s t e r A n a l y s i s Because i t should be p o s s i b l e t o produce an a c c u r a t e and n a t u r a l taxonomic scheme f o r a group o f p l a n t s - f u s i n g d i f f e r e n t methods and d i f f e r e n t types o f s i g n i f i c a n t  information, the r e c u r r i n g features o f the taximetric  a n a l y s e s o f t h e Limnanthaceae possess  special significance.  A f f i n i t i e s that  a r e c o n s i s t e n t l y demonstrated between t a x a by t h e s e methods l i k e l y natural  reflect  relationships. The  c o n s i s t e n t c l u s t e r s o f OTUs (Tables XI and X I I ) produced by t h e  weighted p a i r group method demonstrate c l o s e s i m i l a r i t i e s i n f l a v o n o i d compositions.  These s i m i l a r i t i e s undoubtedly r e f l e c t  OTUs i n t h e s e c l u s t e r s . ters.  I n t h e whole-plant  n a t u r a l a f f i n i t i e s between  However, many OTUs do not f a l l  into consistent clus-  f l a v o n o i d a n a l y s e s , L_. b a k e r i never c l u s t e r s  other t a x a and has been t r e a t e d as a s e p a r a t e group.  Other n o n c l u s t e r i n g  OTUs f l o a t between c o n s i s t e n t c l u s t e r s depending on t h e method o f data sis.  with  analy-  What i s t h e s i g n i f i c a n c e o f t h i s f l o a t i n g phenomenon? There a r e a t l e a s t two p o s s i b l e e x p l a n a t i o n s f o r t h e f l o a t i n g pheno-  menon:  l ) t h e evidence used t o c l u s t e r t a x a i s i n v a l i d o r i n s i g n i f i c a n t ;  2) t h e f l a v o n o i d compositions  o f t h e f l o a t i n g OTUs a r e i n t e r m e d i a t e , and  t h e s e OTUs serve as l i n k s between c l u s t e r s .  Thus, as t h e c o n d i t i o n s o f a n a l y -  s i s change, OTUs w i t h i n t e r m e d i a t e compositions  show v a r i a b l e a f f i n i t i e s .  t h e b a s i s o f t h e dendrograms a l o n e , i t i s i m p o s s i b l e t o determine which o f these explanations i s c o r r e c t .  On  - 99 Because t h e f a c t o r a n a l y s e s dimensional  o r d i n a t e t a x a by a r r a n g i n g them i n t h r e e -  space ( i n t h i s study) at d i s t a n c e s t h a t a r e p r o p o r t i o n a l t o  v a r i a t i o n between them, t h e s e techniques  make a t p o s s i b l e t o e s t i m a t e  imposed, branched r e l a t i o n s h i p s between OTUs.  Such m u l t i - b r a n c h e d  super-  configura-  t i o n s can be e x p l a i n e d i f groups have r e p e a t e d l y d i v e r g e d from an a n c e s t r a l line.  T h i s p a t t e r n o f e v o l u t i o n has  thaceae,  and  a p p a r e n t l y taken p l a c e i n t h e Limnan-  i t i s f o r t h i s reason t h a t some t a x a were c o n s i s t e n t f l o a t e r s .  They are indeed l i n k s between d i v e r g e n t Although  groups.  (1963) r e p o r t e d  S o k a l and Sneath  t h a t t h e r e i s g e n e r a l l y good  agreement between the r e s u l t s o f f a c t o r a n a l y s i s and  c l u s t e r i n g by the weighted  p a i r group method, a comparison o f t h e r e s u l t s o b t a i n e d by t h e s e two i n the p r e s e n t  i n v e s t i g a t i o n i n d i c a t e s t h a t t h i s c o n s i s t e n c y depends on  the v a r i a t i o n between t a x a . i s expressed. r e l a t e d d i r e c t i o n o f v a r i a t i o n expressed t e r i n g cannot adequately  i n one  I f t h e r e i s more than one  how  uncor-  between t a x a , a one-dimensional  convey t h e r e l a t i o n s h i p s between them.  s i n c e most o f the •••variation p r e s e n t for  techniques  clus-  Therefore,  i n the Limnanthaceae cannot be  accounted  dimension, r e l a t i o n s h i p s cannot be p r o p e r l y a s s e s s e d w i t h i n t h i s  group u n l e s s a m u l t i - d i m e n s i o n a l  c l u s t e r i n g technique  i s used.  E. F l a v o n o i d D i f f e r e n c e s between D u p l i c a t e Taxa In many i n s t a n c e s two  o r more OTUs were a n a l y z e d t h a t were i d e n t i f i e d  as t h e same s p e c i e s , s u b s p e c i e s  or v a r i e t y .  By doing  so, i t was  possible to  e s t i m a t e the approximate f l a v o n o i d v a r i a t i o n between p o p u l a t i o n s or taxonomic e n t i t i e s o f Limnanthes and The same was  two  recognized  Floerkea.  OTUs o f L. macounii group t o g e t h e r as would be p r e d i c t e d .  t r u e f o r t h e two  OTUs of  g. laTbajgvar.  alba.  OTUs o f L. f l o c c o s a genera-  a l l y grouped t o g e t h e r , i f somewhat d i f f u s e l y , i n the f a c t o r a n a l y s e s but i n the c l u s t e r analyses.  Although  The  not  t h e s i m i l a r i t i e s were not as g r e a t between-  - 100 -  t h e f o u r OTUs o f F. p r o s e r p i n a c o i d e s , t h e s e t a x a a l s o grouped t o g e t h e r i n t h r e e out o f f o u r c l u s t e r a n a l y s e s  and t h e f a c t o r a n a l y s e s .  OTUs o f F l o e r k e a were chosen t o r e p r e s e n t d i v e r s e geographic  Since these  four  elements o f t h e  s p e c i e s , elements t h a t have p r o b a b l y been i s o l a t e d f o r some t i m e , a c e r t a i n degree o f v a r i a t i o n was expected drift  or d i f f e r e n t  selection  a t t r i b u t a b l e t o divergence  caused by g e n e t i c  pressures.  D i f f e r e n c e s i n f l a v o n o i d p a t t e r n s o f t h e d u p l i c a t e OTUs o f L. douglasii  and L. g r a c i l i s a r e b o t h s i g n i f i c a n t .  i n two r e s p e c t s .  Limnanthes d o u g l a s i i i s unusual  F i r s t , OTU 1 o n l y shows a f f i n i t i e s t o it's r e p l i c a t e , OTU 2,  i n the p e t a l f a c t o r analyses. plant f a c t o r analyses  I n t h e whole-plant  c l u s t e r analyses  and whole-  i t o f t e n c l u s t e r s w i t h more o r l e s s d i s t a n t l y  autogamous d e r i v a t i v e s .  This s i n g l e observation provides  that\) t h e p e t a l f l a v o n o i d s a r e - c o n s e r v e d  further  related  evidence  t o a g r e a t e r degree than t h e whole  plant flavonoids. I t i s p o s s i b l e t o p a r t i a l l y e x p l a i n t h e s t r a n g e a f f i n i t i e s o f OTU 1. These p l a n t s may be descendants o f p l a n t s taken t o Europe by David Douglas i n t h e e a r l y l830's. and have s i n c e been m a i n t a i n e d T h e r e f o r e , over t h e i n t e r v a l o f about 140 y e a r s a t i o n s ) , the f l a v o n o i d composition  as a h o r t i c u l t u r a l v a r i e t y . ( p r o b a b l y somewhat fewer gener-  o f t h i s OTU has d i f f e r e n t i a t e d from o t h e r  members o f t h i s v a r i e t y t h a t n a t u r a l l y occur i n t h e western U n i t e d S t a t e s , even though t h e two remain.very s i m i l a r m o r p h o l o g i c a l l y . may have r e s u l t e d from t h e p r o c e s s e s drift.  This  differentiation  o f i n b r e e d i n g , g e n e t i c s e l e c t i o n , or  Because o f t h e magnitude o f t h e d i f f e r e n c e s between OTU 1 and t h e  n a t u r a l l y o c c u r r i n g OTUs o f L_. d o u g l a s i i , which c l u s t e r c o n s i s t e n t l y w i t h each o t h e r , i t i s p r o b a b l e  t h a t i n b r e e d i n g o r some unknown form o f a r t i f i c i a l  s e l e c t i o n has p l a y e d a g r e a t e r r o l e than random d r i f t menon.  i n c a u s i n g t h i s pheno-  Because OTU 1 c l u s t e r s w i t h autogamous p l a n t s i n t h e whole p l a n t  t a x i m e t r i c a n a l y s e s , i t i s p o s s i b l e t h a t i n b r e e d i n g may l e a d t o d i r e c t e d  - 101 changes, i n whole p l a n t f l a v o n o i d c o m p o s i t i o n .  Why t h e r e s h o u l d he such a  d i r e c t i o n i n f l a v o n o i d change a s s o c i a t e d w i t h autogamy o r i n b r e e d i n g i s not apparent. The  r e s u l t s o f the c l u s t e r analyses  and f a c t o r a n a l y s e s  indicate that  t h e r e i s g r e a t v a r i a t i o n i n t h e f l a v o n o i d p a t t e r n s w i t h i n both L. g r a c i l i s and L. d o u g l a s i i .  The observed  v a r i a t i o n i n both o f t h e s e s p e c i e s i s much  g r e a t e r than t h a t observed w i t h i n other s p e c i e s o f Limnanthes, w i t h t h e p o s s i ble  e x c e p t i o n o f L. f l o c c o s a .  compositions  Providing that the divergence  i n flavonoid  between segments o f a s p e c i e s ( o r any r e l a t e d group o f p l a n t s )  r e s u l t s mainly  from t h e random process, o f g e n e t i c d r i f t  s e l e c t i o n p r e s s u r e s , t h e extent o f t h e d i v e r g e n c e  o r from comparable  between elements o f t h e  s p e c i e s d o u g l a s i i , g r a c i l i s and f l o c c o s a has i m p l i c a t i o n s w i t h r e g a r d t o both t h e r e l a t i v e ages o f t h e s e s p e c i e s , and t o ranks o f t h e components o f t h e s e species.  TAXONOMY' OF THE LIMNANTHACEAE A.  Introduction The  c u r r e n t l y accepted  taxonomic scheme o f t h e Limnanthaceae i s a  s l i g h t l y expanded form o f t h e system proposed by Mason i n 1952 (Table I ) . There a r e t h r e e primary guished  f e a t u r e s o f t h i s scheme,  l ) Floerkea i s d i s t i n -  from Limnanthes by numbers o f f l o r a l p a r t s and by having hypogeal •  cotyledons.  2) The genus Limnanthes i s s p l i t  i n t o two s e c t i o n s , R e f l e x a e and  I n f l e x a e , based on p e t a l p o s i t i o n a f t e r p o l l i n a t i o n . t a x a , i n c l u d i n g some f o r m e r l y r e c o g n i z e d as d i s t i n c t morphological  3) V a r i o u s Limnanthes species with c l e a r c u t  and g e o g r a p h i c a l d i f f e r e n c e s , a r e reduced  s p e c i f i c rank l a r g e l y on t h e b a s i s o f i n t e r ' f e r t i l l t y  t o v a r i e t a l o r sub-  in artificial  crosses.  P l a n t taxonomists have been r e l u c t a n t . t o r e v i s e c l a s s i f i c a t i o n a r r i v e d a t by b i o s y s t e m a t i c methods when c o n f l i c t i n g  evidence  schemes  i s produced by  - 102 phenetic analyses.  The  type o f evidence  based, whether m o r p h o l o g i c a l  on which the p h e n e t i c a n a l y s i s i s  or c h e m i c a l , does not seem t o m a t t e r ;  the  i n g i s t h a t b i o s y s t e m a t i c s t u d i e s more c l e a r l y i n d i c a t e p h y l o g e n e t i c  feel-  rela-  tionships. Mason's treatment  o f Limnanthes, based on m o r p h o l o g i c a l  c a l comparisons, and the r e s u l t s o f i n t e r b r e e d i n g t r i a l s ,  and  i s g e n e r a l l y con-  s i d e r e d t o be an a c c u r a t e p h y l o g e n e t i c arrangement o f the genus. not a l l o f the a v a i l a b l e evidence of evidence  cytologi-  supports t h i s c o n c l u s i o n .  However,  Of the t h r e e  c o n s i d e r e d by Mason, h i s c o n c l u s i o n s depend most h e a v i l y on  r e s u l t s of a r t i f i c i a l  crosses.  h i s arrangement o f t h e genus.  type  the  H i s o t h e r r e s u l t s do not n e c e s s a r i l y support S i n c e t h e r e was  no v a r i a t i o n i n karyotype mor-  phology o f the genus, Mason's r e v i s i o n o f Limnanthes was  not  cytological results.  s t u d i e s o f t h e genus  Other m o r p h o l o g i c a l treatments  c o n f l i c t w i t h Mason's c o n c l u s i o n s . 1910;  Jepson,  1936)  and  P r e v i o u s authors  (Howell,  differences.  net  distinct  In a d d i t i o n , the p h e n e t i c  o f Ornduff and C r o v e l l o (1968), based on m o r p h o l o g i c a l support  I89T; Rydberg, •  c o n s i d e r e d s e v e r a l o f Mason's v a r i e t i e s t o be  s p e c i e s based on m o r p h o l o g i c a l  does not  inf-luenced by h i s  analysi  characters, generally  a l l t h e d e t a i l s o f Mason's taxonomic scheme.  Therefore,  the  e f f e c t has been t o a t t a c h s p e c i a l s i g n i f i c a n c e t o t h e r e s u l t s o f h y b r i d i -  zation t r i a l s  i n Limnanthes.  Three a d d i t i o n a l o b s e r v a t i o n s suggest  t h a t t h e phyl'ogenetic  cance o f e x p e r i m e n t a l  crossing t r i a l s  l ) Ornduff  r e p o r t e d t h a t g e n e t i c i s o l a t i n g mechanisms a p p a r e n t l y  (197T) has  i n Limnanthes may  signifi-  need r e e v a l u a t i o n .  have a r i s e n between Limnanthes taxa,' not on t h e b a s i s o f d i s t a n c e o f r e l a t i o n s h i p , but r a t h e r on a p u r e l y s p a t i a l b a s i s ; d i s t i n c t two  t h e more g e o g r a p h i c a l l y  t a x a a r e , the more l i k e l y t h a t no g e n e t i c b a r r i e r t o c r o s s i n g  w i l l have developed  between them.  2)  UV  f l o w e r photography demonstrates t h a t  an i s o l a t i n g mechanism o f an e c o l o g i c a l n a t u r e may  e x i s t between v a r i e t i e s  - 103  o f L. d o u g l a s i i , and existence  that a r t i f i c i a l  o f such a mechanism.  w i t h c e r t a i n key  f e a t u r e s o f the  -  i n t e r f e r t i l i t y does not p r e c l u d e  3) The  f l a v o n o i d data c o n s i s t e n t l y  t h e r e has  l ) S i n c e the o r i g i n a l d e s c r i p t i o n of the  "been disagreement r e g a r d i n g  i n the f a m i l y a r e . g r e a t o f two not  genera.  new  Limnanthaceae,  enough or d i s c o n t i n u o u s  enough t o warrant  recognition  2) The p h e n e t i c a n a l y s i s o f Ornduff and C r o v e l l o (1968) does  do not  3) The  c y t o l o g i c a l characters  d i s t i n g u i s h i t from Limnanthes (Ornduff,  f l a v o n o i d d a t a does not The  Floerkea  whether the m o r p h o l o g i c a l d i f f e r e n c e s  support the maintenance of s e p a r a t e genera.  of Floerkea  disagrees  existing classification.  There i s evidence i n d i c a t i n g t h a t the taxonomic s t a t u s o f requires reevaluation.  phenetic  evidence t h a t may  support the  separation of Floerkea  1971)and  4)' The  Limnanthes.  s i m i l a r i t i e s between OTUs based on f l a v o n o i d s be a p p l i e d t o the s y s t e m a t i c s  o f the  provide  Limnanthaceae.  Because the f l a v o n o i d evidence stands i n d e p e n d e n t l y o f t h e r e s u l t s o f taxonomic t r e a t m e n t s , t h i s  evidence p r o v i d e s  c a t i o n of the family.  a key  n i f i c a n c e o f the warrant any w i t h i n the  new  But  the b a s i s f o r a new  q u e s t i o n must be  f l a v o n o i d evidence:  other  classifi-  answered r e g a r d i n g  the  sig-  do the r e s u l t s o f the f l a v o n o i d a n a l y s i s  conelus'ions"."? or r e q u i r e a r e i n t e r p r e t a t i o n of r e l a t i o n s h i p s  family?  The  d e m o n s t r a t i o n of f l a v o n o i d a f f i n i t i e s does j u s t i f y the r e v i s i o n  o f a c l a s s i f i c a t i o n p r o v i d i n g t h a t two  conditions  a b l e evidence supports such a r e v i s i o n , and equivocal.  the  Both o f t h e s e c o n d i t i o n s  2)  are met:  l ) other  avail-  the f l a v o n o i d r e s u l t s are  are f u l f i l l e d  i n the  context  of  un-  the  Limnanthaceae. A comparison o f the a l l other  sources has  f l a v o n o i d evidence w i t h the r e l e v a n t  l e d to a r e c o n s t r u c t i o n of a h y p o t h e t i c a l  h i s t o r y o f the Limnanthaceae and I n the  a new  evidence from evolutionary  proposed c l a s s i f i c a t i o n of the  f o l l o w i n g s e c t i o n s , the .proposed h i s t o r y and  family.  classification will  be  - 104  presented  first,  these p r o p o s a l s . and  -  f o l l o w e d by a d i s c u s s i o n o f t h e evidence  f o r and  F i n a l l y , t h e need f o r taxonomic r e v i s i o n w i l l be  a s y n o p s i s o f the f a m i l y w i l l be p r e s e n t e d  against discussed,  i n c l u d i n g the necessary  nomen-  c l a t u r a l changes.  B. H y p o t h e t i c a l E v o l u t i o n a r y H i s t o r y of t h e Limnanthaceae '  The  information presented  i n t h i s t h e s i s , taken t o g e t h e r w i t h  a v a i l a b l e i n f o r m a t i o n r e g a r d i n g d i s t r i b u t i o n s and  e c o l o g i c a l requirements  members o f the Limnanthaceae, has many i m p l i c a t i o n s c o n c e r n i n g history.  the family's  However, t h i s h i s t o r y i s p u r e l y specu-  since i t s evaluation requires nonexistent  fossil  In the e a r l y T e r t i a r y , Limnanthes p r o b a b l y o f i t s modern descendents.  I t was  a predominantly  information.  c l o s e l y resembled some outcrossing spring  annual,  w i t h a h a p l o i d number of f i v e , and had medium s i z e d w h i t e f l o w e r s and leaves with s l i g h t l y d i v i d e d l e a f l e t s .  streamside  compound  S i n c e the g r a s s l a n d s i t e s o c c u p i e d  most modern c o u n t e r p a r t s had not y e t appeared, Limnanthes was  probably  herb o f the woodlands, o c c u r r i n g at moderate e l e v a t i o n s , and  ducing a determinate was  of  These i m p l i c a t i o n s have made i t p o s s i b l e t o r e c o n s t r u c t a h y p o t h e t i -  c a l h i s t o r y which i s p r e s e n t e d below. lative  the  probably  number o f r e l a t i v e l y l a r g e n u t l e t s .  reasonably  by  a pro-  I t s distribution'.-'  e x t e n s i v e i n western North America, and perhaps i t  extended a c r o s s the c o n t i n e n t .  With the u p l i f t  the p o p u l a t i o n o f Limnanthes p r o b a b l y was  split  o f t h e c o a s t a l mountain in  ranges,  two.  I t i s l i k e l y t h a t o c c a s i o n a l t r e n d s t o autogamy were ongoing i n e a r l y elements of Limnanthes, as they are today.  One  such element, g e n e t i c a l l y  i s o l a t e d by autogamy, and b e t t e r adapted t o t h e c o o l i n g t r e n d o f t h e was  Tertiary,  p r o b a b l y a b l e t o p e r s i s t f u r t h e r n o r t h than the parent p o p u l a t i o n .  autogamous^ Limnanthes r a p i d l y e v o l v e d reduced such as c l e i s t o g a m y .  I t s d i s s e m i n a t i o n was  This  f l o w e r s w i t h accompanying t r a i t s  probably  somewhat improved,  and  - 105 i t may  have become s l i g h t l y weedy. Three modern members o f t h e Limnanthaceae most c l o s e l y resemble  t h r e e p o s t u l a t e d a n c e s t r a l elements. by F. p r o s e r p i n a c o i d e s which s t i l l of  i t s ancestor.  have p r o b a b l y the ancestor  The  the  autogamous d e r i v a t i v e i s r e p r e s e n t e d  o c c u p i e s the deciduous woodland h a b i t a t  Since the s e l e c t i o n pressures  stayed f a i r l y constant, t h i s  associated with t h i s habitat  species probably  s p l i t o f f m i l l i o n s o f y e a r s ago.  closely  resembles  The secondmajor element, i s o -  l a t e d t o the east o f the c o a s t range, i s r e p r e s e n t e d today by_L_. s t r i a t a , a s p e c i e s which may  still  occupy moderate e l e v a t i o n streamside  western element.'.persisting from the s p l i t  sites.  i s r e p r e s e n t e d today o n l y  The  by  L_. b a k e r i , a narrow endemic of C a l i f o r n i a a s s o c i a t e d w i t h moderately h i g h elevation vernal pools. With the g r a d u a l c o o l i n g t h a t o c c u r r e d d u r i n g t h e Miocene Epoch, each of  the t h r e e a n c e s t r a l elements o f Limnanthes was  pushed southward.  In i t s  southward m i g r a t i o n , the p o p u l a t i o n r e p r e s e n t e d today by L_. s t r i a t a was  divided  by t h e c e n t r a l v a l l e y o f C a l i f o r n i a , one p a r t proceeding, down t h e west s i d e of  the newly u p l i f t e d S i e r r a Nevadas, and t h e o t h e r down t h e east s i d e of  the coast range.  Elements of t h i s m i g r a t i o n a p p a r e n t l y p e r s i s t today.  Although  the main range o f L. s t r i a t a : , i s east of the c e n t r a l v a l l e y i n the S i e r r a Nevadas, Ornduff  ( O r n d u f f . C r o v e l l o , 1968;  1971)  has r e c e n t l y d i s c o v e r e d a v a r i -  ant western element o f t h i s s p e c i e s i n t h e n o r t h e r n C a l i f o r n i a c o a s t a l moun- . t a i n s , which he at f i r s t (from T r i n i t y It  thought was  a new  s p e c i e s and r e f e r r e d t o as  County).  i s l i k e l y t h a t Limnanthes and.Floerkea  p e r s i s t e d v i r t u a l l y unchanged  as woodland p l a n t s through the T e r t i a r y u n t i l t h e c o m p a r a t i v e l y ment o f g r a s s l a n d s t h e s e new  "trinity"  i n the P l e i o c e n e Epoch.  recent  However, w i t h the a v a i l a b i l i t y  s i t e s , i t i s c o n c e i v a b l e t h a t elements o f L_. s t r i a t a invaded  r a p i d l y evolved adaptations  develop-  a p p r o p r i a t e t o the new  life  style.  and  In c o a s t a l  of  - io6 a r e a s , t h e g r a s s l a n d i n v a d e r s were p r o b a b l y  the forerunners o f the Reflexae,  c h a r a c t e r i z e d by t h e L." d o u g l a s i i t y p e , w h i l e t h e e a s t e r n i n v a d e r s were probab l y the ancestors The  o f t h e I n f l e x a e , c h a r a c t e r i z e d by t h e L. g r a c i l i s  o l d e s t western elements occupying  t h e range west o f t h e c o a s t  type.  (mountains  were a p p a r e n t l y unable t o compete s u c c e s s f u l l y i n . t h e new s i t e s and have i a r g e l y d i s a p p e a r e d w i t h t h e e x c e p t i o n o f L_. b a k e r i . In the comparatively  modern P l e i s t o c e n e , t h r e e r e p e a t i n g  processes  may have taken p l a c e which account, f o r both t h e d i s t r i b u t i o n s and d i v e r s i t y o f extant forms o f Limnanthes.  l ) A l l p o p u l a t i o n s , i n c l u d i n g F l o e r k e a , prob-  •gably have advanced and r e t r e a t e d n o r t h and south w i t h v e g e t a t i o n a l changes accompanying p e r i o d s o f warming and c o o l i n g a s s o c i a t e d w i t h During t h e c o o l p e r i o d s , continuous groups.  glaciation.  p o p u l a t i o n s may have been s p l i t  i n t o many  2) G e o g r a p h i c a l l y i s o l a t e d p o p u l a t i o n s may have become g e n e t i c a l l y  d i s t i n c t due t o r a p i d a d a p t a t i o n s t h e new g r a s s l a n d h a b i t a t s .  t o t h e d i f f e r i n g niches, made a v a i l a b l e by  In t h i s fashion populations of the sections  R e f l e x a e and I n f l e x a e may have d i v e r g e d on an e c o l o g i c a l b a s i s and a c c o r d i n g t o a l t i t u d i n a l and l a t i t u d i n a l  zonation.  i n v a r i o u s new s p e c i e s , s u b s p e c i e s  T h i s process  or v a r i e t i e s .  c o u l d have r e s u l t e d  3) The r e c u r r i n g t r e n d t o  autogamy i n Limnanthes, coupled w i t h t h e g e n e r a l l y b e t t e r t o l e r a n c e o f t h e s e d e r i v a t i v e s o f s l i g h t l y c o o l e r c o n d i t i o n s , probably day p e r s i s t e n c e o f these p l a n t s i n s i t e s m a r g i n a l o f Limnanthes.  has l e d t o t h e p r e s e n t  f o r most o u t c r o s s i n g t a x a  I f s o , t h e s e p l a n t s can be i n t e r p r e t e d as r e l i c s o f e a r l i e r  northward m i g r a t i o n s . • I t i s l i k e l y that rapidly, occurring adaptations  t o t h e modern g r a s s -  l a n d h a b i t a t s i n the •'Reflexae. hare l e d t o t h e c r e a t i o n o f L. v i n c u l a n s and the f o u r v a r i e t i e s o f L_. d o u g l a s i i .  Similar d i f f e r e n t i a t i o n i n the Inflexae  may have l e d t o t h e c r e a t i o n o f L_. montana,. the v a r i e t i e s o f L_. g r a c i l i s , t h e v a r i e t i e s o f L. a l b a and t h e v a r i e t i e s o f L. f l o c c o s a .  The p e r s i s t e n t auto-  -  107 -  gamous r e l i c s o f t h e northward m i g r a t i o n s  are.L. macounii, probably  from an a n c e s t o r resembling- L. douglasii,:'arid t h e s u b s p e c i e s d e r i v e d from an a n c e s t r a l form o f t h i s  o f L_. f l o c c o s a ,  species.  F i g u r e XIX, t h e h y p o t h e t i c a l p h y l o g e n e t i c summarizes t h e h i s t o r y o f t h e f a m i l y .  t r e e o f t h e Limnanthaceae,  No attempt has been made to, i n d i c a t e  which o f the: •species, L. b a k e r i o r F. p r o s e r p i n a c o i d e s , f i r s t the main l i n e o f Limnanthes. roughly  The amount o f d i v e r g e n c e  comparable f o r b o t h , and i t i s p r o b a b l e  approximately  t h e same time.  derived  diverged  from  from t h i s main l i n e i s  t h a t t h e s p l i t happened a t  S e c t i o n a l d i v s i o n s have.been made i n F i g u r e XIX,  and t h e s e w i l l be d i s c u s s e d below.  C. Assumptions t h a t Led t o t h e H y p o t h e t i c a l H i s t o r y Processes  o f e v o l u t i o n i n Limnanthes have made i t d i f f i c u l t t o under-  s t a n d r e l a t i o n s h i p s i n t h i s group. served such as k a r y o t y p e ,  C e r t a i n f e a t u r e s have been s t r o n g l y con-  f r u i t t y p e , and b a s i c f l o r a l  structure.  However,  o t h e r f e a t u r e s may have changed r a p i d l y i n c l u d i n g number o f f l o r a l p a r t s , f l o w e r and p l a n t s i z e , b r e e d i n g result  e i t h e r from a d a p t a t i o n s  crossing habit.  system, and l e a f l e t morphology.  These changes  t o new n i c h e s , or from t h e l o s s o f t h e out-  Because o f e v o l u t i o n a r y convergence, t a x a p o s s e s s i n g  common  t r a i t s o f t h e s o r t e a s i l y m o d i f i e d by e v o l u t i o n may not n e c e s s a r i l y have natural  affinities. The  use o f f l a v o n o i d c h a r a c t e r s f i l l s  the taxonomy o f t h e Limnanthaceae. s i o n s r e s u l t from d i f f e r e n t information.  C o n f l i c t s i n previous  i n t e r p r e t a t i o n s o f incomplete  The f l a v o n o i d data p r e s e n t e d  u s e f u l because i t b a s i c a l l y supports  taxonomic  conclu-  or insubstantial  i n t h i s t h e s i s i s taxonomically  t h e taxonomy o f t h e group, as f a r as i t  i s p r e s e n t l y known, and s i m u l t a n e o u s l y , which the' present  an obvious gap i n e v a l u a t i n g  provides  a d d i t i o n a l i n f o r m a t i o n by  system can r e e v a l u a t e d and r e f i n e d .  FIGURE XIX H y p o t h e t i c a l Phylogenetic. Tree o f t h e Limnanthaceae  Section  Line  Taxon  Section  bakeri  Bakera  striata.  Limnanthes  macounii doug. d o u g l a s i i doug.  sulphurea  Reflexae  vinculans doug. rosea doug. n i v e a Hypothetical Ancestor  grac. alba  parishii alba  alba v e r s i c o l o r montana grac.  Inflexae  gracilis  floe. bellingeriana floc. floccosa j—floe. grandiflora  1—floe, c a l i f o r n i c a floe.  pumila  proserpinacoides  Floerkea  -  109 -  The taxonomic i m p l i c a t i o n s o f t h e f l a v o n o i d d a t a a r e a l l r e l a t e d t o amount o f d i v e r g e n c e i n f l a v o n o i d p a t t e r n s between t a x a . must be made r e g a r d i n g t h i s d i v e r g e n c e .  The f i r s t  S e v e r a l assumptions  i s that divergence i n  f l a v o n o i d p a t t e r n from t h a t o f a common a n c e s t o r i s a random p r o c e s s t h a t a r i s e s from g e n e t i c d r i f t ;  or-, i f t h e changes a r e caused by s e l e c t i o n p r e s -  s u r e s , t h e s e p r e s s u r e s w i l l be much t h e same f o r a l l t a x a .  This  assumption  p r o b a b l y cannot be made, f o r h o r t i c u l t u r a l v a r i e t i e s which a r e t h e products of  artificial The  selection.  second  assumption  i s t h a t t h e r a t e o f d i v e r g e n c e o f f l a v o n o i d com-  p o s i t i o n i s approximately uniform f o r c l o s e l y r e l a t e d o u t c r o s s i n g annuals, such as most t a x a o f Limnanthes. that the r a t e o f divergence w i l l heterozygosity. all  A f t e r c o n v e r s i o n t o autogamy, i t i s p r o b a b l e slow down w i t h t h e accompanying l o s s o f  However, s u b s t a n t i a l amounts o f v a r i a t i o n were d e t e c t e d i n  f o u r c o m p l e t e l y autogamous members o f t h e f a m i l y .  This v a r i a t i o n  indi-  c a t e s t h a t d i v e r g e n c e does o c c u r i n autogamous p l a n t s , and t h a t t h e convers i o n t o autogamy does not p r e c l u d e t h e r e t e n t i o n o f some v a r i a b i l i t y .  However,  whether v a r i a t i o n c o n t i n u e s t o i n c r e a s e i n d e f i n i t e l y a t t h e same r a t e i n t h e s e autogamous t a x a i s i m p o s s i b l e t o determine The t h i r d assumption  i s c l o s e l y r e l a t e d t o t h e second.  f l a v o n o i d c o m p o s i t i o n proceeds of  the plant.  on t h e b a s i s o f t h e a v a i l a b l e data. Divergence i n  a t a slower r a t e i n t h e p e t a l s than i n t h e r e s t  T h i s c o n s e r v a t i o n i n t h e p e t a l s r e s u l t s d i r e c t l y from t h e  a b i l i t y o f t h e s e compounds t o a t t r a c t . p o l l i n a t o r s . Once'the above t h r e e assumptions a r e a c c e p t e d , f o u r c o n c l u s i o n s can be drawn which suggest  t r e n d s i n t h e e v o l u t i o n o f Limnanthes.  The f i r s t i s  t h a t t h e amount o f d i v e r g e n c e between two t a x a i s r o u g h l y p r o p o r t i o n a l t o t h e l e n g t h o f time t h a t has passed ancestor.  s i n c e t h e s e t a x a were d e r i v e d from a common  On t h i s b a s i s , F_. p r o s e r p i n a c o i d e s and L. b a k e r i each s e p a r a t e d  from Limnanthes l o n g b e f o r e t h e c r e a t i o n o f any o f t h e remaining t a x a , w i t h  - 110  the p o s s i b l e e x c e p t i o n The  of L_.  striata.  second c o n c l u s i o n  a t i o n are more a n c i e n t  -  i s t h a t t a x a w i t h much i n t e r n a l f l a v o n o i d v a r i -  than those with l i t t l e v a r i a t i o n .  For  instance,  the  amount o f v a r i a t i o n i n L. g r a c i l i s i n d i c a t e s t h a t t h i s taxon i s r e l a t i v e l y more a n c i e n t present  than L_. a l b a .  Likewise,  i n members o f the s e c t i o n s I n f l e x a e and  s t r i a t a and mately the  bakeri) same  The  ancient  Reflexae- ( e x c l u d i n g the  species  approxi-  age.  t h i r d conclusion  i s t h a t d i v e r g e n c e i n autogamous d e r i v a t i v e s of i n outcrossers  o r i g i n o f the autogamous p l a n t s .  populations  comparable t o t a l v a r i a t i o n  i n d i c a t e s t h a t t h e s e s u p r a s p e c i f i c t a x a are of  the same magnitude as t h a t present  taxa  the roughly  of Floerkea,  p r o b a b l y i n d i c a t e s a more  For t h i s r e a s o n the v a r i a t i o n i n the  an amount n e a r l y as g r e a t  as t h a t observed f o r a l l  o f Limnanthes ( e x c l u d i n g Ly b a k e r i ) , i n d i c a t e s t h a t F.  has been a d i s t i n c t taxon s i n c e w e l l b e f o r e  the  proserpinacoides  e v o l u t i o n of most other  family  members. The  fourth conclusion  are more t a x o n o m i c a l l y and  implications of p e t a l  separation  o f the  section Inflexae  s e p a r a t e the two Floerkea  and  flavonoids^  This conclusion  from the R e f l e x a e  L_. s t r i a t a and L_. b a k e r i ) by the f l a v o n o i d analyses  whole p l a n t m a t e r i a l .  flavonoids  s i g n i f i c a n t t h a n t h o s e of the whole p l a n t  thus suggest a more n a t u r a l c l a s s i f i c a t i o n system.  supported by' the out  i s t h a t the  sections recognized  not  do  not  the s i n g l e Limnanthes s p e c i e s b a k e r i from the main f a m i l y day  t h i s theory.  line.  d i s t r i b u t i o n s of a l l  members o f the Limnanthaceae support t h e h y p o t h e t i c a l h i s t o r y o f the and were u s e f u l i n f o r m u l a t i n g  of  by Mason, t h e y c l e a r l y d i s t i n g u i s h  L i k e the f l a v o n o i d e v i d e n c e , the p r e s e n t  may  (with-  o f p e t a l s but  A l t h o u g h the whole p l a n t f l a v o n o i d a n a l y s e s  is  group  It i s true that d i s t r i b u t i o n s  change r a p i d l y and thus suggest m i s l e a d i n g  e v e r , the c l o s e agreement between f l a v o n o i d and  evolutionary  conclusions.  distributional  evidence  How-  - I l l-  suggests t h a t present  day  l o c a t i o n s are s i g n i f i c a n t  clues u s e f u l i n t r a c i n g  the f a m i l y h i s t o r y . Great emphasis has  been p l a c e d  change c r e a t e d by r e l o c a t i o n i n new Limnanthes can be r e g a r d e d as r e c e n t versely, certain.extant  grassland  niches.  evolutionary  Most modern t a x a  e x i s t today p r o b a b l y l i t t l e  changed f o r m i l l i o n s  and.changes are l a r g e l y the r e s u l t o f g e n e t i c  Several pieces  drift.  l ) the whole-plant f l a v o -  n o i d f a c t o r a n a l y s i s shows t h i s s p e c i e s . c e n t r a l l y l o c a t e d between L_. baker 1;  any  s e c t i o n I n f l e x a e and  while simultaneously  other t a x o n ;  piriacoides and  3) L.  showing a g r e a t e r  L_. montana,, which may  represents  As  a f f i n i t y t o L_. b a k e r i  discussed  above the  covery o f a f a r removed, m o r p h o l o g i c a l l y  The  continued  a comparatively  change,  ancient  species  The  v a r i e d d i s j u n c t of t h i s species  dissug-  origin.  Comparison o f the Proposed C l a s s i f i c a t i o n t o t h e . E x i s t i n g  i.  Above the' S p e c i e s  Classification  Level'  c l a s s i f i c a t i o n p r e s e n t e d i n F i g u r e XIX c l u s t e r analyses'of  v i s e d to provide  k)  than  pro-  presence o f a  D.  f a c t o r ; and  than  proser-  streamside h a b i t a t  i n t h i s t y p e o f s i t e might t e n d t o minimize e v o l u t i o n a r y  The  section  occupy a s t r e a m s i d e h a b i t a t r a t h e r  the a n c e s t r a l c o n d i t i o n .  gests t h a t i t indeed has  forming  a reduced v e r s i o n of the  s t r i a t a i s the o n l y f a m i l y member, except F_.  the newer g r a s s l a n d . s i t e s . bably  Floerkea  2) the p e t a l f l a v o n o i d f a c t o r a n a l y s i s shows L_. s t r i a t a  a common l i n k between the Reflexae,  rela-  o f evidence suggest t h a t L_. s t r i a t a p r o b a b l y most  c l o s e l y resembles the a n c e s t r a l Limnanthes t y p e :  and  Con-  stream-  Here, the f o r c e s of n a t u r a l s e l e c t i o n have p r o b a b l y stayed  t i v e l y constant,  of  d e r i v a t i v e s moulded by t h i s f o r c e .  f a m i l y members have r e t a i n e d t h e more a n c i e n t  s i d e or woodland h a b i t a t and of years.  on the d r i v i n g f o r c e f o r  the'best  f l a v o n o i d data.  f i t utilizing  i s l a r g e l y the product  However, t h i s scheme was  of de-  a l l the a v a i l a b l e e v i d e n c e , i n c l u d i n g  .  t h a t p r e s e n t e d by Mason, Ornduff i n f o r m a t i o n was t h e new  - 112  -  and A r r o y o .  Since a l l of the a v a i l a b l e  used, and a l l f a m i l y members were c o n s i d e r e d  .  simultaneously,  c l a s s i f i c a t i o n p r o b a b l y more c l o s e l y approximates a c t u a l r e l a t i o n -  s h i p s i n the Limnanthaceae than d i d the o l d scheme. The  h y p o t h e t i c a l h i s t o r y and  c l a s s i f i c a t i o n o f the Limnanthaceae  depart from the accepted taxonomy of t h e f a m i l y i n c e r t a i n key p o i n t s . ever, both finement  systems share many f e a t u r e s , and t h e new  o f the o l d .  information^ taxa.with are'of p a r a l l e l The  scheme i s a c t u a l l y a r e -  The ,major d i f f e r e n c e s a r i s e because o f d i f f e r i n g  p r e t a t i o n s 0% group ranks.  T h i s p r e s e n t s a problem, s i n c e i n l i g h t o f  comparable a f f i n i t i e s  How-  internew  s h o u l d be a d j u s t e d so t h a t they  rank.  proposed c l a s s i f i c a t i o n o f t h e Limnanthaceae has been drawn.with  five sectional divisions.  An a l t e r n a t e , but l e s s d e s i r a b l e , c h o i c e would have  been d i v i s i o n o f t h e f a m i l y i n t o t h r e e s e c t i o n s or t h r e e genera c o n s i s t i n g of F l o e r k e a , Limnanthes b a k e r i , and the remaining  t a x a of Limnanthes.  From t h e f l a v o n o i d evidence t h e r e i s l i t t l e  doubt t h a t b o t h L. b a k e r i  and F. p r o s e r p i n a c o i d e s have d i v e r g e d from t h e remaining comparatively conclusion;  great extent.  f a m i l y members t o a  This p a r a l l e l divergence leads t o a  necessary  i f F l o e r k e a i s r e c o g n i z e d as a genus a p a r t from Limnanthes, then  L. b a k e r i s h o u l d a l s o be  so r e c o g n i z e d .  However, t h e m o r p h o l o g i c a l d i f f e r e n c e s  between t h e t h r e e groups a r e r e l a t i v e l y t r i v i a l , and L_. b a k e r i has been demons t r a t e d to, be i n t e r f e r t i l e w i t h v a r i o u s other members of Limnanthes. i n t e r f e r t i l i t y makes a t h r e e g e n e r i c s p l i t t h e f a m i l y c o n s i s t s of o n l y one Although two  genera,  o f the f a m i l y u n t e n a b l e .  This Therefore,  genus.  the accepted c l a s s i f i c a t i o n of the Limnanthaceae r e c o g n i z e s  i t i s u n l i k e l y t h a t many taxonomists  o f F l o e r k e a i n t h e same genus w i t h Limnanthes. t h a t t h e y a r e not s e p a r a t e genera.  w i l l object t o the A l l t h e hard.data  inclusion  suggests  The m o r p h o l o g i c a l d i f f e r e n c e s , i n c l u d i n g  - 113 -  l o s s o f f l o w e r p a r t s , a r e e a s i l y accounted  f o r by t h e c o n v e r s i o n t o autogamy,  p a r t i c u l a r l y s i n c e p a r a l l e l changes have taken p l a c e i n L_. m a c o u n i i . one  good m o r p h o l o g i c a l t r a i t  s e p a r a t e s . F l o e r k e a from Limnanthes;  hypogeous, w h i l e Limnanthes i s epigeous. suggested  that t h i s t r a i t  Ornduff  Only  Floerkea i s -  and C r o v e l l o (1968) have  a l o n e does not c o n s t i t u t e s u f f i c i e n t grounds f o r  t h e maintenance o f two genera,  and I agree.  Although  extensive interbreeding  t r i a l s have been made between a l l Limnanthes t a x a , a p p a r e n t l y no attempts have.been made t o c r o s s F l o e r k e a w i t h Limnanthes.  I f such a cross- produced  t i l e h y b r i d s , t h i s would f u r t h e r support t h e j o i n i n g o f t h e s e two t a x a a s i n g l e genus.  U n f o r t u n a t e l y , t h e t i n y cleistogamous  p i n a c o i d e s would make a r t i f i c i a l c r o s s p o l l i n a t i o n s S i n c e t h e f a m i l y Limnanthaceae has p r o b a b l y t i n c t p h y l e t i c l i n e s of approximately  extremely  difficult.  e v o l v e d from t h r e e  Reflexae.  pending  dis-  equal rank, t h e . d e c i s i o n t o r e c o g n i z e and t r a d i t i o n .  s e c t i o n s , I n f l e x a e and R e f l e x a e , r e c o g n i z e d by Mason a r e most  n a t u r a l groupings  into  f l o w e r s o f F_. p r o s e r -  f i v e s e c t i o n s i n s t e a d ofj t h r e e was based on convenience two  fer-  The  probably  t h e removal o f L_. s t r i a t a and L_. b a k e r i from t h e  S i n c e Mason's two groups have been w e l l a c c e p t e d , and c o n t a i n a l l  but t h r e e o f t h e s p e c i e s i n t h e f a m i l y , i t i s i n a p p r o p r i a t e t o merge t h e s e two  sections:  a merger t h a t would be n e c e s s a r y ,  i f t h e f a m i l y were t o be  d i v i d e d i n t o t h r e e groups o f p a r a l l e l e v o l u t i o n a r y rank. Having made t h e d e c i s i o n t o p r e s e r v e Mason's s e c t i o n a l d i v i s i o n t o t h e maximum extent a l l o w e d by t h e new evidence,  i t i s necessary  to erect a  s e c t i o n i n a d d i t i o n t o t h e I n f l e x a e and R e f l e x a e c o n s i s t i n g o n l y o f L. s t r i a t a . T h i s s e c t i o n i s named Limnanthes, s i n c e i t i s p r o b a b l e t h a t L_. s t r i a t a most c l o s e l y resembles t h e a n c e s t r a l f a m i l y t y p e .  This d i v i s i o n into three sections  keeps t h e R e f l e x a e , I n f l e x a e and Limnanthes p a r a l l e l and i s a n e c e s s a r y since these three probably  step  e v o l v e d from a common a n c e s t o r some time a f t e r t h e  s e p a r a t i o n o f s e c t i o n s F l o e r k e a ( c o n t a i n i n g F. p r o s e r p i n a c o i d e s ) and Bakera  - 114  (containing t h e y are  L_. b a k e r i ) .  not  S i n c e the  o f an be  family  However, i n view o f the  into five  sections  i s the most  old,if;two-sectional  of evidence:  l ) p e t a l p o s i t i o n a f t e r p o l l i n a t i o n , and  i n t e r p r e t e d t o support the  p r o b a b l y c o n t r o l l e d by  one  This  or a few  be  The  following  alleles.  itself  It i n the  a t r i v i a l character,  selected  v o r e s or o t h e r a n i m a l s .  ..  the  the  a v a i l a b l e evidence i n d i c a t e s t h a t  i t probathere  i n f l e x e d flower type i n  the  p e t a l i n f l e x i o n , which tends t o r e t a i n n u t l e t s  f o r and  m a i n t a i n e d i n the  invading  might a i d d i s s e m i n a t i o n of n u t l e t s by  section Inflexae  also evolved i n f l e x i n g sepals,  s c i s s i o n l a y e r below the  grassland  species  herbi-  I f t h i s h y p o t h e s i s i s c o r r e c t , L_. f l o c c o s a . i s t h e most  e v o l v e d member o f t h e  c i e s has  ancestor with  i s a h y p o t h e s i s a t t e m p t i n g t o e x p l a i n why  o f Limnanthes s i n c e t h i s t r a i t  highly  well  Limnanthes t a x a .  i s possible that  f l o w e r , was  equally  It i s possible that i n f l e x i o n  a s e l e c t i o n p r e s s u r e f o r maintenance o f the  grassland-inhabiting  presence  Whether such a m u t a t i o n might have.happened.more t h a n  once i s h a r d t o determine, but not.  the  evidence can  o f p e t a l s might have r e s u l t e d from a s i n g l e m u t a t i o n o f an r e f l e x e d p e t a l type.  2)  two  proposed f i v e - s e c t i o n d i v i s i o n .  P e t a l p o s i t i o n a f t e r p o l l i n a t i o n i s by  may  desirable  d i v i s i o n o f Limnanthes i s supported, by  i n t e r s e c t i o n a l b a r r i e r to hybridization.  b l y has  circum-  available alternatives.  The pieces  f i v e groups d i v e r g e d at d i f f e r e n t t i m e s ,  c o m p l e t e l y p a r a l l e l i n rank.  s t a n c e s , a d i v i s i o n o f the among the  -  i n t h i s r e g a r d s i n c e t h i s spe-  and.most s u b s p e c i e s possess an  c a l y x which tends t o cause d i s p e r s a l of the  ab-  flower  as a u n i t . Dispersal  of n u t l e t s by  c l i n g i n g t o f u r , may  overcome one  comparatively large n u t l e t s w i t h woodland p l a n t s  a n i m a l s , e i t h e r by  or by the  flowers  problem o f d i s s e m i n a t i o n i n Limnanthes.  of t h i s genus are  (Salisbury,  ingestion  1942;  of a s i z e n o r m a l l y  Stebbins,  1974).  . Although  The  associated apparently  - 115 nece s s a r y  -  f o r c o m p e t i t i o n i n shady s i t e s , g r a s s l a n d . i n v a d e r s would he put a t  a disadvantage  by t h i s t r a i t , and thus  Besides  compensating, mechanisms-./might;, e v o l v e .  i n f l e x i o n of:;flower p a r t s , n a t u r a l s e l e c t i o n p r o b a b l y  has  f a v o r e d i n c r e a s e d p r o d u c t i o n o f seed by g r a s s l a n d i n h a b i t a n t s t o p r o v i d e p o t e n t i a l f o r r a p i d c o l o n i z a t i o n o f t h e s e newer s i t e s . i s s i g n i f i c a n t t h a t H i g g i n s et . a l . were more determinate  the  In t h i s regard, i t  ( l 9 T l ) found t h a t L_. s t r i a t a and L_. b a k e r i  i n t h e i r f l o w e r i n g than o t h e r Limnanthes t a x a .  This  shared c h a r a c t e r i m p l i e s t h a t b o t h o f t h e s e p l a n t s are more p r i m i t i v e i n t h i s r e s p e c t t h a n o t h e r members o f the genus. How^well do t h e r e s u l t s o f a r t i f i c i a l c r o s s e s support  d i v i s i o n of the  Limnanthaceae i n t o t h e f i v e s e c t i o n s , F l o e r k e a , . Bakera, Limnanthes, and.Inflexae?  Reflexae  ' Because of b a r r i e r s t o c r o s s i n g , the I n f l e x a e i s d i s t i n c t .  S i n c e no i n t e r b r e e d i n g t r i a l s have been conducted no c o n c l u s i o n s can be drawn.regarding  u s i n g F l o e r k e a as  this section.  R e f l e x a e and Limnanthes are p a r t i a l l y i ' n t e r f e r t i l e . c r o s s e s between L_. m a c o u n i i ,  The  a parent,  s e c t i o n s Bakera,  However, the s u c c e s s f u l  L_. v i n c u l a n s and the v a r i e t i e s of L.  douglasii  g e n e r a l l y produce more f e r t i l e hybids than t h o s e between t h e s e t a x a and L. s t r i ata. ( O r n d u f f ,  1971).  A l s o , h y b r i d s between L. b a k e r i and the p r e v i o u s  taxa  showed the' lowest f e r t i l i t y . On the b a s i s o f f l a v o n o i d evidence  a s t r o n g case can be made.for •  t r e a t i n g the' s e c t i o n s Limnanthes and Bakera as groups d i s t i n c t In a d d i t i o n , other evidence  does not c o n t r a d i c t t h i s s e p a r a t i o n .  d i t i o n a l p i e c e o f evidence, p r e s e n t e d such a s e p a r a t i o n .  earlier in this thesis, also  Limnanthes macounii  One  Reflexae.  ad-  supports  and t h e v a r i e t i e s o f L_. d o u g l a s i i have  each e v o l v e d U V - v i s i b l e f l o r a l p a t t e r n i n g . t a x a t o g e t h e r , j u s t ,as  from t h e  This character holds these  i t ' s e p a r a t e s L. b a k e r i and L — s t r i a t a from t h i s  five group.  - 116 ii.  -  S p e c i e s L e v e l and Below Three o f the f i v e newly proposed  s e c t i o n s , Bakera, Limnanthes  and  F l o e r k e a ,' are monotypic , c o n t a i n i n g the s p e c i e s L. b a k e r i , L. s t r i a t a  and  r  F. p r o s e r p i n a c o i d e s , r e s p e c t i v e l y . a l l y been r e g a r d e d as a d i s t i n c t have been, or w i l l now  Each o f t h e s e t h r e e t a x a has  species.  tradition-  C e r t a i n elements o f doubt, e i t h e r  be expressed c o n c e r n i n g t h e a c c e p t e d s t a t u s and  o f the remaining Limnanthes t a x a c o m p r i s i n g t h e s e c t i o n s I n f l e x a e and  rank Reflexae.  I n t h e i n t e r v e n i n g y e a r s s i n c e Mason p u b l i s h e d h i s scheme, one  of  his- c o n c l u s i o n s t h a t has been q u e s t i o n e d i n v o l v e s . r e l a t i o n s h i p s between t h e t h r e e s p e c i e s L_. montana, L_. a l b a and L_. g r a c i l i s .  Mason h y p o t h e s i z e d t h a t  t h e t a x a , L_. g r a c i l i s v a r s . g r a c i l i s and p a r i s h i i , and L_. montana, once were p a r t o f a continuous  p o p u l a t i o n o f a s i n g l e s p e c i e s which became d i s c o n t i n u -  ous and d i v e r g e d i n t o t h e t h r e e d i s t i n c t t a x a . cific  s t a t u s s i n c e i t was  not i n t e r f e r t i l e w i t h the o t h e r two  r e c o g n i z e d as v a r i e t i e s o f L. g r a c i l i s . t h a t t h i s was L_. g r a c i l i s vello  Mason accorded L_. montana spe-  an i n c o s i s t e n t treatment  Gentry and M i l l e r  t a x a which he  (1965) thought  and.suggested t h a t t h e v a r i e t i e s  s h o u l d a l s o be d e s i g n a t e d as s e p a r a t e s p e c i e s .  Ornduff and  of Cro-  (1968) c o n c l u d e d t h a t - t h e t h r e e t a x a s h o u l d a l l be r e c o g n i z e d as e i t h e r  v a r i e t i e s or s p e c i e s , s i n c e e i t h e r treatment h y p o t h e s i s which they c o n s i d e r e d c o r r e c t . d u f f and C r o v e l l o support a d i f f e r e n t  would be c o n s i s t e n t w i t h Mason's  I n t e r e s t i n g l y , the r e s u l t s o f Orn-  combination:  t h a t L. a l b a v a r . v e r s i -  c o l o r , L_. - g r a c i l i s v a r . p a r i s h i i and L. montana, a r e a l l v a r i e t i e s o f one i e s , and t h a t L_. a l b a v a r . a l b a and L. g r a c i l i s v a r . g r a c i l i s t o t h e rank o f s p e c i e s .  s h o u l d be  spe-  raised  However, t h e y chose not t o f o l l o w t h e s e r e s u l t s which  they i n t e r p r e t e d t o be t a x o n o m i c a l l y  insignificant.  A f t e r c o n s i d e r a t i o n of t h e - e v i d e n c e p r e s e n t e d by t h e .above a u t h o r s ,  and  a n a l y s i s . o f t h e f i v e t a x a , t h e b e s t s o l u t i o n i s p r o b a b l y t o r e c o g n i z e each of t h e two  v a r i e t i e s o f L_. g r a c i l i s , t h e two  v a r i e t i e s o f L. a l b a and L. montana  -  at t h e same rank. h i g h e r rank.than  At any  -  r a t e , L. montana s h o u l d notObe m a i n t a i n e d  at a  the o t h e r f o u r t a x a , s i n c e the f l a v o n o i d evidence i n d i c a t e s  a very recent divergence The  H 7  o f t h i s taxon  a v a i l a b l e d a t a support  from t h e v a r i e t y v e r s i c o l o r . .  the h y p o t h e s i s  t h a t the p u t a t i v e  ancestor  o f L_. montana. and the v a r i e t i e s g r a c i l i s , p a r i s h i - i , - a l b a and, v e r s i c o l o r p i e d a continuous  range east of the c o a s t a l mountains from southern  f o r n i a t o c e n t r a l Oregon.  S i n c e elements o f L_. g r a c i l i s have t h e  occu-  Calimost..diverse  f l a v o n o i d p a t t e r n s and  share a f f i n i t i e s w i t h L_. s t r i a t a and. the s e c t i o n Re-  f l e x a e , i t i s probable  that the p u t a t i v e ancestor of the f i v e taxa  L_. g r a c i l i s .  At some c o m p a r a t i v e l y  p o p u l a t i o n was  split  r e c e n t time t h i s continuous  into three parts.  V a r i e t y g r a c i l i s was  resembled  ancestral  d e r i v e d from t h e  n o r t h e r n p o p u l a t i o n and v a r i e t y p a r i s h i i from t h e s o u t h e r n p o p u l a t i o n .  But  the c e n t r a l p o p u l a t i o n r a p i d l y d i v e r g e d i n a more r a d i c a l f a s h i o n , c r e a t i n g t h r e e d i s t i n c t t a x a , L. montana and.the v a r i e t i e s a l b a and v e r s i c o l o r , lowing t h e i r s u c c e s s f u l i n v a s i o n s i n t o d i f f e r i n g h a b i t a t s , . T h i s suggests  hypothesis  t h a t the f i v e t a x a s h o u l d be d i v i d e d i n t o t h r e e s p e c i e s and  varieties;  fol-  three  i_. e_. , t h e v a r i e t i e s g r a c i l i s , p a r i s h i i and a l b a .become s p e c i e s ,  and t h e h y p o t h e t i c a l new  s p e c i e s o f JJ. a l b a c o n s i s t s o f t h e t h r e e v a r i e t i e s  a l b a , v e r s i c o l o r , and montana. the morphological  However, t h i s i s an i m p r a c t i c a l s o l u t i o n , s i n c e  d i f f e r e n c e s d i s t i n g u i s h i n g t h e v a r i e t i e s would be of a g r e a t e r  magnitude t h a n those s e p a r a t i n g , the species'. WJhen a l l t h e f a c t o r s a r e c o n s i d e r e d , t h e r e i s l i t t l e but  confusion  t o be gained by r e c o g n i z i n g any- o f t h e above f i v e t a x a . at d i f f e r e n t ranks). By t h i s c r i t e r i o n , t h e f i v e - m i g h t be c o n s i d e r e d as v a r i e t i e s o f one g r a p h i c a l l y d i v e r s e , polymorphic s p e c i e s . e c o l o g i c a l , g e o g r a p h i c a l , and.morphological  geo-  However, i n view o f the pronounejed d i f f e r e n c e s between t h e s e  taxa,  and t h e l a c k o f n a t u r a l h y b r i d i z a t i o n even where b a r r i e r s t o c r o s s i n g a r e . i n complete, treatment  as f i v e v a r i e t i e s  i s .unsatisfactory.  T h e r e f o r e , t h e most  -  r e a s o n a b l e way L_. a l b a and  -  118  t o t r e a t t h i s group i s t o e l e v a t e  L_. g r a c i l i s t o s p e c i f i c s t a t u s  ating five distinct  each o f the v a r i e t i e s o f  a l o n g w i t h L. montana, thus  cre-  species.  Past taxonomic treatments o f the L_. f l o c c o s a group have drawn conf l i c t i n g conclusions.  Mason ( 1 9 5 2 ) r e c o g n i z e d t h r e e v a r i e t i e s of L.  i n c l u d i n g f l o c c o s a , and recognition he was  the  r a r e endemics p u m i l a and  o f t h e s e t a x a . a t v a r i e t a l s t a t u s was  bellingeriana.  r e c o g n i z e d as a d i s t i n c t  species.  bellingeriana. interf e r t i l i t y  A r r o y o ( 1 9 7 3 a ) a l s o has  and  since  each  was  Mason's judgement i n t h i s case  r e s t e d on the m o r p h o l o g i c a l s i m i l a r i t i t e s a n d . d i s t r i b u t i o n s S i n c e Mason's work, O r n d u f f ( 1 9 7 1 ) has  His  s l i g h t l y inconsistent,  unable t o demonstrate i n t e r f e r t i l i t y between the t h r e e ,  formerly  floccosa,  o f the  three.  produced h y b r i d s between f l o c c o s a indicated that  and  a c e r t a i n amount o f  e x i s t s between some u n i d e n t i f i e d elements of the L_. f l o c c o s a  group. R e c e n t l y , A r r o y o ( l 9 7 3 a ) has  changed the treatment o f L_. f l o c c o s a , '  r a i s i n g each o f Mason's t h r e e v a r i e t i e s t o s u b s p e c i e s and d i t i o n a l subspecies, g r a n d i f l o r a a n d . c a l i f o r n i c a . based on the conditions,  r e s u l t s of a t a x i m e t r i c considering  various  This  a n a l y s i s of plants  floral  and\vegetative  designating  further division  riate.  By her  a n a l y s i s , the  form a d i s c r e t e . g r o u p i n g species' flj3ccosa_ and separated since the  was  characters. r e s u l t s , as •  a two-way s p l i t would have been more approp-  s u b s p e c i e s g r a n d i f l o r a , c a l i f o r n i c a and  distinct  ad-  grown under u n i f o r m  A l t h o u g h A r r o y o d i v i d e d L_. f l o c c o s a ' i n t o f i v e t a x a , her she p r e s e n t s them, i n d i c a t e t h a t  two  pumila  from a second group c o n s i s t i n g of the  bellingeriana.  each r e p r e s e n t s one  Arroyo explains o f two  t h a t the two  groups  subare  d i s c o n t i n u o u s l e v e l s o f autogamy i n  species. One  o f A r r o y o ' s major premises i s t h a t L.  as an autogamous d e r i v a t i v e o f L. a l b a  (1973a,  floccosa recently  1973b).  evolved  I t i s upon t h i s  pre-  - 119  mise t h a t she bases, at l e a s t t i o n i n L_. f l o c c o s a .  -  in p a r t a l l  i s an i n t e r e s t i n g one and worthy o f  con-  Although Arroyo does not p r e s e n t her r a t i o n a l e , presumably she  sees  sideration  T h i s premise  o f her c o n c l u s i o n s about e v o l u -  here.  the h i g h l y autogamous forms o f L_. f l o c c o s a ' l i n k e d t o L. a l b a by t h e  subspecies  grandiflora:-, c a l i f o r n i c a and p u m i l a , which are more or l e s s i n t e r m e d i a t e i n f l o w e r s i z e , degree o f pubescence, and  supposedly,  l e v e l of autogamy.  This  h y p t h e s i s i s not c o n s i s t e n t w i t h the o t h e r a v a i l a b l e i n f o r m a t i o n . Ornduff and C r o v e l l o (1968) found L. f l o c c o s a d i s t i n c t  from a l l o t h e r  t a x a of Limnanthes i n t h r e e out o f f o u r of t h e i r phenetic- a n a l y s e s . s i s t e n c y was  This  con-  shared by o n l y one o t h e r group c o n s i s t i n g o f F l o e r k e a and L_. m a c o u n i i .  In t h e ' f o u r t h a n a l y s i s , o n l y one  s u b s p e c i e s , p u m i l a , showed any a f f i n i t y t o  L_. a l b a , w h i l e the s u b s p e c i e s f l o c c o s a and b e l l i n g e r i a n a . f o r m e d a d i s t i n c t , separate c l u s t e r .  (The s u b s p e c i e s g r a n d i f l o r a and c a l i f ornica,- ;:had; riot ;,y et.,  been d e s c r i b e d . ) There are few m o r p h o l o g i c a l c h a r a c t e r s which possess much taxonomic utility  i n d i s t i n g u i s h i n g members of t h e Limnanthaceae.. G e n e r a l l y , t h e  char-  a c t e r s t h a t a r e s t r o n g l y conserved by e v o l u t i o n are a l l u n i f o r m between t a x a , w h i l e the p l a s t i c c h a r a c t e r s have e v o l v e d r a p i d l y many times c e s t r a l l i n e s , o f t e n c r e a t i n g s i m i l a r i t i e s by convergence.  in different  an-  Interestingly  enough, the L_. f l o c c o s a group i s w e l l d i s t i n g u i s h e d from o t h e r t a x a of Limnanthes by what appear t o be e x c e l l e n t taxonomic c h a r a c t e r s . guishing characters are:  These d i s t i n -  t h e .sepals have a c h a r a c t e r i s t i c a p i c u l a t e t i p ,  the s e p a l s become v a l v e - l i k e , i n f o l d i n g as the n u t l e t s mature, and an s i o n l a y e r i s formed t o some degree between r e c e p t a c l e and p e d i c e l , d i s p e r s a l o f the n u t l e t - c o n t a i n i n g - f l o w e r as a u n i t . c h a r a c t e r i s t i c s i n L_. f l o c c o s a suggests t h a t t h i s Limnanthes t a x a i n c o m p a r a t i v e l y a n c i e n t t i m e s .  The presence  abscis-  promoting of these  s p e c i e s d i v e r g e d from  other  C e r t a i n l y , t h e s e morpho-  - 120 -  l o g i c a l differences  do not support a c l o s e r e l a t i o n s h i p w i t h any o t h e r members  of the section Inflexae,  i n c l u d i n g L_. a l b a .  Mason (.1952) p l a c e d  L_. f l o c c o s a , i n t h e I n f l e x a e  petal position after pollination.  on t h e b a s i s  o fits  The f l a v o n o i d d a t a c l e a r l y support.;:; t h i s  placement s i n c e i n t h e p e t a l f a c t o r a n a l y s e s t h e s u b s p e c i e s b e l l i n g e r i a n a and pumila occur w i t h i n t h e grouping formed by o t h e r members o f t h e s e c t i o n XVII &  (Figures  XVIII). Most o f t h e s u b s p e c i e s o f L. f l o c c o s a were a n a l y z e d from d r i e d  which i n c l u d e d  s e v e r a l o f Arroyo's c o l l e c t i o n s .  S i n c e i n s u f f i c i e n t p e t a l ma-  t e r i a l was a v a i l a b l e from t h e s e c o l l e c t i o n s , i t was, t h e r e f o r e , t o perform whole p l a n t  material,  only  possible  f l a v o n o i d a n a l y s e s on t h e e n t i r e L_. f l o c c o s a . group.  These a n a l y s e s have s e v e r a l t a x o n o m i c a l l y s i g n i f i c a n t i m p l i c a t i o n s . Figure of whole p l a n t species  XX i s an expanded view o f t h e P r i n c i p a l Components flavonoids  o f t h e OTUs o f L. f l o c c o s a .  f l o c c o s a (23,24,25) form a d i f f u s e - grouping.  pumila (i2Q" "&.4:'2lj..jaM^iellingeriana and  dried material,  (18 & 1 9 ) ,  form two d i s t i n c t  The t h r e e OTUs o f subBoth o f t h e s u b s p e c i e s  a n a l y z e d on t h e b a s i s  of fresh  c l u s t e r s s e p a r a t e d c l e a n l y by t h e a p p r o x i -  mate t o t a l v a r i a t i o n p r e s e n t w i t h i n t h e e n t i r e s p e c i e s . species  Analysis  o f c a l i f o r n i c a (22) a n d . g r a n d i f l o r a  However, t h e sub-  (26) f a l l v e r y c l o s e t o t h e OTUs  o f s u b s p e c i e s b e l l i n g e r i a n a i n d i c a t i n g t h a t t h e s e f o u r OTUs have.very s i m i l a r flavonoid  compositions.  The  f l a v o n o i d d a t a support t h e f o l l o w i n g  vergence i n t h e s u b s p e c i e s f l o c c o s a i s e q u i v a l e n t d i v e r g e n c e w i t h i n t h e whole s p e c i e s  hypothesis.  Since the d i -  i n magnitude t o t h e t o t a l  complex, and a l l o t h e r s u b s p e c i f i c  taxa  f a l l w i t h i n t h e l i m i t s o f t h i s v a r i a b i l i t y , s u b s p e c i e s f l o c c o s a most c l o s e l y approximates t h e a n c e s t r a l t y p e . the  The l a r g e d i s c r e p a n c y between pumila and  c l u s t e r formed by t h e r e m a i n i n g  s u b s p e c i e s g r a n d i f l o r a , c a l i f o r n i c a and  b e l l i n g e r i a n a , suggests t h a t t h e s e two groups r e p r e s e n t s e p a r a t e l i n e s o f  FIGURE XX P r i n c i p a l Components A n a l y s i s  o f 9 OTUs o f  L. f l o c c o s a Based on Whole.Plant F l a v o n o i d s  i  ro H  -  122 -  d i v e r g e n c e from t h e a n c e s t r a l form o f L . f l o c c o s a . Because o f t h e presence  o f two m o r p h o l o g i c a l c h a r a c t e r s , i t . i s  that* subspecies pumila was t h e f i r s t These c h a r a c t e r s a r e :  probable  t o d i v e r g e from t h e L_. f l o c c o s a . group.  l ) l i k e other t a x a o f Limnanthes, t h e s t i g m a t i c branches  o f s u b s p e c i e s p u m i l a a r e s p l i t o n l y p a r t way down t h e - s t y l e , w h i l e t h e y a r e split  a l l t h e way down i n t h e . o t h e r f o u r s u b s p e c i e s ;  2) t h e a b s c i s s i o n  layer  t h a t forms beneath t h e r e c e p t a c l e does not develop t o as g r e a t .an extent i n pumila as i t does i n t h e remaining  subspecies.  pumila do n o t r e a d i l y d i s a r t i c u l a t e as a u n i t 1  Therefore, the flowers of (Arroyo, 1973a).  B e f o r e a d e c i s i o n can be made r e g a r d i n g whether t h e f l o c c o s a group r e q u i r e s taxonomic r e v i s i o n , i t i s n e c e s s a r y t o c o n s i d e r t h e v a l i d i t y o f t h e . s u b s p e c i f i c erections o f Arroyo.  Although h e r t a x i m e t r i c treatment was based  on 42 c h a r a c t e r s , by h e r own admission t h e emphasis i s l a r g e l y on f l o r a l a c t e r s , thus emphasizing gamy has e v o l v e d many  t h e taxonomic s i g n i f i c a n c e o f autogamy.  char-  S i n c e auto-  times i n d i f f e r e n t l i n e s o f t h e Limnanthaceae, t a x o -  nomic c o n c l u s i o n s based  on t r a i t s a s s o c i a t e d w i t h s e l f i n g p r o b a b l y w i l l be  erroneous, o r a t b e s t m i s l e a d i n g . B e s i d e s t h e w e i g h t i n g o f c h a r a c t e r s l i n k e d t o autogamy, A r r o y o ' s a n a l y s i s i s f u r t h e r p r e j u d i c e d on t h e b a s i s o f two t r a i t s , pubescence and stature.  Of t h e 42 c h a r a c t e r s determined,  plant parts.  7 o f t h e s e concern pubescence o f  The c h a r a c t e r used by Mason t o d i s t i n g u i s h two o f t h e v a r i e t i e s  o f L_. f l o c c o s a , rows o f h a i r s a t t h e p e t a l b a s e s , i s i n c l u d e d i n h e r a n a l y s i s as an e i g h t h c h a r a c t e r .  A l s o , o f t h e 42 c h a r a c t e r s , l 6 o r almost  hQ%, r e l a t e  s t r i c t l y t o p h y s i c a l s t a t u r e , b e i n g measurments o f l e n g t h s and widths o f various plant parts. l a p i s present..  B e s i d e s t h e s e redundancies,  1  a d d i t i o n a l c h a r a c t e r over-  F o r i n s t a n c e , " f r u i t i n g c a l y x a b s c i s s i o n zone" and "mode  of n u t l e t d e h i s c e n c e " a r e p r e s e n t e d as s e p a r a t e c h a r a c t e r s , when i n f a c t . t h e y are 100%. c o r r e l a t e d .  - 123  -  Because o f t h e u n j u s t i f i e d w e i g h t i n g o f c e r t a i n c h a r a c t e r s i n her a n a l y s i s , Arroyo's taxonomic c o n c l u s i o n s are p r o b a b l y o f l i t t l e v a l u e . her i n t e r p r e t a t i o n of her own I n s t e a d o f her two  r e s u l t s appears  data.  p r i n c i p a l groups b e i n g s e p a r a t e d by d i s c o n t i n u o u s l e v e l s  o f autogamy, i t i s p r o b a b l e t h a t t h e s e two the b a s i s of s i z e ;  i n c o n s i s t e n t w i t h her  Even  groups were s e p a r a t e d simply  on  one group has l a r g e r component p a r t s than t h e o t h e r .  A f t e r c o n s i d e r a t i o n o f the a v a i l a b l e i n f o r m a t i o n , i t i s c l e a r t h a t c e r t a i n changes s h o u l d be made i n the c l a s s i f i c a t i o n o f t h e L_. f l o c c o s a group. S i n c e t h e evidence  i n d i c a t e s t h a t the s u b s p e c i e s pumila d i v e r g e d from L_. f l o c -  cosa a t an e a r l i e r time than the o t h e r t a x a , t h a t i t i s w e l l d i f f e r e n t i a t e d m o r p h o l o g i c a l l y and c h e m i c a l l y from the r e l a t e d t a x a , and t h a t i t i s a p p a r e n t l y not c r o s s f e r t i l e w i t h the o t h e r s u b s p e c i e s , t h i s taxon s h o u l d be  reinstated  as a . separate., s p e c i e s . The  s u b s p e c i e s b e l l i n g e r i a n a - , g r a n d i f l o r a and c a l i f o r n i c a a l l appear  t o be c l o s e l y r e l a t e d and p r o b a b l y can be c o n s i d e r e d as v a r i e t i e s or L_. f l o c c o s a , or a s p e c i e s d i s t i n c t  from L_. f l o c c o s a .  l i n g e r i a n a .is m o r p h o l o g i c a l l y d i s t i n c t  from g r a n d i f l o r a and c a l i f o r n i c a , which  are v e r y c l o s e i n t h e i r m o r p h o l o g i c a l and t h e s e l a t t e r two  ecological characteristics.  However,  s u b s p e c i e s a r e d i s t i n g u i s h e d by a wide d i s c o n t i n u i t y i n t h e i r  ranges, and each l i e s a t a d i f f e r e n t cosa.  Of t h e three^'subspecies, b e l -  e x t r e m i t y o f t h e range o f s u b s p e c i e s  floc-  S i n c e A r r o y o has r e p o r t e d t h a t t h e r e i s a c e r t a i n amount o f n a t u r a l  g e n e t i c exchange w i t h i n t h e L_. f l o c c o s a - group, i t i s p r o b a b l y b e s t t o i n c l u d e t h e t a x a b e l l i n g e r i a n a , g r a n d i f l o r a , c a l i f o r n i c a and f l o c c o s a i n t o one s p e c i e s . The d i f f e r e n c e s between g r a n d i f l o r a . and c a l i f o r n i c a a r e p r o b a b l y not g r e a t enough t o p r e s e r v e t h e s e as s e p a r a t e e n t i t i e s , and t h e y s h o u l d be merged i n t o one  taxon. I t i s suggested t h a t members o f t h e s p e c i e s L_. f l o c c o s a s h o u l d be r e -  i n s t a t e d at v a r i e t a l rank, r a t h e r than a t the s u b s p e c i e s l e v e l chosen by  Arroyo.  - 124  -  T h i s i s because: l ) a c e r t a i n amount o f g e n e t i c exchange o c c u r s between t h e s e t a x a , as demonstrated by i n t e r g r a d i n g o f c h a r a c t e r s i n some a r e a s ; Mason's c h o i c e o f rank i s adequate, and t h e r e was  no apparent  her d e c i s i o n t o change the. t a x a t o s u b s p e c i e s from  and.2)  reason f o r  varieties.  The R e f l e x a e has been regarded as a n a t u r a l g r o u p i n g o f s p e c i e s cont a i n i n g L. b a k e r i , L. s t r i a t a , L_. m a c o u n i i , L. d o u g l a s i i , L. v i n c u l a n s , and perhaps F l o e r k e a p r o s e r p i n a c o i d e s (Ornduff & C r o v e l l o , 1 9 6 8 ) .  For t h e r e a -  sons a l r e a d y p r e s e n t e d , L. b a k e r i and L_. s t r i a t a . s h o u l d be d e l e t e d from  this  s e c t i o n , and become t h e monotypic members o f t h e s e c t i o n s Bakera and Limnanthes. Limnanthes m a c o u n i i , L. v i n c u l a n s , and t h e f o u r v a r i e t i e s  of L_. doug-  l a s i i t o g e t h e r form a group encompassing a g r e a t range of m o r p h o l o g i c a l sity.  In f a c t t h e s e t a x a are h e l d t o g e t h e r l a r g e l y on the b a s i s o f r e f l e x e d  p e t a l p o s i t i o n a f t e r p o l l i n a t i o n and demonstrated i n t e r f e r t i l i t y . all  diver-  However,-  o f t h e s e t a x a c l u s t e r e d t i g h t l y t o g e t h e r i n one c l u s t e r a n a l y s i s based  on  " v e g e t a t i v e c h a r a c t e r s " (Ornduff & C r o v e l l o , 1 9 6 8 ) . Mason d i d not have f r e s h m a t e r i a l o f L. macounii taxonomy o f Limnanthes. mined t h a t t h i s taxon was  when he r e v i s e d  T h e r e f o r e , on t h e b a s i s o f d r i e d m a t e r i a l he d e t e r a s e p a r a t e s p e c i e s , and i f i t belonged  t o the genus  Limnanthes ( i n s t e a d o f Floerkea)-,' i t s r e f l e x e d p e t a l s p l a c e d i t i n the Reflexae.  the  With t h e r e d i s c o v e r y o f t h i s taxon i n n a t u r e , both o f t h e s e  section judge-  ments have been upheld. A f t e r h a v i n g grown f r e s h m a t e r i a l of L. macounii it  i s e v i d e n t t h a t t h i s s p e c i e s i s more c l o s e l y l i n k e d t o t h e R e f l e x a e  i s apparent  i f i t i s c o l l e c t e d from i t s n a t u r a l h a b i t a t .  nanthes t a x a a r e p h e n o t y p i c a l l y p l a s t i c , L. macounii it  from two p o p u l a t i o n s ,  Although  than  a l l Lim-  i s p a r t i c u l a r l y so.  i s found i n n a t u r e , t h i s s p e c i e s -is v e r y i n c o n s p i c u o u s , b e i n g t o t a l l y  cumbent, or i f u p r i g h t , no t a l l e r than the s u r r o u n d i n g herb u s u a l l y no l a r g e r than a few c e n t i m e t r e s .  elements,  As pro-  and  When grown i n t h e greenhouse under  - 125  -  normal c o n d i t i o n s , t h i s s p e c i e s a t t a i n s an u n n a t u r a l l y l a r g e s i z e mating t h a t o f the o t h e r Limnanthes t a x a . d o u g l a s i i v a r s . sulphurea t a l l y , or p a r t i a l l y ,  I t s f o l i a g e resembles t h a t .'of L.  or d o u g l a s i i , and  5-merous f l o w e r s .  approxi-  The  i t not i n f r e q u e n t l y produces t o s i z e of i t s f l o w e r s , however, r e -  mains s m a l l . The  e v o l u t i o n a r y consequences o f autogamy have been extreme i n  L. m a c o u n i i , i n c l u d i n g l o s s o f f l o r a l p a r t s a n d . r e d u c t i o n These changes n o t w i t h s t a n d i n g ,  grows n a t u r a l l y .  relic  c o n d i t i o n s under which  of Vancouver I s l a n d , cut o f f by the S t r a i t  from some r e c e d i n g p o p u l a t i o n of d o u g l a s i i - l i k e p l a n t s pushed  ward by a p e r i o d of c o o l i n g .  This hypothesis  o f L_. macounii i n more southern  i s supported  Limnanthes t a x a i s made p o s s i b l e l a r g e l y by I f so, t h i s  macounii appears t o be i n l i t t l e  Although  gether  The  two  s p e c i e s a r e l i n k e d by  i n f l a v o n o i d composition.  vergence supports  the hypothesis  from t h e main R e f l e x a e  the  un-  taxon. varieties  similarities in  OTUs of L_. macounii,• which c l u s t e r t o -  i n b o t h whole'plant.and p e t a l a n a l y s e s ,  of divergence  to  L.  several  p e t a l f l a v o n o i d d a t a c l e a r l y l i n k s L_. macounii w i t h t h e  U V - v i s i b l e flower patterning.  of  s i t u a t i o n p a r a l l e l s the a b i l i t y of  danger of i m m e d i a t e . e x t i n c t i o n ,  Furthermore, t h e two  (Figure- I ) .  i t s conversion  u s u a l l y c o o l , or l a t e s p r i n g s might decimate what remains o f t h i s  o f L_. d o u g l a s i i .  south-  f a r t o the n o r t h  t o t a l l y autogamous F l o e r k e a t o grow/in more n o r t h e r n a r e a s .  The  of  r e g i o n s and t h e p r e s e n t - d a y d i s j u n c t i v e d i s -  I t i s l i k e l y t h a t t h e a b i l i t y of L. macounii t o p e r s i s t  autogamy.(Mosquin, 1 9 6 6 ) .  a  both by t h e absence  t r i b u t i o n of L_. d o u g l a s i i a l o n g t h e west c o a s t o f t h e U n i t e d S t a t e s  t h e remaining  size  I t i s almost c e r t a i n t h a t t h i s p l a n t p e r s i s t s o n l y as  i n the warmest areas  Georgia  size.  i t i s apparent t h a t the o v e r a l l d i m i n u t i v e  o f t h i s p l a n t can be a t t r i b u t e d l a r g e l y t o the m a r g i n a l it  i n flower  exhibit a f a i r l y  small.amount  T h i s r e l a t i v e l y s m a l l amount o f d i -  t h a t L_. macounii has  only r e c e n t l y diverged  l i n e p r o v i d i n g t h a t t h e r e has been t h e r e t e n t i o n o f  -  -  126  some v a r i a b i l i t y by t h i s s p e c i e s . Although  two  were f o r m e r l y c o n s i d e r e d s e p a r a t e  t h e t a x a s u l p h u r e a , n i v e a , r o s e a and L_. d o u g l a s i i .  s p e c i e s , Mason merged  d o u g l a s i i as v a r i e t i e s o f t h e s p e c i e s  However, t h e s e v a r i e t i e s have m o r p h o l o g i c a l  same magnitude as t h o s e  d i f f e r e n c e s of the  s e p a r a t i n g o t h e r s p e c i e s o f Limnanthes.  With the-  e x c e p t i o n o f p a r t i a l i n t e r g r a d i n g o f v a r i e t i e s rosea, and n i v e a i n an a r e a o f o v e r l a p , n a t u r a l h y b r i d s a p p a r e n t l y a r e not formed between t h e s e f o u r v a r i e£Ip's>>  However, a l l f o u r are e x p e r i m e n t a l l y  a l l o p a t r i c populations  s e r v e as p a r e n t s .  inter fertile,-  T h e r e f o r e , Mason r e c o g n i z e d the f o u r  as v a r i e t i e s s i n c e the b i o l o g i c a l s p e c i e s concept taxonomic treatment.  p a r t i c u l a r l y when  was  uppermost i n Mason's  With t h e evidence made a v a i l a b l e h e r e , t h i s  treatment  requires reconsideration. All  the v a r i e t i e s o f L_. d o u g l a s i i a r e predominantly b e e - p o l l i n a t e d  outcrossers.  UV  e t i e s t h a t may  f l o r a l photography d i s c l o s e s d i f f e r e n c e s . between the  l e a d t o i s o l a t i o n r e s u l t i n g from e t h o l o g i c a l , r a t h e r than  genetic f a c t o r s . The lasii  the f a m i l y .  •  '  f l a v o n o i d f a c t o r analyses  are.as  distinct The  from one  of  one  as most o f t h e r e c o g n i z e d s p e c i e s i n  e x c e p t i o n s a r e . t h e v a r i e t i e s r o s e a and.nivea  which  fall  analyses.  t h e t a x a rosea-, n i v e a , s u l p h u r e a and d o u g l a s i i are t o b;e' v a r i e t i e s  s p e c i e s , i t i s apparent  different  i n d i c a t e - t h a t most v a r i e t i e s o f L_. doug-  another  c l o s e l y t o g e t h e r i n a l l the f a c t o r If  vari-  ranks.  The  t h a t e q u a l l y d i s t a n t . t a x a have been a s s i g n e d  divergence  i n f l a v o n o i d composition  e t i e s i s no l e s s than the d i v e r g e n c e these varieties-.  o f L";: m a c o u n i i ,  of these four v a r i -  a distinct  s p e c i e s , from  A s i m i l a r s i t u a t i o n a l s o e x i s t s f o r the r e c e n t l y described  s p e c i e s L. v i n c u l a n s . When O r n d u f f  d e s c r i b e d L.. v i n c u l a n s (1969a), he c i t e d - l e a f l e t  ' t e r i s t i c s t h a t he i n t e r p r e t e d t o show t h a t t h i s  s p e c i e s was  charac-  intermediate  - 127 "between L_. b a k e r i and L. d o u g l a s i i . l e a f l e t s o f L_. b a k e r i and l u t i o n a r y processes. i s probable  However, the s i m i l a r i t i e s between the  L_. v i n c u l a n s p r o b a b l y  Although the e f f e c t  r e s u l t from d i f f e r e n t  evo-  on t h e morphology i s s i m i l a r , i t  t h a t the l e a f l e t s o f L. v i n c u l a n s have.become e n t i r e by l o s s o f  l a t e r a l l y d i v i d e d segments, w h i l e the l e a f l e t s o f L.- b a k e r i remain m o s t l y undivided.  This hypothesis  i s supported  by the l e a f morphology of t h e  l i n g s o f L_. v i n c u l a n s , s i n c e at t h i s stage nanthaceae h a v i n g  non-compound l i n e a r  Similarities probably sures  result  i t i s the o n l y s p e c i e s o f t h e  i n l e a f l e t margins between L_. b a k e r i and  from analogous a d a p t a t i o n s  L_. v i n c u l a n s  caused by comparable s e l e c t i o n p r e s species.  In  other  c h a r a c t e r s L_. v i n c u l a n s c l o s e l y resembles t h e L_. d o u g l a s i i group, and i t s closest natural a f f i n i t y with v a r i e t y nivea.  i n d i c a t e d by t h e s i m i l a r i t i e s between these two tween a l l o p a t r i c  taxa  probably  This a f f i n i t y i s  i n f l o r a l c h a r a c t e r s and by h i g h  (Ornduff,  Lim-  leaves.  c h a r a c t e r i s t i c of the c o a s t a l h a b i t a t s of t h e s e  shares  seed-  interfertility  1971), a l t h o u g h h y b r i d s a r e o n l y formed be-  populations.  A l t h o u g h f r e s h m a t e r i a l o f L_. v i n c u l a n s was  not a v a i l a b l e f o r t h i s  i n v e s t i g a t i o n , i t s whole p l a n t f l a v o n o i d s were determined from d r i e d m a t e r i a l and  compared t o the o t h e r t a x a .  I n t h e ' c l u s t e r and  f a c t o r analyses, " L . v i n -  culans . c l u s t e r e d w i t h OTUs of L_. d o u g l a s i i i n d i c a t i n g a c l o s e r e l a t i o n s h i p . I n a d d i t i o n , the d i s t a n c e s e p a r a t i n g L_. v i n c u l a n s from t h e v a r i e t i e s  of  L_. d o u g l a s i i i s no g r e a t e r t h a n t h e d i s t a n c e s e p a r a t i n g t h e v a r i e t i e s themselves. On t h e ' b a s i s of t h e a v a i l a b l e i n f o r m a t i o n , i t i s p o s s i b l e t o hypot h e s i z e how  e v o l u t i o n proceeded i n t h e R e f l e x a e .  that hypothesized  f o r the I n f l e x a e l i n e ,  In a. process  parallel  elements of a d o u g l a s i i - t y p e a n c e s t r a l  p o p u l a t i o n became i s o l a t e d g e o g r a p h i c a l l y and/or e c o l o g i c a l l y and into d i s t i n c t  to  diverged  e n t i t i e s as a . r e s u l t o f d i f f e r e n t s e l e c t i o n p r e s s u r e s  i n the  - 128 v a r i o u s new h a b i t a t s .  S i x of these divergent  e n t i t i e s e x i s t t o d a y , 'the  f o u r v a r i e t i e s o f L_. d o u g l a s i i , and.the s p e c i e s L_. macounii and L. v i n c u l a n s . Probably  a l l s i x diverged comparatively  recently.  However, among t h e s e s i x  t a x a a r e some o f ' t h e most h i g h l y adapted forms i n t h e f a m i l y . There i s l i t t l e s e c t i o n Reflexae n i v e a and r o s e a .  j u s t i f i c a t i o n f o r maintaining the s i x taxa of the  at d i f f e r e n t r a n k s , w i t h t h e e x c e p t i o n s S i n c e these t a x a have been found  of the v a r i e t i e s  to intergrade i n a region  o f sympatry, perhaps they s h o u l d be r e c o g n i z e d as v a r i e t i e s o f one s p e c i e s . S i n c e L. macounii- and L. v i n c u l a n s have been r e c o g n i z e d and m a i n t a i n e d r a t e s p e c i e s , and t h e r e a r e good d i s t i n g u i s h i n g m o r p h o l o g i c a l , and  as sepa-  distributional  e c o l o g i c a l c h a r a c t e r s s e p a r a t i n g t h e v a r i e t i e s d o u g l a s i i , s u l p h u r e a and  r o s e a , i t would p r o b a b l y be most c o n s i s t e n t t o e l e v a t e t h e s e l a t t e r t a x a . t o t h e rank o f s p e c i e s .  three  The proposed s p e c i e s r o s e a would c o n s i s t o f  ' t h e two v a r i e t i e s n i v e a and r o s e a .  E. Summary o f P r o p o s a l s i.  f o r R e v i s i o n o f t h e Limnanthaceae  Introduction A f t e r c o n s i d e r a t i o n o f t h e a v a i l a b l e evidence  regarding r e l a t i o n s h i p s  i n t h e Limnanthaceae, i t i s apparent t h a t t h e f a m i l y r e q u i r e s i n t e r n a l nomic r e v i s i o n a t every l e v e l .  Some r e s e a r c h e r s w i l l i n s i s t t h a t a f a m i l y  s h o u l d not be r e v i s e d s o l e l y on t h e b a s i s o f c h e m i c a l , c h a r a c t e r s ; and t h i s  taxo-  i s not what i s b e i n g proposed.  I agree,  I t i s t r u e that the f l a v o n o i d s pro-  v i d e a s e t o f c h a r a c t e r s o f g r e a t taxonomic u t i l i t y  i n t h e Limnanthaceae., and.  t h e r e f o r e were depended on h e a v i l y , i n f o r m u l a t i n g a new c l a s s i f i c a t i o n . e v e r , i n no i n s t a n c e does t h e o t h e r a v a i l a b l e evidence flavonoid information.  Although  improbable t h a t d i f f e r e n t  How-  c o n f l i c t with the  i n t e r p r e t a t i o n s o f d a t a may v a r y , i t i s  i n t e r p r e t a t i o n s would g r e a t l y a l t e r t h e proposed  f a m i l y c l a s s i f i c a t i o n , a t l e a s t u n t i l new i n f o r m a t i o n from o t h e r sources i s  - 129 -  brought t o bear on t h e problem.  ii.  Genus L e v e l F l o e r k e a and Limnanthes should be merged i n t o one genus.  The d i s -  s i m i l a r i t i e s Let-ween these two genera a r e simply not g r e a t enough, or d i s c o n tinuous  enough, t o p r e s e r v e them as s e p a r a t e genera.  On t h i s  issue, the evi-.  dence i s c o n c l u s i v e . With r e g a r d t o t h e m e r i t o f m a i n t a i n i n g F l o e r k e a as a genus d i s t i n c t from Limnanthes on t r a d i t i o n a l grounds, t h e f o l l o w i n g passage from Jepson (1951, P- 14) has a p p l i c a t i o n : ... I t i s , however, n e c e s s a r y t h a t t h e l i m i t s o f genera s h o u l d , w i t h i n c r e a s e o f knowledge o f t h e i r s t r u c t u r e , p r o b a b l e phylogeny, geog r a p h i c h i s t o r y and ecology, be s u b j e c t t o r e v i s i o n and m o d i f i c a t i o n . No genus has any v e s t e d r i g h t s on account o f l o n g usage or a p p r o v a l by t h e g r e a t masters. Continued r e s e a r c h , i n c r e a s e d knowledge and an e n l a r g e d viewpoint must c o n t i n u a l l y f i n d vent i n new g e n e r i c expressions. The  g e n e r i c name s h o u l d become F l o e r k e a s i n c e t h i s genus was d e s c r i b e d 32  y e a r s b e f o r e Limnanthes, a n d . t h e r e f o r e  iii.  takes  precedence.  Section Level The  previous  c l a s s i f i c a t i o n , which r e c o g n i z e d F l o e r k e a and two s e c t i o n s  o f Limnanthes s h o u l d be amended t o a s i n g l e genus c o n t a i n i n g f i v e s e c t i o n s : Bakera, F l o e r k e a , I n f l e x a e , R e f l e x a e and Limnanthes.  The primary  reason f o r  t h i s a l t e r a t i o n is. t o p r o v i d e i n t e r n a l c o n s i s t e n c y o f rank.  i v . S p e c i e s and V a r i e t a l L e v e l s With t h e o b j e c t o f p r o v i d i n g u n i f o r m i t y i n r a n k i n g p a r a l l e l t a x a , i t i s necessary  t o r e v i s e d e s i g n a t i o n s o f many t a x a i n t h e Limnanthaceae a t t h e  s p e c i e s l e v e l and.below.  I t i s a l s o necessary  d i f f e r e n c e s as i t i s a p p l i e d i n t h i s c o n t e x t .  t o r e v i s e t h e concept The b i o l o g i c a l s p e c i e s  of species concept  - 130 has been found wanting as applied t o the Limnanthaceae.  Since t h i s c r i t e r i o n  was previously assumed to hold the utmost i n taxonomic s i g n i f i c a n c e , other sorts o f evidence were deemphasized i n e a r l i e r treatments.  °  The Limnanthaceae should be r e v i s e d t o consist of 15 species, the species rosea containing two v a r i e t i e s and the species f l o c c o s a containing three v a r i e t i e s .  With one exception, a l l of the taxa designated by previous  authors would remain d i s t i n c t , but the ranks of many of these taxa would be changed.  I t i s recommended that the subspecies  of L. f l o c c o s a , g r a n d i f l o r a  and c a l i f o r n i c a , as described by Arroyo, be merged i n t o the s i n g l e taxon f l o c c o s a var. g r a n d i f l o r a .  F. Synopsis o f the Family Limnanthaceae A s i n g l e genus designated Floerkea W i l l d . , Neue S c r i f t , Geselschaft Nat. 3:449-1801. [Limnanthes R. B r . , London and Edin. P h i l o s . Mag. IXI,2:70.. 1833] Section Limnanthes 1. F. s t r i a t a (JepVbh^Earker, comb. nov. [L. s t r i a t a . J e p s o n , F l . C a l i f . 2:1*11. 1936.] Section Bakera 2/ F_. bakeri (J.T. Howell) Parker, comb. nov. [L_. bakeri J.T. Howell, Plantae Occi'dentales. I I I . L e a f l . West. Bot. 3:206. 1943.] . Section Floerkea 3. F. proserpinacoides W i l l d . , Neue S c h r i f t . Ges. Nat. 3:449. 1801. '[F. occidentalis. Rydberg, Mem. N.Y. Bot, Gard. 1:268. 1900.] Section Inflexae 4. F. alba Greene, F l . Fran. 100. 1891. [L. alba Bentham, P I . Hartw. 301. 1848.] IL. alba var. aetonsa Jepson, F l . C a l i f . 2:411. 1936..] ,[L. alba var. alba (Benth.) C T , Mason, Univ. C a l i f . Publ. Bot. 25:455. 1952."]  - 131 Section Inflexae — 5.  continued  F. f l o c c o s a (Howell) v a r . f l o c c o s a P a r k e r , comb. nov. [L. f l o c c o s a H o w e l l , F l . NW Amer. 1:108. I897.] [L. f l o c c o s a v a r . f l o c c o s a (Howell) C.T. Mason, Univ. C a l i f . P u b l . Bot. 25:455- 1952.] [L. f l o c c o s a ssp. f l o c c o s a (Howell) A r r o y o , B r i t t o n i a 25:177. 1973.] v a r . b e l l i n g e r i a n a (M.E. Peck) P a r k e r , comb. nov. [L- b e l l i n g e r i a n a M.E. Peck, P r o c . B i o l . Soc. Wash. 50:93. 1937.] [L. f l o c c o s a v a r . b e l l i n g e r i a n a (M.E. Peck) C.T. Mason, Univ. C a l i f . P u b l . Bot. 25:455- 1952.] [L. f l o c c o s a ssp. b e l l i n g e r i a n a (M.E. Peck) A r r o y o , B r i t t o n i a 25:177- 1973.] ........... v a r . g r a n d i f l o r a (Arroyo) P a r k e r , comb. nov. [L. f l o c c o s a ssp. g r a n d i f l o r a A r r o y o , B r i t t o n i a 25:177. 1973.] [L_. f l o c c o s a ssp. c a l i f o r n i c a A r r o y o , B r i t t o n i a 25:177. 1973.]  6.  F. g r a c i l i s (Howell) P a r k e r c o m b . nov. [L. g r a c i l i s H o w e l l , F l . WW Amer. 1:108. I897.] I L . g r a c i l i s v a r . g r a c i l i s (Howell) C.T. Mason, Univ. C a l i f . P u b l .  Bot. 25:455- 1952.]  7-F_.  8.  montana (Jepson) P a r k e r , comb. nov. I L . montana Jepson, F l . C a l i f . 2 : 4 l 2 .  1936.']"  F. p a r i s h i i (Jepson) P a r k e r , comb. nov. [L. v e r s i c o l o r v a r . p a r i s h i i J e p s o n , F l . C a l i f . 2 : 4 l l . 1936.] [L. g r a c i l i s v a r . p a r i s h i i (Jepson) C.T. Mason, Univ. C a l i f . P u b l .  Bot. 25:455- 1952.]  9.  F. pumila (Howell) P a r k e r , comb. nov. [L. p u m i l a H o w e l l , F l . NW Amer. 1:108. I897.] [L. f l o c c o s a v a r . p u m i l a (Howell) C.T. Mason, U n i v . C a l i f . P u b l .  Bot. 25:455- 1952.]  [L.- f l o c c o s a 10.  ssp. pumila'(Howell) A r r o y o , B r i t t o n i a  25:177. 1973.]  F. v e r s i c o l o r Greene, E r y t h e a 3:62. 1895. J L . v e r s i c o l o r (Greene) Rydberg, N. Amer. F l . 25:99. 1910.] J L . a l b a v a r . v e r s i c o l o r . ( G r e e n e ) C.T. Mason, Univ. C a l i f . Publ..  Bot. 25:455- 1952.]  Section Reflexae 11.  F. d o u g l a s i i B a i l l o n , A d a n s o n i a y 1 0 : 3 6 2 . . I 8 7 S . [L. d o u g l a s i i R. B r . , London E d i n . P h i l o s . Mag. 111,2:70.. 1833.] ,[L_. s u l p h u r e a o d o r a t a Loud. , Enc. P I . 1543. 1855.] ["L. s u l p h u r e a (Loud.)" Rydberg, N. Amer. F l . 25:98. 1910.] [L_. h o w e l l i a n a Abrams, Madrono 6:27.' 1 9 4 l . ] [L. d o u g l a s i i v a r . d o u g l a s i i (R. Br.) C.T. Mason, Univ. C a l i f . P u b l . Bot,  _ 25:455. 1952.] 12. F. macounii ( T r e l e a s e ) A. Gray, Syn. F l . 1:363. 1897. [F. p r o s e r p i n a c o i d e s Macoun, Cat. Canad. P I . 1:90. 1883.] [L. d o u g l a s i i Macoun, Cat. Canad. P I . 3:502. 1884.] [L. macounii T r e l e a s e , Mem.  Boston Soc. Nat. H i s t . 4:85.  I887.]  - 132 -  Section Reflexae —  continued  13. F. r o s e a Greene v a r . r o s e a , F l . F r a n . 100. 1891. [L. r o s e a Bentham, PI.- Hartw. 302. 1848.] [L. p u l c h e l l a Hartweg, J o u r . H o r t . Soc. London 3:220.'1848.] .[L. r o s e a v a r . Candida J e p s o n , F l . C a l i f . 2:411. 1936.] [L. d o u g l a s i i v a r . r o s e a (Benth.) C.T. Mason, Univ. C a l i f . P u b l . Bot. 25:455- 1952.]  IL.  v a r . n i v e a (Mason) P a r k e r , comb. nov. d o u g l a s i i v a r . n i v e a C.T. Mason, Univ. C a l i f . P u b l . B o t . 25:455- 1952.]  14. F. s u l p h u r e a (Mason) P a r k e r , comb. nov. [L. d o u g l a s i i v a r . s u l p h u r e a C.T. Mason, Univ. C a l i f . 25:455. 1952] 15. F. v i n c u l a n s (Ornduff) P a r k e r , comb. nov. [L. v i n c u l a n s Orncluff, B r i t t o n i a 21:11. I969.]  Publ. Bot.  - 133 -  CONCLUSIONS  1)  The f l a v o n o i d s  found i n t h e Limnanthaceae a r e f l a v o n o l  glycosides.  They a r e unusual w i t h r e g a r d t o type o f a g l y c o n e , i n c l u d i n g v a r i o u s t i v e s o f s y r i n g e t i n and l a r i c y t r i n . g a r d i n g extent o f g l y c o s y l a t i o n ; are found n a t u r a l l y o c c u r r i n g  2)  These f l a v o n o i d s  a r e a l s o unusual r e -  d i - r u t i n o s i d e s and r h a m n o s y l - r u t i n o s i d e s  f o r the f i r s t  On t h e b a s i s o f p r e s e n t l y  deriva-  time.  known f l a v o n o i d  s i m i l a r i t i e s , t h e Limnan-  thaceae i s d i s t i n c t ; no f a m i l i e s appear t o share any special, a f f i n i t i e s w i t h i t .  3)  Because o f s i m i l a r i t i e s i n f l a v o n o i d c o m p o s i t i o n between a l l members  of the L i m n a n t h a c e a e i t  k) •  i s apparent t h a t they a r e a l l c l o s e l y r e l a t e d .  The a p p l i c a t i o n o f t a x i m e t r i c  t e c h n i q u e s t o f l a v o n o i d taxonomy o f  the Limnanthaceae i n d i c a t e s t h a t t h e f l a v o n o i d s c a t i o n o f r e l a t i o n s h i p s -in t h i s  5) "  'Flavonoids o c c u r r i n g  are useful f o r the c l a r i f i -  family.  i n r e l a t i v e l y greater-concentrations  are better  i n d i c a t o r s o f r e l a t i o n s h i p s between members o f t h e Limnanthaceae t h a n t h o s e occurring  6)  i n t r a c e amounts.  When f l a v o n o i d c h a r a c t e r s  a r e a n a l y z e d by c o n v e n t i o n a l c l u s t e r i n g  t e c h n i q u e s Ci.e_. , t h e weighted p a i r group method), i t makes l i t t l e whether o r not mutual absences a r e - c o n s i d e r e d .  difference  - 134  7) use  No t a x o n o m i c a l l y  -  s i g n i f i c a n t d i f f e r e n c e s were d e t e c t e d  o f d r i e d p l a n t m a t e r i a l when f r e s h m a t e r i a l was  8)  due  the  unavailable.  C l a s s i f i c a t i o n s based on p e t a l f l a v o n o i d s more c l e a r l y i n d i c a t e  n a t u r a l r e l a t i o n s h i p s between t a x a o f the Limnanthaceae than do based on whole p l a n t  9) '  The  ( i n c l u d i n g flowers)  t e c h n i q u e s of f a c t o r a n a l y s i s p r o v i d e  t e r i n g techniques. conflict..  c l e a r e r i n d i c a t i o n s of  evolution i n  10) .  The  standard  However, the r e s u l t s d e r i v e d from the two  Apparently, o r d i n a t i o n of taxa  m u l t i p l e branching of evolutionary  classifications  flavonoids.  r e l a t i o n s h i p s between t a x a i n the Limnanthaceae than do the  not  to  l i n e s and  clus-  techniques  i s r e q u i r e d to understand  do the  the d i f f e r e n t ongoing r a t e s  of  the-family.  flavono.fd p a t t e r n o f the h o r t i c u l t u r a l v a r i e t y o f L. d o u g l a s i i  cannot be d i r e c t l y compared w i t h other t a x a o f Limnanthes i n chemosystematic s t u d i e s .-because o f the  11)  light.  d i s t i n g u i s h one  A new  This character  i s taxonomically  significant  s u p r a s p e c i f i c group from t h e r e s t of the  visible  and h e l p s  to  family.  other a v a i l a b l e  information.  I t i s apparent from d i s c u s s i o n o f the t a x o n o m i c a l l y  formation  . .  c l a s s i f i c a t i o n of the Limnanthaceae i s proposed a f t e r i n t e g r a t i o n  o f the f l a v o n o i d d a t a and  13)  s e l e c t i o n and/or i n b r e e d i n g .  C e r t a i n members 'of the Limnanthaceae have f l o r a l p a t t e r n i n g  o n l y under UV  12) '  e f f e c t s of a r t i f i c i a l  known f o r the Limnanthaceae, t h a t t h i s f a m i l y  r e v i s i o n at a l l l e v e l s .  significant i n -  r e q u i r e s taxonomic  - 135 LITERATURE CITED:  1.  Abrams, L. 1941.  2.  A r r o y o , M.T.K. 1973a. A t a x i m e t r i c study o f i n f r a s p e c i f i c v a r i a t i o n i n autogamous Limnanthes f l o c c o s a (Limnanthaceae). B r i t t o n i a 25:177-191-  3  4.  A new  Limnanthes  from Oregon. Madrono  6:29.  1973b. 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P r i r . S o e d i n . -197M2) , 157-160.  64.  Von F r i s c h , K. T967. The Dance, Language and O r i e n t a t i o n o f Bees. HarvardU n i v e r s i t y P r e s s , Cambridge.  65. Warburg, E.F. -1938. Taxonomy and r e l a t i o n s h i p i n t h e G e r a n i a l e s i n t h e light  o f t h e i r .cytology. New P h y t o l o g i s t  37:130-159,189-209-  66. Willdenow.- 1801. Neue S c r i f t . G e s e l s c h a f t Nat. 3:449-  - 139 -  APPENDIX I  Mason's C l a s s i f i c a t i o n o f Limnanthes R. B r .  1  Section Inflexae 1. L_. f l o c c o s a Howell v a r . p u m i l a (Howell) Mason  2.  "  var. hellingeriana  "  var. floccosa  (M.E. Peck) Mason  L_. a l b a Benth. var.- a l b a "  var. versicolor  (Greene) Mason  3. L_. g r a c i l i s Howell v a r . g r a c i l i s " k.  var..parishii  (Jepson) Mason  L. montana Jepson  Section Reflexae 5. L_. d o u g l a s i i R. B r . v a r . . d o u g l a s i i "  v a r . n i v e a Mason  "  v a r . s u l p h u r e a Mason  " 6.  . v a r . r o s e a (Benth.) Mason  L; s t r i a t a Jepson  7. L. b a k e r i J.T. Howell 8.  1 —  L. macounii T r e l e a s e  Mason, C T . 1952. A s y s t e m a t i c study o f t h e genus Limnanthes U n i v e r s i t y o f C a l i f o r n i a P u b l i c a t i o n s i n Botany  R.  25:455-512.  - 140 -  APPENDIX I I Voucher Specimens  OTU  o f Limnanthes Taxa Grown from Seed'  Taxon Name  U.S.D.A. P l a n t A c c e s s i o n No.  W-.H. P a r k e r C o l l e c t i o n No.  L. d o u g l a s i i v a r . d o u g l a s i i  278170  114,116,138  L. d o u g l a s i i v a r . d o u g l a s i i  283708  105,165  3  L. d o u g l a s i i v a r . n i v e a  283713  100,166,117  1+  L. d o u g l a s i i v a r . r o s e a  283715  104,169,118  5  L. d o u g l a s i i v a r . s u l p h u r e a .  283718  101,170  7  L. b a k e r i  283706  102,120  8  L. s t r i a t a  283727  106,119  9  L. m a c o u n i i  315048  112  10  L. macounii  11  L. a l b a v a r . a l b a  283701  111,160  12  L. a l b a v a r . a l b a  B55689  113  13  L. a l b a v a r . v e r s i c o l o r  283705.  108,157  14  L. g r a c i l i s v a r . g r a c i l i s  283722  164,168  15  L. g r a c i l i s , v a r .  283723  107.  16  L. g r a c i l l ' s v a r . p a r i s h i i  283724  103,159  17  L. montana  283725  109,158,161  18  L. f l o c c o s a ssp.-. b e l l i n g e r i a n a . :  283720  115,162  20  L. f l o c c o s a s s p . p u m i l a  283721  110,163  1  Voucher specimens a r e d e p o s i t e d i n t h e Herbarium o f t h e U n i v e r s i t y o f  1  128,167  British  gracilis-  Columbia.  - 141 -  APPENDIX I I I  TLC Map o f P e t a l F l a v o n o i d s 1  7  of  L. d o u g l a s i i v a r . d o u g l a s i i , OTU 1  -p  a >  H O CQ  fl ai hO  O,-  rH  o -p fl CL)  H o H 0)  >  Q  fl OJ  R ,-.  Sr-,  CD Origin  1st Development, Aqueous S o l v e n t  l ) See M a t e r i a l s  —  and Methods f o r d e t a i l s o f e x t r a c t i o n s and developments  - 142 APPENDIX IV  TLC  Map o f Whole P l a n t  Flavonoids of  L_. d o u g l a s i i v a r . d o u g l a s i i , OTU 1  -  143  APPENDIX  TLC  Map  -  V  of P e t a l Flavonoids  . d o u g l a s i i var.  of  d o u g l a s i i , OTU  2  - 144 -  APPENDIX VI  TLC  Map o f Whole P l a n t  Flavonoids of  L_. d o u g l a s i i v a r . d o u g l a s i i , OTU 2  - 145  -  APPENDIX VII TLC Map  of Petal Flavonoids of  L. douglasii var. nivea, OTU  3  -  146 -  APPENDIX V I I I  TLC Map o f Whole P l a n t F l a v o n o i d s o f L_. d o u g l a s i i v a r . n i v e a , OTU 3  - 147  -  APPENDIX IX  TLC Map  of P e t a l Flavonoids  L. d o u g l a s i i v a r . r o s e a , OTU  of 4  - 148  -  APPENDIX X TLC Map  o f Whole PlantsFlavonoids  L. d o u g l a s i i v a r . r o s e a , OTU  k  of  APPENDIX .XI  TLC Map  of P e t a l Flavonoids  of  L. d o u g l a s i i v a r . s u l p h u r e a , OTU  5  - 150 -  APPENDIX XII  TLC Map o f Whole Plant: F l a v o n o i d s o f ' L_. d o u g l a s i i v a r . s u l p h u r e a , OTU 5  - 151 APPENDIX X I I I  TLC  Map o f Whole P l a n t F l a v o n o i d s o f L. v i n c u l a n s , OTU 6  4  - 152  APPENDIX  TLC  Map  -  XIV  of P e t a l Flavonoids L. b a k e r i , OTU  7  of  - 153 APPENDIX XV  TLC  Map o f Whole P l a n t F l a v o n o i d s L. b a k e r i , OTU 7  of  - 154. -  APPENDIX XVI  TLC Map o f P e t a l F l a v o n o i d s o f L. s t r i a t a , OTU 8  - 155 APPENDIX XVII  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. s t r i a t a , OTU 8  - 1 6 5  -  APPENDIX XVIII  TLC Map  of P e t a l Flavonoids of  L. m a c o u n i i , OTU  9  -  157-  APPENDIX XIX  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. m a c o u n i i , OTU 9  - 158 APPENDIX XX  TLC Map o f P e t a l F l a v o n o i d s L. m a c o u n i i , OTU 10  of  - 159 APPENDIX XXI  TLC Map o f Whole P l a n t F l a v o n o i d s o f L_. m a c o u n i i , OTU 10  - i6o APPENDIX XXII  TLC  Map L-  of P e t a l Flavonoids  alba var.  a l b a , OTU  of 11  -  161  -  APPENDIX XXIII  TLC Map o f Whole P l a n t . F l a v o n o i d s o f L. a l b a v a r . a l b a , OTU  11  -  162  -  APPENDIX XXIV  TLC  Map  of P e t a l Flavonoids  L. a l b a v a r .  a l b a , OTU  of 12  -  163  -  APPENDIX XXV  TLC  Map o f Whole P l a n t F l a v o n o i d s L. a l b a v a r , a l b a , OTU 12  4  of  - 164 -  APPENDIX XXVI  TLC  Map o f P e t a l  L_. a l b a v a r .  Flavonoids of  v e r s i c o l o r , OTU 13  - 165 APPENDIX XXVII  TLC Map o f Whole P l a n t . F l a v o n o i d s o f L. a l b a v a r . v e r s i c o l o r , OTU  13  - 166  -  APPENDIX XXVIII TLC L.  Map  of P e t a l F l a v o n o i d s  gracilis  var.-  g r a c i l i s , OTU  of ih  - 16? APPENDIX XXIX  TLC L.  Map o f Whole P l a n t F l a v o n o i d s o f gracilis  v a r . g r a c i l i s , OTU 14  ©  10  (Ti?  - 168  -  APPENDIX  TLC  Map  XXX  of P e t a l F l a v o n o i d s  L_. g r a c i l i s  var.  g r a c i l i s , OTU  of 15  - .169 APPENDIX XXXI  TLC  Map o f Whole P l a n t F l a v o n o i d s o f  L_. g r a c i l i s  v a r . g r a c i l i s ,-• OTU 15  -  170  -  APPENDIX XXXII  TLC L.  Map  of P e t a l F l a v o n o i d s  g r a c i l i s var.  p a r i s h i i , OTU  of 16  - 171 APPENDIX XXXIII  TLC L.  Map o f Whole P l a n t F l a v o n o i d s o f g r a c i l i s var.' p a r i s h i i , OTU  16  - 172 APPENDIX XXXIV  TLC Map o f P e t a l F l a v o n o i d s o f L. montana, OTU  •V,'  17  - 173 APPENDIX XXXV  TLC Map o f Whole P l a n t F l a v o n o i d s o f L. montana, OTU  17  - 174 APPENDIX XXXVI  TLC  Map o f P e t a l F l a v o n o i d s o f  L_. f l o c c o s a  s s p . b e l l i n g e r i a n a , OTU 18  -  175-  APPENDIX XXXVII  TLC  Map o f Whole P l a n t  Flavonoids of  L_. f l o c c o s a ssp. b e l l i n g e r i a n a , OTU  18  - 176 -  APPENDIX XXXVIII  TLC Map o f Whole P l a n t . F l a v o n o i d s p f L_. f l o c c o s a s s p . b e l l i n g e r i a n a , OTU 19  - 177 -  APPENDIX XXXIX  TLC  Map o f P e t a l F l a v o n o i d s o f  L_. f l o c c o s a s s p . p u m i l a , OTU 20  - 178 APPENDIX XL  TLC  Map o f Whole P l a n t F l a v o n o i d s < L_. f l o c c o s a s s p . p u m i l a , OTU 20  - 179 APPENDIX XL,!  TLC  Map o f Whole P l a n t F l a v o n o i d s  of  L_. f l o c c o s a s s p . p u m i l a , OTU 21 .  Z  - l8o APPENDIX XLII  TLC  Map o f Whole P l a n t  Flavonoids of  L. f l o c c o s a s s p . g r a n d i f l o r a , OTU 22  - 181 APPENDIX X L I I I  TLC L.  Map o f Whole P l a n t floccosa  Flavonoids of  s s p . - f l o c c o s a , OTU 23  - 182 -  APPENDIX XLIV  TLC  Map o f Whole P l a n t  L_. f l o c c o s a  Flavonoids of  s s p . f l o c c o s a , OTU 24  - 183 -  APPENDIX XLV  TLC L.  Map o f Whole P l a n t floccosa  Flavonoids of  s s p . f l o c c o s a , OTU 25  - 184 -  APPENDIX XLVI  TLC  Map of. Whole P l a n t F l a v o n o i d s  of  L_. f l o c c o s a s s p . c a l i f o r n i c a , OTU 26  - 185 APPENDIX XLVII  .TLC Map o f Whole P l a n t F l a v o n o i d s o f F. p r o s e r p i n a c o i d e s , OTU 27  - 186 APPENDIX XLVIII  TLC Map o f Whole .Plant ..Flavonoids o f F. p r o s e r p i n a c o i d e s ,  OTU  28  - 187- APPENDIX XLIX  TLC Map o f Whole P l a n t F l a v o n o i d s o f F. p r o s e r p i n a c o i d e s , OTU 29  - 188 APPENDIX L  •  TLC Map o f Whole P l a n t . F l a v o n o i d s o f F. p r o s e r p i n a c o i d e s , OTU 30  APPENDIX LI  J  1 1 1  1  I • ...  1  U-1  I . . . .  1  i  1  i  I • ...  !  1  1  1  !  l_J  I . . . .  1  I •  1  i  ...  i_4 I u—I ; i • • I • ... I • . . . I • ... I  1 : 1  I . . . . I  APPENDIX  • • - i .... t  . . . .  i  1  . . . .  i  . . . .  I . . . .  i . . . .  LII  I  .... i .... I  . . . .  i  . . . .  I . . . .  t .  APPENDIX L I I I •100 mHz | NMR  i  . . .  •  ; I  ; •  . . . .  i  I  .  .  .  .  I  .  • .  i .  .  Spectrum o f TMS  • i  •  I  .  .  • .  .  1  ;  i  I  .  i .  i .  .  • i •  1  I  .  .  .  .  E t h e r of Isorhamnetin  1  • i • i • I  .  .  .  .  3-0-yft-D-Rutinoside  • i • i • i • i •  I . . . .  I  . . . .  I . . . .  I , . . .  1  • • . . . .  t  . . . .  t . . . .  t  . 100 mHz  1  4 0 0  3 0 0  NMR  Spectrum o f TMS  APPENDIX LIV • Ether o f M y r i c e t i n 3-0-/3 -D-Rut inos i d e  2 0 0  100  i  0  Hz  . APPENDIX LVI 100 mHz NMR  Spectrum of TMS Ether of Syringetin S-O-^-D-Rutinoside  - 195 -  APPENDIX LVIT.  •' .  Matrix of S i m i l a r i t y C o e f f i c i e n t s  Calculated  for  30 OTUs by J a c c a r d C o e f f i c i e n t : High C o n c e n t r a t i o n s  0. 0. 0. 0.  789474 714236 714236 714236  0. 9 0 4 7 6 7 0. 7 3 9 1 3 0 0. 7 3 9 1 3 0 0. 7 5 0 0 0 0 -077533' O. 7 6 1 9 0 5 650000 0. 6 5 0 0 0 0 611111 0. 7 0 0 0 0 0 " 5 7 3 9 4 7 " 0. 7 3 9 1 3 0 565717  ~orsoooocr 0. 0. 0. 0.  "10 11 —  13 14 -T516  O 65000tr 0. 8 5 7 1 4 3 0. 739474.. -0.--7391300. 3 1 2 5 0 0 0. 4 5 3 3 3 3 -  "O. 7ST90T7  0. 8 2 6 0 8 7 0. 3 2 6 0 3 7 0. 3 3 3 3 3 3  0. 909091  "07-5555560. 8 5 7 1 4 3 0. 7 7 2 7 2 7 0. 6 6 6 6 6 7 0. 6 6 6 6 6 7 -07714234.- 0." 6 3 6 3 6 4 ' 0. 7 5 0 0 0 0 0. 6 1 5 3 3 5 u. 6V5652  OTSaOOOO  0. 8 0 9 5 2 4 0. 7 3 9 1 3 0 0. 6 0 0 0 0 0 -07 8 3 0 0 0 0 .0. 6 5 0 0 0 0 0. 666667 i66667 0. 5 2 3 3 1 0 0. 6 5 0 0 0 0 0 T £ S 4 7 1 T " "07"7 T 4 2 8 6 -07"E52T740. 8 4 2 1 0 5 0. 7 2 7 2 7 3 0. 8 9 4 7 3 7 0. 7 7 2 7 2 7 0. 5 9 0 9 0 9 0. 86:3636 • Or 7 8 2 6 0 9 " 0: -652174- -0.-625000' 0. 7 6 1 9 0 5 0. 6 5 0 0 0 0 0. 6 9 5 6 5 2 O. 3 9 4 7 3 7 0. 7 7 2 7 2 7 0. 8 5 0 0 0 0 ""OTTJUOOOO "0. 8095i'4 • 0. 6 5 2 1 7 4 07 5 9 0 9 0 9 0. 5 6 5 2 1 7 p. 68421. 1 0. 7 1 4 2 3 6 0. 7 2 7 2 7 3 ""07652174" "07 7 50000" "07-8000000. 7 5 0 0 0 0 0. 634211 0. 6 1 9 0 4 3 0. 5 4 5 4 5 5 0. 6 3 1 5 7 9 0. 684211 "07-66666 7" -0752"r739 0. 5 9 0 9 0 9 0. 68471 1 0. 7 3 6 8 4 2 0. 6 1 7 0 5 9 0. 73634 2 0. 7 6 1 9 0 5 0. 6 9 5 6 5 2 0. 5 6 0 0 0 0 07 6 2 5 0 0 0 "~0.~71'4?.86'~ "0".""3'500'0'0'' " 0 7 6 8 4 ? IT 0. 7 7 7 7 7 8 0. 7 0 5 8 3 2 0. 6 5 0 0 0 0 0.590909 0.521739  0. 7 6 0 0 0 0 -07-6T53T3TJ 0. 7 7 2 7 2 7 0. 6 6 6 6 6 7 "07 6 3 6 3 6 4 O. 6 1 5 3 3 5  (.1. 5 / 1 4 2 V 0. 7 0 3 3 3 3 0. 5 6 0 0 0 0 0. 6 0 8 6 9 6 0. 5 9 0 9 0 9 -07652174- "0.-565217 0. 692.303 0. 4 4 8 2 7 6  OrSSOOOCT—O7"6"10O0O~ ~ur>ppnrTA  UTTWZBB—077454-51  0. 6 0 0 0 0 0  0. 6 3 0 0 0 0  0. 4 8 1 4 3 1  0. 6 0 8 6 9 6  O. 5 0 0 0 0 0  0. 5 4 5 4 5 5  0.666667  0.608696  0.590909  0.600000  0.647059  0.611111  07"4-6"r5'T8  07-590909—07"4T6T9O—0775233T0~  5- -07-500000  -  —  r  17  0. 6 0 0 0 0 0 0 7 5 7 1 4 29" -07"9333330. 7 7 2 7 2 7 0. 521 739 0. 565217  0.576923  0.30957.4  0.619048  0.666667  0. 5 0 0 0 0 0  0. 6 3 6 3 6 4  0. 5 2 3 3 1 0  0. 5 7 1 4 2 9  -  20  07-65-2T74— ~07"342103" ~0. /22772 O. 5 9 0 9 0 9 O. 5 2 0 0 0 0 0. 6 6 6 6 6 7 O. 4 7 6 1 9 0 •O7"75OO0Cr- "07700000~ 0. 5 4 5 1 5 5 0. 6 3 1 5 7 9 0. 5 3 3 2 3 5 0. 6 3 1 5 7 9 0. 7 6 4 7 0 6 0. 7 6 1 7 0 6 0."7?58 3 3.T" ~Q. 6OO0OO" 0. 7 7 7 7 7 8 0. 5 5 0 0 0 0 0. 7 2 2 2 2 2 0. 5 0 0 0 0 0 0_565217 0. 5 2 3 3 1 0 0. 59~0?"0"9~""I~"84"2~l"0?~  0r>J2"6-3T?T 0. 6 8 7 5 0 0 0. 5714 29 ~07'7Z22Z7~  0. 6 0 8 6 9 6 CrzrpVtTtS  0.521739 07"777773"  0.600000 07"E3"i  ~57"8"0750W"  0^600000 0. 8 0 0 0 0 0  0.680000 0:~6"956"5'2  0. 4 8 1 4 8 1 07947368  0.6818)3 0. 5 0 0 0 0 0 0. 515155 0 7 5 6 5 2 1 7 " '0"7'7T4236" 0. 6 0 0 0 0 0  0. 5 6 5 2 1 7 0.634211  0. 6 5 2 1 7 4 0.750000  J65217  0. 736342  0. 526316  0. 578947  0.650000  0.777778  0. 7 7 7 7 7 8  0. 7 5 0 0 0 0  0^ 5 2 0 0 0 0 0. 8 1 2 1 0 5 "  0. 5 9 0 9 0 9  0. 5 5 0 0 0 0 0. 8 8 8 8 3 9  _0. 6 0 0 0 0 0 PT "764706"  0. 2 6 3 1 5 3  0. 315789 0. 533333  r  -  orsooooo""  0.5217;  T~~750~~Cr TX"72  0. 7 5 0 0 0 0 0. 7 7 7 7 7 3 0. 7 0 0 0 0 0 O. 8 0 9 5 2 4 0. 7 3 9 1 3 0 0.600000 "07:313132" 0: 9 4 7 3 6 8 " "07"9"0d'00"0'*" "07"571'4?9 0. 7 3 6 3 1 2 O. 7 6 1 9 0 5 0. 7 3 6 3 4 2 0. 5 7 3 9 4 7 0. 6 1 9 0 4 8 0. 714236. 0. 5 6 5 2 1 7 0. 6 5 0 0 0 0 ™ 0. 6 1 9 0 4 8 0. 7 0 5 3 3 2 0. 7 0 5 8 8 2 0. 6 5 0 0 0 0 0. 7 0 5 3 8 2 0. 61 9 0 1 3 0. 77T^778_ 0. 8 0 0 0 0 0 '"a" 6 5 7 1 7 4 ""67 7 5 0 0 0 0 " "0'~394737"' 0 /2227.7 0. 8 2 3 5 2 9 0. 9 4 4 4 4 1 0. 8 2 3 5 2 9 0. 8 0 0 0 0 0 27 0. 4 7 0 5 3 3 0. 5 7 6 3 1 6 0. 4761_90_ 0. 3 4 7 S 2 6 0. 476190 0. 5 5 5 5 5 6 ~0. 5 2 6 3 1 6 0. 4 1 1 7 6 5 0. 600000 0. 5 5 5 5 5 6 0. 5 0 0 0 0 0 0. 5 2 6 3 1 6 0. 368421 0. 428571 0. 3 9 1 3 0 4 0. 2 3 0 0 0 0 "07391304" "07450000" 0. 428571"" "0731578:'" 0. 5 6 2 5 0 0 0. 4 5 0 0 0 0 0. 3 3 3 3 8 9 0. 428571 0. 312500 0. 315.789 0. 2 3 5 7 1 4 0. 2 2 7 2 7 3 "CT2837T4~ 0. 3 3 3 3 3 3 0. 3 1 5 7 8 ? 0. 3 3 3 3 3 3 0. 333333 0. 3 3 3 3 3 3 0. 428571 0. 3 1 5 7 8 9 0. 470533' 0. 5 2 6 3 1 6 0. 4 7 6 1 9 0 0. 3 1 7 8 7 6 0. 4 0 9 0 9 1 " "0. 4 7 3 6 3 V 0 7 5 2 6 5 1 6 07 4117 6 5 0. 714286 0. 5 5 5 5 5 6 0. 5 0 0 0 0 0 0. 5 2 6 3 1 6  :  0._666667_ "0. 681 818  0. 63421 1 0. 3 4 7 8 2 6 0. 3 7 5 0 0 0 .0. 2 9 1 6 6 7 0. 5 3 8 2 3 5 ~0". 5 0 0 0 0 0 " 0. J5556 0. 5 6 2 5 0 0 0. 5 3 3 2 3 5 0. 2 8 0 0 0 0 0. 3 0 7 6 9 2 0. 2 3 0 7 6 9 "07473631 ' 0."409091" 0 7 4 5 0 0 0 0 0. 4 4 4 1 4 1 0. 4 7 3 6 8 1 0. 7 5 0 0 0 0 0. 2 2 7 2 7 3 0. 2 6 0 8 7 0 0. 2 2 7 2 7 3 ' "0. 332941"" 0. 3 0 0 0 0 0 0. 4 0 0 0 0 0 0. 3 5 2 9 4 1 0. 6 0 0 0 0 0 0. 3 1 7 8 2 6 0. 3 7 5 0 0 0 0. 2 9 1 6 6 7 "0. 5 0 0 0 0 0 " " 0 7 5 0 0 0 0 0 " "07 5 5 5 5 5 6 ' 0. 4 7 0 5 8 8 0. 5 8 8 2 3 5 0. 6 6 6 6 6 7  0. 47365  0. 421053 0^ 3 8 0 9 5 2 0. 3333:33  0. 5 5 5 5 5 6  0. 583735  0. 5 2 3 3 1 0  0. 555556  0. 5 2 6 3 1 6  0. 190476_ 0. 23S093_ ' 0 7 4 7 3 6 8 4 "0'411765  0. 3 3 3 3 3 3 0. 2 S 0 0 0 0 0. 352941 0. 35294"! 0. 4 1 6 6 6 7 0. 4 0 0 0 0 0 0. 3 3 3 3 3 3 0 7 3 5 0 0 0 0 " 07500000'" 0. 6 1 5 3 8 3 0. 451545  0. 235294 07TM538~  0. 3888S9 07437500"  - 196 -  APPENDIX LVIIl Matrix of S i m i l a r i t y C o e f f i c i e n t s  Calculated f o r  30 OTUs by Simple Match C o e f f i c i e n t : High C o n c e n t r a t i o n s  8 9 TCT 11 12 13 14 ~T~ 16 17 18 ] 19 20  22  124.  | J28  29  30  0. 9 1 3 0 1 3 0. 8 6 9 0 6 3 0. 069:365 0. & 6 9 S 6 5 0. 7 8 2 6 0 9 ~0. 7 3 9 1 3 0 0. 3 1 7 3 2 6 0. 8 4 7 8 2 6 0. 8 2 6 0 3 7 0. 7 8 2 6 0 9  0. 9 3 6 3 2 2 0. 9 1 3 0 4 3 0. 8 6 9 3 6 5 0. 9 1 3 0 1 3 0. 0. 3 6 9 3 6 5 0. 9 1 3 0 1 3 0. 9 1 3 0 1 3 0. 3 6 9 5 6 5 0. 7 3 9 1 3 0 ~6~78^609 67739130 0. 3 9 1 3 0 4 0. 9 3 4 7 8 3 0. 891304 0. 3 4 7 8 2 6 0. 8 1 7 3 2 6 0. 8 4 7 8 2 6 ' 07 8 6 9 3 6 5 07 3 6 9 3 6 5 ""0. 826037" 0. 3 6 9 3 6 5 0. 8 6 9 5 6 5 0. 7 3 2 6 0 9  0. 8 6 9 5 6 5 0. 7 3 9 1 3 0 0. 7 3 2 6 0 9 0. 8 4 7 8 2 6 0. 7 6 0 8 7 0 0. 3 9 1 3 0 4 0. 8 0 ' ! 3 4 8 0.804348 0.82603/ 0. 8 4 7 8 2 6 0/826037' " o r 8 2 6 0 3 7 - " 0. " 7 8 2 6 0 9 6 7 8 0 1 3 4 8 " 07 973261 0. 8 2 6 0 8 7 0. 6 5 2 1 7 4 0. 3 9 1 3 0 4 0. 7 6 0 8 7 0 0. 7 8 2 6 0 9  0. 7 8 2 6 0 9  a 391304 0.347826 0. 7 6 0 3 7 0 ~ 0. 7 6 0 S 7 0 0.304348 0.673913 0.869365 0.739130 0.760870 0. 3 4 7 8 2 6 0. 9 3 1 7 3 3 782609 _0. 326087 0. 6 9 5 6 5 2 p. 8 0 4 3 4 8 0. 760870 0. 782609 0.913043 0. 913043__0._86936S_ 0 . 7 8 2 6 0 9 0. 3 6 9 5 6 5 •'"07934783 "'"' 847826 0.801348 0.804318 0.826037 0.869363 0.817826 0. 9 3 4 7 3 3 0. 3 4 7 8 2 6 0. 8 4 7 3 2 6 0. 8 4 7 8 2 6 0. 3 4 7 8 2 6 0. 7 1 7 3 9 1 0. 7 8 2 6 0 9 0. 7 8 2 6 0 9 0. 6 9 5 6 5 2 0. 8 0 4 3 4 3 0. 7 6 0 3 7 0 0. 7 8 2 6 0 9 0.. 882266008877 67369-.6S 0 0 . 7 3 9 1 3 0 "07739130 0. 8 6 9 5 6 5 0. 9 3 1 7 3 3 0. 9 3 6 5 2 2 0. 8 1 7 8 2 6 0. 8 6 9 3 6 5 01_8/j782& O. 760870 0. 913043 0. 826087 0 _847326 0. 8 1 7 8 2 6 0. 3 9 1 3 0 4 0. 9 3 4 7 3 3 0. 8 4 7 8 7 6 0. 8 0 4 3 1 3 8 2 6 0 8 7 " " 0". 801343" '67326037"' "0." 3 0 4 3 4 3 " "6." 3 9 1 3 0 4 804348 0.717391 0.32608/ 0.782609 804348 0. 7 6 0 8 7 0 0. 3 9 1 3 0 1 0. 8 1 7 8 2 6 0. 7 6 0 3 7 0 0. 3 4 7 8 2 6 869565 0. 9 3 1 7 8 3 0. 9 3 6 5 2 2 0. 9 3 4 7 8 3 0. 826087 0. 8 9 1 3 0 4 0.931783 0.891301 0. 8 6 9 5 6 5 0. "869565 0.869365 0.' 8 6 9 3 6 5 " 0. 8 2 6 0 8 7 0. 8 2 6 0 3 7 " "6. 8 6 9 3 6 5 ~ 0 7 9 1 3 0 4 3 0. 8 0 4 3 4 3 0.804343 0.782609 0.891301 0.32608/ 0.891304 0. 8 2 6 0 8 7 0. 8 6 9 5 6 5 _0._86936 • J S 2 6 0 9 _0,_7S2609_. ._JL.S26037__0. 7 3 9 1 3 0 O. 86'; Q, 7 6 0 8 7 0 0. 7 3 2 6 0 9 0. 8 6 9 5 6 3 0.936522 0 . 8 4 7 8 2 6 0. 8 9 1 3 0 4 0 ." 8 9 1 3 0 4 '. o7s26"087 0~8913d4~""' 0. 9 1 3 0 4 3 0. 8 2 6 0 8 7 0. 8 4 7 8 2 6 0. 8 4 7 8 2 6 0. 7 8 2 6 0 9 0. 8 6 9 5 6 5 0. S 2 6 0 8 7 0. 7 8 2 6 0 9 0. 7 8 2 6 0 9 0. 7 8 2 6 0 9 0. 9 1 3 0 1 3 0. 9 1 3 0 1 3 0. 9 1 3 0 4 3 0. 84 7 8 2 6 0.347326 0.369563 0.934783 0.913043 0..304348 0. 7 3 2 6 0 9 0. 8 0 1 3 1 8 782609 826087 0. 7 6 0 3 7 0 0. 7 1 7 3 9 1 0. 3 9 1 3 0 4 0. 8 4 7 8 2 6 0. 7 6 0 3 7 0 0. 7 6 0 8 7 0 0. 3 9 1 3 0 4 0. 891 304 891304 804318 0. 8 6 9 3 6 5 0. 9 1 3 0 1 3 0. 3 6 9 3 6 5 0. 3 9 1 3 0 1 0. 8 6 9 3 6 3 0. 8 9 1 3 0 4 7826019_ .Q,_801318.. :2609_. 0=. 0^391304 0. L.1782.6 0 76 0870 0^760870. _ a.801.34 8 O. 8 9 1 3 0 1 0. 8 9 1 3 0 1 0. 8 0 1 3 1 8 0. 9 3 4 7 3 3 0. 8 2 6 0 8 7 0. 9 1 3 0 4 3 0. 3 6 9 3 6 5 0. 9 3 4 7 8 3 0. 8 6 9 3 6 5 0. 9 3 4 7 3 3 0. 8 0 4 3 4 8 0. 9 1 3 0 1 3 0. 8 4 7 3 2 6 0. 8 0 4 3 4 3 0. 7 6 0 3 7 0 0. 7 6 0 3 7 0 0. 8 0 4 3 4 3 0. 7 6 0 3 7 0 0. 8 2 6 0 8 7 0. 3 2 6 0 8 7 0. 804318. .0. 3 9 1 3 0 4 _ 8 _9 _ _1_3_0_4 .2 _6 .0 —3 -7 __•_8 1 .—. -t 6 t \ I 0.891304 i~i 1 •* • r~\0/l . 9ft 891304 0. 9 1 3 0 4 3 0. 0_ . 8 0___. 9.-.1 3 0 1.i 3 y-. 0 . 7 8n 2 3 4 7O'.'/! 33 / C ' ? 0. 8 6 9 3 6 5 0. 8 0 1 3 4 3 0. 9 1 3 0 1 3 0. 9 1 3 0 4 3 0^847826__0^608a0___J? .782609 . 0._8J.9565.. _0. 9 1 3 0 4 3 _ 0 ^ 8 6 9 5 6 ^ _ j 3 . _ 7 S 2 c ^ _ _ 0 _ 7 i _ 3 . 6 0 9 _ ^ a 0. 7 8 2 6 0 9 0. 8 6 9 5 6 5 0. 3 2 6 0 8 7 0. 8 4 7 8 2 6 0. 9 7 8 2 6 1 0. 9 7 3 2 6 1 0. 9 5 6 5 2 2 0. 304.313 0. 9 1 3 0 4 3 0. 9 1 3 0 4 3 0. 8 9 1 3 0 4 0. 8 9 1 3 0 1 0. 8 9 1 3 0 4 O. 7 8 2 6 0 9 0. 7 S 2 6 0 9 0_S5l6£____2 QJZS2&.Q2 (UtSlS-OA Jj_..826CJE___ •O. 8 2 6 0 8 7 "P. S 6 9 S 6 S 0. 8 6 9 5 6 5 0. 8 9 1 3 0 4 0. 8 4 7 8 2 6 0. 9 1 3 0 4 3 0. 9 1 3 0 4 3 0. 9 1 3 0 1 3 O. S 4 7 S 2 6 0. 3 2 6 0 3 7 0. 391304 0. 8 4 7 8 2 6 O. 3 9 1 3 0 1 0. 8 2 6 0 8 7 0 . 9 1 3 0 1 3 _ 0. 8 6 9 3 6 5 „0^7S26.09.___.0.,_782.609-.__0._.826037 .0.773.9.1.3.0. 0. 302..343 0._8Q13.4.& Q_S260.SZ_ 0. 8 2 6 0 8 7 0. 9 3 6 5 2 2 0. 9 1 3 0 1 3 a 891 S O l " 0 . 9 3 6 5 2 2 0.891301 0.95652.2 0.847826 0.934733 0 973261 0. 9 3 4 7 8 3 0. 9 1 3 0 4 3 0. 8 6 9 5 6 5 0. 7 8 2 6 0 9 0 6936"-' O. 717391 0, 7 A O « 7 0 0. 6 7 3 9 1 3 0. 6 7 3 9 1 3 .0_-6:/.3i_13. ft 6 3 0 4 3 5 0. 8 0 4 3 4 8 0. 8 4 7 8 2 6 0. 8 1 7 3 2 6 0. 7 6 0 3 7 0 0. 8 2 6 0 8 7 0. 7 8 2 6 0 9 0. 8 1 7 3 2 6 0. 7 8 2 6 0 9 0. 8 2 6 0 8 7 0. 8 0 1 3 4 8 0. 7 6 0 8 7 0 O 826087 0. 8 0 4 3 4 3 0. 8 1 7 8 2 6 0. 8 1 7 8 2 6 0. 3 2 6 0 3 7 0. 3 6 9 3 6 5 Q_jS__J04_S_ A-&XXZ&0. 7 3 9 l 3 0 _ 0. 7 3 9 1 3 0 0. 6 9 3 6 5 2 _ 0. 608696. 0. J . 0 3 6 9 6 ._0.._60_8696_. _0._S65217 O J 17391 6.739130 0.717391 0. 7 S 2 6 0 9 0.717391 0. 7 6 0 8 7 0 "o7"693652 6. 7 6 0 8 7 0 0. 6 9 5 6 5 2 0. 7 8 2 6 0 9 0. 7 8 2 6 0 9 0. 7 3 9 1 3 0 0. 7 8 2 6 0 9 0. 7 8 2 6 0 9 0. 9 3 4 7 8 3 0. 7 6 0 8 7 0 0 7 6 0 8 7 0 0. 8 4 7 3 2 6 0 . 6 7 3 9 1 3 0.63043.J 0.63043!. 0. 6 3 0 T 3 3 . 0. 7 6 0 8 7 0 0. 7 1 7 3 9 1 0.630435 0.739130 0.739130 0.717391 0. 7 6 0 S 7 0 0. 8 4 7 8 2 6 0 739130 0.717391 0. 7 8 2 6 0 V 0.760370 0.693632 0 . 7 3 9 1 3 0 0. 7 6 0 8 7 0 0. 6 7 3 9 1 3 0. 8 1 7 8 2 6 0 . 7 3 9 1 3 0 0 . 8 2 6 0 3 7 0.717391 0 . 8 0 4 3 1 3 0 . 7 6 0 8 7 0 0 . 9 1 3 0 4 3 0. 7 8 2 6 0 9 P,7/3?J30.__i?J60870... 0 804318 0. 7 6 0 8 7 0 0. 6 7 3 9 1 3 0. 6 7 3 9 1 3 0. 6 7 3 9 1 3 .0,,630435. 0. 7 3 9 1 3 0 0. 8 0 4 3 4 8 0. 7 1 7 3 9 1 0. 8 0 1 3 1 8 0. 8 0 1 3 4 3 07 7 8 2 6 0 9 0 . ' 8 0 1 3 4 S " 0. 7 8 2 6 0 9 " 0. 8 0 1 3 1 3 0. 7 8 2 6 0 9 0. 8 2 6 0 8 7 0. 717391 0. 8 6 9 3 6 5 0 826087 0. 8 2 6 0 3 7 0. 8 0 1 3 4 8 0. 3 0 4 3 4 3 0. 8 4 7 8 2 6 0. 9 1 3 0 4 3 0. 8 9 1 3 0 1 0. 9 1 3 0 1 3 :  _a_81782&  1  ;  APPENDIX LIXV -  Matrix of S i m i l a r i t y C o e f f i c i e n t s Calculated f o r 30 OTUs. by Jaccard C o e f f i c i e n t : A l l Concentrations  r""~ .  2  3  1 11 21  4 14 24  13  12  6 16 26  15  7 17 27  8 18 28  9 IV 29  197 "  10 20  0. 791667J 0. 730769  0. 783714  '"0." 826037"~o: 74074* f" 0.' "689653"*' 5  0, 730769  0. 703714  0. 733333  0. 881613  6  0. 666667  0. 724138  0. 793103  0. 814315  07640000" 0 586207  ' 07 313337  ' "7  S ' 0. 850000  _.b'bbbo""  646667 '" a 600000*' " a  0. 692308 _0. 78260V  0. 692308  0. 629630  0. 600000 0. 636361  0. 600000  9  0. 736812  0.  0. 538462  0. 603696  0. 538167  0. 338162  10  0. 727273  0. 653316  0. 607143 7 ™ 615383  0. 607143  0. 607143  0, 610000  0. 608696  0. 736842  11  0. 666667  0. 666667  0. 7 33333  0. 580645  0. 6 2 3 0 0 0  0. 6774 1V  0. 451513  0. 629630  0. 4814S1  0. 666667  0. 680000 _0. 678571. "6."8S4615  0. 750000  0. 336207  0. 633333  0. 689655  0. 451613  0. 703333  0. 480000  0. 615335  0. 807692 0. 769231  0. 311815  0. 703701  0. 7 5 0 0 0 0  0. 750000  0. 531774  0. 708333  0. 608696  0. 680000  _12_  i  0. 680000  "a  0. 8 3 7 1 4 3  13  0. 326087 0. 314315  0, .613385. _<?. 675000 538162 0 500000 " 6. 625000  _ J . -..P.-666667 _ 0 4  0. 500000  i 15 1  0 629630  0 625000 0. 703704  0 720000-  0 703701 0. 720000  „P. _53.5556_ ..0  0 33:3714  0 33___Q___  ,615385.._..0 _5_83333_ JX_6.190.48 __,-_ 573?47_ _<_• 590909  0 612837  0...863636. 0. 769231.. 0,.777.778 . 0. 800000_.. 0.. 7 77.77.S_ 0...714286 O. 6133S5 O. 714286 6. 64.6667 0. 375000 " 0.7,52 J 74 O. 708333 _0__703_10A__J?_-J77_777J3._ _0_--.<_ZL_.. 0. "75900 0. 800000 0. 8 4 6 1 5 4  19  0. 826087  0...6.29_:.30.  _.0_'7142.._. .0.._..66_..&7 0. 692303  CL.S.1.8.1.8.2 .0._63.6361. _0. 7.08333— O._6.6.66.6.'„_0__3_O0.0.0.Q  .0. _-7_6._2.___  0.,730769... 0. 826.0-7.__0. .730769-.0. .730769_ -0..708333.. .0. .830000.....0..7-684 2..._0._/2727_-_ 0. 863636 O. 640000 O. 560000 0. 730000 0. 750000  O. 782609. 0___i„ 31 O. 714286 0. 730769  0. 81.___54_ 0. 875000  0. 730769 0. 727273  0. 777778 0. 730000  O. 789474 0. 625000 O. 518319 "" 0. 5S3333  0. 576923 0. 652174  0... 6.521.74 .0. 722722  0...576923.. 0. 576923 .. .0. 603696, ..0.-.736842... 0._703S32 .0. 7 00000. 0. 681818 0. 631318 0. 608696 0. 789474 O. 590V09  O 600000 6T"560000  O. 615380,__0._ 625000 0. 625000" 0. 777778  0. 652174 0. 633346 0 66666'/ " 0. 666667" 0. 615333 " C. 750000 0. 750000  ; 24 0. 750000 0. 693652  _a_..07,__ 0. 769_31 0. 750000  0. 355556 0. 750000  0. 6 1 5 3 8 5 "O. 7*14286  20.-777.7_/8_ _0._629630.. O. 739130 0. 333333 O. 760000 0. 782609  536 "O. 727273  0. 607143. O. 607143 0. 8 3 7 1 4 3 o7_10000"  O. 520000 0. 632174  0. 700000 0. 666667  0-._63636__. J__.7_?i_3_l3_  0. 500000 _O_590909_ O. 727273 0. 6*31379  0. 464286. 0. 511.667.__0._500.QOO_ _0._63717 4_ 0. 703333 6. 583333 6. 782609 " 6. 619018  0. 500000 0. 625000  0. 521739 0. 636364  0. 350000 0. 695652  0. 365217 0. 684211  0^81481.5_ _0._607_143 0. 769231_ _qj314S 1.5 O. 8316IE ' 6. 72000b' 6. 875000 0." 800000 0 71666'/ **0. 693652 0. 6 6 6 6  0^732609 0. 826037  0. 608696 ,6572 0  0. 630000 0.637174  0. 666667 "6. 730769 0. 703333 0. 695652  0. 576923 0. 610000  O. 681818 0. 727273  07571429 0. 863636  0. 632174 0. 700000 0. 5S3333 0. 619048  O. 576923 0. 592393 0. 538462. 0. 666667 0. 800000  0. 636361 0. 533714 0. 650000  .38167 333333  "67652174* 0. 607143 0. 666667  0 653316 " 0. 730769 "0. 615333 0. 750000 0. 750000 0 680000 0. 714286 0. 826037 0. 727273  0 652174 O. 666667 ' O. 750000  0. 653316 0. 680000 O. 900000  0. 5 9 2 5 9 3 0. 761903  592593 560000  0. 555556 0. 730000 0. 750000  0. 607) 43 0. 950000 0. 727273  O. 666667 O. 640000  0. 464286 0. 782609  0. 511667 0. 583333  0. 500000 0. 782609  0. 619043_C^5600JX\. 0_. 31 S 3 ) ' ? , P. 520000 0. 464236 O. 161533 " a " 533333 0. 631579 0. 72.772 0. 700000 0 600000 0. 583333  0.318319 0. 666667 0. 5 4 5 1 5 5  0. 464286 0. 608696 O. 700000  0.423077 a 511*667  0. 500000 O. 315155  0. 450000 _0_5154__i_ Oj?.' 0.608696 0. 578917  0. 573!. 10 0. 431783  0. 0. 500000  0. 4 3 S . 8 3 0. 526316  0. 291667 0. 0. 317826 . 0. 317826 0. 400000 " 0. 500000  0. 0. 352941 0. 421053 0. 400000.. 0. :47S26 0. 612837  0. 333333 0. 300000  0. 461338  0 513153.. 0. 0. 652174 0. 590909 0. 4703SSS 0. 761706  0. 590909 0. 631579  0. 730769 0. 750000 0. 800000  0. 433077 0. 42837 l' 0. 379310 0. "6. 500000 '0.46 1338 0. 428371 0. 500000 0. 5 4 5 4 5 5 0. 300000 ' 0. 300000 0. 480000 0 480000 0. 423571 703882 0._666667 ____.5Zi.429_ _9, 476190_ _o_ 4 8 0 0 0 0 . , _ _ • _5000po_ _P: _37_1129, _o.  •|_..:  o__-._o.ab_o  0_900000_ _Q, 720000 0. 615383 o7607143  0. 6666.67 ' 0. 553556  27  0 148_7.6___C-__520.0_OQ .0._4.7_826J_  0 714286  0. 296296 0. 330932  0. 296296  0 360000 0 5 3 5 5 5 6 6 0 0 0 0 0 _ 0. "•.'•.',•.56 ' 0 625000' 0 68 '• 21 1 " 0. 7 1 4 286 0 . , 5 0 0 0 0 0 6-5000 0 5 9 0 9 0 9  0. 500000 0. 652171 0. 750000  0. 400000 0. 320000 0. 296296. 0. 333333 0. 170503 0 100000 0. 666667 _ "6. 300000  0. 296296 a 333333 .0. • . 1 4 1 4 1  0. 333333 0. 4 70588 0. 333333  b. 100000  0. 0. 937300  APPENDIX LX  - 198 -  —. -Matrix—of-Similarity-Coefficients Calculated f o r 30 OTUs by Simple Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s -  *  1 11 21  2 12. 22  _2  0. 891304  '3  0. 84 7826  .*.+* »  s  3  <?TKs, Uk»le  0  13  5  O. 847826  0. S 6 9 S 6 g _ 0 . 826087  0. 934783  0. 804343  0. 826087  0. 391304  0. 869365  ~ '° 'S°4348"'"~07~73?^  3  18 28  10 20  29  _ 8 _ 0 . 934783  0.826087  0.891.301  0. 913043 "''0:7391.30'"" 07739130  :  9  7 17 27  0. 869565  0. 913013 '" 6. 817826'"' 0. 804343 '  7  r  16 26  4  6  A _ * , S;~ .&_ AvicU  4 14 24  0. 8260S7_,0. 391304  0.782609 .0.739130  0.804348  0.826037  0.782609  0. 782609  0.739130  0.739130  0.826087  0.82608/  jTT--b:m956S---re04348--o; 760870-b:'782609" ' 0. "760870 "~07760870 '767 801318"" a'804348 ^ " ^ T ^ l " II  -  f  ^  0.804348  -  -  ^  f  g  Q.,732609  ^  ^  l  ^  ^  0.826037  0.717391  O. 7 3 9 1 _  -  ° ^ °  0-^-^-0-47826  ^  '  ^  "0-71 7 3 9 ? " o : 8 0 4 3 4 - - " - -  4  8  S  7  , S  -- ° ^ '  S  p. 869365 rt  U ^ . ..0,934783 _ 0,369565 j 0. 826087 6. 804343 1  8OJ304  1  3  0  -  o v ^ c  0  -  7  2  3  24  ^  ° ° 0*1304-  0.913013  0.891301  i  :  S  7  ^  0 •=•/-•=.".,< 5  ^  .  0  0.8043,8  C,^^  .. 913.043__a_826087 _p._S4787.6,  ^  1  ^  ^  ^  6087  0--84j'-32_  » ' - ^  s  °7 ®^!T 0 8478-6  7  ^  / 8 2 & 0 9  0.847826  0. 931783"" 0.'326dS7""'"o.7S26oV"'  0.347326  " 15:5 *  0.71739.  ,-  ^ ° * - 0...78>609_ .0. 8 2 6 0 3 7 _ _ a 826087 .. 0..804 3,8_  ^ - - O - ^ - ^ O I — 0 _ ^ . 9 3 6 5 _ CX-9.13043_0.-34-/826_0.869365—O^S6  0.326037  0  0  0  ^370 ° - ° 7  S  : 6  : 3 V  0.934783  0. 72:9130 a  V  3  /  !  7  8  0. 760370 0 891301  3  0.869565  0. 7 6 0 8 7 0 0. 7 6 0 8 7 0  0.891304  0.891301  0. 717391 0.801318  0. 760870 0. 826087  0.760870  0. 804348 0. 847826  0.89130.1  0 7S"60* o! 869363  0. 801348  0 826037  o.jtltH o.ii^jl  „ | j g ?  0. 826037 0^804343 0. 760870"" 6. 826037 0. 847826 0. 869563  0. 847826 0. 78260? 0. 801343 0.""869565"0. 891301"'~07'847876" 0. 869565 0. 913043  a 847826 _0. 760870 a 817326  6.' 347826' "oTS01348" "0. "869365"  0± 30434S 67 934783  0. 82608"/ 0. 869365  26  0. 826087" 0. S0434S 0. 80131S 0. S260S7 0. 891304 0. 956522  0. S47S26 0. 869565 0. 9130'.3  0. 801318 0.801318  0. 673913 0.891304  0. 760870 0.782609  0. 760870 0.891301  0. 782609 0. 826037  27  0. 826087 0. 760870 0^ 673913 0. 6956S2 6.891301" 0. S69363  0. 717391 0. 739130 0. 717391 0. 673913 a 782609 _0. 8 4 7 8 ? 6 _ 0 . 817826 _ O, 804318 6.S26CS7" 0~7S2609 67732609 0. 8 6 9 3 6 5  0. 673913 0. 760370  0. 760870 0. 782609  0. 760870 0. 804348  0. 782609 0. 826037  a  8  ,  7  8  3  6  0. 739730 0. 891301 0. 869365  .0, 760870 0.695652 _0. 652174 .0, 673913 a' 632174" 0. 7173*1" 67 71739l'~ 0. 732609 0. 86V565 0. 804318 0. 760S70 0. 717391 •29  0. 739130 0. 386937 0. 801348  30  0. 847826 .0 693657 0. 918013  ft8478ie6  0. 760870 0. 978261 0. 369365  a  *  3  *  7  8  3  ft8M  *"  0  9  1  3  0  ,  1  3  .0. 652174 , 0. 6 0 8 6 9 6 _ .0. 608696_.._0,782609, .p..693602._ 0, 71 7391 0. 782609 0. 7 3 9 1 3 0 " 0. 739130 0. 717391" 0. 739130 0. S01318 0. 760870 0. 801318 0. 891304  0. 430435 0. 5 6 9 5 7 0.. 652 f? 4 0. 652174 0. 652174 0. 801318 0. 739130. .0,732609. 0. 652174  0. 586937 0. 717391 0. 739130  0. 586957 0. 630133 0. 760870 0. 673913 0. 673913 0. 739130 0 , 7 3 9 1 3 0 . .0. 826087 . 0. 891304  0. 73760? 0, 7173'1 0. SV1304  0. 739130 0. 369363 0. 801313  0. 693657 0. 826087 0. 891301  0 73»i;:0 0. 760370 0 80 * 7 <8 .0. 369365 "0 3478:6" 0. 801318  0. 760870 0. 673913  0. 693652 0. 826087  0. 695632 0. 782609 0. 782609 0. 804318 0.. 782609__0. 801318. .0. 626037_0.817826 0. 978:61 0. VI3013 0. 80131S  -  199 -  APPENDIX LXI Matrix of S i m i l a r i t y C o e f f i c i e n t s  Calculated f o r  18 OTUs by J a c c a r d C o e f f i c i e n t : High  Concentrations  ( 16  13  7 17  2  1 1  12  4 )5  10 1 4  ]  9  !8  0. 4 3 0 0 0 0  >-— ^  7  0. 4 4 4 4 4 4  0. 6 5 5 1 7 ?  0. 6 6 6 6 6 7  0. 5 6 0 0 0 0  0. 5 1 3 3 ) 9  5  0. 7 3 6 3 1 2  0. 5 5 5 5 5 6  0. 5 1 7 2 1 )  0. 7 5 0 0 0 0  4  0. 3 5 7 1 4 3  0. 4 0 0 0 0 0  0. 4 2 3 0 7 7  0. 6 4 7 0 5 9  0. 6 3 1 5 7 9  10  0. 6 3 1 5 7 ?  0. 6 0 0 0 0 0  0. 6 1 5 3 8 3  0. 7 3 6 3 1 7  0. 7 1 4 2 8 6  0. 6 1 1 1 ) 1  1  0. 3 2 3 5 2 9  0. 428571  0. 4 0 0 0 0 0  0. 5 7 1 4 7 9  0. 6 3 6 3 6 1  0. 7058.37  0. 5 4 5 1 5 5  0. 5 2 6 3 1 6  0. 5 7 0 0 0 0  0. 6 0 0 0 0 0  0. 6 3 1 5 7 9  0. 6 1 9 0 1 8  0. 5 0 0 0 0 0  0. 7 7 7 7 7 8  0. 4 5 1 3 1 5  IS  0. 4 6 6 6 6 7  0. 2 9 1 6 6 7  0. 269231  0. 4.1 1765  0. 3.50000  0. 428571  0. 3 8 8 8 8 9  0. 1 7 0 3 8 8  16  0. 7 1 4 2 8 6  0. 4 3 4 7 8 3  0. 4 0 0 0 0 0  0. 6 2 5 0 0 0  p. 5263.1 6  0. 6 9 2 3 0 8  0. 5832.35  0. !. '83783 0. 4 7 0 3 8 8  0. 6 3 6 3 6 1  13  0. 2 6 6 6 6 7 0. 3 6 3 6 3 6  0. 2 i 7 3 9 1  0. 2 0 0 0 0 0  0. 3 ) 2 5 0 0  0. 2 6 3 ) 5 3  0. 3 0 7 6 9 2  0. 2 9 4 1 1 8  0. 2 2 2 2 7 7  0. 250000  0. 414 411  17  0. 2 0 0 0 0 0 0. 2 7 2 7 2 7  0. 1 7 3 9 ) 3 0. 8 0 0 0 0 0  0. 160000  0. 2 5 0 0 0 0  0. 2 1 0 3 2 6  0. 2 3 0 7 6 9  0. 2 3 5 2 9 4  0. 166667  0. 187300  0. 3 3 3 3 3 3  1 1  0. 4 4 4 4 4 4 0. 5 7 1 4 7 9  0. 5 2 1 7 3 9 0. 4 1 6 6 6 7  0. 4 3 0 0 0 0 0.  0. 473684  0. 4 0 9 0 9 )  0. 5 0 0 0 0 0  0. 4 5 0 0 0 0  0. 3 8 0 9 5 2  0. 35CCC0  0. 4 7 8 3 7 j  12 • 0. 5 0 0 0 0 0 0. 6 4 2 8 3 7  0. 5 6 5 2 ) 7 0. 384 6 1 5  0. 5 2 0 0 0 0 0. 3 0 7 6 9 2  0. 5 7 6 3 1 6 0. 9 2 3 0 7 7  0. 4 5 4 5 4 3  •0. 4 7 0 5 8 8  0. 3 0 0 0 0 0  0. 128571  0. 4 0 0 0 0 0  0. 5 0 0 0 0 0  15  0. 4 2 8 5 7 1 0. 6 0 0 0 0 0  0. 2 6 0 8 7 0 0. 5 7 1 4 2 9  0. 2 4 0 0 0 0  J L 47837!  0. 3 7 5 0 0 0 0. 500000_  0. 3 ) 5 7 8 9 0. 4 6 1 5 3 3  0. 5 0 0 0 0 0  0. 3 3 2 9 4 1  0. 352941  0. 2 3 5 2 9 4  0. 5 5 5 5 5 6  14  0. 3 6 3 6 3 6 0. 4 4 4 1 4 1  0. 6 9 5 6 5 2 0. 3 1 2 5 0 0  0. 6 1 0 0 0 0 0. 2 5 0 0 0 0  0. 5 2 3 8 1 0 0. 7 5 0 0 0 0  0. 4 5 3 3 3 3 0. 7 0 5 8 8 2  0. 4 0 0 0 0 0 0. 3 7 5 0 0 0  0. 5 7 1 4 2 9  0. 3 2 0 0 0 0  0. 4 7 6 1 9 0  0. 3 3 3 3 3 3  20  0. 0. 4 2 8 5 7 1  0. 4 3 4 7 8 3 0. 2 5 0 0 0 0  0. 4 0 0 0 0 0 0. 2 7 2 7 2 7  0. 4 4 4 4 4 4 0. 6 9 2 3 0 3  0. 3 3 0 9 5 2 0. 6 1 2 8 5 7  0. 3 7 5 0 0 0 0.  0. 421 OSS 0. 6 7 5 0 0 0  0. 2 8 5 7 1 4  0. 3 1 5 7 3 9  0. 2 3 5 7 ) 1  0. 352941  - 200 -  APPENDIX LXII  ;  . Matrix of S i m i l a r i t y Coefficients  Calculated  for  '  18 OTUs by Simple Match C o e f f i c i e n t : High C o n c e n t r a t i o n s  j  !  r 3  .  16  \ s r  s 53  7 17  2 1 1  5 17  4 13  10 14  9  18  0. 7 1 7 3 9 1  7  0. 6 7 3 9 1 3  0. 7 8 2 6 0 9  •7  0. 3 6 9 5 6 5  0. 7 6 0 3 7 0  0. 717391  0. 3 9 1 3 0 4  0. 7 3 9 1 3 0  0. 6 9 3 6 5 7  0. 891.304  4  0. 9 5 6 5 2 2  0. 6 7 3 9 1 3  0. 6 7 3 9 1 3  0. 8 6 9 5 6 5  0. 8 4 7 8 2 6  10  0. 3 4 7 3 2 6  0. 7 8 2 6 0 9  0. 7 8 7 6 0 9  0. 891304  0. 8 6 9 3 6 3  0. 84 7 8 2 6  1  0. 9 3 4 7 3 3  0. 6 5 2 1 7 4  0. 603696-  0. 3 0 4 3 4 8  0. 8 2 6 0 8 7  0. 8 9 1 3 0 4  0. 7 3 2 6 0 9  9  0. 8 0 4 3 4 8  0. 7 3 9 1 3 0  0. 7 8 2 6 0 9  0. 8 4 7 8 7 6  0. 8 2 6 0 8 7  0. 8 0 4 3 4 8  0. 9 1 3 0 4 3  "IS  1  0. 826037"""  "6Sf0".3_-  0758-6937  ""07".77391  0. 7 3 9 1 3 0  0. 827-ro"87 " .377'67.870  0. 80434 8  0. 7 6 0 8 7 0  16  0. 9 1 3 0 1 3  0. 717391  0. 6 7 3 9 1 3  0. 8 6 9 3 6 3  0. 80134(-.  0. 9 1 3 0 4 3  0. 8 4 7 8 7 6  0. 34 732.6  0. 3 0 1 3 1 8  0. 9 1 3 0 1 3  13  0. 7 6 0 3 7 0 0. "847826"  0. 6 0 8 6 9 6  0. 5 6 5 2 1 7  0. 7 6 0 8 7 0  0. 6 9 3 6 3 7  0. 8 0 4 3 4 3  0. 7 3 9 1 3 0  0. 6 9 5 6 5 2  0. 7 3 9 1 3 0  0. 8 9 1 3 0 1  17  0. 7 3 9 1 3 0 0. 8 2 6 0 8 7  0. 5 8 6 9 5 7  0. 5 1 3 4 7 8  0. 7 3 9 1 3 0  0. 6 7 3 9 1 3  0. 7 8 2 6 0 9  0. 7 1 7 3 9 )  0. 6 7 3 9 1 3  0. 717391  0. 8 6 9 3 6 3  0. 7 8 2 6 0 9 0. 8 6 9 3 6 5  0. 7 6 0 3 7 0 0. 8 4 7 8 2 6  0. 717391  0. 7 8 7 6 0 9  0. 717391.  0. 8 2 6 0 3 7  0. 7 6 0 8 7 0  0. 717391  0. 717391  0. 8 2 6 0 8 7  0. 8 0 4 3 4 8 0. 3 9 1 3 0 4  0. 7 8 2 6 0 9  0. 7 3 9 1 3 0 0. 8 0 1 3 1 8  0. 8 0 1 3 4 8 0. 978261  0. 7 3 9 1 3 0  .0. 3 0 4 3 4 8  0. 7 8 2 6 0 9  0. 7 8 9 1 8 0  0. 7 3 9 1 3 0  0. .34 787.6  0. 8 7 6 0 3 7  15  0. 8 2 6 0 8 7 0. 9 1 3 0 4 3  0. 6 3 0 4 3 5 0. 9 3 1 7 8 3 '  0. 5 8 6 9 3 7 0. 9 1 3 0 1 3  0. 7 8 2 6 0 9 0. 7 1 7 3 9 1 07869365"" 0. 8 1 7 8 2 6  0. 8 6 9 5 6 5  0. 7 6 0 8 7 0  0. 7 6 0 8 7 0  0. 717391  0. 9 1 3 0 4 3  14  0. 6 9 5 6 5 2  0. 8 4 7 8 7 6 0. 7 6 0 3 7 0  0. 8 0 4 3 4 3 0. 7 3 9 1 8 0  0. 7 8 2 6 0 9 0. 9 1 3 0 1 3  0. 7 1 7 3 9 1 0. 8 9 1 3 0 1  0. 7 3 9 1 3 0 0. 7 8 7 6 0 9  0. 8 0 4 3 1 8  .0. 6 3 0 4 3 5  0. 7 6 0 8 7 0  0. 7 8 9 1 3 0  0. 7 8 2 6 0 9 0. 7 3 9 1 3 0 0. 8 2 6 0 8 7  0. 7 1 7 3 9 1 0. 8 0 1 3 4 8  0. 6 7 3 9 1 3 0. 8 2 6 0 3 7  0. 7 8 7 6 0 9 0. 9 1 3 0 4 3  0. 717391 0. 8 9 1 3 0 1  0. 7 8 7 6 0 9  760870 869365  0. 6 7 3 9 1 3  0. 7 ) 7 3 9 1  0.  11  12  70  0. 97877-rr 67 '826087  0. 3 2 6 0 8 7  -  -  201 -  APPENDIX L X I I I Matrix of S i m i l a r i t y C o e f f i c i e n t s  Calculated f o r  18 OTUs by J a c c a r d C o e f f i c i e n t : A l l C o n c e n t r a t i o n s  7 8  0. 481431  7  0. 451613  -3 5 j  0.- 6666.1,7- ~0.~500000— 0.-468750O. 636364 °" 837143  4  10—0...4 6 ^  16  n  7  0. 538824  0. 407407  0. 387097  0. 617C59  0. 545435  0.312857  0.625000  0.607143  0.321739  0.634211. 0.371429  0.380643  0.789474  0.818)82  0.666667' 0.680000  0.576923  0.750000  0.45,613  0.666667  0.5652,7  0.733333  0.608696  0.588462  ft  4  6  ,  9  3  8  °*«™\  o-^oooo  I S  -^:.550S0.J::7038-..  ° '  l  ^ ^ S ^ ^ S ^ ^ X l  0. 652174  0.580613  . - _ q . l - 0 1  0. 548387  VSaa^^UUO^  0 603696  9  0. 727273  ^  ^  ^  Z™™  :  ^  0.-00000  ° ^ 5 0 0  0.366667  0.383333  0 . 5 0 0 0 0 0  0.438333  0 . 5 2 1 7 3 9  0.47826,  ^  :  0.68121,0  0.12337,  - - : - - o „ _ ? : ^ ^ ^ ^ ^ ^ ^ ^ ™ ° < »  0.625000  0.652,74  *^QQQ  a 0.482759  66666  0.617059  0.880957  ° 0,^0000.0.^00000  '  0.388233  < > • •  0.37,179  0.777227  ',  0. 6 1 7 0 3 9  0.^0000.500000  - 202 APPENDIX LXIV  .  ' Matrix of S i m i l a r i t y C o e f f i c i e n t s  . Calculated f o r  18 OTUs by Simple Match C o e f f i c i e n t : A l l C o n c e n t r a t i o n s  3 16  0 13  7 17  7 11  5 12  A ' I S  10 11  O. 693652 7  0. 630135  0. 301318  -_—0-369565-—Or-6 93652—0r-63043S-  4  0.826087  0.695657  0. 673913  0.826087  0.936522  0.652174  0.586957  0.869365  0.782609  10—0,-69565-2—Ox-*S260S»—0.-7-1-739-1—0^-739-1^0—0^-7-3-9-1^:0—0t-A*S*S21  0.804348  0.7173?)  0.652174  0.304348  0.760870  0.760870  0.717391  9  0.369365  0.739130  0.717391  0.9)3013  0.913013  0.869563  0.87.6087  0. 8260S7-....-0-695652.  0-5369.37  0.-7.32.609  0.-652O.4—0.-S7.6037-—0.7394 30  0.-84-7826—0.-739130 -  16  0. 913043  0. 739)30  0. 630433  0. 869363  0. 787609  0. 91.3013  0. 782609  0. 804318  0. 869365  0. 869365  13  0.782609  0.69365?  0.536937  0.782609  0.637171  0.826087  0.739130  0.760870  0.739)30  0.869563  17  0.760870 0. 847826  0.673913 CL_—2&1  0.5657)7  0.760870  0.630433  0.801348 :  0.717391  0.739130  0.7)73?)  C. 817826  11  0.717.39) 0.71739) 0.608696 0. 80.43.48. -0...89130-4—.0.-869.365  0.7)7391 : ;  0.630433  0.760870 :  0.760870  0.787609  0.7)73?)  0.817826  I 12 j  0.804348 fl 391.304  0.760870 n 93478.3  0.804318 O 91304.3  0.7)739)  0.804348 \  0.801348  0.826087  0.801348  0.89)801  • 15  0.732609 0. 3 6 9 3 6 5  0.695652 —-000000  0.586957 0.787609 0.6321710...973261—0„.8?.1.30.4—0_i>3.4.233  0.876087 :  0.739130  0.760870 •  0.739130  0 369365  0.652174 0. 7?9) :o  0.826087 0 826Q37  0.630133 0.693632 0.632)74 n 804348 _ , i y — . 1.3——34732.6  0.693652 CU-82-60S7  0.782609  0.6739)3  0.739)30  0.739)30  0.695652  0.787609  0.536937 0.695632 0.603696 0.739)30 0.739)30 I-•JO"—0.-S9.1304—0^-39-1304—0.-869363—0^-$'4S04S  0.717391  0.693652 .  0.737609  „L8  14  ,  20  0.657)74 O 913043  :  0.760870  ..  - 203 -  APPENDIX LXV  C o o r d i n a t e s o f 30 OTUs P l o t t e d i n F i g u r e XV  FULL EVJDI-NCF: 85 OHARAO'TFR 30 TAXON IJHOL.F PLANTS FACTOR ANALYSIS 3 8  (  2  5' 6 4 10 1  5. 1 0 4 7 2 2. 3 8 1 9 0 12. 5 6 7 6 3 2. 6 8 3 2 7  6. 4 9 2 5 6 7. 9 6 0 7 5  5. 8 6 2 9 0 -3. 9 2 4 3 3  -J.  9 21 25  61403 -2. 77445 -3. 41676 --0. 8 9 3 9 4  19 24 18  0. 6 4 4 3 2 2. 4 4 7 8 1 1. 6 4 4 7 8  16 13 17  4. 1 2 7 2 0 2. 3 4 4 6 5 i. 47492  11 12  5. 1 4 3 2 9 i. 62888  15 14 22 20 26 23  -1. 35048 1. 3 4 9 4 9 16. 1 8 7 3 3 -1. 55822 0. 5 0 3 7 4 -1. 9 0 3 7 4  3.74875 3. 6 3 3 5 2 -7. 0 8 1 3 8 0. 3 6 5 2 5  87779 91848 72009 32734 03864 978.1 4  4. 0 2 5 0 3 - 0 . 3.8532 -2. 56179 - 0 . 513 3 8  - w. -2. -1. -3. 0. -0.  2 37778 5. 3 6 7 2 7  - 3. 6 8 5 5 : " : -0. 47168  -0. 88189  -0.  96837  -1. 2 2 5 4 J  0.  07238  -1. 87514  - ] . 33 3 0 0  0. 8 8 2 3 2 . 2. 2 3 2 4 2 -0. 58143  -1.  09444 -•). 3 2 0 0 5 -1. 03165  -0.  49022  3.  25865  0. 5 5 2 8 3  -0. 99326  -0.  95467  27061  -2.  63492  39721  -.1. 9 8 1 5 1 -3. 23 4 2 4  H i 73 0. 3 2 8 9 4 -2. 39296  -1. 81081 - 1 . 174 36 -1. 80789  -3.  -.1. 0 5 2 1 4  29  -16.  13764  6. 3 3 9 9 3  28  - a  22529  2. 5 0 8 0 4  27  -10.  99766  5. 3 2 9 0 8  30  -11. 02921  4. 8 0 7 8 8  C<=-rcL\w\iedr o f  0. 2 6 3 7 3  -3. 2 7 7 7 3 -2.  40283  -3.83 768 -2. 28976 2.  13874  -0.  90234  3. 8 6 7 J 3 3. 03 3 53  OlX^i  - 204 -  APPENDIX LXVI C o o r d i n a t e s o f 30. OTUs P l o t t e d i n F i g u r e XVI'  fHJLL 3 8 7 2  4 10 I 9 21 25 19 24 18 16 13 17 11 12 15 14 22 20 26 23 29 28 27 30  K-:VIDFMI::F£ 3. 21928 1. 50211 2. 52230 2.55308 2. 75269 3. 68256 2. 87802 0.50069 1721282 0. 3 "279 -1.40398 0.21078 3.40729 2. 25668 i . 69776 2. 18008 2: 56977 1. 83000 2.35664 1. 51780 1.40422 0. 94048 2. 24218 0.20707 1.79377 0.61740 -15.08429 -6.33614 - 1 1 . 3.3691 - 1 0 . 43195  31 CHARACTER 30 I AXON WHO. E PLAN. 8 H ALT. IR ANAI 0.62447 1.49291 -0.57834 2.753 7... 21. 45496 1. 86687 - 0 . 11239 1.76760 2. 56790 5. 82146 0. 30923 8. 94659 - 0 , 23809 6. 76683 -3.21428 -3.29830 - 1 . 27731 =!T^63T - 3 . 827-.4 - 2 . 06476 -0.95383 -3.41115 - 1 . 0 5 3 47 - 0 . 9 3 03 4 - 0 . 3 03 09 -0.37409 0. 08534 3. 54 88 3 - 1 . 0912"/ ' 1. 71099" 0. 26560 4. 26759 0. 5.8476 0. 96630 - 0 . 05950 0. 51094" 0.33 730 5 . 7 3 596 - 0 . 19365 3. 20201 -0.23906 -0.41786 - 1 . 72693 - 3 . 45868 0. 0747.1 - 0 . 3 6 3.85 -2.09082 - 3 . 784 00~ - 0 . 19609 -5.23886 -1.79867 -3.25904 -3.04708 - 8 . 13 638 -1.38548 -5.71783 - 3 . 27503 - 5 . 23 33 7 - 1 . 42650 - 5 . 90945  - 205 APPENDIX LXVII Coordinates o f 18 OTUs P l o t t e d i n F i g u r e XVII  7 FULL 8 7 2  (  4  10~ 1  9 18 16 13 17 11 12 1.5 14 20  - T0.K56728 fll—  10. 15. 3. 6. -0. 5. 3. 3. -7. -3. - 5.0. - 1.1. -2. -i. -8. 3. -4.  82163 55555. 47656 08708 8339.. 03554 21599 05944 5.5.308 97997 335 32 52882 26424 59037 85.080 02657 39254  -2. -5. 6. -2. -3. -2. -3. -4. 0. 0. -0. 3. -v  0. 0. 1. J. 3.  94 474 20182 72893 56263 973 78 5 5 3.05. 88063 38083 23383 727 5 1 89735 0879.5 79703 55 5 95. 22936 64442 4 5 857 57670  - 5 . 7 3 675 6. 18045 —2. 47233 - 5 . 20709 - J . 85070 - 1 . 39448 - 1 . 24277 - 3 . 774 43 - 3 . 34 4 5.2 -0. 5 3 984 - 0 . 94380 5. 035 06 1. .32 J. 6 3 5. J 8 5.9 5 0. 854 29 0. 80772 2. 08492 5. 5 5384  - 206 -  • APPENDIX LXVIII C o o r d i n a t e s o f 18 OTUs P l o t t e d i n F i g u r e XVIII  f FULL 8 7 2_.  10  1  9 18 16 13 17 11 12 15 14 20  (f-F-A^V  80535 46086 53850 46101 40208 21 04 1 1 4429 60514 44199 45416 95039 ' 58709 97436 324 62 - 1 . 51074 - 7 . 37026 0. 601 15 -4. 46916  2. 6. 7. 4. 7. 1. 5. 5. 2. --5. --5L -9. -10. --2.  39534 - 1 . 45724 • 86507 10. 38435 4 2979 5. 31503 30339 0. 34355 0. 86824 841 45 64758 - 1 . 75707 68-5 3 4 1. 06514 0 . 89649 05267 28316 0. 2 1 2 9 8 " ' . ~ - 3 . 29506 24 556 -2. 70632 531 21 8. 584 07 54636 74321 •.*>. 82487 0. 0554 9 - 1 . 19221 0. 04995 - 0. 96795 - 3 . 62824 - 3 . 59780 ^/ 05435 27592 - 0 . 871 29 - 1 . 54248  2. 14. 0. 0. -1. 1. -1. 2. -3. —2. -3. -3.  

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