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Polyacetylenes from Bidens Marchant, Yu Yoke 1985

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POLYACETYLENES FROM  BIDENS  by  YU YOKE MARCHANT B. Sc., U n i v e r s i t y of B r i t i s h Columbia, 1972 M. Sc., U n i v e r s i t y of B r i t i s h Columbia, 1974  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Botany  Department  We accept t h i s t h e s i s as conforming . i i ^ t h e regu-i*r?ed standard  TH&"UNIVERSITY OF BRITISH COLUMBIA August, 1985 © Yu Yoke Marchant, 1985  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by the head o f  department or by h i s o r her r e p r e s e n t a t i v e s .  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T  E-6  (3/81)  1Y3  written  ABSTRACT The  Hawaiian s p e c i e s of Bidens  are m o r p h o l o g i c a l l y  e c o l o g i c a l l y d i v e r s e taxa which have evolved ancestral  species.  the e v o l u t i o n isolating  of  Adaptive  a  single  r a d i a t i o n has o c c u r r e d  without  physiological  or  from  and  genetic  interspecific  mechanisms s i n c e a l l s p e c i e s are i n t e r f e r t i l e  and  g e n e t i c d i s t a n c e s among p o p u l a t i o n s , based on  isozyme  show  d i f f e r e n c e s or  little  correlation  with m o r p h o l o g i c a l  taxonomic  classification.  This  evolution  of morphological  and  it  disparity  i n secondary m e t a b o l i t e s  Leaves and Hawaiian Bidens  Polyacetylenes  three C can  were  of  has  been  species.  six  subspecies  be not  thiophenyl d e r i v a t i v e s , 1 7  in Bidens  has  one  derived detected  in the r o o t s  from  oleic  in  B.  tort a.  of  all  above the s p e c i e s l e v e l  was  distribution  in t h i s group.  i i  k  and  species.  reported.  found  of  The  acetoxymethyl  There appears to be no  s i g n i f i c a n t p a t t e r n to the  1 3  i n the l e a v e s of 13 taxa  not been p r e v i o u s l y  variation  C  acid.  c o u l d be d i s t i n g u i s h e d by t h e i r complement of l e a f no  of  C,  hydrocarbons were i s o l a t e d  2-(2-phenylethyne-1-yl)-5  root a c e t y l e n e s and except  and  they are found  thiophene taxa  and  All  occurrence  the  were examined f o r p o l y a c e t y l e n e s . Eleven  tetrahydropyran  although  there  i n these  r o o t s of 19 s p e c i e s and  hydrocarbons, aromatic  identified.  between  b i o c h e m i c a l c h a r a c t e r s makes  of i n t e r e s t to determine whether or not  divergence  loci,  within  Most and taxa  taxonomically polyacetylenes  The  complexity  assessed  using  hybrids.  Crosses  of  polyacetylene  experimentally  inheritance  produced  was  interspecific  between s p e c i e s which do not produce l e a f  a c e t y l e n e s r e s u l t e d i n F,  individuals  Crosses  which produce l e a f a c e t y l e n e s and  between  those which do always  not y i e l d e d  identical  with d i f f e r e n t the  species  major  hybrids  with  acetylenes.  acetylenes  to p a r e n t a l a r r a y s . Progeny from  s e t s of a c e t y l e n e s expressed  compounds  nonparental  without  found  acetylenes  biosynthetically  closely  parents  a combination of  i n both p a r e n t s . In a l l cases,  in  the  F,  generation  i n the i n the  i n d i v i d u a l s from Type B c r o s s e s .  2  De novo b i o s y n t h e s i s of p o l y a c e t y l e n e s was  investigated  a c e t y l e n e s were administered 1 2  were  r e l a t e d t o compounds found  p a r e n t s . P o l y a c e t y l e n e s y n t h e s i s was not segregated F  not  C0  1  from  2  ene-tetrayne-ene plants,  recovered  " C 0 and subsequently  f o r 12,  2  i n pulse-chase  24  and  was  indicating  also  168  i n Bidens  studies.  three  species  allowed hours.  isolated  1 0  leaves  C-labelled of  Bidens  t o metabolize i n Radioactive  C  1 3  from the roots of a l l  that . t r a n s l o c a t i o n  of  1  "C-labelled  p r e c u r s o r s from a e r i a l t i s s u e s o c c u r r e d . Phenylheptatriyne seedlings  of  B.  (PHT) alba,  was d e t e c t e d suggesting  i n two that  polyacetylene  b i o s y n t h e s i s begins during germination  or  Quantities  t o i n c r e a s e up t o and  i n the leaves  continue  soon  day o l d  thereafter.  beyond 24 days while amounts i n the h y p o c o t y l s peak a t seven days.  R e l a t i v e PHT values i n the r o o t s a r e 100 times  higher  than  those  i n the a e r i a l t i s s u e s f o r the f i r s t  there i s a l s o a gradual d e c l i n e i n these two  weeks  and c o n t i n u i n g  Phenylheptatriyne  i s absent  24 days, but  l e v e l s beginning at  beyond the experimental from  the r o o t s  period.  of mature  B.  alba. Many p o l y a c e t y l e n e s are t o x i c t o b i o l o g i c a l systems i n the  presence of UV-A r a d i a t i o n . These in vitro  led  t o speculation  polyacetylenes  about  from  yeasts  and y e a s t - l i k e  a l l these  Sporobolomycetaceae,  organisms,  Cryptococcaceae  I m p e r f e c t i , were p h o t o s e n s i t i v e t o some resistant presence  t o others, or absence  of l e a f  of these  Nevertheless,  i t is significant  isolated  1 3  aromatic  p h o t o s e n s i t i v e in  fungi  were  without  members of and  Fungi  p o l y a c e t y l e n e s and  polyacetylenes  saprophytes  in  this  gloeospori odes , d i d not c o l o n i z e C  of  there was no c o r r e l a t i o n between the  distribution  species  functions  of Hawaiian Bidens with and  species  l e a f a c e t y l e n e s . Although the  putative  in' the organisms which produce them. Nineteen  s p e c i e s of p h y l l o p l a n e isolated  the  e f f e c t s have  acetylenes  to  vitro.  iv  and the  among s p e c i e s of Bidens.  that  t h e only study,  Bidens which  pathogenic  Col Ietol ri chum leaves  containing  i t i s extremely  Table of Contents Chapter  Page  ABSTRACT  i i  LIST OF TABLES  viii  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS  xii  I. GENERAL INTRODUCTION  1  A. POLYACETYLENES  1  B. BIBLIOGRAPHY  11  I I . POLYACETYLENES IN HAWAIIAN BIDENS  17  A. INTRODUCTION  17  B. MATERIALS AND METHODS  22  PLANT MATERIAL  22  ISOLATION AND POLYACETYLENES  IDENTIFICATION  OF 22  C. RESULTS  36  POLYACETYLENES IN BIDENS TAXA  36  POLYACETYLENES IN BIDENS HYBRIDS  45  D. DISCUSSION  54  POLYACETYLENES IN BIDENS TAXA  ..54  POLYACETYLENES IN BIDENS HYBRIDS  64  E. CONCLUSION  69  F. BIBLIOGRAPHY  71  I I I . BIOSYNTHESIS OF POLYACETYLENES FROM  1 a  C0  74  2  A. INTRODUCTION  74  B. MATERIALS AND METHODS  77  BIOSYNTHESIS  1  OF  POLYACETYLENES FROM *C0  IN BIDENS  2  77 v  PLANT MATERIAL  77 1a  ADMINISTRATION OF C 0 MEASUREMENT OF POLYACETYLENES  77  2  1  *C  UPTAKE  INTO 80  KINETIC STUDIES  83  STATISTICAL ANALYSIS  '..83  ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS ..84 C. RESULTS  86  BIOSYNTHESIS OF POLYACETYLENES IN BIDENS LEAVES  FROM  1  "C0  2  86  ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS .101 D. DISCUSSION  106  BIOSYNTHESIS OF POLYACETYLENES IN BIDENS LEAVES  FROM  1tt  C0  2  106  ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS .110 E. CONCLUSION  112  F. BIBLIOGRAPHY  114  IV. PHOTOTOXICITY  OF  POLYACETYLENES  TO PHYLLOPLANE  FUNGI  117  A. INTRODUCTION  117  B. MATERIALS AND METHODS  120  PLANT MATERIAL  120  ISOLATION AND IDENTIFICATION OF FUNGI  120  PHOTOTOXICITY ASSAYS COMPARISON OF PHOTOTOXICITY  128 OF  POLYACETYLENES TO CRYPTOCOCCUS  LAURENT11  TEST COMPOUNDS  SELECTED ... 131  132  C. RESULTS  134 vi  ISOLATION, IDENTIFICATION AND OF PHYLLOPLANE ORGANISMS PHOTOSENSITIVITY ACETYLENES  OF  DISTRIBUTION 134  MICROORGANISMS  TO 146  COMPARISON OF PHOTOTOXICITY POLYACETYLENES TO CRYPTOCOCCUS  OF SELECTED LAURENT11 ...155  D. DISCUSSION  163  ISOLATION, IDENTIFICATION AND OF PHYLLOPLANE MICROORGANISMS PHOTOSENSITIVITY  DISTRIBUTION  OF  MICRORGANISMS TO POLYACETYLENES  163 PHYLLOPLANE 166  E. CONCLUSION  173  F. BIBLIOGRAPHY  174  GENERAL CONCLUSION A. BIBLIOGRAPHY  179 180  LIST OF TABLES  I.  Naturally occurring acetylenes  3  II.  Bidens  III.  Polyacetylenes  from Hawaiian Bidens  37  IV.  Polyacetylenes  from Hawaiian Bidens  38  V.  Polyacetylenes  i n the leaves of Hawaiian  taxa examined f o r p o l y a c e t y l e n e s  Bi dens  VI.  39  Polyacetylenes  i n the r o o t s of Hawaiian  Bi dens  VII. VIII.  Bidens  40  h y b r i d s examined f o r p o l y a c e t y l e n e s . . . 46  • Polyacetylenes  from Bidens  hybrids  IX.  Polyacetylenes  i n B. hawaiensis  X.  Polyacetylenes  i n B. cosmpides  XI.  Polyacetylenes  i n b. macrocarpa  XII.  Polyacetylenes  in F  XIII.  Percent quenching of r a d i o a c t i v i t y by  2  hybrids hybrids hybrids  plants  XIV.  C0  2  XVI.  E f f i c i e n c y of  1ft  of B. alba  87  C uptake  88  Twelve hour uptake of "C i n t o PHT 90  Twelve hour uptake of '*C i n t o ene-tetrayne-ene of B. hi 11 ebr andi ana leaves 1  Twelve hour uptake of *C i n t o Twelve hour uptake of  1 ,  C  93  One week uptake of *C i n t o PHT by leaves  94 1  One week uptake of *C i n t o ene-tetrayne-ene (1)  XXII.  leaves  1  B. alba XXI.  92  i n t o MeOH and PE  f r a c t i o n s by B. molokaiensis XX.  91  acetylenes  1 and 5 of B. cosmoi des leaves XIX.  50  1  (1) XVIII.  49  82  by B. al ba l e a v e s XVII.  48  uptake d u r i n g 60 minutes  pulse-labelling XV.  47  52  p o l y a c e t y l e n e s 1,4 and 5 1<t  23  of B. hi 11 ebr andi ana leaves  One week uptake of  1 ,  C  into  acetylenes  1 and 5 of B. cosmoi des leaves  viii  95  96  XXIII.  One week uptake of  1 0  C i n t o MeOH and PE  f r a c t i o n s of B. molokaiensis XXIV.  97 1  Twenty-four hour uptake of *C i n t o PHT by B. al ba leaves  XXV.  1ft  XXVI.  1fl  98  C uptake i n t o MeOH and PE f r a c t i o n s of  B. alba  l e a v e s i n 24 hours  99  {4):B.alba  C uptake i n t o PHT  leaves i n 24 hours XXVII.  100  " ^ - l a b e l l e d ene-tetrayne-ene in roots of Bidens  XXVIII.  (1)  1  given * C 0  102  2  Accumulation and d i s t r i b u t i o n of phenylheptatriyne  ( 4 ) i n B. alba s e e d l i n g s . . 1 0 3  XXIX.  Bidens  XXX.  P l a n t s a s s o c i a t e d with Bidens  taxa sampled  f o r p h y l l o p l a n e fungi...121 sampled f o r  p h y l l o p l a n e fungi XXXI.  124  Composition of malt e x t r a c t  (MYPT) c u l t u r e  medium XXXII.  126  Yeasts and y e a s t - l i k e fungi  isolated  from  Hawaiian p l a n t s XXXIII.  129  P o l y a c e t y l e n e s used f o r p h o t o t o x i c i t y assays  XXXIV.  133  D i s t r i b u t i o n of fungi  i s o l a t e d by the  spore f a l l method XXXV.  D i s t r i b u t i o n of f u n g i i s o l a t e d with the leaf  XXXVI.  135  impression method  139  D i s t r i b u t i o n of f u n g i i s o l a t e d w i t h the l e a f d i s c method  XXXVII.  143  D i s t r i b u t i o n of y e a s t s and y e a s t - l i k e  fungi  among Hawaiian Bidens XXXVIII.  P h o t o s e n s i t i v i t y of microorganisms t o e x t r a c t s of Hawaiian Bidens  XXXIX.  leaves  .149  P h o t o s e n s i t i v i t y of microorganisms t o e x t r a c t s of Hawaiian Bidens  XL.  147  leaves  P h o t o s e n s i t i v i t y of C. laurentii  from  d i f f e r e n t host p l a n t s t o p o l y a c e t y l e n e s  ix  150  152  XLI.  P h o t o s e n s i t i v i t y of p h y l l o p l a n e  fungi  to p o l y a c e t y l e n e s XLII.  153  E f f e c t s of changes  in polyacetylene  c o n c e n t r a t i o n and length of UV exposure on percent XLIII.  s u r v i v a l of C. laurentii  S u r v i v a l curves f o r C. laurentii to p o l y a c e t y l e n e s  i n UV l i g h t  x  156 exposed 161  LIST OF FIGURES 1.  The Hawaiian I s l a n d s  18  2.  L o c a l i t i e s of Kauai Bidens  populations  sampled 3.  24  L o c a l i t i e s of Oahu Bidens  populations  sampled 4.  26  L o c a l i t i e s of Maui Bidens  populations  sampled 5.  28 Bidens  L o c a l i t i e s of Molokai  populations  sampled 6.  30  L o c a l i t i e s of Hawaii Bidens  populations  sampled 7.  32  B i o g e n e t i c r e l a t i o n s h i p s of p o l y a c e t y l e n e s from Hawaiian Bidens  42  from Cor eops i s  8.  Phenylthiophenes  44  9.  Dendogram of taxa based on s i m i l a r i t y of polyacetylenes  10.  1ft  11.  1  12.  C0  2  - f e e d i n g apparatus  14.  78  *C - E f f i c i e n c y curve Accumulation Bidens  13.  55  alba  Accumulation  81  and d i s t r i b u t i o n  of PHT i n  seedlings  104  and d i s t r i b u t i o n  Bidens  alba  Effect  of a - t e r t h i e n y l  of PHT i n  seedlings  105 (21)  and UV-A on  the 24 hour s u r v i v a l of Crypt ococcus 15.  Effect  laurentii  157  of p h e n y l h e p t a t r i y n e  on the 24 hour s u r v i v a l 16.  Effect  of C. laurentii  of phenylheptadiyne-ene  UV-A on the 24 hour s u r v i v a l 17.  Effect UV-A  (4) and UV-A ( 5 ) and of C. laurentii  of h e p t a d e c a - t e t r a e n e - t r i y n e  on the 24 hour s u r v i v a l  xi  158 159  (8) and  of C. laurentii  160  ACKNOWLEDGEMENT S  I wish t o express my a p p r e c i a t i o n have  contributed  their  time,  behalf throughout the past  to  effort  five  the people  who  and e x p e r t i s e on my  years.  I thank the members of my r e s e a r c h committee, D r s . Fred Ganders, Jack Maze and Anthony G l a s s , and Towers,  my  inimitable supervisor, for their  c o l l e c t i v e advice and c r i t i c a l Dr.  Lily  encouraging and  Wat  and  i n d i v i d u a l and  Zyta  as w e l l as generous  Abramovski  with  were  technical  always  assistance  suggestions.  matters  mycological,  to  Dr.  p o l y a c e t y l e n e chemistry,  to  Fortran  Vince  programmes,  c o n s t r u c t i o n of the care  of  1tt  to C0  greenhouse  who helped prepare  this  the U n i v e r s i t y  Jjrfrgen  Dr.  Lam  Kermit  Grant  for help  in  f o r advice  on  Ritland  forhis  and Ken J e f f r i e s f o r t o Bob Kantymir  p l a n t s , t o James B j e r r i n g f o r h i s a n a l y s i s and  to  Len  Marchant,  manuscript.  Finally., the f i n a n c i a l by  R.J. Bandoni  feeding apparatus,  2  a s s i s t a n c e with s t a t i s t i c a l  and  Neil  support.  I am a l s o g r a t e f u l t o Dr.  for  especially,  support  of  p r o v i d e d by Mr. Marchant  B r i t i s h Columbia i s g r a t e f u l l y  acknowledged.  xi i  I.  A.  GENERAL INTRODUCTION  POLYACETYLENES The m a j o r i t y of n a t u r a l  polyacetylenes.  not s t r i c t l y  group  of secondary  today a r e  metabolites,  poly-ynes (Jones and T h a l l e r ,  which o r i g i n a t e from ole ic - a c i d found  known  The name encompasses what now appears t o be  a b i o g e n e t i c a l l y uniform usually  acetylenes  (Bu'Lock,  1978),  1966) and  i n the roots and a e r i a l p a r t s of p l a n t s , and i n fungi  (Bohlmann et al., 1973), algae (de N a p o l i sponges  (Cimino  Faulkner, insects  1981),  et  al.,  1981),  sea hares  (Moore and Brown,  natural  product  was  et  al.,  nudibranchs et  (Schulte  1981),  (Walker and  al.,  1981)  first  clearly  triple  bond  in a  e s t a b l i s h e d by Arnaud  (1902) i n h i s study o f " t h e monoacetylenic a c i d , t a r i r i c I ) , a component of the seed f a t of Picramnia  (Table  (Simaroubaceae). The f i r s t  oxide who,  c o n s i d e r i n g the n a t u r a l occurrence  correct  proposed an a l l e n i c  compound,  elucidation  pol ^ a c e t y l e n e was f i r s t recognized  LachnophylI um  of  a  et  al.  gossypinum acid  tariri carlina (1906),  triple  bond  (1933). The  naturally-occurring  a c h i e v e d by Vil'yams et  the lachnophyllum  dec-2-ene-4,6-diynoic  of a  acid  formula f o r the compound. The  s t r u c t u r e was g i v e n by Gilman  structural  who  aromatic  (Table I ) , was i s o l a t e d and s t u d i e d by Semmler  unlikely,  and  1978).  H i s t o r i c a l l y , the occurrence of a  DC.  are  ester  al.  isolated  Bge. as the methyl (Table I ) . These  1  (1935)  first  ester  from of  compounds  2 were  found  reasonable  accidentally  because  amounts and e a s i l y  they  were  present  purified.  Seven a c e t y l e n e s were d e s c r i b e d between 1902 since  then,  1976)  primarily  discovered  over  700  aliphatic  e x t i n c t i o n c o e f f i c i e n t s , thus a l l o w i n g (Jones,  1950.  (Thaller,  polyacetylenes  to show very c h a r a c t e r i s t i c UV  q u a n t i t i e s of substance  and  more have been i d e n t i f i e d  because  in  1959;  were  s p e c t r a with high  detection  of  small  Bohlmann el  1966;  al  .,  1973). Around  1950,  Basidiomycetes structure,  which,  al;  when  by  comparison  tentatively  structures  substances  were c h a r a c t e r i z e d by UV  Compositae,  et  antibiotic  et  (Anchel  their  Anchel,  1950). S e r i o u s a t t e n t i o n was  focussed  Celmer  and the  mycomycin  a l l e n e , d i a c e t y l e n e and diene groupings When Jones investigations  and  the UV  i s largely  Sjrfrensen,  on  co-workers  as  Kavanagh  this isolated  one  of  (Table I ) . started  their  broad  from  an  antibiotic  test  s p e c t r a of c u l t u r e f l u i d s  through  the  concurrent  to  (Jones,  efforts  of  one  were  isolated  of  1959). Jones,  Bohlmann, Anchel and t h e i r a s s o c i a t e s i n the  these  and  containing  30 years that so many p o l y a c e t y l e n e s are known today. 85%  area  i n t o a c e t y l e n e s from f u n g i , they changed the  s c r e e n i n g technique determining  his  fine  acetylene  1953;  Solomon (1952a; 1952b; 1953) antibiotic  by  with p o l y a c e t y l e n e s from  1950;  identified  It  s p e c t r a showing  established al ,  produced  last About  from higher p l a n t s . They are  f a i r l y widespread amongst the Campanulaceae  and  Araliaceae  3  TABLE I . NATURALLY OCCURING ACETYLENES  CH. -(CH )^ 3  2  C=C-(CH ) COOH 2  tariric Picramnia  A  acid  tariri  carlina  DC.  oxide  Car Ii na a caulis L.  CH -(CH ) -(C=C) -CH=CH-COOCH 3  2  2  2  lachnophyllum  3  ester  LachnophylI urn gossypinum Bge.  H(C=C) -CH=C=CH-(CH=CH) CH COOH 2  2  2  mycomycin  Nocardia  acidophilus  5-(3-buten-1-ynyl)-2,2'-bithienyl Taget es pat ula L .  phenylheptatriyne Bi dens alba L .  ichthyothereol lchthyothere  terminal is  HO-CH -(CH ) -(C=C) -(CH=CH) -CH 2  2  2  2  3  cicutoxin Ci cut a vi r osa L .  Spreng.  (OH)-(CH > ~CH 2  2  3  CH -CH=CH 3  (C=C) -CH=CH A  2  C , j-ene-tetrayne-ene Heliantheae:  Coreopsidinae  CH -(C=C)-CH=CH 3  2  Cij-pentayne-ene Heliantheae:  Coreopsidinae  CH-CO-(C=C) -CH-CH=CH-(CH )-CH 2  2  dehydrofalcarinone Heliantheae: Galinsoginae  "  ^-C-(C=C) -CH 2  3  0  capillin  Artemesia capi 11 or i s Thnb.  a  w o-terthienyl  Tageies pat ula L.  6  and  have  found  sporadically  (Bohlmann et al.,  families found  been  i n s e v e r a l other p l a n t  1973). They are  most  frequently  i n members of the U m b e l l i f e r e a e and the Compositae, i n  which  they  occur  in  a l l 13  tribes,  especially  H e l i a n t h e a e , Anthemideae and Cynareae (S^rensen,  the  1977; Swain  and W i l l i a m s , 1977) . As  distinct  from  the  mainly  related plant f a m i l i e s , acetylenes characterized  by  cyclic,  a l i p h a t i c a c e t y l e n e s of of  aromatic  the or  Compositae  are  heterocyclic  end  groups. Some of these complex s t r u c t u r e s are r e s t r i c t e d t o a single  tribe  while  some  heterocyclic  thiophenes,  have been found  occurrence  seemingly  (S^rensen, the  i n the m a j o r i t y of t r i b e s ,  u n r e l a t e d to morphological  their  characters  1977). In f a c t , the d i s t r i b u t i o n of a c e t y l e n e s i n  Compositae does not o f t e n c o r r e l a t e w e l l with b o t a n i c a l  classification. extensive it  compounds, such as  Bohlmann  study  appears  discrete,  that,  some  polyacetylenes  (1973)  have  made  an  of the p o l y a c e t y l e n e s from t h i s f a m i l y and although  particular  subtribes,  al.  et  distribution  f e a t u r e s are mostly  genera may  acetylene  be  or useful  some  is  r e s t r i c t e d t o some  sections,  so  that  taxonomically  at d i f f e r e n t  the  Heliantheae:  l e v e l s below the t r i b e . For  example,  Coreopsidinae (Table  I)  are  members  of  characterized  and i t s aromatic  by  subtribe C  1 3  ene-tetrayne-ene  d e r i v a t i v e s , together with  less  unsaturated compounds. The s u b t r i b e H: G a l i n s o g i n a e c o n t a i n s dehydrofalcarinone  and  other  C  1 7  acetylenes  (Table  I)  7 et  (Bohlmann  al.,  Bidens and Dahlia, acetylene  arrays  Scrfrensen,  Lam  related  (Bohlmann  genera,  have  and Zdero, 1968; 1966;  Coreopsis, similar  Bohlmann and 1964;  S^rensen  1966; 1958a, 1958b, 1958c; Sjrfrensen et al . , that Dahlia  1961), except 1973;  closely  Bohlmann et al . , 1967;  Bornowski, 1966; and  1973). W i t h i n C o r e o p s i d i n a e ,  a l s o has C  and Kaufmann, 1971;  1 7  acetylenes  (Lam  et al., 1970)  Chin  1971;  which are  c h a r a c t e r i s t i c of two other r e l a t e d genera Glossocardia Isostigma The and  (Bohlmann et al., first  Solomon,  properties.  1973).  fungal p o l y a c e t y l e n e s , l i k e mycomycin  and i s o l a t e d In the same  because  of t h e i r  Thus the  of p o l y a c e t y l e n e s  antifungal  compounds  from  R e i s c h et al. (1967) fungistatic  synthetic alcohols,  Artemesia  1978;  acetylenes, aldehydes  of a  dermal  Wagner, 1977).  large  including  and ketones  is  a c e t y l e n i c ketones such  investigated  effects  the  capillar  (Table I) which i s h i g h l y a c t i v e a g a i n s t  mycoses (Jones and T h a l l e r ,  and  has l e d to  as the a c t i v e compounds.  Thunb. were i d e n t i f i e d as conjugated as c a p i l l i n  antibiotic  way, the d i s c o v e r y of a n t i b i o t i c  p r o p e r t i e s of p l a n t s or of p l a n t e x t r a c t s identification  (Celmer  (Bohlmann et al., 1969),  1953) and agrocybin  were d e t e c t e d  and  the.  bacteriostatic  number  of  hydrocarbons, with  simple acids,  one or two t r i p l e  bonds, as w e l l as the C, -ene-tetrayne-ene and pentayne-ene 3  compounds. In general t h e i r with  aromatic  fungicidal  substituents  f i n d i n g s suggest were  most  that acetylenes  active  and  e f f e c t s i n c r e a s e with p o l a r i z a t i o n of the  that triple  8 bond  and  compounds  degree  of  which  were  bacteriocidal  unsaturation more  in  the  molecule while  hydrophilic  tend  to  be  agents.  S e v e r a l p o l y a c e t y l e n e s are known to be g e n e r a l l y t o x i c . The as  p l a n t e x t r a c t used by n a t i v e s of the Lower Amazon Basin fish  poison  tetrahydropyran  on  et  al.,  Cicut a  of  arrowheads  i c h t h y o t h e r e o l and  its active principles toxicity  their  (Cascon et virosa  i t s acetate  al . ,  L.  contains  is  1965) due  (Table I) as  and  the  potent  to c i c u t o x i n (Anet  1953). In  1973,  Gommers  nematocidal  and  Geerligs  activities  of  reported  (Table  s i g n i f i c a n t l y enhanced by UV  These  subsequently phototoxic  isolated  to  Chan et  1965;  Candida al.,  light.  from Tagetes  patula  albicans  (Robin)  and were  compounds  L. and  found  were to be  Berkh.  (Daniels,  1975). T h i s d i s c o v e r y l e d to a  systematic  thiophene  derivatives  from  Towers and h i s a s s o c i a t e s (e.g., Camm et 1980;  the  I)  i n v e s t i g a t i o n of the p h o t o t o x i c p r o p e r t i e s of their  that  a-terthienyl  5-(3-buten-1-ynyl)-2,2'-bithienyl  and  the  Towers and Wat,  1978;  Towers et  the al,,  al.,  polyacetylenes Compositae by 1975;  1977;  Towers, et  Wat  a/.,  1980). It  is  now  w e l l e s t a b l i s h e d that many p o l y a c e t y l e n e s ,  notably a - t e r t h i e n y l and p h e n y l h e p t a t r i y n e toxic  to  biological  systems  in  (320-400nm) r a d i a t i o n . In a d d i t i o n (Arnason et  al . , 1980;  DiCosmo et  al.,  the to  (Table presence  bacteria  I),  are  of  UV-A  and  fungi  1982), these compounds  9  kill  human  f i b r o b l a s t s and e r y t h r o c y t e s  MacRae et al., 1980b; Towers et  al . ,  1980),  adult  et al., 1979), c e r c a r i a  nematodes,  insect  (Arnason et al., 1981; Kagan and Chan, 1981)  (Wat et al . ,  1977;  (Graham  l a r v a e and eggs Wat et  1983;  al.,  v i r u s e s (Warren et al., 1980; Hudson  and d e a c t i v a t e  et al., 1982), but they are not genotoxic  et  (MacRae  al.,  1980a). U n l i k e the effects  linear  furanocoumarins,  whose  phototoxic  can be e x p l a i n e d by the photo-induced m o d i f i c a t i o n  of DNA (Song and Tapley,  on  cell  membranes. S p e c i f i c a l l y , a - t e r t h i e n y l a c t s as a t y p i c a l  Type  II  photodynamic  activity  sensitizer,  aerobic  by p h e n y l h e p t a t r i y n e  and anaerobic  in  vitro  for  and, although  there in  putative  i n the organisms  does not preclude  is vivo  and  their  considerable functions, at  practical  which produce them. T h i s  application  of t h e i r  b i o c i d a l properties. Polyacetylenes  are n o t o r i o u s l y  and decompose r a p i d l y  solution  Bohlmann  both  no obvious p h y s i o l o g i c a l r o l e can be a l l o c a t e d t o  polyacetylenes  (e.g.,  and  1984).  effects  their  its  el al ., 1980; Arnason  are known about p o l y a c e t y l e n e s  about  cells  under  details  speculation present,  oxygen  occurred  c o n d i t i o n s (Wat  et al., 1981; McLachlan et at., Many  requiring  act  the p h o t o s e n s i t i z a t i o n of E. coli  while  erythrocytes  1979), p o l y a c e t y l e n e s  et  Anchel et  al.,  biodegradability  i n aqueous  a/., 1950;  Celmer  1973; Towers,  potent unstable  and i n l i g h t  and Solomon,  1980).  This  1953;  rapid  may c e r t a i n l y be e x p l o i t e d to advantage i n  10  the  search  for  biological control  effective  environmentally  nontoxic  agents.  The purpose of t h i s study of  and  i s to e x p l o r e v a r i o u s aspects  p o l y a c e t y l e n e s i n one group of p l a n t s ,  Hawaiian  Bidens,  in order t o e s t a b l i s h p r e l i m i n a r y i n f o r m a t i o n on t h e i r 1.  occurrence and e v o l u t i o n a r y s i g n i f i c a n c e ,  2.  b i o s y n t h e t i c pathways, and  3.  antibiotic properties.  Such p r e l i m i n a r y data are necessary those  hypotheses  knowledge  which  concerning  information  will  be  will  these  lead  in to  chemicals.  order a  identify  r a p i d growth of As  well,  u s e f u l i n determining t h e i r  u s e f u l n e s s and d e f i n e the l i m i t s of m a n i p u l a t i o n utilization.  to  this  potential for  human  11 B. BIBLIOGRAPHY  Anchel, M. 1953. Identification of an antibiotic polyacetylene from Cliiocybe di at ret a as a suberamic a c i d ene-diyne. Am. Chem. Soc. J . 75: 421 - 462. Anchel, M., J . P o l a t n i c k and F. Kavanagh. 1950. I s o l a t i o n of a p a i r of c l o s e l y r e l a t e d a n t i b i o t i c substances produced by three s p e c i e s of Basidiomycetes. Arch. Biochem. 25: 208 - 220. Anet, E., B. Lythgoe, M.H. S i l k and S. T r i p p e t t . 1953 Oenanthotoxin and c i c u t o x i n . I s o l a t i o n and s t r u c t u r e s . J . Chem. Soc. 309 - 322. Arnason, T., J.R. S t e i n , E. Graham, C.K. Wat, G.H.N. Towers and J . Lam. 1981. P h o t o t o x i c i t y t o s e l e c t e d marine and freshwater a l g a e of p o l y a c e t y l e n e s from s p e c i e s i n the A s e t e r a c e a e . Can. J . Botany 59: 54 - 58. Arnason, T., C.K. Wat, K. Downum, E. Yamamoto, E. Graham and G.H.N. Towers. 1980. P h o t o s e n s i t i z a t i o n of E. col i and S. cerevisiae by p h e n y l h e p t a t r i y n e from Bidens pilosa. Can. J . M i c r o b i o l . 26: 698 - 705. Arnason, T., T. Swain, C.K. Wat, E.A. Graham, S. P a r t i n g t o n and G.H.N. Towers. 1981. Mosquito l a r v i c i d a l a c t i v i t y of p o l y a c e t y l e n e s from s p e c i e s i n the Asteraceae. Biochem. S y s t . E c o l . 9: 63 - 68. Arnaud, A. 1902. Sur l a c o n s t i t u t i o n de l ' a c i d e tarririque. C R . Hebd. Seanc. Acad. S c i . , P a r i s 134:473 - 482. Bohlmann, F., T. Burkhardt and C. Zdero. 1973. N a t u r a l l y O c c u r r i n g A c e t y l e n e s . Academic P r e s s , London. Bohlmann, F., R. Jente and R. Reinecke. 1969. Polyacetylenverbindungen. Uber d i e biogenese der n a t u r l i c h e n C - und C - acetylenverbindungen. Chem. Ber. 102: 3283 - 3292. 1 3  1 0  Bohlmann, F. and C. Zdero. 1968. Polyacetylenverbindungen, CLVII. Uber d i e i n h a l t s s t o f f e von Coreopsis nuecensis A. H e l l e r . Chem. Ber. 101: 3243 - 3254. Bohlmann, F., M. Grenz, M. Wotschokowsky and E. Berger. 1967. Polyacetylenverbindungen CXXXIV. Uber neue thiophen acetylenverbindungen. Chem. Ber. 100: 2518 2522. Bohlmann, F. and H. Bornowski. 1966. Polyacetylenverbindungen XCVIII. Uber phenolisch substituierte naturliche acetylenverbindungen. Chem.  12 Ber.  99:1223 - 1228.  Bohlmann, F., K.M. K l e i n e and H. Bornowski. 1966. Polyacetylenverbindungen XCI. S t r u k t u r und synthese eines C - k e t o a c e t a t s aus Cosmos sulphureus cav. Chern. Ber. 99: 142 - 147. 1 8  Bohlmann, F., H. Bornowski and K. M. K l e i n e . 1964. Uber neue polyne aus dem T r i b u s H e l i a n t h e a e . Chem. Ber. 97: 2135 2138. Bu'Lock, J.D. 1966. The b i o g e n e s i s of n a t u r a l a c e t y l e n e s , pp. 79-95 i n Comparative Phytochemistry T. Swain (Ed.). Academic Press, London. Camm, E.L., G.H.N. Towers and J . C. M i t c h e l l . 1975. UV-mediated antibiotic activity of some Compositae s p e c i e s . Phytochemistry 1 4 : 2007 - 2011. Cascon, S . C , W.B. Mors, B.M. Tursch, R.T. A p l i n and L . J . Durham. 1965. I c h t h y o t h e r o l and i t s a c e t a t e ; the a c t i v e polyacetylene constituents of Icht hyoi here terminal is (Spreng.) Malme, a f i s h poison from the Lower Amazon. Amer. Chem. Soc. J . 8 7 : 5237 - 5241. Celmer, W.D. and I.A. Solomon. 1952a. The s t r u c t u r e of the antibiotic mycomycin. Amer. Chem. Soc. J . 7 4 : 1870 1871 . Celmer, W.D. and I.A. Solomon. 1952b. Mycomycin. I . Isolation, . crystallization and chemical c h a r a c t e r i z a t i o n . Amer. Chem. Soc. J . 7 4 : 2245 - 2248. Celmer, W.D. and I.A. Solomon. 1953. Mycomycin. I I I . The s t r u c t u r e of mycomycin, an a n t i b i o t i c c o n t a i n i n g a l l e n e , d i a c e t y l e n e and cis, trans-cMene groupings. Amer. Chem. Soc. J . 75 1372 - 1376. Chan, G.F.Q., G.H.N. Towers and J.C. M i t c h e l l . 1975. Ultraviolet-mediated antibiotic activity of thiophene compounds of Tagetes. Phytochemistry 1 4 : 2295 - 2296. Chin, C , M.C. C u t l e r , S i r E.R.H.Jones, J . Lee, S. Safe and V. T h a l l e r . 1970. N a t u r a l acetylenes. Part XXXI. C! -tetrahydropyranye and other p o l y a c e t y l e n e s from the Compositae Dahlia coccinea Cav. v a r . cocci nea. J . Chem. Soc. ( C ) : 314 - 322. a  Cimino, G., A. C r i s p i n o , S. DeRosa, S. DeStefano and G. Sodano. 1981. P o l y a c e t y l e n e s from the sponge Petrosia ficiformis found i n dark caves. E x p e r i e n t i a 3 7 : 924 926. Daniels,  F.  1965. A  simple  microbiological  method f o r  13 demonstrating phototoxic compounds. J . I n v e s t . Dermatol. 44: 259 - 263. De N a p o l i , L., E. F a t t u r u s s o , S. Magno and L. Mayol. 1981. F u r o c a u l e r p i n , a new a c e t y l e n i c s e s q u i t e r p e n i o d from the green algae Caulerpa prolifera. E x p e r i e n t i a 37: 1132. DiCosmo, F., G.H.N. Towers and J . Lam. 1982. Photoinduced fungicidal activity elicited by n a t u r a l l y o c c u r r i n g thiophene d e r i v a t i v e s . P e s t i c . S c i . 13: 589 - 594. Gilman, H., P.R. Van Ess and R.R. B u r t n e r . 1933. The constitution of C a r l i n a - o x i d e . Amer. Chem. Soc. J . 55: 3461 - 3466. Gommers, F . J . and J.W.G. G e e r l i n g s . 1973. L e t h a l e f f e c t of near u l t r a v i o l e t l i g h t on Pratylenchus penetrans from roots of Tagetes. Nematologica 19: 389 - 393. Graham, K., E.A. Graham and G.H.N. Towers. 1980. Cercaricidal activity of phenylheptatriyne and a - t e r t h i e n y l , n a t u r a l l y - o c c u r r i n g compounds i n s p e c i e s of Asteraceae (Compositae). Can. J . Z o o l . - 5 8 : 1955 1958. Hudson, J.B., E.A. Graham and G.H.N. Towers. 1982. The nature of the i n t e r a c t i o n between p h o t o a c t i v e compound phenylheptatriyne (PHT) and animal v i r u s e s . Photochem. P h o t o b i o l . 36: 181 - 186. Jones, S i r E.R.H. 1966. N a t u r a l p o l y a c e t y l e n e s and t h e i r p r e c u r s o r s . Chem. B r i t . 1966: 6 - 13. Jones, S i r E.R.H. 1959 P o l y a c e t y l e n e s . Pedlar L e c t u r e , 12 February 1959, Chemical S o c i e t y , London. Jones, S i r . E.R.H. and V. T h a l l e r . 1978. N a t u r a l a c e t y l e n e s , pp 621-633 i n The C h e m i s t r y of the Carbon-Carbon Triple Bond P a r t 2. S. P a t a i (Ed.). J . Wiley and Sons, New York. Kagan, J . and G. Chan. 1983. The p h o t o o v i c i d a l a c t i v i t y of plant components towards Drosophila melanogasl er . E x p e r i e n t i a 39: 402 - 403. Kavanagh, F., A. Hervey and W.J. Robbins. 1950. A n t i b i o t i c substances from Basidiomycetes, IV. Agrocybe dura. Proc. Nat. Acad. S c i . 36: 102 - 106. Lam,  J . 1973. P o l y a c e t y l e n e s of Dahlia. 1: 83 - 86.  Lam,  J . 1971. P o l y a c e t y l e n e s i n Dahlia imperial is l enuicaulis . Phytochemistry 10: 2227 - 2228.  Bioch.  Systematics and Dahlia  14 Lam, J . and F. Kaufmann. 1971. P o l y a c e t y l e n i c C - e p o x i d e and C tetrahydropyranyl compounds from Dahlia scapigera. Phytochemistry 10: 1877 - 1880. 1lt  1((  MacRae, W.D., G.F.Q. Chan, C.K. Wat, G.H.N. Towers and J . Lam. 1980. Examination of naturally occurring polyacetylenes and a - t e r t h i e n y l f o r t h e i r a b i l i t y t o induce genetic damage. E x p e r i e n t i a 36: 1096 - 1097. MacRae, W.D., D.A.J. I r w i n , T. B i s a l p u t r a and G.H.N. Towers. 1980. Membrane l e s i o n s i n human e r y t h r o c y t e s induced by the n a t u r a l l y o c c u r r i n g compounds a - t e r t h i e n y l and p h e n y l h e p t a t r i y n e . Photobiochem. Photobiophys. 1: 309 318. McLachlan, D., T. Arnason and J . Lam. 1984. The r o l e of oxygen i n p h o t o s e n s i t i z a t i o n s with p o l y a c e t y l e n e s and thiophene d e r i v a t i v e s . Photochem. P h o t o b i o l . 39: 177 182. Moore, B.P. and W.V. Brown. 1978. P r e c o c c i n e l l i n e and related a l k a l o i d s i n the A u s t r a l i a n s o l d i e r beetle Chaul iognat hus pulchellus (Coleoptera: Cantharidae). Insect Biochem. 8: 393 - 395. Reisch, J . , S p i t z n e r , W. and K.E. S c h u l t e . 1967. Zur frage der mikrobiologischen wirk samkeit einfacher acetylenverbingdungen. Arzneim. F o r s c h . (Drug Res.) 17: 816 - 840. S c h u l t e , G.R., M.C.H. Chung and P.J. Scheuer. bicyclic C - e n y n e s from the seahare Aplysia J . Org. Chem. 46: 3870 - 3873. 15  1981 Two oculifera.  Scrimgeour, C M . 1980. N a t u r a l a c e t y l e n i c and o l e f i n i c compounds. Royal Soc. Chem. A l i p h . Nat. Prod. Chem. 3: 1 - 25. Scrimgeour, C M . 1981. N a t u r a l a c e t y l e n i c and o l e f i n i c compounds, e x c l u d i n g marine n a t u r a l products. Royal Soc. Chem. A l i p h . Nat. Prod. Chem. 2: 1 - 19. Semmler, L.T. 1906. Zusammensetzung des a t h e r i s c h e n o e l s der eberwurzewl Carlina acauli s L. Ber. d t . Chem. Ges. 39: 726 - 730. Song, P.S. and K.J. Tapley. photobiology of p s o r a l e n s . 1177 - 1197.  1979. Photochemistry and Photochem. P h o t o b i o l . 29:  S^rensen, J.S. and N.A. Sf^rensen. 1966. S t u d i e s r e l a t e d t o naturally occurring acetylene compounds, XXXIII. A preliminary i n v e s t i g a t i o n of Coreopsis gigantea ( K e l l . ) H.M.Hall. Acta Chem. Scand. 20: 992 - 1002  15 S^rensen, J.S. and N.A. S^rensen. 1958. S t u d i e s related to naturally occurring acetylene copounds, XXIV. 2-phenyl-5(a-propynyl)-thiophene from the e s s e n t i a l o i l s of Coreopsis grandiflora Hogg ex Sweet. Acta Chem. Scand. 12: 771 - 776. Sjzfrensen, J.S. and N.A. S^rensen. 1958. S t u d i e s r e l a t e d t o naturally occurring acetylene compounds, XXII. Correctional studies on the c o n s t i t u t i o n of the p o l y a c e t y l e n e s of some annual Coreopsis s p e c i e s . Acta Chem. Scand. 12: 756 - 764. S^rensen J.S. and N.A. Sfrfrensen. 1958. S t u d i e s related to naturally occurring acetylene compounds, XXIII. 1-phenylhepta-1:3:5-triyne from Coreopsis grandiflora Hogg ex Sweet. Acta Chem. Scand. 12: 765 - 770. S^rensen, N.A. 1977. P o l y a c e t y l e n e s and conservatism of chemical characters i n the Compositae. pp. 385-409 i n The B i o l o g y and Chemistry of t h e Compositae. V o l . I. V.H. Heywood, J.B. Harborne and B.L. Turner ( E d s . ) . Academic Press, New York. S^rensen, S.L. and N.A. S^rensen. 1961. S t u d i e s r e l a t e d t o naturally occurring acetylene compounds, XXIX. Preliminary i n v e s t i g a t i o n s i n the genus Bidens: I. Bidens  radial  a T h u i l l and  Bidens  f erul aef ol i a  (Jacq.)  DC. Acta Chem. Scand. 15: 1885 - 1891. Swain, T. and C.A. W i l l i a m s . 1977. Heliantheae - chemical review. pp. 673-697 i n The B i o l o g y and Chemistry of the Compositae. V o l . I . V.H. Heywood, J.B. Harborne and B.L. Turner, (Eds.). Academic Press, New York. Thaller, V. 1976-77. N a t u r a l acetylenic and olefinic compounds, e x c l u d i n g marine n a t u r a l products. Royal Soc. Chem. A l i p h . Nat. Prod. Chem. 1: 1 - 19. Towers, G.H.N. 1980. P h o t o s e n s i t i z e r s i n p l a n t s and t h e i r photodynamic a c t i o n , pp. 183-202 in Progress in Phytochemistry V o l . 6 . L. R e i n h o l d , J.B. Harborne and T. Swain, ( E d s . ) . Pergamon P r e s s . Towers, G.H.N, and C K . Wat. 1978. B i o l o g i c a l a c t i v i t y of p o l y a c e t y l e n e s . Rev. Latinoamer. Quim. 9: 162 - 170. Towers, G.H.N., C K . Wat, E.A. Graham, R.J. Bandoni, G.F.Q. Chan, J.C. M i t c h e l l and J. Lam. 1977. U l t r a violet-mediated antibiotic activity of s p e c i e s of Compositae caused by p o l y a c e t y l e n i c compounds. L l o y d i a 40: 487 - 496. Towers, G.H.N., T. Arnason, C K . Wat, E.A. Graham, J . Lam and J.C. M i t c h e l l . 1979. P h o t o t o x i c p o l y a c e t y l e n e s and  16  their thiophene d e r i v a t i v e s - effect Contact D e r m a t i t i s 5: 140 - 144.  on human s k i n .  Vil'yams, V.V., V.S. Smirnov and V.P. Golmov. 1935. Nature of the c r y s t a l l i n e substance i n the e s s e n t i a l oil of Lac hnophyl I urn gossypinum Bge. Zhur. Obschei Khim. 5: 1195 - 1203; Chem. A b s t r . 30: 1176 - 1177. Wagner, H. 1977. Pharmaceutical and economic uses of the Compositae. pp. 412-433 i n The B i o l o g y a n d C h e m i s t r y o f the C o m p o s i t a e V o l . I . V.H. Heywood, J.B. Harborne and B.L.Turner, ( E d s . ) . Academic Press, New York. Walker, R. P. and D.J. F a u l k n e r . 1981. C h l o r i n a t e d a c e t y l e n e s from the nudibranch Diaul ul a sandi egensis. J . Org. Chem. 4 6 : 1475 - 1478. Warren, R.A.J., J.B. Hudson, K.R. Downum, E.A. Graham, R. Norton and G.H.N. Towers. 1980. Bacteriophages as indicators of the mechanism of action of photosensitizing agents. Photobiochem. Photobiophys. 1: 385 - 389. Wat,  C.K., S.K. Prasad, E. A. Graham, S. P a r t i n g t o n , T. Arnason, G.H.N. Towers, and J. Lam. 1981. Photosensitization of invertebrates by natural p o l y a c e t y l e n e s . Biochem. S y s t . E c o l . 9: 59 - 62.  Wat,  C.K., T. Johns and G.H.N. Towers. 1980. Phototoxic and a n t i b i o t i c a c t i v i t i e s of p l a n t s of the Asteraceae used in f o l k medicine. J . Ethnopharmacology 2 : 279 - 290.  Wat,  C.K., R.K. Biswas, E.A.Graham, L. Bohm, G.H.N. Towers and E.R. Waygood. 1977. U l t r a v i o l e t - m e d i a t e d c y t o t o x i c a c t i v i t y of p h e n y l h e p t a t r i y n e from Bidens pilosa L. J . Nat. Products 4 2 : 103 - 111.  II.  A.  POLYACETYLENES IN HAWAIIAN BIDENS  INTRODUCTION The  Hawaiian I s l a n d s are u s u a l l y c o n s i d e r e d  to  be  most i s o l a t e d a r c h i p e l a g o on e a r t h . They l i e v i r t u a l l y in the  North  oceanic  Pacific,  islands  separated  such  the  by  4000km  of  which  nearest  ocean  A l l are g i a n t submarine volcanoes  apparently  alone high  as the Marquesas by 3200km, and from  the North American coast 1970).  from  the  s t a t i o n a r y hot spot  beneath  that arose the  has been moving i n a northwesterly  (Carlquist, from an  Pacific  direction  Plate  f o r tens  of m i l l i o n s of years. The i s l a n d c h a i n s t r e t c h e s 2500km from northwest t o southeast  a c r o s s the P a c i f i c , the o l d e r Western  Leeward I s l a n d s having  been reduced  islets  and  atolls,  while  the  Windward I s l a n d s , are mountainous The  major  Kauai  islands  t o l e s s than  Geological  by  erosion  to  main  eastern  group,  and  shoals,  geologically  the  young.  range i n age from 5.6 m i l l i o n years f o r 1.0 m i l l i o n years f o r Hawaii  evidence  indicates  (Figure  1).  that the Hawaiian chain has  never been connected t o a c o n t i n e n t a l  land  mass  (Stearns,  1966). The animals  a n c e s t o r s of a l l arrived  indigenous  Hawaiian  s o i l and temperature c o n d i t i o n s  Islands  make  is  them  exceptionally  variation  in  17  of  the  inviting  by many groups of organisms. In  considerable  and  by a c c i d e n t a l l o n g - d i s t a n c e d i s p e r s a l . The  rainfall,  occupation  plants  Hawaiian  fields  addition,  microclimate  on each  for there  island  KAUAI NIHAU (P  v>OAHU  LANAI  THE MAJOR HAWAIIAN ISLANDS  19  because  of  immigrant  the mountainous  species  environment  with  conditions ideal 1966b;  a  a  species  relative  of  of  one  to  ancestral  life  organisms  in a  such  Fallen),  as  Lipochaeta  (  radiation  i n the Hawaiian  (Asteraceae),  DC.)  commonly  i n North  America  physically  diverse  of  competition,  (Carlquist,  into  numerous  v a r i e t y of h a b i t a t s . Many  p l a n t s (Pelea have  undergone  as  and  The  beggarticks  ko'oko'olau  A. Gray) and spectacular  or  Spanish  i n Hawaii, has numerous  which e x h i b i t g r e a t e r m o r p h o l o g i c a l and e c o l o g i c a l found elsewhere  flies  genus Bidens L.  evolved from a s i n g l e a n c e s t r a l s p e c i e s i n t o  than s p e c i e s of Bidens  1966a;  i s the e v o l u t i o n a r y  lineage  Islands.  known  arrival,  the honeycreepers,  rutaceous  composites  Upon  absence  f o r adaptive r a d i a t i o n  adapted  (Drosophila  needles  new,  1967; 1970). Adaptive r a d i a t i o n  diversification  groups  faced  topography.  taxa  diversity  i n the world  (Ganders  and Nagata, 1983a; 1984). The Hawaiian range of  from a r i d t o s e m i - a r i d l a v a  rainfall  with  s p e c i e s of Bidens  occur i n h a b i t a t s  flows with l e s s than 0.3m  per year, t o dense r a i n f o r e s t and montane  annual  precipitation  that  exceeding  7.0m,  and  bogs  through  e l e v a t i o n s extending from sea l e v e l t o over 2200m. They have diversified  i n growth  trunks over 2m t a l l herbaceous  forms),  habit  to t a l l leaf  (from small t r e e s with woody  shrubs t o e r e c t  shape (from simple t o compound t o  h i g h l y d i s s e c t e d ) , flower head s i z e and shape, and  shape  (from  flat  and p r o s t r a t e  and  straight  achene  to t i g h t l y  size  coiled),  20 presence  and  lengths and wings),  type  of d i s p e r s a l mechanism (awns of v a r i o u s  shapes, pubescence, and presence or  as  well  as  absence  in e c o l o g i c a l tolerances. Differences  between s p e c i e s i n a l l these c h a r a c t e r s are maintained standard  of  under  growing c o n d i t i o n s , i n d i c a t i n g that they are under  s t r o n g g e n e t i c c o n t r o l (Helenurm and Ganders, .1985). Surprisingly,  however,  completely  interfertile  suggests  that  (Ganders  adaptive  e c o l o g i c a l t o l e r a n c e has physiological (Gillett 1983a;  1984).  1970;  The  and  Nagata,  radiation  in  interspecific  Gillett,  different  Bidens  Hawaiian  occurred without  or g e n e t i c  and Lim,  all  1975;  are  1984).  This  morphology  the  and  evolution  i s o l a t i n g mechanisms Ganders  and  Nagata,  s p e c i e s of Hawaiian Bidens  are  as s i m i l a r g e n e t i c a l l y at isozyme l o c i as are p o p u l a t i o n s a  single  species  differentiation genetic  loci  show l i t t l e taxonomic  most  plants.  distances  among  populations  classification  characters  secondary m e t a b o l i t e s In t h i s study,  two  of the subspecies  been  in these  objectives  were were  little  processess,  based on  isozyme  differences  morphological  and  of i n t e r e s t to determine  evolutionary  divergence  in  species. from leaves and  endemic Hawaiian s p e c i e s of Bidens  all  primary  it  of  the p o l y a c e t y l e n e s  of  of  (Helenurm and Ganders, 1985).  makes  whether or not there has  the  exhibit  c o r r e l a t i o n with morphological  T h i s d i s p a r i t y between the e v o l u t i o n biochemical  They  i n isozymes of primary metabolic  and  or  in  of  isolated to  and  determine  roots  and a l l but  identified. the  extent  The of  21 evolutionary Bidens  differentiation  and  characters  to  of p o l y a c e t y l e n e s i n Hawaiian  see whether  i n the group.  they The  are useful  possible  taxonomic  relationship  of  Bidens  p o l y a c e t y l e n e d i s t r i b u t i o n t o the b i o l o g y of Hawaiian i s also considered. Sherff of  (1943) produced a worldwide  the genus Bidens  taxonomic  based on a study of herbarium m a t e r i a l .  In t h i s and subsequent p u b l i c a t i o n s he recognized and  more than  20 i n f r a s p e c i f i c  Ganders and Nagata species  and  in t h i s  interfertile, obtain  The  variation  the extent  i n these  (1983a; 1984) have reduced  plants.  these  8 subspecies. T h e i r c l a s s i f i c a t i o n  of  t o 19  i s followed  thesis.  Since  their F  determined  43 s p e c i e s  taxa endemic t o the Hawaiian  I s l a n d s . He had no i n f o r m a t i o n , however, on environmentally  revision  a l l the  species  of  Hawaiian  Bidens  i n t e r s p e c i f i c h y b r i d s were r e l a t i v e l y easy  are to  e x p e r i m e n t a l l y . S e v e r a l of these h y b r i d s , as w e l l as 2  o f f s p r i n g , were examined for t h e i r  purpose  of  polyacetylenes.  t h i s p o r t i o n of the study was t o determine  the degree of complexity  of p o l y a c e t y l e n e i n h e r i t a n c e .  22  B. MATERIALS AND METHODS  PLANT MATERIAL P l a n t s from 54 p o p u l a t i o n s six  subspecies  polyacetylenes all  endemic  i n leaves and taxa  Figures  1  t o 6.  and  also F  by Ganders and  plants  were  s s p . waihoiensis  were  (1984) St.John  ex S h e r f f  ssp.  f o r a l l p o p u l a t i o n s are shown i n  Voucher  specimens  Columbia  are d e p o s i t e d at  (UBC), and  synthesized  by  the  d u p l i c a t e s of most ( H L A ) . F,  F.R.Ganders  and a l l  grown from seeds or c u t t i n g s i n greenhouses a t  the U n i v e r s i t y of B r i t i s h leaves  Nagata  a t the Harold Lyon Arboretum, Honolulu  hybrids  2  (Table I I ) . T h i s i n c l u d e s  (Drake d e l Cast.) Deg.  . Localities  U n i v e r s i t y of B r i t i s h are  roots  recognized  B. hi 11 ebrandi ana  hi 11 ebrandi ana  19 s p e c i e s and  of endemic Hawaiian Bidens were examined f o r  B. campyl ot heca Schz. B i p .  except and  representing  and  roots  Columbia under n a t u r a l l i g h t , and  of greenhouse  plants  harvested f o r  analysis.  ISOLATION AND IDENTIFICATION OF POLYACETYLENES F r e s h leaves and (MeOH) was with The  (1g t o 10ml  diluted equal  roots  ratio),  were  extracted  ground and  1:1 with d i s t i l l e d volumes of l i g h t  water  with  methanol  f i l t e r e d . The f i l t r a t e and e x t r a c t e d  petroleum ether(PE)  (30-60°C).  combined PE f r a c t i o n s were d r i e d with anhydrous  Solvent  twice  Na SOi,. 2  volume was reduced t o 3ml f o r s p e c t r a l a n a l y s i s . UV  s p e c t r a were recorded  i n s p e c t r a l grade PE  using  either  a  TABLE  1.  8fdens  2.  8.  amplect  ens  asymmet^ica  I I . niCENS  TAXA EXAMINED  Sherff  (Levi.)  Sherff  FOR POLYACETYLENES  12 .  ft.  menziesii  s s p . menzlesi  12a .  P.  menzfesii  s s p . flliformis  I (Gray)  Sherff  (Sherff)  G a n d e r s ft N a g a t a 3. 3a.  8.  campy 1 otheca  B.  campylotheca  Schz . BIp.  s s p . r.impy 1 ofhec.a  s s p . pentamera  (Sherff)  13 .  13b  N a g a t a ft G a n d e r s  P.  micrantha  Gaud.  P.  micrantha  s s p . ctenophylla  B.  cerv i cat a  Sherff  13a .  P.  mi crantha  5.  8.  conjunct  Sherff  14 .  0.  molokalensis  6.  8.  cosmoides  15.  B.  popul  7 .  8.  forbesi r Sherff  16.  P.  sandvIcens<s  8.  forbesiI  7b.  micrantha (Sherff)  N a g a t a ft G a n d e r s  4 .  a  ssp.  (Gray)  Sherff ssp. f o r b e s M  s s p . kahf1fensis  G a n d e r s ft  16a.  Nagata  s s p . kalealaha  ifolia  Ganders  &  Nagata  IHHIebr. ) Sherff Sherff  (i. sandvicensis  Less.  ssp.  s s p . confusa  sandvicensis N a g a t a ft  Ganders  8.  8.  hawaiensis  9.  8.  hi 11ebrandiana  Gray s s p . polycephaij  N a g a t a ft  * 17 .  P.  t o r t a 'Sherff  18.  P.  valIda  19. •  P.  itiebkel  Sherff  Ganders 10.  8.  macrocarpa  11 .  8.  maul ensi r, ( G r a y )  17A  (B18,  B19):  17B  (Gray)  Sherff  Sherff  Sherff  (B36-B41);  17C  ( B 5 5 . B56) : 170  (B110)  K)  FIGURE 2. LOCALITIES OF KAUAI  BIDENS  POPULATIONS SAMPLED  B. cer vi cat a: BB, BB3; B. cosmoides: B9; forbesii: kahiliensis: B112; B54,  B12,  B. forbesii  ssp.  B13, B14, B74, B101, B124: B. forbesii  ssp.  B71, B134: B. sandvi censi s  B. sandvi censi s B131, B 1 3 2 .  ssp.  ssp.  sandvi censis:  confuse. B33, B34; B. val i da:  KAUAI B12,124 X B 74 \ B101  BU  ,B13  \  B 83  B 112  B 9  B 33  cn  B134  B 54,71,132  FIGURE 3. LOCALITIES OF OAHU BIDENS POPULATIONS SAMPLED B.  amplectens:  BI; B. asymmetrical,  ssp.  campylot hecax  B22;  B23; B.  B. sandvi censis  B110 ( D ) .  B195; B. cervicata:  mol okai e ns i s:  B1 1 ;  s s p . sandvicensis:  B43-B47; B. lorla:  B 4 ; B 2 1 1 ; B. campy I ot heca  B18, B 1 9  B.  B88; B. macrocarpax populifolia:  B42;  B5; B 6 ; B7; B20; B 3 5 ;  ( A ) , B36-B41 (B), B55, B56 ( C ) ,  OAHU  B 5t6  B  2  °.  « '  4  6  FIGURE 4.  L O C A L I T I E S OF MAUI  BIDENS  POPULATIONS SAMPLED  B. campyl ot heca s s p . pentamero: B 1 1 4 ; B. conjunct ax B. hi 11ebrandi ana s s p . polycephala: BIO,  B27(cultivated),  menziesii:  B28,  B60-B63;  B67, B66; B. maui ensi s :  B12B;  B. menziesii  ssp.  B 3 1 , BB4; B. mi cram ho s s p . mi cr ant ha: B24, B 2 5 ,  B7B, B79; B. micrantha s s p . kalealaha: B125.  29  30  FIGURE 5. LOCALITIES OF MOLOKAI BIDENS POPULATIONS SAMPLED. B. mol okai ensi s : B72, B73; B. weibkei:  B260.  MOLOKAI  FIGURE 6. LOCALITIES OF HAWAII BIDENS POPULATIONS SAMPLED B.  hawaiensis:  B21, B48-B53; B. menziesii  B30, B32, B130, B163; B. micrantha B150.  ssp.  ssp.  fiiiformis:  cl enophylI a:  B149,  33  HAWAII  34 Pye-Unicam  SP8-100  UV/VIS  or  a  Unicam  SP800A  UV/VIS  spectrophotometer. Samples were evaporated t o dryness nitrogen storage  and  resuspended  in  a t -20°C. Concentrated  Finnigan  1020 Automated  compounds  i n the PE  recorded.  1ml  s p e c t r a l grade MeOH f o r  samples were i n j e c t e d i n t o  GC/MS  and  fraction,  Chromatographic  under  the mass  including  separation  of  a  s p e c t r a of  polyacetylenes, compounds  was  c a r r i e d out with a SE-54 30m x 0.25mm c a p i l l a r y column using a  temperature  helium  g r a d i e n t of 10°/min from 150°C t o 250°C, and  as a c a r r i e r gas.  Polyacetylenes sheets  were separated  c o n t a i n i n g a f l u o r e s c e n t i n d i c a t o r (SG-UV254  N-UV254).  PE  (30-60°C)  d i e t h y l ether(DE), compounds  was used  ( 1968)  to  increasing effect  and Wrang  chromatography on s i l i c a Research) using s i l i c a used  of  DE  systems used f o r proportions  of  the developing  cases,  percentages  separation  of  of the  t o the methods d e c r i b e d  and  Lam  (1975).  g e l 60 and a chromatotron  g e l PF-254 with  PE  (30-60°C)  Column  (Harrison  CaSO««l/2H 0 2  plates  followed  by  increasing  t o e l u t e more p o l a r compounds. The solvent a l l separations PE  were  of  the f o l l o w i n g  t o DE :19:1; 9:1; 17:3; 8:2; 7:3;  5:5. A small a l i q u o t of c o n c e n t r a t e d to  and SG  f o r l a r g e r s c a l e s e p a r a t i o n s . Columns and p l a t e s  were developed with amounts  with  i n each e x t r a c t a c c o r d i n g  by Lam et al .  were  on a n a l y t i c a l s i l i c a g e l  acetic acid  was  13:7;  added  tank f o r t h i n l a y e r chromatography. In a l l  compounds 1 t o 18 were e l u t e d by PE/DE 13:7.  35  I n d i v i d u a l a c e t y l e n e s were i d e n t i f i e d by comparison UV  and  mass  spectra  Extraction, isolation carried  with  and  those  of  known  identification  compounds.  procedures  were  out i n dim l i g h t at 0°C or lower.  I n d i v i d u a l s from one t o f i v e p o p u l a t i o n s of each were  examined  individual  over  a  period  p l a n t s were analyzed  s i x times throughout  of  18  2  populations  f o r p o l y a c e t y l e n e s three  t h i s p e r i o d . Roots were analyzed  sampled  extractions for specific  once.  taxon  months. Leaves from  F, i n d i v i d u a l s were analyzed three times over F  of  This  compounds.  to  twice.  12 months  and  precluded large scale  36 C. RESULTS  IN BIDENS TAXA  POLYACETYLENES  Leaves and r o o t s from 19 s p e c i e s and s i x subspecies Bidens  from  analyzed  Kauai,  Oahu,  for polyacetylenes  (B. campylot heca  taxa  Maui,  Molokai  of  and Hawaii were  (Table I I , F i g u r e s 2 t o 6 ) . Two B i p . ssp. waihoiensis  Schz.  and B. hi I1ebrandi ana (Drake d e l Cast.) Deg. ex  S t . John  Sherff ssp.  hi I1 ebrandiana) were not a v a i l a b l e f o r a n a l y s i s . Compounds 1 to 18 were i s o l a t e d c h r o m a t o g r a p h i c a l l y the  and i d e n t i f i e d  on  b a s i s of UV and mass s p e c t r a (Tables I I I , IV) and t h e i r  d i s t r i b u t i o n among the s p e c i e s recorded Although  (Tables V, V I ) .  a c e t y l e n e s were found i n a l l the root samples  examined, they were absent from the leaves o f 13 of the taxa (Table  V ) . Repeated sampling  of greenhouse p o p u l a t i o n s  a p e r i o d of 18 months r e v e a l e d no q u a l i t a t i v e polyacetylene  production  with  changes  r e p r o d u c t i v e s t a t e of the p l a n t s . T h i s Dahlia  , where  content  and composition  in  consecutive  considerable  over  variation in i n season or  i s in contrast to  variation  i n polyacetylene  were encountered w i t h i n the s p e c i e s  seasons,  and i n the same season i n p l a n t s al. ,  locations  et  a c e t y l e n e s a r e known t o be p h o t o a c t i v e  al. ,1968).  Many  (Chin  et  growing i n d i f f e r e n t  (Towers et al., 1977; Towers, 1980), petroleum  fractions  1970;  and t h e crude  Lam  light  of l e a f and root e x t r a c t s were t e s t e d  f o r p h o t o t o x i c i t y a g a i n s t nine s p e c i e s of f u n g i and b a c t e r i a using  the method of D a n i e l s  (1965). While the root samples  TABLE I I I . POLYACETYLENES FROM HAWAIIAN BIDENS  CHjSCH (CSC) CH=CHCH R  R • H  2  4  1  R « OCOCH CH =CH  (C = C ) C H  2  5  3 3  <Q C C, CH CHC (  S  2  2  3  =  ...  HjR  R • OH  6  R « OCOCH CH CH=CH ( C = C ) ( C H = C H ) ( C H > C H = C H 3  2  2  2  CH (C=C) (CH=CH) (CH ) CH=CH 3  3  2  2  4  4  2  CH =CHCH=CH 2  2  2  (C = C ) C H = CHCH R 2  3  2  B  9  2  CH CH=CH ( C = C ) C H C H = C H ( C H ) C H = C H 3  5  10  2  R • H  H  R « OCOCH  CH CH = 3  7  3  12  3  CH(C=C) CH=CH—L^^J  1 3  2  C C  ^^=^0-  R  R  CH  - 3  I*  R • CH OH  15  R » CHO  16  2  R • CH OCOCH 2  CH,CH=CH ( C S C ) ,CH = CHCHOHCH_OH  1  7  3  18  38 TABLE I V . POLYACETYLENES  FROM HAWAIIAN BIDENS  I, 2  trideca-1,11 diene-3,5,7,9 tetrayne  3  trideca-1  4  1-phenylhepta-1,3,5  5,6,7  1-phenylhepta-l,3  B  heptadeca-2,7,9,16 tetraene-4,6  9  heptadeca-8,10,16 triene-2,4,6  10  heptadeca-2,9,16 triene  I I , 12  trideca-1,3,11  13  tetrahydro-2  14-17  2-  18  trideca-3,11 diene-5,7,9 triyne-1,2  ene-3,5,7,9,11  pentayne  triyne  d i y n e - 5 ene triyne triyne  -4,6 d i y n e  triene-5,7,9  triyne  1,7 d i e n e - 3 , 5 d i y n y l  2-phenylethyne-1  y l -5 m e t h y l  pyran-3-ol thiophene diol  39 TABLE V . POLYACETYLENES IN THE LEAVES OF HAWAIIAN  BIDENS  Compounds Bidens  1  1  2  -  2  _  _  3  +  _  3a  +  -  4  +  _  6  +  -  4  3  _  5  -  -  _  _ -  _  _  +  -  _  _  8  _  _  _  9  +  +  _  _  _  10  +  -  _  _  _  -  -  -  12  _  _  _ _  _  -  _  -  _  _  16a  _  -  17A  _  _  17B  -  17C  _  _  -  17D  _  -  -  _  +  _  18  +  -  _  _  +  _  19  -  _ -  _  +  -  _  _  _  _  _  _  _  _  _  _  _  _  _  -  -  _  +  -  _  -  -  _  -  _  -  -  _  +  +  -  _  -  -  _  -  -  + -  -  +  _  _  _  -  +  +  _ _ _  _  + +  _  • + _ _ _  -  -  _  _  _  -  -  _  _  _  -  -  _  _  _  +  _  _  _  -  _  _  _  +  +  _  _  13a 1 3b 14 15 16  -  _ _  + +  -  -  _ _  + -  -  _  _  13  _  +  12a  _  18  -  _  _  -  12  + +  -  "11  -  +  -  _  -  +  7a  11  -  +  -  10  _  +  7  -  9  -  _  -  _  -  _  _  8  7  -  + +  _  _  40  TABLE V I . POLYACETYLENES IN THE ROOTS OF HAWAIIAN  BIDENS  Compounds / de ns  1  1  +  2  +  2  5  6  7  4  +  +  -  5  +  -  +  +  6  +  +  +  7  +  -  -  7a  +  +  +  8  +  - -  +  3a  +  9 1 1  +  -  12a  -  +  -  +  -  +  13a +  14  +  15  •  •  -  16  +  -  17B  + -  17C  -  17D  •  -  16a  19  -  •  11 12  -  -  -  + +  -  -  -  -  + -  -  +  +  +  -  -  -  •  +  15 16  -  -  -  +  -  -  -  +  -  -  +  13 14  -  +  -  + +  + +  -  -  -  -  +  + -  -  +  1*  + + +  + + + + + +  +  -  -  -  -  -  -  +  -  -  +  -  + + + +  13b  IB  -  +  -  17A  -  +  12 13  10  +  3  10  8  +  -  -  +  -  +  -  •  -  -  -  +  +  •  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  +  -  -  +  -  +  +  -  *  +  +  41 were found  t o be c o n s i s t e n t l y p h o t o t o x i c , only l e a f e x t r a c t s  c o n t a i n i n g a c e t y l e n e s were l e t h a l t o the microorganisms the  presence  of near UV r a d i a t i o n  IV). The absence of p o l y a c e t y l e n e s two et  other Bidens  Bidens.  (320-400nm) (see Chapter i n the l e a v e s of a t l e a s t  s p e c i e s has been p r e v i o u s l y noted  a/.,1973), so t h i s f e a t u r e However,  i t does  in  i s not unique  (Bohlmann  to  Hawaiian  lead to i n t e r e s t i n g speculation  about the p o s s i b l e b i o l o g i c a l  s i g n i f i c a n c e of the compounds  in q u e s t i o n . The C  1 3  p o l y a c e t y l e n e s of Hawaiian  hydrocarbons, aromatic  tetrahydropyran  Bidens  include  eleven  and t h i o p h e n i c d e r i v a t i v e s , a C, „  and three C  1 7  hydrocarbons (Table I I I , I V ) .  Compounds 1 t o 18 can be d e r i v e d from o l e i c a c i d by a s e r i e s of dehydrogenations, Except  o x i d a t i o n s and r e d u c t i o n s  f o r 14 t o 18,  they have p r e v i o u s l y been r e p o r t e d i n  North American and European Bidens, et  (Bohlmann C. grandifl  (Bohlmann  c/,,1973). Compound 16 occurs  has been et  al. , 1966) et  (Anderson  in  although  Coreopsis  respectively  (17)  al. ,1966) while  and  species  15 and 17  have  tinctorius  et  18 L.  Centaurea  of  Bohlmann  al  not been  .,1958). isolated  the isomers 19 and 20 ( F i g u r e 8), a r e found  grandiflora (Bohlmann  and and  C.nuecensis  Zdero,1968;  S^rensen and Sjrfrensen, 1958). Although acetate  i n the leaves of  i n Carthamus  two  Dahlia  and/or  reported  al. ,1977;  N a t u r a l l y - o c c u r r i n g 14, before  Coreopsis  or a Hogg ex Sweet (Bohlmann et  ('safynol')  (Figure 7 ) .  Lam  F. et  the presence  Heller, a/.,1968; of the  presumes the e x i s t e n c e of the p r e c u r s o r s  (14,  FIGURE 7.  BIOGENETIC RELATIONSHIPS  OF  POLYACETYLENES  FROM  HAWAIIAN BIDENS Compounds 1 to IB can be d e r i v e d from intermediate  dehydrocrepenynic  transformations a-oxidations (adapted  which (a),  from Bohlmann et  acid  include  0-oxidations al. ,  oleic by  acid a  via series  dehydrogenations  (0), 1978).  and the a d d i t i o n of  the of (*), HS 2  43  o l e i c a c i d — S — l i n o l e i e acid —CH (CH > CSCCH CHssCH(CH ) COOH 3  2  4  2  2  7  crcpenynic a c i d  1-  C H (CH > CH=CHC~ CCH^CHasCH (CH >COOH 3  2  2  2  ?  dehydroerepenynie a c i d  C,.-C0OH-  C, .-COOH-  CH,(CH.) CH=CHC = CCH.CH=CH(CHj.CHO  22  J  IB  2  CH,CH=CH (CSC) _CH.CH=CH (CH.) ,CH^COOH 2'3 2  • . 9 , 10 13  CH,CH==CH (CSC) ,CH,CH=CH (CH^) CH OH '2 2 3  2 b  2  CH CH =CH (CS C) ^CH^CHsCH (CH ) jCOOH 3  CHjCH =CH (CSC)  CH CH=CH(CSC 3  2  (CH = CH) CH CH CH OH 2  3  2  2  2  5. t.  C H = CHCH CH CH OH 2  I--  2  2  I-  U  CH CH = CH (CSC) CH=CHCH CH OH 3  3  2  2  / CH,CH=CH (CSC).CH,CH,OH 3  1« . 15 . lfc . 17  IB  11. 12 4  22  7  1,  2  3  44  GURE 8. PHENYLTHIOPHENES FROM COREOPSIS  45  15 and 16), extracts. although  these were not d e t e c t e d 17  Compound  i n many  of  the root  i s u b i q u i t o u s i n Bidens  r o o t s , and  i t may not serve as a taxonomic marker, i t s unusual  structure  suggests  that  i t may have p h o t o t o x i c and other  i n t e r e s t i n g b i o l o g i c a l p r o p e r t i e s (Towers, 1979). Compound  3('pentayne-ene'), et  which  al. ,1973),  i s common  Asteraceae  (Bohlmann  quantities  i n the leaves  B. cosmoi des  . T h i s may be due t o i t s extreme i n s t a b i l i t y  to  low  taken  concentrations.  t o prevent  of  Since  only  was  found  one  of  i n the  stringent  i n trace  the s p e c i e s , or  p r e c a u t i o n s were  p o l y a c e t y l e n e degradation d u r i n g l a b o r a t o r y  workup however, and s i n c e the compound has a r e l a t i v e l y 5  extinction coefficient  ( e = l 0 ) , i t would  appear  high  that the  pentayne-ene does not accumulate i n most s p e c i e s of Hawaiian Bidens.  The other h i g h l y conjugated  ('ene-tetrayne-ene'),  a c e t y l e n e hydrocarbon  1  was d e t e c t e d s p e c t r o p h o t o m e t r i c a l l y i n  many l e a v e s and i n a l l crude  root e x t r a c t s  except  that  of  B. tort a A (1 7A) .  POLYACETYLENES IN BIDENS HYBRIDS Leaves  from  21  Bidens  hybrids  p o l y a c e t y l e n e s . Compounds were i s o l a t e d and  identified  on  their distribution of were  F  2  recorded  populations also  the b a s i s  examined  were  chromatographically  of UV and mass s p e c t r a , and  i n T a b l e s VII t o XI. The  from seeds of two s e l f e d (five  analyzed f o r  individuals  of  leaves  individuals selfed  B.  TABLE V I I BIDENS  1.  8. sandvfcensis  s s p . sandvicensis  HYBRIDS EXAMINED  X S.  FOR POLYACETYLENES  2.  molokaiensls  B. sandvicensis mlcrantha  3.  8. v a l f d a  X B. molokalensis  5.  8. m o f o k a f e n s f s  7.  8. m e n z f e s f f  9.  8. s a n d v f c e n s f s  X 8. c o s r o o f d e s  s s p . flllformls  X 8. c o s m o f d e s  ssp. sandvfcensis  X 8.  s s p . sandvfcensis  ssp.  X 8.  micrantha  4.  8. molokalensls  X 8.  macrocarpa  6.  8. forbesii  8.  8. mlcrantha  s s p . mlcrantha  10.  8. menziesii  s s p . flllformls  12.  8. s a n d v f c e n s f s  s s p . forbesii  X 8.  cosmofdes  X 8.  valfda  X 8.  hawalensls  valIda 11.  8. mlcrantha  s s p . mlcrantha  X 8. h a w a f e n s f s  s s p . confusa  X. 8.  sandv f cens f s 13.  8. s a n d v f c e n s f s  s s p . confusa  15.  8. t o r t a  17A X 8. m f c r a n t h a  17.  8. t o r t a  17B X 8. hawalensls  19.  8. valIda  21.  8. c e r v f c a t a  X 8. macrocarpa X 8.  macrocarpa  X 8. m a u f e n s f s ssp.  mlcrantha  14.  8. forbesii  16.  8. populi  foli  18.  8.  17A X 8.  20.  8. c a r v f e a t a  torta  s s p . forbesii a X 8. t o r t a  X 8.  X 8. t o r r a 17C  val/da cosmoides  17A  TABLE V I I I . POLYACETYLENES FROM BIDENS HYBRIDS Compounds Bidens  F,  1  2  Type A Cross 1  3  _  4  _  5  _  8  7  _  _  9  _  10  12  18  _  _  _  _  _  _  _  _  _  +  _  _  _  _  _  _  _  _  _  _  +  _  _  _  +  _  _  _  _  _  4  +  -  -  5  + -  6  +  7  +  2  11  Type B Cross 3  8  -  +  -  +  -  +  -  -  -  +  + +  -  +  -  +  -  -  -  +  + +  -  +  -  +  -  -  -  +  + +  -  +  -  +  -  -  -  _  -  _  _  _  +  +  +  +  +  _  _  +  _  _  _  _  _  _  _  13  -  +  _  _  +  _  _  _  _  _  _  _  14-  -  -  +  -  15  _  _  _  _  +  _  _  _  _  16  _  _  _  _  _  _  _  + _  _  _ +  +  -  _  _  _  -  _  12  +  -  _  _  _  -  -  11  _  + -  _  -  _  - +  +  10  Type C Cross  +  +  _  _ _  _  _  _  TABLE IX. POLYACETYLENES IN  B.HAH'AIENSIS HYBRIDS  Compounds  Bidens F,  1  2  3  4  7  5  11  12  Type B Cross 10 11  _ * « . - * « . « . -  -  *  -  +  -  _  *«.  -  _  *  Type C Cross 17  •  bsent i n parents  \  +  49  TABLE X . POLYACETYLENES  IN B.COSMO IDES  HYBRIDS  Compounds Bidens  F  2  t  3  4  7  8  -  *•  -  +  Type B Cross 5 6 7 Type C Cross 20  •absent  i n parents  +  •  10  TABLE X I . POLYACETYLENES  IN B. MACROCARPA HYBRIDS  Compounds Bidens  F  1  2  +  - - * +  19  •  -  2i  +  - *+  }  3  4  5  7  8  9  10 11  12  Type B Cross 4  • * • - • * • - + - -  Type C Cross  •absent A  -  +  _  i n parents  present  in p a r e n t s but absent  in F ,  +  A  +  -  +  51  sandvicensis  s s p . confusa  B.  X  individuals  of B.  sandvicensis  ssp. sandvicensis,  mauiensis,  sandvicensis  and 22  B185S,  s s p . confusa  B194S,  X  B.  (Table X I I ) .  Type A c r o s s e s between two s p e c i e s which do not produce leaf  a c e t y l e n e s r e s u l t i n F, i n d i v i d u a l s without  (Table V I I I ) . Crosses  between  species  which  acetylenes  produce  leaf  a c e t y l e n e s and those which do not (Type B ) r e s u l t i n h y b r i d s which s y n t h e s i z e l e a f a c e t y l e n e s although not  always . i d e n t i c a l  the compounds a r e  t o the a r r a y s present  i n the parents  (Table VIII t o X I ) . In most c r o s s e s of t h i s category, progeny d i d not produce any compounds absent of  the p a r e n t s .  hawaiensis hybrids  ,  Nevertheless,  in  (Table  Bidens  IX).  from the leaves  crosses  compound 12 i s expressed  the F,  with  B.  i n the l e a v e s of the hawaiensis  leaves  c h a r a c t e r i s t i c a l l y produce compound 18 (Tables I I I , I V ) , one which i s c l o s e l y r e l a t e d t o compound  12  i n the proposed  b i o g e n e t i c scheme shown i n F i g u r e 7. Compound in  the  roots  B.  of  phenylheptatriyne,  hawaiensis.  was found  12 a l s o Compound  i n s e v e r a l h y b r i d s from  which d i d not c o n t a i n i t but which  occurs  d i d have  4,  parents  the c l o s e l y  r e l a t e d compound 5, phenylhepta-diyne-ene. In the three Type B c r o s s e s i n v o l v i n g B.cosmoides, F!  produced the p a r e n t a l a r r a y of C  1 3  a c e t y l e n e s (compounds  1, 3 and 5) and three novel compounds compounds  8  and  biosynthetically acetylenes  (Table  10,  several  C  1 7  steps  the  compound  hydrocarbons removed  from  4  , and  which the  are C  1 3  X ) . Bidens mol okai ensis X B. macrocarpa  TABLE X I I . POLYACETYLENE IN F  Compounds  Bidens  1  Cross 12 (B194)  +  B194S ( F ) 2  Cross 13 (B185) B185S ( F ) 2  + + +  2  2  PLANTS  53  i n d i v i d u a l s s y n t h e s i z e C 3 aromatic V  i n B.  found  macrocarpa  compounds 8 and Type  C  express parents  a  found  combination  result  i n F,  X  by C  of the major In two cases.  individuals acetylenes  B.  1 7  parent  B.  were  expressed.  macrocarpa,  macrocarpa  were  additivity  of  absent parental  the C  from  1 7  in both X B.  , compounds not  In the c r o s s acetylenes  the F, whereas  arrays  which  cervicat a  and B. tort a MB X B. hawaiensis  in either  valida  i s characterized  two s p e c i e s of Bidens which  between  acetylenes  (Table V I I I ) .  cosmoides  5 not  10 (Table X).  crosses  produce l e a f  which  a c e t y l e n e s 4 and  i n B.  from there  cervi cat a  B. B. was  X B.  macrocarpa (Table X I ) . F  2  ( B. sandvicensis B.  from the progeny of two Type B c r o s s e s  individuals  ssp. confusa X B. mauiensis -  sandvicensis  sandvicensis individual  -  B194S)  examined  compounds found sandvicensis  s s p . confusa were analyzed contained  i n a l l the  X  and  B. sandvicensi s s s p .  f o r acetylenes.  Every  compounds 1 and 5, the same  F, and i n the leaves  ssp. confusa (Table X I I ) .  r  B185S,  of B.  54  D. DISCUSSION  POLYACETYLENES IN BIDENS TAXA can be seen i n T a b l e s V and V I , there i s no obvious  As  o v e r a l l p a t t e r n i n the d i s t r i b u t i o n Hawaiian Bidens.  of p o l y a c e t y l e n e s i n  A dendrogram of the taxa produced using the  MIDAS s t a t i s t i c a l package with a simple matching  similarity  coefficient  neighbour)  and  a  single  linkage  c l u s t e r i n g a l g o r i t h m shows l i t t l e the  species l e v e l  taxa  B.mauiensis  do  not  sequentially. often  cluster  Morphologically together, e.g.,  and B. mol okai e ns i s , B. cervical a and B. forbesii,  B.conjuncta subspecies  h i e r a r c h i c a l p a t t e r n above  ( F i g u r e 9 ) . Many taxa c l u s t e r at the same  l e v e l , and most l e v e l s added similar  (nearest  and  B.micrantha  s s p . micrantha,  of B.menzi esi i , B.micrantha,  B. sandvi censis.  The r e l a t i v e  or  the  B. campyl ot he ca and  constancy  of p o l y a c e t y l e n e s  w i t h i n taxa and the absence of h i e r a r c h i c a l  s t r u c t u r e i n the  classification  provides  evidence  above  the s p e c i e s  Moreover, the r e l a t i v e l y derived  oleic  acid  Hawaiian  (Figure Bidens  7).  Asteraceae,  aromatic  s m a l l array of compounds can  Certainly  share  as w e l l  in  from a common a n c e s t o r .  from a common unsaturated  dehydrogenating the o l e f i n i c the  little  of r e l a t i o n s h i p s among taxa but may be expected  a case of m u l t i p l e divergences  be  level  fatty acid this  the enzyme  precursor,  means  system  that the capable  of  bond, a c a p a c i t y widespread i n as  the means t o s y n t h e s i z e the  a c e t y l e n i c and t h i o p h e n i c systems, a l r e a d y  notable  55  FIGURE  9.  DENDROGRAM  OF  TAXA  BASED  ON  SIMILARITY  OF  POLYACETYLENES  ro to  o ro  O  14  hill  6 cosm IB  vali  11 Bieme 9 macr 3 caca 17a t o r i 11a mefi 3a cape 17c t o r 3 17b t o r 2 12 mimi 6 hawa 5 conj 4 cerv 12a mict 7a foka 16b saco 7  fofo  10 n a u i 2 asym I7d tor4 16 sasa 15 popu 13 n o l o fc 12b n i k a 19 weib I 1 ampl  56  i n the T r i b e Heliantheae  (Heywood et al . , 1977).  A s p e c i a l f e a t u r e which separates from  i t s relatives  phenylthiophene supports  t h e Hawaiian  i s the c o n s i s t e n t  presence  derivative. This i s s i g n i f i c a n t  the b i o s y s t e m a t i c  Hawaiian Bidens evolved  evidence  group of a  i n that i t  indicating  that a l l  from a s i n g l e a n c e s t r a l s p e c i e s . I t  would appear, however, t h a t - t h e r e has been l e s s e v o l u t i o n a r y diversification characters in  i n polyacetylenes  although  than  more d i f f e r e n t i a t i o n  i n morphological than  has  occurred  isozymes. All  except  four Hawaiian taxa can be d i s t i n g u i s h e d by  unique a r r a y s of l e a f and root a c e t y l e n e s . B.mi cr ant ha s s p . kalealaha  and B.mol okai ensi s  both  produce only 1 and 18;  B. ampl eel ens and B. wi ebkei produce 1, 2 and 17 (Tables IV).  These  four  taxa  produce  a c e t y l e n e s of any of the Bidens, similar  morphologically,  p r o f i l e s probably  do not  the s m a l l e s t  reflect  number of  and s i n c e they are not  their  identical  III,  very  polyacetylene  taxonomic r e l a t i o n s h i p s but  a r e , r a t h e r , a r e s u l t of the p a u c i t y of compounds. Similarities correlate  well  i n polyacetylenes with  morphological  r e c o g n i t i o n of r e l a t e d polyacetylenes  does  species  among  many  (1975). patterns  do not  similarities,  groups  and the  on t h e b a s i s of  not appear t o be p o s s i b l e . The  compounds seem t o be randomly a s s o r t e d among are  taxa  of the morphological  t h e taxa,  d i f f e r e n c e s noted  Distributions  of p a r t i c u l a r  of taxonomic  significance,  various  acetylenes  as  by G i l l e t do show  however. Q u a l i t a t i v e  57  variation Although  i n p o l y a c e t y l e n e s was found w i t h i n only one taxon. only one p o p u l a t i o n of some taxa was a v a i l a b l e  analysis, the  two t o f i v e p o p u l a t i o n s were analyzed  taxa.  sandvicensis  A l l populations  one forma a c c o r d i n g t o S h e r f f Bidens molokaiensis  species,  two v a r i e t i e s  was t r e a t e d  as two s p e c i e s  ( F i g u r e 5), and  r e s t r i c t e d t o the top of Diamond Head on Oahu a r e decumbent,  low spreading  herbs  teeth  on the leaves  disappeared  c u l t i v a t e d under the same c o n d i t i o n s 1983b)  No  acetylenes  a c e t y l e n e s are Bidens except and  were  B.cuneata,  whose supposed the  number  when the p l a n t s were  (Ganders  found  and Nagata,  i n the l e a v e s and  mauiensis  variable  i s very  to B.mol okai ens i s  similar  Bidens,  l e a f shape. S h e r f f recognized s e v e r a l  v a r i e t i e s based on l e a f shape d i f f e r e n c e s , but they are considered  untenable  (Ganders  B. mauiensis  specimens  examined  occurrence  and  distribution  and Nagata, were  the presence of compounds 10, 12 and V a r i a t i o n w i t h i n taxa was variable  uniform  from  found  taxon  now  1983b).  A l l  i n the  of p o l y a c e t y l e n e s .  mauiensis does d i f f e r c o n s i s t e n t l y  morphologically  root  identical.  that i t has winged achenes unique in Hawaiian  highly  by  (Figure 3 ) .  d i f f e r e n c e s i n the shape of t h e i r l e a f bases and of  taxon  (1937).  S h e r f f , B.mol okai ensis on Molokai  They  ssp.  have i d e n t i c a l compounds even though t h i s  as i n t e r p r e t e d here i n c l u d e s seven and  f o r 16 o f  B. sandvicensis  of  for  Bidens  B.mol okai ensi s by  13 i n i t s r o o t s . only endemic  i n B. tort a,  a  t o Oahu and  58  widespread feature the  in distribution.  Although  i s a t w i s t e d or c o i l e d aohene, p l a n t s  degree  of  coiling  of  achenes  p o p u l a t i o n . They can a l s o vary of  leaf  do  not appear  to  from  have  distinct  different  the Koolau  roots  17B  are  geographical  combinations  Range  also  and  hydrocarbons.  The  compounds.  (Figure  3 ) . Of  these,  highly  (PHT)  unusual 5  17A being  i s found. i n that  was  no  present.  acetylenes  combinations  i n the l e a v e s , only the C of a c e t y l e n e s  1 7  i n the roots  throughout  the p e r i o d  of the study.  B. tort a i s as v a r i a b l e i n i t s p o l y a c e t y l e n e s as i t i t s morphology,  characteristic variation  the p o l y a c e t y l e n e s  of s p e c i f i c  populations  while  appear  t o be  morphological  i s not. contrast,  interpopulation endemic  a were  f o r a l l four p o p u l a t i o n s . These d i f f e r e n c e s  consistent  Although  In  ranges.  17D accumulate only 5 i n the l e a v e s ; 17C d i d  were d i s t i n c t i v e remained  of  (1) was d e t e c t e d , although  not have any aromatic  in  coloration  appear t o be unique among Hawaiian Bidens  ene-tetrayne-ene  is  w i t h i n the same  i n the amount and  the only p l a n t s i n which p h e n y l h e p t a t r i y n e  Both  in  17A, 17B, and 17C a r e from the Waianae Range and  individuals  Their  can vary  from four d i f f e r e n t p o p u l a t i o n s of B. tori  t o possess  Populations 17D  even  distinctive  pubescence and i n the number of l e a f l e t s . V a r i a n t s  Individuals found  i t s most  no  taxon  differences.  t o Hawaii,  distinctive  other  species  is a  showed  F o r example,  morphologically  although  i t occurs  in  intra-  or  B. hawai  ensis,  uniform  and  ecologically  59 v a r i a b l e s i t e s . I t may r e a d i l y be recognized on the b a s i s of leaves  or  flowers  populations  alone.  examined  A l l individuals  were  p o l y a c e t y l e n e s accumulated  consistent  from  three  i n the p a t t e r n of  i n the leaves  and r o o t s . The  leaves c o n t a i n e d only compound 18 and the r o o t s produced a l l c l a s s e s except In  all  differed  the aromatic cases  acetylenes.  examined  in polyacetylene  subspecies  which  morphological  have  the subspecies distribution.  rather  In  subtle  c h a r a c t e r s can e a s i l y  be  of a s p e c i e s some  cases,  distinctions separated  in  on the  b a s i s of p o l y a c e t y l e n e d i f f e r e n c e s . Bidens which  sandvicensis  inhabits  i s an  found  to bipinnately  (1937)  according  on  the basis  t o Ganders  B. sandvi censi s  and  These  i s presently  two  taxa  and Oahu.  compound  leaf  recognized  forms  or  (1983b;  by  are i n v a l i d 1984) and  c o n s i d e r e d t o c o n s i s t of ssp. (Ganders  and Nagata,  are often d i f f i c u l t to t e l l  morphologically - subspecies restricted  of Nagata  sandvi censi s and ssp. confusa 1984).  taxon  i n t o narrow u l t i m a t e segments, and a l l forms may be  i n the same p o p u l a t i o n . Many segregates  Sherff  taxon  variable  a wide range of h a b i t a t s on Kauai  Leaf shapes can be t r i f o l i o l a t e divided  extremely  confusa  is a  high  1983b; apart  elevation  t o the edge of Waimea Canyon i n Kauai and  tends t o have l a r g e r narrower l e a f l e t s than  r a y flowers  and flower  heads, and  ssp. sandvi censi s . They may, however,  be d i s t i n g u i s h e d by the presence of phenyl-diyne-ene (5) i n the  leaves  of  s s p . confusa  and the t o t a l  absence  of  60  acetylenes  in  populations  of s s p . sandvicensis.  the leaves  from Oahu and one p o p u l a t i o n from Kauai  sandvicensis,  of  ssp.  of s s p . confusa were  and two p o p u l a t i o n s  examined. The root a c e t y l e n e s  Five  of the two subspecies a r e  ident i c a l . Bidens menziesii obviously  more  ssp. menziesii  closely  related  and ssp. filiformis  are  t o each other than to any  other Hawaiian Bidens (Ganders and Nagata,1983b). They characteristic  leaves  which  vary  i n size  have  but which are  b i p i n n a t e l y d i v i d e d i n t o long l i n e a r segments l e s s than wide.  Both  areas,  i n c l u d i n g the c i n d e r cones and lava flows  (Figures  4  favour  relatively  arid,  sunny  and windswept of Hawaii  t o 6 ) . The two subspecies are q u i t e d i s t i n c t  m o r p h o l o g i c a l l y however, and a r e a l l o p a t r i c Molokai  (ssp. menziesii)  on Maui and  (ssp. filiformis).  and Hawaii  are f u r t h e r d i s t i n g u i s h e d by the absence of l e a f in ssp. filiformis in  5mm  and the presence of C  s s p . menziesii.  There  a r e no  where  leaf  1 7  They  acetylenes  compounds 8 and 9  differences  in  root  acetylenes. Another example subspecies  Subspecies similar  only  consists  and s s p . pentamera  both  of a c e t y l e n e s i n t h e leaves except  subspecies  of  two of which were examined i n t h i s  campylot heca  range  campylot heca  pentamera a l s o accumulates 5 and 7, aromatic latter  separate  two  while root a c e t y l e n e s are i d e n t i c a l can be found  in B. campyl ol heca. Bidens subspecies,  acetylenes  i s restricted  three study.  had a  that ssp.  a c e t y l e n e s . The  t o the foggy  rainforests  61 above  1500m  on  morphologically  East  Maui  (Figure  campylot heca  from ssp.  4)  and  differs  primarily - in  leaf  subspecies,  ssp.  shape. Bidens forbesii,  forbesii which  occurs  Acetylenes  to  populations  a  the v i c i n i t y  a r e absent although  two  montane  of  Mt.  in  (Figure 2).  but not i n the r o o t s  are separable  of p o l y a c e t y l e n e d i f f e r e n c e s . For example, i s sympatric  B. val ida  with  B.forbesii  of s s p .  and  interfertile  achene  but  they  flower a t d i f f e r e n t lack  characters. remain  times  of  polyacetylenes,  s e v e r a l compounds, notably  The  discrete  B.forbesii  two  distinct  species are  taxa because they  the year. those  on 'the  on M t . K a h i l i ,  They are very s i m i l a r v e g e t a t i v e l y although  floral  leaves  form  .  ssp. kahiliensis Kauai.  Kahili  forest  two compounds, 5 and 12 , are found i n  Most s p e c i e s of Hawaiian Bidens basis  wet  from the l e a v e s of a l l s i x  the r o o t s of ssp. kahiliensis for be sii  of  p r i m a r i l y along the north coast of  kahiliensis,  Kauai, and ssp. restricted  consists  B.forbesii  While  of  B.valida  contain  5, the 'phenyl-diyne-ene'.  grown from a mixed c o l l e c t i o n of c u t t i n g s from t h i s  Plants locality  were d i s t i n g u i s h e d by a n a l y s i s of l e a f a c e t y l e n e s and identities  later  r o o t s of B.valida distinguished this basis.  confirmed  when  d i d not c o n t a i n  from  their  the p l a n t s flowered. The compound  those of B.forbesii  5  and  may  s s p . kahiliensis  be on  62 Bidens cervicata forbesii.  Both  quadrangular flower  i s c l o s e l y r e l a t e d t o B.forbesii  taxa have i d e n t i c a l achenes and prominently  stems, although  heads,  ssp.  terminal  B. cervi cat a has s l i g h t l y l a r g e r  inflorescences,  and s m a l l e r , l e s s  succulent and more numerous l e a f l e t s which are narrower and more  deeply  serrate  than  p o p u l a t i o n s of B.forbesii of  Kauai  appear  (B12, B74,  somewhat  B. cervi cat a  those  of B.forbesii.  ssp. forbesii  B101 and B124)  intermediate  Some  from the north coast have i n d i v i d u a l s which B.forbesii  between  and  in morphology. The two s p e c i e s , however, can be  separated  on the b a s i s of p o l y a c e t y l e n e d i f f e r e n c e s .  forbesii  does  not accumulate a c e t y l e n e s  Bidens  i n the leaves but  B. cervi cat a leaves c o n t a i n compounds 5 and 7. The roots of all  B.forbesii  populations  tetrahydropyran, Analysis  a  compound  of m o r p h o l o g i c a l l y  they c o n t a i n e d the a c e t y l e n e s forbesii  , suggesting  be  separated  c o n t a i n 12,  not found  on  of  typical  morphologically  B.forbesii  also  intergrade.  similar  species  Both  occur  on  i n q u a n t i t a t i v e c h a r a c t e r s , but  d i f f e r e n t polyacetylenes  in their roots.  produces compound 8 i n i t s leaves while  are absent  ssp.  the b a s i s of p o l y a c e t y l e n e s i n c l u d e  Maui and d i f f e r mainly  possess  n  intermediate p l a n t s showed that  B. conjunct a and B. mi crantha ssp. mi crantha. West  the C, -  B. cervicat a.  in  that the two s p e c i e s do not  Other examples where can  sampled  B.conjuncta  polyacetylenes  i n the leaves of B.micrantha.  Morphologically, d e f i n e d taxon  B. asymmel ri ca  and i s sometimes d i f f i c u l t  is a  rather  poorly  t o d i s t i n g u i s h from  63 B. sandyicensis contiguous  sandvi censi s  ssp.  i n the southern  where  Koolau  their  Range on Oahu. I t i s a l s o  r a t h e r s i m i l a r t o B. tort a , which occurs Koolau  Range.  variation  B. tort a  Although  i n polyacetylenes,  ranges a r e  i n the northwestern  e x h i b i t s great  these  three  interplant  species  can be  separated on the b a s i s of t h e i r p o l y a c e t y l e n e s . Finally, species  B. cosmoides  endemic  is a  t o Kauai.  achenes  subtending (Ganders  which  20 - 25mm beyond  a r e permanently  and Nagata,1983a).  the monotypic  enveloped  s p e c i e s that S h e r f f (1937)  section  t o the Hawaiian  B. cosmoides, This  with  Degeneria.  Islands,  was c o n s i d e r e d  Gillett  all  other Hawaiian Bidens.  from  B. cosmoides  a  single  indicate  unlikely  roots  (1975) l a t e r  i n t r o d u c t i o n s of  ancestor. a  close  of B. cosmoi des,  by Ganders and can be  at least  seven  other  supports a monophyletic  crossed likely  The p o l y a c e t y l e n e s of relationship found  with  other  i n leaves  only compound 3 was not found i n  other Hawaiian taxa. Each of the other compounds in  species.  T h e r e f o r e they most  Hawaiian t a x a . Of the nine p o l y a c e t y l e n e s and  placed i t  one which gave r i s e t o  B. cosmoides  (1983b; 1984), because  evolved  their  different  and the other t o a l l the other Bidens  hypothesis  Nagata  by  It is sufficiently  proposed that there had been two separate Bidens  the anthers,  c h a f f y b r a c t s , both unique f e a t u r e s i n the genus  from a l l other Bidens in  unique  I t has l a r g e flower heads with  e x s e r t e d s t y l e s which extend and  morphologically  taxa.  origin  was  The p o l y a c e t y l e n e  f o r the Hawaiian  found data  s p e c i e s of  64 Bi dens .  POLYACETYLENES IN BIDENS HYBRIDS Few  studies  production Carey et  al .  truncicolor  (1974) which  inheritance  examined  of  polyacetylene  2  and  (1966) and Clitocybe  the basidiomycete  synthesizes  CHCH OH,  3  homokaryons  the  have been r e p o r t e d . B i s t i s and Anchel  CH -(C=C) CH 3  on  trans-dehydromatricarianol,  i t s methyl  ether.  Individual  e x h i b i t e d d e f i n i t e and r e p r o d u c i b l e d i f f e r e n c e s  in p o l y a c e t y l e n e p r o d u c t i o n . These d i f f e r e n c e s were in  the progeny  and  the l e v e l s of p o l y a c e t y l e n e s  with  evident  of c r o s s e s between d i s t i n c t i v e homokaryons produced  s p e c i f i c mating types. The study  to provide experimental  evidence  were  correlated  i n 1966 was the f i r s t  f o r genetic  c o n t r o l of  polyacetylene synthesis. Norton Bidens pilosa  (1984) performed a s i m i l a r  alba  L.  v a r . minor  v a r . radial a (Blume) S h e r f f .  phenylheptatriyne  was found  from B. alba half  Bidens  synthesizes  (PHT or compound 4 i n t h i s paper) i n i t s leaves.  i n the l e a v e s of a l l F, i n d i v i d u a l s  resulting  X B. pilosa  of t h a t i n B. alba.  expected  alba  from B. pilosa  g e n e r a t i o n although with  using  (Schz. B i p . ) B a l l a r d and B.  leaves whereas a c e t y l e n e s a r e absent PHT  investigation  but a t l e v e l s which were l e s s PHT s y n t h e s i s segregated  the r a t i o s of segregants  did  than  i n the F not  agree  values and i n d i v i d u a l values were much lower  than a n t i c i p a t e d i f  PHT  levels  are a  function  of  gene  2  65 dosage. In  the present  study,  the i n h e r i t a n c e of p o l y a c e t y l e n e  b i o s y n t h e s i s i n Hawaiian Bidens Bidens  produce  express  this ability  and  roots  occurs  acetylenes  appear  was examined.  i n t h e i r r o o t s , but only  i n the l e a v e s . B i o s y n t h e s i s t o be independent  de novo i n the leaves  in  (Van F l e e t ,  leaves  1970) and  on the b a s i s of t h e i r  a c e t y l e n e a r r a y s and s e l e c t e d h y b r i d s Quantitative  15 taxa  (see Chapter I I I ) . Most of the  Hawaiian s p e c i e s can be separated  analysis.  A l l Hawaiian  levels  were  used  leaf  for this  of a c e t y l e n e s produced were  not measured. The  only other study of t h i s nature was r e p o r t e d by Van  F l e e t ( l 970). He genetics roots,  of stems  grandiflora  worked  with  polyacetylene and leaves  formation of C.  and examined the by the endodermis of  saxicola  Alexander,  Hogg ex. Sweet and t h e i r a r t i f i c i a l  h y b r i d s . The stems and leaves produced  mainly  in  of  most  Cor eopsi s  of  trideca-triene-triyne  the two parent  hybrids,  (compounds  11  a  mixture  and 12)  phenylhepta-diyne-ene (compounds 5 and 6) was entity  Van F l e e t  calls  a  i n t e r p r e t e d here as n a t u r a l mixtures The  of compounds  roots  predominantly  and n a t u r a l species  PHT but i n some forms of C. grandiflora  the a r t i f i c i a l  of C.  'general hybrids  C.  of the and the  produced.  ecotype', i n general,  and  An  which i s contains  4, 5 and 6 i n i t s stems and l e a v e s .  saxicola  and C.  gr andi flora  the t r i d e c a - e n e - t e t r a y n e - e n e  produce  (compounds 1 and  2) and "....compounds produced i n the r o o t s of  the h y b r i d s  66  are predominantly  the same as the p a r e n t s . "  Biosynthesis v a r i a b l e than could  i n the a e r i a l  Coreopsis  i n the r o o t s .  not be  t i s s u e s seems t o be more  distinguished  or  'hybrid  separated  ecotypes'  on the b a s i s of  a c e t y l e n e s produced i n the r o o t s but c o u l d be d i s t i n g u i s h e d from  parental  types  on  the b a s i s of l e a f a c e t y l e n e s . The  v a l i d i t y of Van F l e e t ' s data may be questioned information  provided  i s descriptive  nonspecific.  Nevertheless,  i t does  and  because the statistically  indicate  that  the  g e n e t i c s of p o l y a c e t y l e n e b i o s y n t h e s i s i n higher p l a n t s i s a c h a l l e n g i n g problem. Data Bidens in  from  a  preliminary  h y b r i d s suggests  leaves  is  a  investigation  of  that a c e t y l e n e b i o s y n t h e s i s heritable  P o l y a c e t y l e n e s were found  and  dominant  number  of  F  2  s y n t h e s i s was not segregated  individuals  examined  Instead, a l l p l a n t s produced sandvicensis significant  from  se  phenotype.  (Table  i n the  (Tables VII  i n the small  Type B c r o s s e s .  the a c e t y l e n e s  s s p . confusa variation  per  i n the leaves of progeny from Type  B and Type C c r o s s e s but not from Type A c r o s s e s to X I ) . Acetylene  Hawaiian  found  i n B.  X I I ) . Whether there was  quantitative  levels  of  compounds produced i s not known. In g e n e r a l , Hawaiian Bidens acetylenes,  consisting  of C  to the scheme proposed here, derived  from  oleic  acid  1 7  produce a l i m i t e d a r r a y and C  1 3  of  compounds. According  these compounds may be commonly but a r e subsequently  elaborated  along b i o s y n t h e t i c a l l y d i v e r g e n t pathways (Figure I I - 7 ) .  In  67  the l e a v e s , most s p e c i e s tend t o produce or C  1 7  compounds  in B. macrocarpa  Type B  progeny  produced  only  i n the parents were s y n t h e s i z e d , and  hybrids, C  aromatic  1 3  the p a r e n t a l a r r a y were observed. is  1 3  i n the p a r e n t a l c l a s s . In B. cosmoi des h y b r i d s ,  compounds not found  1 7  C  compounds. With the exception of c r o s s e s i n v o l v i n g B.  cosmoi des and B. macrocarpa,  C  predominantly  compounds absent  Data from Type  C  from  crosses  s i m i l a r . There was a d d i t i v i t y of p a r e n t a l p o l y a c e t y l e n e s  in F, progeny but there was a l s o s y n t h e s i s of C when  only  C  1 3  and vice  a r r a y s were expected  compounds  1 7  versa.  This i s  not s u r p r i s i n g s i n c e one would'expect that the complete s e t of  f o r de novo a c e t y l e n e s y n t h e s i s e x i s t s i n  instructions  the l e a f genome. Moreover, r e g u l a t i o n and c o n t r o l of g e n e t i c expression  i s f u r t h e r complicated  of Hawaiian Bidens 1974;  Gillett That  significant  (2N=72;  and Lim,  only  amounts  X=12)  1970;  certain  by the p o l y p l o i d c o n d i t i o n (Mears,  Skottsberg, compounds  i n each  1953). are  species  number of f a c t o r s . C e r t a i n enzymes along have  depressed  1980; Fedorov,  expressed  c o u l d be due t o any the sequence  a c t i v i t y or may be absent,  end products  would a f f e c t  be  exist  of  the sequence  does  i n i s o l a t i o n and the l e v e l of i t s a c t i v i t y would  i n f l u e n c e d by the s t a t e  of  primary  processes  f a t t y a c i d metabolism. Whatever the governing c l e a r that  rates  the d i r e c t i o n of e q u i l i b r i u m  in the s y n t h e t i c sequence. In a d d i t i o n , not  may  the pool s i z e of  key p r e c u r s o r s and i n t e r m e d i a t e s , and the turnover the  in  the status  quo  i s altered  such  as  factors, i t i s  when  Bidens  are  hybridized.  69 E. CONCLUSION The  leaves  of Hawaiian s p e c i e s of Bidens  and r o o t s  accumulate a moderate d i v e r s i t y of p o l y a c e t y l e n e s which all  be  biosynthetically  phenylthiophenes to  related.  may  Of these compounds, the  14 t o 17 appear t o be u b i q u i t o u s and  unique  the s p e c i e s . T h i s i s c o n s i s t e n t with other evidence  that  the Hawaiian s p e c i e s are a l l d e r i v e d from a s i n g l e a n c e s t r a l immigrant  t o the Hawaiian  evolutionary morphology  diversification and ecology  d i f f e r e n t i a t i o n than Polyacetylenes  i n Hawaiian  i s found  are  has been l e s s  Bidens,  than but  i n isozymes. constant  within  specific  difficult  i n roots  and leaves  a given  although  compounds are r a r e . Even subspecies  to d i s t i n g u i s h identified  morphologically on  the  basis  populations  studied  s t r o n g l y supports  of Ganders and Nagata (1983b,1984)  of  ecogeographical Subspecies  differences  the s p e c i e s  based  on  which  can be  p o l y a c e t y l e n e s . The d i s t r i b u t i o n of p o l y a c e t y l e n e s  their in  the  concepts  morphological  data.  of the same  i n polyacetylenes  Above the l e v e l of subspecies were  greater  accumulated. Nearly a l l taxa can.be d i s t i n g u i s h e d  unequivocally  and  in  exhibited interpopulational variation in  by the a r r a y of a c e t y l e n e s species  There  i n polyacetylenes  are usually  taxon. Only B.torta compounds  Islands.  species  exhibited  as d i d d i f f e r e n t and s p e c i e s ,  as many species.  polyacetylenes  not c o r r e l a t e d with r e l a t i o n s h i p s based on morphology.  Therefore,  i t i s not yet p o s s i b l e t o d e f i n e  species  groups  70  w i t h i n Hawaiian Bidens based on c o r r e l a t e d morphological and chemical  characters.  r a d i a t i o n i n Hawaiian Bidens  Adaptive  has produced a group of s p e c i e s that combine of  morphological  and chemical  an  assortment  c h a r a c t e r s which occur  in a  l a r g e number of combinations. The  de  novo  synthesis  of p o l y a c e t y l e n e s  l e a v e s i s a h e r i t a b l e and dominant expressed  all  Acetylenes  were  i n the l e a v e s of h y b r i d s with at l e a s t one l e a f  acetylene-producing segregated  trait.  i n Bidens  parent.  Synthesis,  i n the s m a l l number of F  2  however,  was not  i n d i v i d u a l s examined,  of which produced the p a r e n t a l a r r a y s .  71 F. BIBLIOGRAPHY Andersen, A.B., J . Lam and P.Wrang. 1977. P o l y u n s a t u r a t e d compounds of Centaurea scabiosa. Phytochem. 16: 1829-1831. B i s t i s , G. and M. Anchel. 1966. Evidence f o r g e n e t i c c o n t r o l of polyacetylene production in a Basidiomycete. Mycologia 58: 270-274. Bohlmann, F., F.T. Burkhardt and C. Zdero. 1973. N a t u r a l l y O c c u r r i n g A c e t y l e n e s . Academic Press, London. Bohlmann, F. and C. Zdero. 1968. Uber d i e i n h a l s s t o f f e von Coreopsis nuecensis A. Heller. Chem. Ber. 101: 3243-3254. Bohlmann, F., M. Grenz, M. Wotschokowsky and E. Berger. 1967. Polyacetyleneverbindungen, CXXXIV. Uberneue thiophenacetyleneverbindungen. Chem. Ber. 100: 2518-2522. Bohlmann, F., S. Kohn and C. Arndt. 1966. Polyacetylenverbindungen, CXIV. Die p o l y i n e der gattung Carthamus L. Chem. Ber. 99: 3433-3436. Bohlmann, F., S. Postulka and J. Ruhnke. 1958. Polyacetylenverbindungen, XXIV. Die p o l y i n e der gattung Centaurea, L. Chem. Ber. 91: 1642-1656. Carey, S., M. Anchel and G. B i s t i s . 1974. P o l y a c e t y l e n e production in Clilocybe truncicola : E f f e c t of mating-type combination. Mycologia 66 : 327-332. Carlquist, S. 1970. Hawaii: H i s t o r y Press, New York.  A  Natural  H i s t o r y . Natural  C a r l q u i s t , S. 1967. The b i o t a of l o n g - d i s t a n c e D i s p e r s a l . V. Plant d i s p e r s a l to P a c i f i c i s l a n d s . Bull. Torrey Bot. Club 94 : 129-162. C a r l q u i s t , S. 1966a. The b i o t a of l o n g - d i s t a n c e dispersal. I. P r i n c i p l e s of d i s p e r s a l and e v o l u t i o n . Quart. Rev. B i o l . 41 : 247-270. Carlquist, S. 1966b. The B i o t a of l o n g - d i s t a n c e d i s p e r s a l . II. Loss of d i s p e r s i b i l i t y in Pacific Compositae. E v o l u t i o n 20 : 30-48. C h i n , C , M. C. C u t l e r , E. R. H. Jones, J . Lee, S. Safe and V. Thaller. 1970. N a t u r a l A c e t y l e n e s . Part XXXI. C, i,-tetrahydropyranyl and other p o l y a c e t y l e n e s from the Composite Dahlia cocci nea Cav. v a r . cocci nea. J . Chem.  72  Soc.  (C): 314-322.  Daniels, F. 1965. A simple m i c r o b i o l o g i c a l method f o r demonstrating phototoxic compounds. J . I n v e s t . Dermat. 44 : 259-263. Fedorov, A. A. 1974. Chromosome Numbers o f F l o w e r i n g P l a n t s . Otto K o e l t z Science P u b l i s h e r s , K o e n i g s t e i n . Ganders, F. R. and K. M. Nagata. 1984. The r o l e of h y b r i d i z a t i o n i n the e v o l u t i o n of Bidens on the Hawaiian I s l a n d s . P.179-194 i n P l a n t B i o s y s t e m a t i c s W. F. Grant (Ed.). Academic Press, Canada. Ganders, F. R. and K. M. Nagata. 1983a. R e l a t i o n s h i p s and f l o r a l b i o l o g y of Bidens cosmoides ( A s t e r a c e a e ) . Lyonia 2: 23-31. Ganders, F. R. and K. M. Nagata. 1983b. New taxa and new combinations i n Hawaiian Bidens ( A s t e r a c e a e ) . Lyonia 2 : 1-16. G i l l e t t , G. W. 1975. The d i v e r s i t y and h i s t o r y of P o l y n e s i a n Bidens . Harold L. Lyon Arboretum Lecture No. 6. U n i v e r s i t y of Hawaii, Honolulu. G i l l e t t , G. W. and E. K. S. Lim. 1970. An experimental study of the genus Bidens (Asteraceae) i n the Hawaiian I s l a n d s . U n i v e r . C a l i f . P u b l . Bot. 56 : 1-63. Helenurm, K. and F.R. Ganders. 1985. Adaptive g e n e t i c d i f f e r e n t i a t i o n i n Hawaiian Bidens (in press).  r a d i a t i o n and . Evolution  Heywood, V. H., J . B. Harborne and B. L. Turner (Eds.) 1977. The B i o l o g y and Chemistry o f t h e Compositae . Volumes I and I I . Academic Press, New York. Lam,  J . , F. Kaufmann and 0. Bendixen. 1968. Chemical C o n s t i t u e n t s of the genus Dahlia . I I I . A chemotaxonomic evaluation of some Dahlia cocci nea s t r a i n s . Phytochem. 7: 269-275.  Marchant, Y. Y. and G. H. N. Towers. Unpublished  Results.  Mears, J . A. 1980. Chemistry of p o l y p l o i d s : A summary with comments on Parthenium (Asteraceae-Ambrosiinae). Pages 77-101 i n W. H. Lewis (Ed.) P o l y p l o i d y : Biological R e l e v a n c e . Plenum Press, New York. Norton, R. A. 1984. S t u d i e s of P o l y a c e t y l e n e p r o d u c t i o n i n normal and transformed t i s s u e c u l t u r e s of Bidens alba . Ph.D. D i s s e r t a t i o n , U n i v e r s i t y of B.C.  73 S h e r f f , E. E. 1937. The genus Bidens F i e l d Mus. Bot. Ser. 16 : (I and I I ) .  Nat. H i s t .  Skottsberg, C. 1953. Chromosome Numbers i n Hawaiian Flowering P l a n t s . P r e l i m i n a r y Report. A r k i v . f o r Bot. 3: 63-70. SpVensen, J.S. and N. A. S^rensen. 1958. S t u d i e s r e l a t e d t o naturally-occurring Acetylene compounds. XXIV. 2-Phenyl-5(a-propynyl)-thiophene from the e s s e n t i a l o i l s of Coreopsis grandiflora Hogg Ex Sweet. A c t a . Chem. Scand. 12 : 771-776. Stearns, H. T. 1966. Geology of the State P a c i f i c House Inc. Palo A l t o , C a l i f .  of Hawaii  S t u r t e r v a n t , A. H. 1965. Row, New York.  . Harper and  A H i s t o r y of G e n e t i c s  Towers, G. H. N. 1980. Photosynthesizers from p l a n t s and t h e i r photodynamic a c t i o n . Prog. Phytochem. 6 : 183-202. Towers, G.H.N., C.K. Wat, E.A. Graham, R.J. Bandoni, G.F.Q. Chan, J . M i t c h e l l and J . Lam. 1977. U l t r a v i o l e t - m e d i a t e d a n t i b i o t i c a c t i v i t y of s p e c i e s of Compositae caused by p o l y a c e t y l e n i c compounds. L l o y d i a 40 : 487-498. Van  F l e e t , D.S. 1970. Enzyme l o c a l i z a t i o n and the g e n e t i c s of polyenes and p o l y a c e t y l e n e s i n the endodermis. Advancing F r o n t i e r s of P l a n t Science 26: 109-143.  Wrang, P.A. and J . Lam. 1975. P o l y a c e t y l e n e s from Chrysanthemum I eucanl hemum. Phytochem. 14 : 1027-1035.  1  I I I . BIOSYNTHESIS OF POLYACETYLENES FROM * C 0  A.  INTRODUCTION Natural  polyacetylenes  comprise  combinations of d i f f e r i n g c h a i n lengths of. unsaturation,  and  groups and c y c l i c  a  The chains  almost from  C  varying  Thaller,  exclusive down  1 8  a (C  6  wide - C  1 8  range  ),  support  occurrence  of  that  straight  Bu'Lock et 1966;  al.,  1961;  Bohlmann and currently  This  d i s t a l half  1966;  was  primarily  (C  1 0  ~ C )  Subsequent  for  first  involves  carbon  evidence  1963; al.,  1959; Jones , 1967).  by  Bu'Lock  d e s a t u r a t i o n of  acid  to  the b i o g e n e s i s of  proposed  the  synthesis  Gregory,  F a i r b r o t h e r et  of o l e i c  1 8  and  Smith, 1962;  hypothesis  plants  system of crepenynic  (usually  Bu'Lock and  accepted  polyacetylenes in (1966).  (e.g.,Bu'Lock  Jente,  the  the b i o s y n t h e s i s of  i s abundant experimental  t h i s assumption  to  1978).  suggests  there  degrees  relationship  p o l y a c e t y l e n e s i s a v a r i a n t of that of f a t t y a c i d from a c e t a t e , and  of  c o n s i d e r a b l e number of f u n c t i o n a l  systems i n  chromophores (Jones and  The  2  via  the  the  a-en-6-yne  acid.  transformations  include  chain-shortening  by the c l a s s i c a l a- or ^ - o x i d a t i o n s of f a t t y a c i d s  at the c a r b o x y l end), conjugated  system,  c h a i n - s h o r t e n i n g at decarboxylation, and T h a l l e r ,  rearrangement and/or o x i d a t i o n of extension the  distal  of end  functionalization  1978). The  exact  the by  and  the  chromophore,  deformylation cyclization  or  (Jones  sequence of r e a c t i o n s would be  74  75  c h a r a c t e r i s t i c of the organism and a  variety  of a c e t y l e n e s  i t s physiology,  depending  on  the  producing type  and  a v a i l a b i l i t y of enzymatic s u b s t r a t e s . Hawaiian Bidens s p e c i e s s y n t h e s i z e a l i m i t e d C  1 3  and C  p o l y a c e t y l e n e s in t h e i r  1 7  leaves and r o o t s  I I I , IV, V, V I ) . A l l these compounds derived  from  outlined  acid 7.  i n Figure  may  be  i n the sequence The  presence  of  (Tables  theoretically of r e a c t i o n s non-parental  i n the leaves of F, progeny from Bidens c r o s s e s  acetylenes may  oleic  a r r a y of  be e x p l a i n e d using t h i s b i o g e n e t i c scheme (Chapter  thus  demonstrating  i t s u t i l i t y . Although  only h a l f the  of Hawaiian Bidens s y n t h e s i z e l e a f a c e t y l e n e s , t h i s has  been  from  a  common  1984;  Helenurm and Ganders, 1985; presumably  ancestor  had the g e n e t i c  Marchant  t o have  evolved  et  al.,  1984),  information for acetylene i n the l e a v e s .  of the o b j e c t i v e s of t h i s study was t o e s t a b l i s h  that de novo p o l y a c e t y l e n e s y n t h e s i s occurs Bidens  ability  (Ganders and Nagata, 1983a; 1983b;  b i o s y n t h e s i s which some taxa do not express One  taxa  shown here as a dominant and h e r i t a b l e phenotype.  Since a l l the n a t i v e s p e c i e s are b e l i e v e d  all  II),  independently  f o l l o w e d by  of the system in the r o o t s . T h i s was  investigation  accumulation  i n the leaves of  i n intact  of  the k i n e t i c s  of  acetylene  p l a n t s with comparisons of s p e c i e s  producing  different  relative  e f f i c i e n c y of a c e t y l e n e s y n t h e s i s from * C 0 . T h i s  would  1  2  provide  synthesizing  l e a f a c e t y l e n e s and an assessment of the  some 1  indication  "C-labelled  of the p r a c t i c a l i t y  acetylenes  using  of  whole p l a n t s .  76 Finally, in Bidens  the accumulation and d i s t r i b u t i o n seedlings  representative  was  species.  determined  using  of p o l y a c e t y l e n e s B.  alba  as a  77 B. MATERIALS  AND METHODS  BIOSYNTHESIS OF POLYACETYLENES  PLANT  1  *C0  I N BIDENS  2  MATERIAL Bidens  B.  FROM  cosmoides,  molokaiensis  B. hi I I ebr andi ana  and B. alba  ssp.  v a r . radiata  polycephala,  were grown  i n UBC  greenhouses under standard c o n d i t i o n s . With the e x c e p t i o n of B.  molokaiensis,  p l a n t s used  were approximately  experiments  s i x months o l d .  OF " C 0  2  Whole, f r e s h l y watered photosynthetic soil  feeding  2  1  ADMINISTRATION  and  1  for * C 0  chamber  p l a n t s were  illustrated  s u r f a c e s were f i r s t  placed  inside  the  i n F i g u r e 10. The pots  covered  with  aluminum  foil.  P l a n t s were allowed t o e q u i l i b r a t e i n the c l o s e d chamber f o r 30 minutes t o one hour before each  experiment.  chamber  hood  was  s e t up  in a  fume  f l u o r e s c e n t l i g h t s switched on. white cm  -2  light  with  of  water,  source and the fluctuations within  the  tungsten  entire  the overhead lamp  emitting  an i n t e n s i t y of 303.57 ± 48 j o u l e s s e c "  i n the chamber was the major  tray  A  with  The  source  of  radiation.  1  A  50 cm i n depth, was kept between the l i g h t plants  i n order  to  minimize  temperature  w i t h i n the box. A small fan c i r c u l a t e d the a i r chamber  and  temperature  a d m i n i s t r a t i o n . r e m a i n e d constant at 23°C.  during  1  *C0  2  FIGURE  10.  '  'COi  -  FEEDING APPARATUS  79  All  valves  experiment  were  closed  at  the  of  and the chamber then p a r t i a l l y evacuated  stopcock 3 f o r 1.5 minutes. Stopcock the  beginning  3 was then  each  through  closed  for  r e s t of the f e e d i n g . Three hundred m i c r o l i t r e s or 500ML 1  of aqueous NaH "C0 was  pipetted  (53.0 j/Ci/umole,  3  into  flask  s t i r b a r . Equimolar added  to  quantities  NaH *C0  England  Nuclear)  A equipped with a small magnetic  1  the  New  of  concentrated  solution  3  and  HC1  g e n e r a t i o n of  were i a  C0  2  a c c o r d i n g t o the e q u a t i o n : 14  NaH C0  1  3  + HCl(conc) —>-H 0 + *C0 2  2  + NaCl  allowed to proceed. The r e a c t i o n f l a s k was kept water 1  *C0  bath  (35-40°C).  Stopcocks  1  atmospheric  chamber  by  allowing  1  2  with  atmospheric  pressure,  1 was c l o s e d .  P l a n t s were allowed to metabolize f o r one hour 1 a  C0 -enriched 2  environment,  at  stopcock  3 into  18  3  period  was  measured  by  1  1  unused  *C0  2  u t i 1 i z a t i o n d u r i n g each taking  three  0.2  *C0  2  was  dissolved  evacuated  in Oxifluor-C0  2  feeding  mL samples of  chamber a i r through a rubber septum at 15 minute 1  2  2  1.0M NaOH, where i,t was trapped as  1  N a H C 0 . The r a t e of " C 0  i n the  the end of which the * C 0  generator was d i s c o n n e c t e d and the through  warm  a i r i n v i a the * C 0 - g e n e r a t i n g apparatus. When  chamber p r e s s u r e was e q u a l i z e d stopcock  a  and 2 were opened and  f l u s h e d i n t o the p h o t o s y n t h e t i c  2  in  intervals.  (New England Nuclear)  and subsequently counted f o r r a d i o a c t i v i t y .  80 1  MEASUREMENT OF "C UPTAKE INTO  POLYACETYLENES  In a l l experiments a 60-minute p u l s e of to p l a n t s i n the chamber. Leaf samples the  plants  immediately  after  1fl  C0  1fl  C0  2  was given  were then taken  from  a d m i n i s t r a t i o n and at  2  predetermined time i n t e r v a l s t h e r e a f t e r . P l a n t s were allowed to  photosyhthesize  hours  after  periodically extracted  i n atmospheric  the r a d i o a c t i v e as  plants  a i r f o r 12, 24 and 168  pulse.  Roots  were  sampled  became a v a i l a b l e . A l l samples were  for polyacetylenes  according  to  ,the  method  d e s c r i b e d i n Chapter I I . A l i q u o t s of MeOH and PE radioactivity The  light  fractions  were  counted f o r  i n 10 mL of Aquasol 2 (New England N u c l e a r ) .  petroleum  (PE) e x t r a c t s  p r e p a r a t i v e TLC p l a t e s  were  fractionated  on  (Merck, SG 60 F-254 0.25mm, 0.5mm and  2.0mm t h i c k , 20 X 20cm) and r e s p e c t i v e p o l y a c e t y l e n e s e l u t e d off with  the s i l i c a purified  acetylenes  concentrations the and  g e l with PE (30-60°C). A l l samples  (C) of  absorbance  as  reference  compounds.  The  p o l y a c e t y l e n e s were c a l c u l a t e d  (A) at the wavelength  the molar  were run  extinction  of  maximum  coefficient  of  from  absorbance  the compounds  a c c o r d i n g t o the formula e = AX/C.l where '1' i s the length of the sample c e l l (Parikh,  1cm  1974).  Samples were d r i e d and resuspended and  and equals  placed  scintillation  in  a  PDS/3-ISOCAP/300  i n 1OmL of Aquasol 2 (Searle)  liquid  counter. R a d i o a c t i v i t y , measured as counts per  18  XIII.  PERCENT  QUENCHING  POLYACETYLENES 1,  Compound  OF 4 AND  RADIOACTIVITY 5.  Percent decrease  i n dpm.  4  5  1  0  0.16  0.00  0.17  10  0.46  0.38  0.00  50  0.00  0.22  0.00  100  0.29  0.16  0.05  300  0.00  0.23  0.00  0.00  0.00  0.00  800  0.00  0.02  0.07  1000  4.54  0.00  0.36  ug  500  •  83 minute  (cpm) and the d i s i n t e g r a t i o n s  each sample, standard of  1 4  per minute  c o r r e c t e d f o r background,  (dpm) of  was c a l c u l a t e d from a  C - e f f i c i e n c y curve ( F i g u r e 11). Quenching  effects  the three a c e t y l e n e s examined here (1,4 and 5) were a l s o  prepared and are shown i n Table X I I I .  KINETIC STUDIES 1  The uptake of *C i n t o the methanol fractions  and p o l y a c e t y l e n e s  and l i g h t petroleum  i n l e a v e s of the four  Bidens  s p e c i e s was determined f o r 12 hours and 168 hours (one week) after  1  *C0  2  a d m i n i s t r a t i o n . In the 12 hour s t u d i e s ,  were taken a t two s t u d i e s , samples  hour  intervals  and  i n the week  taken once a day. S p e c i f i c a c t i v i t y  MeOH and PE f r a c t i o n s was expressed per u n i t and  f o r acetylenes  as  dpm/mg  values  expressed  fresh  long  f o r the weight  compound. T o t a l amounts of  a c e t y l e n e s e x t r a c t e d were c a l c u l a t e d and  samples  for  each  per gram of l e a v e s .  time  Bidens  period alba  was  used i n 24 hour time course s t u d i e s where samples were taken at two hour  intervals.  STATISTICAL ANALYSIS Raw  data  experiments using analysis  from  the 24  hour  time  B.alba  were transformed and  course  tracer  subjected  of v a r i a n c e using the programme UBC Anovar  to  (1978).  In each experiment, 3 p l a n t s and 3 r e p l i c a t e s of MeOH and PE  84 f r a c t i o n s per plant were a v a i l a b l e f o r Samples  for  each p l a n t  13  all thus  Acetylene  complicated  radioactivity  c a l c u l a t i o n s are based on readings from  1:  2:  by  uneven  cell  sizes  was  and two separate  comparisons were made.  T h r e e - l e v e l nested anova design f o r  MeOH and PE Run  of  p l a n t s at each time i n t e r v a l sampled. Data a n a l y s i s  statistical Run  intervals.  subsequently had to be combined f o r  i s o l a t i o n of PHT because of the low l e v e l present.  time  1 B  C  uptake  into  1 4  C  uptake  into  fractions.  Two-level  nested  anova design f o r  PHT. Four  species  cosmoides, hour  and  B.  of  alba and B.  Bidens,  B.  molokaiensis  hi I I e b r andi ana, were used i n  B.  the  12  168 hour s t u d i e s . Only one p l a n t was a v a i l a b l e per  experiment and 3 r e p l i c a t e s f o r MeOH and PE f r a c t i o n s had to be combined  i n t o one a l i q u o t  Subsequently, acetylenes  only  at  each  one  for polyacetylene  set  time  of  data  was  purification. available  for  sampled and was not analyzed f o r  var i a n c e .  ACCUMULATION  AND  DISTRIBUTION  OF  PHENYLHEPTATRIYNE  IN  BIDENS  ALBA SEEDLINGS Seeds  of  B.alba  collected  germinated on damp f i l t e r paper d i s h e s at 2 2 ° C .  from greenhouse p l a n t s were in  covered  sterile  petri  S e e d l i n g s were kept i n the dark f o r one week  and then allowed to  develop  under  white  light  (15  hour  85 photoperiod/day, " U n i v e r s a l White" f l u o r e s c e n t t u b e s ) . Whole seedlings and All  were  harvested  l e a v e s , hypocotyls samples  were  and  at days 2, 4 and  5 of  roots at days 7,  15,  weighed  and  30-60°C)  fractions  described  i n Chapter I I . D r i e d PE  in  1.0mL  prepared  crude  20  and  24.  l i g h t petroleum  according  s p e c t r a l grade MeOH and  germination  to  the  (BP  method  f r a c t i o n s were resuspended 20uL  samples  fractionated  by high performance l i q u i d chromatography (HPLC) on a V a r i a n MCH-10 reverse phase column and V a r i a n Model 5000 HPLC V a r i a n S e r i e s 634 The  UV  wavelength  v a r i a b l e wavelength UV  detector of  maximum  phenylheptatriyne was  achieved  minutes.  c o n d i t i o n s was eluted  at  a  tissue.  at  detector.  250nm  absorption  was and  performed  is  the  167,000)  crude  for  fractions  1.0mL/min with a s o l v e n t  3  mins. (=* 93%  =  of the  r a t e of  time  which  (e  from 70% CH CN/20% H 0  11.94  (Spectra-Physics) weight of  flow  Retention  peaks  set  (PHT). Separation  g r a d i e n t proceeding 15  was  with  2  (R_,  for  to 100% PHT  CH CN). an  3  under  SP4100  these values expressed  in  these  Quantitation  3  by  CH CN  of  Integrator  per u n i t  fresh  86 C. RESULTS  Three  species  of  FROM *C0  radiat a  were  used  2  Bidens,  Hawaiian  hi 11 e br andi ana ssp. polycephala, var.  was  limited  by  quantities.  B.  and  B.alba  i n a s e r i e s of time course  tracer  Choice  of  Hawaiian  the a v a i l a b i l i t y of h e a l t h y young  p l a n t s which produce e a s i l y d e t e c t a b l e acceptable  B.cosmoides,  B. molokaiensis  s t u d i e s of p o l y a c e t y l e n e b i o s y n t h e s i s . species  IN BIDENS LEAVES  1  BIOSYNTHESIS OF POLYACETYLENES  Bidens  leaf  acetylenes  cosmoides  leaves  measurable amounts of compounds 1 and 5 (Table V)  contain and  1  used  i n order t o assess the p a r t i t i o n i n g of *C i n t o  acetylenes.  Bidens  hi 11 ebrandi ana  compound  (Table  V ) , and B. molokaiensis,  1  which  in  produces  were these  mainly  which does not  have l e a f a c e t y l e n e s , were used as comparisons. Bidens produces grown  from  Hawaiian four  PHT  (4) i n i t s l e a v e s and numerous p l a n t s were  seeds  greenhouses.  s p e c i e s were repeated  a l l experiments,  photosynthesize atmosphere  the  data 2  total  f o r 60  within  disappearance  *C0  i n UBC  Experiments  with  twice and those with B.  alba  times. In  1  alba  plants  minutes  in  were  of  1 4  i n Table XIV. Conversion  C  into  the p l a n t s  to  C0 -enriched 2  d u r i n g the 60 minute pulse was  was n e a r l y complete (99.42%) f o r a l l  incorporation  1 4  a  the e n c l o s e d chamber ( F i g u r e 10).  presented  uptake  allowed  monitored  1<t  C0  2  and  1ft  of NaH C03 t o  experiments and  and percent  i n t o p o l y a c e t y l e n e s shown i n Table XV.  1ft  C  TABLE  XIV. '*C0  UPTAKE DURING 60 MINUTE PULSE-LABELLING OF  2  B. ALBA*  Time  Total  (mins)  1 a  C0  i n Chamber  2  (dpm)  0  5.44 X 10  s  + 6.2%  15  4.54 X 10  6  +  30  2.80 X 10  s  + 7.2%  45  2.12 X 10  s  + 2.2%  60  1 .32 X 10  6  +  0.20mL a i r / s a m p l e (0.032% C 0 ) 2  or 0.0064 mL C0 /sample 2  t o t a l volume  1 S  C0  therefore: total  2  1<l  i n chamber (80L) = 25.6mL C02 i n chamber at sampling =  dpm/0.0064mL X 25.6mL  * average of 4 experiments  3.5%  3.8%  1  TABLE XV. EFFICIENCY OF *C  UPTAKE*  Percent c o n v e r s i o n of NaH^COa t o 1 4  C0  99.4%  2  Total  1  *C0  2  i n chamber:  from 300 uCi NaHC0  6.73 X 10  3  8  dpm  (298 uCi) from 500 uCi NaHC0  11.28 X 10  3  8  dpm  (499 uCi)  Percent t o t a l  1 4  C  uptake  1.0% (0.1% - 2.4%)  Percent  i n c o r p o r a t i o n of  1fl  C  into  2.9%  acetylenes (0.1% - 12.9%)  * data from  15 experiments  89 The the  1  uptake of *C i n t o the MeOH and PE  fractions  i n leaves of the four Bidens  polyacetylenes  s p e c i e s was  determined f o r 12 hours and 168 hours (one week) a f t e r administration. at  In  gram  f o r each time p e r i o d  fresh  weight  of  2.0  percent  of  and values  leaves.  Tables XVI t o XXIII and F i g u r e s  2  samples  the  expressed  percent  12 t o 21. In g e n e r a l , 0.1 t o  administered  of t h i s recovered  Bidens where  alba  samples  radioactivity  i n t o B.alba  percent  0.02  was  t o 12.9  i n the p o l y a c e t y l e n e s .  was used i n 24 were  per  These r e s u l t s a r e shown i n  i n c o r p o r a t e d i n t o the methanol f r a c t i o n s and  C  C0  once a day. T o t a l amounts of a c e t y l e n e s e x t r a c t e d were  calculated  1 4  i a  the 12 hour s t u d i e s , samples were taken  two hour i n t e r v a l s and i n the week long s t u d i e s ,  taken  and  hour  time  course  studies  taken at two hour i n t e r v a l s . Uptake of  leaves i s shown i n Table XXIV. Less than 0.1  of the administered  methanol f r a c t i o n ,  l a b e l was i n c o r p o r a t e d i n t o the  from which 0.22 percent  went  into  PHT.  A n a l y s i s of v a r i a n c e f o r t h i s data  i s r e p o r t e d i n Tables XXV  and XXVI. The 24 hour accumulation  of  leaves  was  statistically  samples w i t h i n components  were  each  time  also  1  "C-PHT  the Bidens  period  significant.  f o r both  MeOH  i n the r o o t s .  De  procedure. s t u d i e s produce novo  b i o s y n t h e s i s i n r o o t s and leaves seem t o occur (Van F l e e t ,  and PE  T h i s i s c l e a r l y due t o  s p e c i e s used i n these  the ene-tetrayne-ene (1)  alba  s i g n i f i c a n t . D i f f e r e n c e s between  t e c h n i c a l problems inherent i n the e x t r a c t i o n All  i n B.  1970) but t h i s does not preclude  acetylene  independently translocation  TABLE  X V I . TWELVE HOUR UPTAKE  1  OF  « C INTO PHT BY B.  ALBA  LEAVES  T I me  MeOH  (hours)  dpm/gFW  dpm/gFW  dpm/mg  1  3.38 X 10' ± 5.7%  15.26 X 10' + 1.9%  400 ± 2.4%  308  3  3.32 X 10' ±  2 6 0 0 ± 1.9%  230  5  1.16 X 10" ± 2.4%  600 ± 8.1%  298  7  1.31 X 10' 1 4 4/„  26. B1 X 10' ± 6.5%  6000 ± 2.5%  237  9  1.17 X 10' ± 5.1/.  5.68 X 10' ± 8.4%  38700 ± 6 . 3 %  266  3.27.  5.54 X 10' ± 7.4%  4 100 ± 2 . 9 %  156  30.77 X 10' ± 6.0%  62500 + 4.5%  189  PHT  PE  6.57.  11  0.77 X 10* ±  13  0.75 X 10' ± 6.27.  9.79 X 10' : 3.0% 3.96 X 1 0  Total  percent  ''C I n c o r p o r a t i o n  i n t o MeOH f r a c t i o n  Total  percent  "'C I n c o r p o r a t i o n  into  PHT f r a c t i o n  1  ± 8.9%  PHT ug/gFW  • 2.02 =0.38  VO O  TABLE XVII. TWELVE HOUR UPTAKE OF ''C INTO ENE-TETRAYNE-ENE  Time (hours)  MeOH  PE  dpm/gFW  dpm/gFW  (1) OF 3. HILLEORANOtANA LEAVES  1 dpm/mg  1 ug/gFI  1  5 32 X 10'  +  6 . 1%  424  +  8 . 3%  600 ± 2.1%  5. 12  3  3 . 84 X 10'  +  3 .27.  227  +  4 .47.  1400 ± 2.5%  3.31  5  1 .6 , 1 X 10'  +  3 .4'/.  197 * 4 . 5%  4000 ± 4 . 87.  1 .62  7  0. 92 X 10*  +  2 . 1%  309  +  5 . 27-  52500 ± 5.27.  1 .08  9  2 28 X 10'  •  1 .97.  87  +  3 , 17.  9200 ± 3.1%  0.95  11  2 .26 X 10'  +  3  229  +  6 97  12600 ± 3.2%  0.93  13  1 .19 X 10'  +  3 4%  197  ±  4 .27  640O ± 5.5X  1.91  T o t a l percent  1 4  1  C Incorporation  OX  i n t o MeOH f r a c t i o n = 0.457=  T o t a l percent 'C i n c o r p o r a t i o n i n t o Compound 1 = 0.023%  TABLE X V I I I . TWELVE HOUR UPTAKE OF •«C INTO ACETYLENES 1 AND 5 OF  Time  MeOH  PE  1  B. COSM01DES  5  LEAVES  total acetylenes  (hours)  dpm/gFW  dpm/gFW  dpm/mg  dpm/mg  1  4.89 X 10' ± 3.5/  27 ± 1.5%  100 + 4.8%  600 • 4.8%  3  9.16 X 10' ± 6.1%  565 ± 3.4%  600 ± 3.1%  1600 ± 6.7%  5  4.65 X 10» ± 2.7%  675 ± 2.4%  400 ± 2.9%  800 • 2.8%  7  5.41 X 10 ± 3.2%  19J6 ± 2.2%  1900 i 6.9%  2100 ± 5.2%  9  2.84 X IO' i 3.11%  322 1 + 1.3%  5600 ± 7.2%  6200 + 5 . 9 %  19  11  1.04 X 10' ± 5.2%  1936 ± 6.7%  1800 ± 7.1%  2600 ± 6.5%  47  13  0.91 X 10' ± 4.8%  2153 ± 8.7%  4700 ± 5.8%  9000 ± 4.5%  20  s  4  T o t a l percent ' C i n c o r p o r a t i o n  into MeOH f r a c t i o n = 1.01  T o t a l percent ''C i n c o r p o r a t i o n  into Compound 1 = 0.089  T o t a l percent '*C I n c o r p o r a t i o n i n t o Compound 5 =0.044 T o t a l percent ''C i n c o r p o r a t i o n  into t o t a l a c e t y l e n e s = 0.13  ug/gFW  102 33 128 28  TABLE XIX. TWELVE HOUR  UPTAKE  OF  1  "C  INTO  MEOH  AND  FRACTIONS OF B. MOLOKAIENSES LEAVES  Time (hours)  MeOH  PE  dpm/gFW  dpm/gFW  5  496 ± 2.6%  2.73 X 10 ± 2.4%  5  933 + 4.8%  ± 5.8%  240 ± 9.2%  1  4.64 X 10 ± 5.9%  3 5  3.81 X 10  7  1.06 X 10 ± 4.1%  1115 ± 5.8%  9  1.79 X 10 ± 5.9%  393 ± 9.7%  11  3.27 X 10 ± 3.1%  13  2.09 X 10 ± 4.4%  T o t a l percent  5  5  5  5  5  '"C i n c o r p o r a t i o n  2633 ± 8.9% 3090 ± 6.3%  i n t o MeOH f r a c t i o n = 0.41  TABLE XX. ONE WEEK UPTAKE OF « C INTO PHT BY B. ALBA LEAVES 1  Time  MeOH  (hours)  PE  PHT  dpm/gFW  dpm/gFW  25  1.43 X 10' ± 2.6%  3618 ± 4.5%  4700 ± 2.5%  59  49  2.51 X 10* ± 2.3%  3486 ± 7.5%  12100 ± 3.8%  42  73  3.97 X 10* ± 7.6%  12920 ± 7 . 5 %  23800 ± 3 . 4 %  157  97  2.57 X 10* ± 4.7%  7311 + 1.4%  17700 ± 5.9%  70  169  0.36 X 10* ± 2.4%  111 + 3.2%  20300 ± 6.2%  109  T o t a l percent  dpm/mg  PHT ug/gFW  ''C I n c o r p o r a t i o n Into MeOH f r a c t i o n =0.15 1  T o t a l percent *C I n c o r p o r a t i o n Into PHT = 12.8  VJD  TABLE XXJ. ONE WEEK UPTAKE OF •*C INTO ENE-TETRAYNE-ENE M ) OF P. HILLfPKANOiANA  Time (hours)  PE  dpm/gFW  dpm/gFW  25  1 28 X  10'  49  1 5B X  10'  +  73  O. 89 X  10'  145  2 49 X  169  O 79 X  4 . 1%  464  J  2595 t  + 6.97.  1  dpm/mg  ug/gFW  6 .2%  0 .37  8.57-  9 2 1 0 0 • 3 .57-  0 80  * 3 .97,  1 105 • 5 . 37-  4 0 6 0 0 • 9 .2%  0 97  10'  * 9 8"/-  128  i  2 5 8 0 0 • 6. .27.  0 . 94  10'  * 3 5/.  794  * 7 37.  .5/,  T o t a l percent ''C I n c o r p o r a t i o n Into MeOH f r a c t i o n « 0.17 1  1  W=OH  LEAVES  T o t a l percent 'C I n c o r p o r a t i o n Into Compound 1 =0.20  7.07  4 1300  46100  3  o 38  TABLE XXII. ONE WEEK UPTAKE OF '«C INTO ACETvLENES 1 AND 5 OF 8.  Time  MeOH  PE  1  COSMOWtS  LEAVES  5  Total  dpm/gFW  dpm/gFW  dpm/mg  dpm/mg  ug/gFW  25  a. 59 X 10' + 4.97.  2.25 x 10* ± 4.4y.  1.6 X 10' ± 2.3%  2.6 X 10' ± 1.8%  35  49  6.66 x 10* ± 8 0"  1.26 x IO' ± 6.87  6.7 X 10" ± 5.2%  4.52 X 10' ± 3 . 9 %  73  3 10 x 10" ± 5 27.  0.91 x 10' ± 6.8%  3.54 x 10* ± 8.1%  2.88 X 10' ± 4.5%  145  0.42 X 10" ± 1.8%  0.18  x  10' ± 1.1%  3 15 X 10' ± 6.6%  0.49 X 10' ± 4.8%  47  169  3.10 X 10* 1 9.2%  1.4 1  X  10* ± 2.4%  3.48 X 10' • 6.1%  0.72 X 10' ± 6.2%  24  (hours)  T o t a l percent  • -c i n c o r p o r a t i o n Into MeOH f r a c t i o n » 0.55  T o t a l percent • 'C I n c o r p o r a t i o n i n t o Compound 1 = 0.59 T o t a l percent • 'C I n c o r p o r a t i o n i n t o Compound 5 = 0.33 T o t a l percent ' 'C I n c o r p o r a t i o n i n t o t o t a l a c e t y l e n e s = 0.93  9 13  97  TABLE X X I I I . ONE  WEEK  UPTAKE  OF  1 B  C  FRACTIONS OF B. MOLOKAJENSIS  Time (hours)  MeOH dpm/gFW  AND  LEAVES  49  1.49 X 10 ± 8.5%  73  0.51 X 10 ± 9.1%  97  7.38 X 10 ± 9.0%  169  0.86 X 10 ± 2.8%  5  1042 ± 6.2%  5  547 ± 7.5%  s  324 + 1.9%  5  907 ± 5.4%  s  8686 ± 1.0%  '"C i n c o r p o r a t i o n  PE  dpm/gFW  1.15 X 10 ± 4.4%  Percent  MEOH  PE  25  Total  INTO  i n t o MeOH f r a c t i o n = 0.07%  TABLE XXIV. TWENTY-FOUR HOUR UPTAKE OF " C INTO PHT BY S. ALBA LEAVES  T Ime  MeOH  (hours)  PE  dpm/gFW  PHT  PHT  dpm/gFW  dpm/mg  1224 ± 5.2%  11.23 ± 6 .4%  373  ug/gFW  1  2 .94 X 10'  3  1 .91 X 10' + 2 .8%  614 ± 3.7%  10.2 ± 5 .0%  325  5  1 .95 X 10' + 8 .3%  1271 ± 8.5%  19.2 ± 3 .2%  455  7  2 .48 X 10' + 6 .3%  4042 ± 6.8%  54.8 ± 5 .8%  474  9  1 .55 X 10' + 6 . iy.  2269 ± 6.7%  65.4 ± 11 . 1%  446  11  1 .49 . X 10' + 7 .9%  2669 ± 4.3%  234.0 ± 8 .5%  222  13  O..83 X 10'  + 5 .9%  761 ± 7.1%  122.1 ± 5 .6%  193  15  0..77 X 10' + 4..4%  1147 ± 8.1%  45.7 ± 6 . 0%  474  17  1 .. 15 X 10'  2.,9%  852 ± 2 . 6 %  70.9 ± 2..5%  237  19  0. 75 X 10* + 5. 9%  408 ± 3 . 2 %  47.8 ± 11 .3%  214  21  0. 34 X 10' ± 2. 7%  139 ± 3 . 5 %  15.7 ± 2.0%  150  23  0. 17 X 10' ± 3. 8%  87 ± 2.3%  46.6 ± 1. 5%  65  25  0. 17 X 10' ± 2. 2%  156 ± 1 . 9 %  7.6 ± 1.4%.  180  T o t a l percent T o t a l percent  1  "C i n c o r p o r a t i o n ''C i n c o r p o r a t i o n  7 .8%  Into MeOH f r a c t i o n = 0.09 Into PHT » 0.22  vo co  TABLE XXV. " C UPTAKE INTO MEOH AND PE FRACTIONS OF P. ALBA LEAVES IN 24 HOURS*  ANOVA: f o r dpm MeOH/gFW Source  DF  s.s.  M.S.  TIME  12  8 34 x 10' '  6 95 X  SAMPLE  26  9 57 x 10''  3.68 X 10''  78  121 x 1 0 "  1.44 X 10"  AL I ERROR  0  TOTAL  1 16  10'"  F value  F prob.  Tested against  1.8873  O.0851  2  256.0845  O.OOOO  3 4  0 1.80 X  lO''  ANOVA f o r dpm PE/gFW: SOURCE  D. F .  S.S.  M.S.  TIME  12  1.45 X 10'  1.21 X 10  SAMPLE  26  1.07 X 10'  4 . 12 X 10"  AL I  78  5.05 x 10'  6.47 X 10*  ERROR  0  0  TOTAL  116  2.57 x 10*  F value  F  prob.  Tested against  2 9323  0.0106  2  63.6992  0.0000  3 4  • T h r e e - l e v e l nested design. Run *1.  VO  vo  100  TABLE XXVI. '*C UPTAKE INTO PHT(3):  B. ALBA  LEAVES  IN  24  HOURS*  ANOVA f o r dpm/mg ( 3 ) : Source  D.F.  S.S.  M.S.  F value  F prob.  Tested against  TIME  12  138012  11501  SAMPLE  26  116337  .4474  ERROR  0  TOTAL  38  2.5704  0 254349  Two-level nested d e s i g n , Run# 2.  0.0214  2 3  101  of  precursor  molecules  from  leaf  to  root.  Roots  were  harvested and e x t r a c t e d f o r compound 1 at the end of the hour  and  week  long experiment and the data  shows s u b s t a n t i a l  i n Table  12  XXVII  1  i n c o r p o r a t i o n of *C i n t o the a c e t y l e n e .  ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE  IN BIDENS  ALBA SEEDLINGS Two-day  to  24-day s e e d l i n g s of B. alba  f o r PHT i n the l e a v e s , hypocotyls and amounts  Phenylheptatriyne levels  in  leaves  was  present  increased  period. Concentrations  and  The  Figures  12  and  13.  i n two day o l d s e e d l i n g s and throughout  100 times higher  the a e r i a l t i s s u e s i n i t i a l l y  to d e c l i n e a f t e r two weeks.  relative  u s i n g HPLC and  the  experimental  i n the h y p o c o t y l s decreased  week. The r o o t s c o n t a i n e d than  roots.  of PHT i n each sample was determined  the r e s u l t s shown i n Table XXVIII  were e x t r a c t e d  amounts  a f t e r one of  PHT  although q u a n t i t i e s began  1  TABLE XXVII. •OLABELLED ENE-TETRAYNE-ENE BIDENS GIVEN  Time  Plant  B.  13  B. B.  B.  alba  B. molokai e ns i s B.  169 169  cosmoi des  hi 11 ebr andi ana  169 169  alba  B. molokai ensi s  13 13  C0 . 2  1 dpm/mg  (hours)  13  1 S  (1) IN ROOTS  B.  cosmoi des  hi I I ebr andi a na  ug/gFW  12078  9.0  70000  0.1  268  11.3  25000  1.0  2235  14.7  6650  7.1  2692  2.7  4689  1 .0  1 03  TABLE  XXVIII.  ACCUMULATION  PHENYLHEPTATRIYNE  Days  FW mg/lOOOuL  (4)  AND IN B.  mg/20uL sample  DISTRIBUTION  OF  ALBA SEEDLINGS  % area  Rel.amt.  PHT/sample  PHT/mg sample  Seedlings: 2  122.4  2.45  49.31  20  4  125.3  2.51  77.05  31  5  100.8  2.02  76.88  39  7  152.4  3.05  65.59  22  15  140.8  2.82  76.86  28  20  156.5  3.13  95.59  31  24  159.8  3.20  96.54  30  7  150.1  3.00  81.43  27  15  180.0  3.60  58.78  16  20  199.3  3.99  63.42  16  24  169.9  3.79  64. 15  17  7  0.560  0.0112  26.37  2355  15  0.625  0.0125  46. 17  3694  20  0.231  0.0046  12.46  2709  24  0.821  0.0164  30.64  1666  Leaves:  Hypocotyls:  Roots:  901  106 D. DISCUSSION  IN BIDENS  1  BIOSYNTHESIS OF POLYACETYLENES FROM * C 0  2  LEAVES  The term b i o s y n t h e s i s i s d e f i n e d by Swain (1965) as the in  vivo  endothermic  production  of more complex molecules  from simpler ones. N a t u r a l p o l y a c e t y l e n e s are thought t o be derived  from f a t t y a c i d p r e c u r s o r s which are s y s t e m a t i c a l l y  transformed i n t o a d a z z l i n g which  have  Bohlmann et  potent  array  of compounds,  photobiocidal  al. , 1973;  Jones  effects  some of  (Bu'Lock, 1966;  and T h a l l e r ,  1978; Towers,  1979). At p r e s e n t , no in  vivo  real  evidence  exists  c o n c e r n i n g the  formation of the carbon-carbon t r i p l e bond although  d i r e c t dehydrogenation v i a cis speculatively  (Bu'Lock  double  and Smith,  bonds  was favoured  1967),  and appears  probable  (Haigh et al., 1968; Jones et al., 1975; Jones and  Thaller,  1978).  Nevertheless,  the  interrelationships the  chain  of a c e t y l e n e s  lengths,  rearrangements,  various  of  c o n t a i n i n g groups and c y c l i z a t i o n s great  detail  i n the l a b o r a t o r i e s  Ewart R.H. Jones, labelled  precursors include and  mostly  precursors  linoleic  1 4  with  obtained  C - and/or  acids,  such as m o d i f i c a t i o n s of  introduction  introduction  of  triple  oxygenhave  been  bonds,  and sulphurstudied i n  of F. Bohlmann and S i r  the use of by  3  biosynthetic  total  H-labelled  specifically  synthesis. acetate,  These oleic  crepenynic a c i d and d e h y d r o m a t r i c a r i a  107 ester  variously  administered  v i a the i n t a c t root or l e a f  s u r f a c e or the fungal c u l t u r e medium (Bohlmann et Bohlmann and  and Schulz,  Richter, Unlike  precursors system, often  1968; F a i r b r o t h e r et al.,  1976; Jones,  al . , 1968; 1967; Jente  1966).  the s i t u a t i o n with microorganisms where p u t a t i v e can be e a s i l y and n a t u r a l l y  the uptake difficult  necessarily  of  true  defined  l a b e l l e d substances i n t o p l a n t s i s  and u n s a t i s f a c t o r y ,  a  fed to a  reflection  of  the  results  not  the in vivo s i t u a t i o n  (Swain, 1965; Brown and Wetter, 1972; F l o s s , 1977). The only way  1  i n which  physiological  "C  can be  administered  f a s h i o n i s as  incorporation  into  1tt  complex  C0  even  2  secondary  r a t h e r low. T h i s has not been  to plants though  rates  metabolites  previously  in a of  may be  demonstrated f o r  polyacetylenes. In the present  i n Bidens  polyacetylenes  the de  study,  leaves  novo  biosynthesis  was i n v e s t i g a t e d i n time  1  course s t u d i e s . " C - l a b e l l e d p o l y a c e t y l e n e s from  three  of Bidens  species  first  1 2  to metabolize i n C 0  168  XVI  (Tables  incorporated  0.1  administered  t o XXIV).  t o 2.4  radioactivity  which 0.1 t o 12.9 percent polyacetylenes be due t o  percent  2  recovered 1 0  C O and 2  f o r 12, 24 and  In g e n e r a l , the p l a n t s (mean  =  0.9%) of the  i n t o the methanol f r a c t i o n (mean  -  2.9%) went  from  i n t o the  examined (Table XV). The range i n values may  individual  photosynthetic  were  administered  subsequently allowed hours  of  rates  and/or since  interspecific environmental  variations in factors (C0  2  108  c o n c e n t r a t i o n s , H 0, l i g h t and temperature) were e s s e n t i a l l y 2  uniform  for a l l  pronounced  experiments.  between  B.  These  alba  differences  and B. mol okai enses i n the 12  hour experiments. The B. molokaiensis o l d e r and e v i d e n t l y l e s s v i g o r o u s In a l l methanol  experiments,  fractions  a r e most  peak  preceded  plants available  than B. alba p l a n t s . specific  that  activity  i n the l i g h t  generally  expected 1  to rise i n i t i a l l y  2  that  with  intermediates  2  C0  with  of  the l i g h t  petroleum  S p e c i f i c a c t i v i t i e s of these components would be  *C0 -derived  1 2  including  a r e found. Peak a c t i v i t y of p o l y a c e t y l e n e s  coincided  fractions.  i n the petroleum  f r a c t i o n s where most of the l i p o p h i l i c compounds, polyacetylenes,  were  increasing  and then  fall  formation  from  as f l u s h i n g  with  proceeded. Total  amounts  p e r i o d are expressed leaves.  This  rate  (1) i n B.  alba  (Tables XVI,  acetylenes  remain  compounds  fluctuates. 1,  suggests  that  are synthesized  respectively  activity  i s o l a t e d at each time  as micrograms per gram f r e s h weight  information  ene-tetrayne-ene constant  of p o l y a c e t y l e n e s  at  and XX,  a  relatively  essentially  uniform  cosmoides  while  leaves  of  specific synthesize  3 and 5 but o n l y 1 and 5 were e a s i l y  l e v e l s i n B. cosmoides  leaves,  XXI). Levels  in the system used here (TLC p u r i f i c a t i o n ) . acetylene  (4) and the  and B. hi 11ebrandiana  XVII  Bidens  PHT  of  detected  The data f o r  i s i n c o n c l u s i v e and may be  due t o the e x c l u s i o n of the pentayne-ene from the a n a l y s i s .  109 According are  t o the scheme i n F i g u r e 7, compounds 1 and 5  s y n t h e s i z e d i n p a r a l l e l but separate pathways; one does  not precede the other along the same sequence of r e a c t i o n s . In  B.  cosmoides,  maximum  specific  a c t i v i t i e s of the two  compounds occurs a t the same time and decreases Either  conversion  extremely Compound  from  rapid 3  or  one  both  acetylene  to  similarly.  the other  are synthesized  is  concurrently.  was not i n v e s t i g a t e d i n these experiments but a 1  comparison of i t s *C-uptake r a t e with that of 1 and  5 may  have been more e n l i g h t e n i n g . The B.  alba  1U  h i g h e s t s p e c i f i c a c t i v i t y of C-PHT s y n t h e s i z e d by was 0.0275 /zCi/mg PHT (6.25  a f t e r 300 MCi of Figure  1ft  C0  2  was  X 10" dpm/mg), 12 hours  administered  (Table  14). In the 24 hour experiments, 500 uCi of  fed t o the p l a n t s and peak a c t i v i t y was even nCi/mg one  PHT  or  XVI and 1 4  C0  lower  2  was  (0.0001  234 dpm/mg) (Table XXIV) while that f o r the  week experiments was 0.0105 nCi/mg  PHT  or  2.38  X  10*  dpm/mg (Table XX). These r e s u l t s r e f l e c t incorporated (2.0%,  in total  1  *C  i n t o the p l a n t s d u r i n g each of the experiments  0.1% and  controlling  the d i f f e r e n c e s  0.2%) and  illustrate  the d i f f i c u l t y  of  1  the a c t u a l dose of *C f i x e d p h o t o s y n t h e t i c a l l y  by whole p l a n t s . Other f a c t o r s c o n t r i b u t i n g t o the v a r i a n c e may  include  r e l a t i v e d i f f e r e n c e s i n the metabolic  activity  of the b i o s y n t h e t i c s i t e s as w e l l as d i f f e r e n c e s i n the pool sizes and  of the a c e t y l e n e p r e c u r s o r s , p o s t u l a t e d  those of the f i n a l products.  In any case  intermediates  i t appears that  1 10 the n a t u r a l s y n t h e s i s of * C - l a b e l l e d P H T with a s i g n i f i c a n t 1  amount  of  radioactivity  may  not be  p o s s i b l e using  this  method. At roots  t h e end of were  the 12 hour and 168 hour experiments,  extracted  ene-tetrayne-ene  for  (1) was  detected  XXVII), i n c l u d i n g B. mol okai ensi s , into i t s  M e O H  of the f a c t This  and  that  P E  indicates  in a l l which  1  t r a n s l o c a t e d from a e r i a l  plants  XXIII),  not s y n t h e s i z e  that  "C-labelled (Table  incorporated  f r a c t i o n s (Tables XIX,  i t does  1  polyacetylenes.  leaf  "C-labelled  1  "C  in spite  acetylenes.  precursors  were  t i s s u e s t o s i t e s i n the r o o t s where  de novo s y n t h e s i s of root compounds takes p l a c e .  ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS In mature B. alba p l a n t s , leaves c o n t a i n mainly P H T (4) while  the  stems  have  comparable  phenylhepta-diyne-ene (5) and the r o o t s 5 ene-tetrayne-ene polyacetylenes previously  1984).  B. al ba  i n developing  P H T and  as  well  as the  The accumulation of  plants  has not been  reported.  Detectable seedlings  (1) (Norton,  of  amounts  of  l e v e l s of P H T B.  alba,  were  found  suggesting  i n two-day o l d  that  b i o s y n t h e s i s begins during  germination  (Table  12, 13). Q u a n t i t i e s i n the leaves  continue  XXVII  and F i g u r e s  t o i n c r e a s e up t o 24  days  or  polyacetylene  soon  thereafter  and presumably  beyond  111 that t o a d u l t l e v e l s while amounts i n the hypocotyls seven  days  and  subsequently  c o n c e n t r a t i o n . T h i s i s probably  decline  to  a  peak at lower  accompanied by a concomitant  i n c r e a s e i n the l e v e l s of compound 5 (Figure 12). PHT plants  i s absent from the (Towers,  roots  of  1980; Norton, 1984)  but i s present  s e e d l i n g s . R e l a t i v e PHT l e v e l s i n the r o o t s higher  than  those  beginning  experimental is  there at  by  a  i s a l s o a gradual  two  time p e r i o d  accompanied  a r e 100  alba i n the times  i n the a e r i a l t i s s u e s f o r the f i r s t 24  days. N e v e r t h e l e s s , levels  Bidens  mature  weeks  d e c l i n e i n these  and c o n t i n u i n g beyond the  (Table XXVII and F i g u r e concomitant  compounds 1 and 5 (data not shown). d i s t r i b u t i o n of PHT i n B. alba by one month a f t e r the onset  increase  i n l e v e l s of  I t appears  reaches i t s a d u l t  of germination.  13). T h i s  that the proportions  1 12 E. CONCLUSION The  complete  elucidation  r e q u i r e s the a p p l i c a t i o n of According  to  Adelberg  l a b e l l i n g with p r e c u r s o r s , use  of  main  a  several  source  of  current  b i o s y n t h e t i c pathway  different  techniques.  (1953),  these  include  isotopic  in  enzyme s t u d i e s  and the  vitro  microorganisms with blocked  acetylene  s y n t h e t i c pathways. The  knowledge  of  the pathways  b i o s y n t h e s i s a r e experiments of the f i r s t  (e.g., Bu'Lock, et  of  and  Smith,  1963; Jones,  of  category  1966; Bohlmann  al., 1968). In f u n g a l c u l t u r e s , b i o s y n t h e t i c experiments a r e e a s i e r  and  give  although  higher  incorporations  than  those  with p l a n t s ,  a v a r i e t y of a l t e r n a t i v e sequences may be a v a i l a b l e  for both types of organisms (Jones and T h a l l e r , 1978). In  this  study,  polyacetylenes Bidens of all  1<t  de  novo  alba  C incorporated  was demonstrated using  i n t o the f i n a l products  three a c e t y l e n e s  of  *C0 . 2  Levels  were minimal but labelled.  v a l i d i t y of p o s t u l a t e d b i o s y n t h e t i c sequences must  confirmed  by  the d e t e c t i o n  c a t a l y z i n g key steps  are  and  ideally  and  isolation  i n the pathway.  Hawaiian  be u s e f u l organisms f o r in  should  s e v e r a l reasons: which  1  i s o l a t e d were s i g n i f i c a n t l y  be t e s t e d by more than one method,  Bidens  biosynthesis  i n the leaves of s e l e c t e d s p e c i e s of Hawaiian  and Bidens  The  the  vitro  they produce a l i m i t e d a r r a y of  closely  related,  different  should of  be  enzymes  species  of  studies for acetylenes  species  produce  d i f f e r e n t a r r a y s i n leaves and roots and may be s e l e c t e d f o r  1 13 particular  compounds,  interspecific  hybrids  are  easily  produced and the p l a n t s are r e l a t i v e l y easy to propagate and maintain under standard greenhouse  conditions.  11 4 F. BIBLIOGRAPHY  Adelberg, E.A. 1953. The use of m e t a b o l i c a l l y blocked organisms f o r the a n a l y s i s of b i o s y n t h e t i c pathways. B a c t e r i o l . Rev., 17: 253 - 267. Bohlmann, F., biogenese 860.  H. Bonnet and R. Jente. 1968. Uber d i e des p h e n y l h e p t a t r i i n s . Chem. Ber. 101: 855 -  Bohlmann, F. and R. Jente. 1966. Zur biogenese p h e n y l p o l y i n e . Chem. Ber. 99: 995 - 1001. Bohlmann, F. and H. Schulz. polyinen mit zellfreien L e t t e r s , 4795 - 4798.  der  1968, Uber d i e b i l d u n g von homogenaten. Tetrahedron  Brown, S.A. and L.R. Wetter. 1972. Methods f o r i n v e s t i g a t i o n of b i o s y n t h e s i s i n higher p l a n t s . pp1-45 i n Progress i n Phytochemistry: Volume I I . L. Reinhold and Y. L i w s c h i t z ( E d s . ) . John Wiley and Sons, London. Bu'Lock, J.D. 1966. The b i o g e n e s i s of n a t u r a l acetylenes. pp79-95 i n Comparative Phytochemistry. T. Swain (Ed.). Academic Press, London. Bu'Lock, J.D., D.C. A l l p o r t and W.B.Turner. 1961 (Part I I ) . The biosynthesis of Polyacetylenes. Part III. Polyacetylenes and triterpenesin in Polyporus ant hracophi I us . J . Chem. Soc. 1654 - 1662. Bu'Lock, J.D. and H. Gregory. 1959. The b i o s y n t h e s i s of p o l y a c e t y l e n e s . 2. O r i g i n of the carbon atoms. Biochem. J . 72: 322 - 325. Bu'Lock, J.D. and G.N. Smith. 1963. A c e t y l e n i c fatty acids in seeds and s e e d l i n g s of sweet quandong. Phytochemistry 2: 289 - 296. Bu'Lock, J.D. and G.N. Smith. 1962. P o l y a c e t y l e n i c a c i d s i n d e v e l o p i n g s e e d l i n g s of sweet quandong. Biochem. J . 85: 35P. F a i r b r o t h e r , J.R.F., S i r E.R.H. Jones and V. T h a l l e r . 1967. Natural acetylenes. Part XXV. The b i o s y n t h e s i s of benzenoid p o l y a c e t y l e n e s . J . Chem. Soc. ( C ) : 1035 1039. F l o s s , H.G. 1977. R a d i o t r a c e r s i n b i o s y n t h e t i c s t u d i e s , pp. 689-732 i n R a d i o t r a c e r Techniques and A p p l i c a t i o n s . V o l 2. E.A.Evans and M.Muramatsu ( E d s . ) . M. Dekker Inc. Ganders,  F.R.  and  K.M.  Nagata.  1984. The  role  of  115 h y b r i d i z a t i o n i n the e v o l u t i o n of Bidens on the Hawaiian I s l a n d s . P. 179-194 i n P l a n t B i o s y s t e m a t i c s . W.F. Grant (Ed.). Academic P r e s s , Canada. Ganders, F.R. and K.M. Nagata. 1983a. R e l a t i o n s h i p and floral b i o l o g y of Bidens cosmoides ( A s t e r a c e a e ) . Lyonia 2: 23 - 31. Ganders,F.R. and K.M. Nagata. 1983b. New taxa and new combinations i n Hawaiian Bidens ( A s t e r a c e a e ) . Lyonia 2: 1 - 16. Haigh, W.G., L . J . M o r r i s and A.T.James. 1968. A c e t y l e n i c a c i d b i o s y n t h e s i s i n Crepis rubra. L i p i d s 3 : 307 - 312. Helenurm, K. and F.R. Ganders. 1985. Adaptive r a d i a t i o n and genetic differentiation i n Hawaiian Bidens. E v o l u t i o n (in press). Jente, R. and E. R i c h t e r . 1976. Zur biosynthese des dehydromatricanaesters. Phytochem. 1 5 : 1673 - 1679. Jones, S i r E.R.H. 1966. N a t u r a l p o l y a c e t y l e n e s and t h e i r p r e c u r s o r s . Chem. i n B r i t . 1966: 6 - 13. Jones, S i r E.R.H. and V. T h a l l e r . 1978. N a t u r a l a c e t y l e n e s , pp. 621-633 i n T h e C h e m i s t r y o f t h e C a r b o n - C a r b o n T r i p l e Bond. Part 2. S. P a t a i (Ed.). John Wiley and Sons, New York. Jones, S i r E.R.H., V. T h a l l e r and J.L. Turner. 1975. N a t u r a l a c e t y l e n e . Part XLVII. B i o s y n t h e t i c experiments with the fungus Lepista di emi i ( S i n g e r ) . B i o g e n e s i s of the C a c e t y l e n i c cyanoacid d i a t r e t y n e 2. J . Chem. S o c , Perkin Trans. I : 424 - 428. Marchant, Y.Y., F.R. Ganders, C K . Wat and G.H.N. Towers. 1984. P o l y a c e t y l e n e s i n Hawaiian Bidens. Biochem. Syst. E c o l . 1 2 : 167 - 178. Norton, R.N. 1984. S t u d i e s of P o l y a c e t y l e n e normal and transformed t i s s u e c u l t u r e s of Ph.D. D i s s e r t a t i o n , U n i v e r s i t y of B.C. Parikh,V.M. 1974. A b s o r p t i o n Spectroscopy M o l e c u l e s . Addison-Wesley, O n t a r i o .  production i n Bidens alba. of  Organic  Swain, T. 1965. Methods used i n the study of b i o s y n t h e s i s , pp. 9-36 i n B i o s y n t h e t i c Pathways i nHigher P l a n t s . J.B.Pridham and.T. Swain ( E d s . ) . Academic Press, New York. Towers, G.H.N. 1980. P h o t o s y n t h e s i z e r s from p l a n t s and t h e i r photodynamic a c t i o n . Prog. Phytochem. 6:183 - 202.  8  116  UBC  ANOVAR. 1978. Analysis Adapted by M.Greig and Centre.  of D.  variance and c o v a r i a n c e . Osterlin. UBC Computing  Van  F l e e t , D.S. 1970. Enzyme l o c a l i z a t i o n and the genetics of polyenes and p o l y a c e t y l e n e s i n the endodermis. Adv. F r o n t i e r s P i . S c i . 2 6 : 109 - 143.  Wiermann, R. 1981. Secondary p l a n t products and c e l l and t i s s u e d i f f e r e n t i a t i o n , pp. 86-116 i n The Biochemistry of P l a n t s . A Comprehensive T r e a t i s e . V o l . 7 . P.K. Stumpf and E.E.Conn ( E d s . ) . Academic Press, New York. Weiss, U. and J.M. Edwards. 1980. The B i o s y n t h e s i s of Aromatic Compounds. J . Wiley and Sons. Toronto.  IV.  A.  PHOTOTOXICITY OF POLYACETYLENES TO PHYLLOPLANE FUNGI  INTRODUCTION A l l Hawaiian Bidens  only et  15  taxa  al .,1984).  possess  synthesize  them  acetylenes  i n the roots but  i n the l e a v e s  (Marchant  There appears t o be no s i g n i f i c a n t  correlation  between the presence or absence of l e a f a c e t y l e n e s Bidens,  other  feature  of  pers.  comm.).  Nearly  cosmoides  and B.  a l l rainforest  macrocarpa)  s p e c i e s with and without habitats and  have  leaf  evolved  from  a  1983a; 1983b; 1984;  species leaf  acetylenes  al.,1984),  common ancestor  Helenurm  species  a/.,1962), as  Artemesia  well  al.,1973), B. pilosa as  vulgaris  (Bohlmann et although  occur  in  drier  genetic Bidens  (Ganders and Nagata,  and Ganders,  i s not unique  t o Bidens  even el  (Bohlmann  1984),  a c e t y l e n e s , but  1985; Marchant  do  so  in  species  Hawaiian  Chrysanthemum  do not s y n t h e s i z e i n the r o o t s .  fortuitous.  117  L.  (Bohlmann  Compositae douglasii leaf This  Bidens  cernua L.  (Blume) S h e r f f of  Bidens  taxa.  i n g e n e r a l . Bidens  v a r . minor  L.,  to  B. tripartita  other  al.,1973)  they  present  i n the l e a v e s of c e r t a i n  phenomenon or  B.  the g e n e t i c i n f o r m a t i o n f o r de novo a c e t y l e n e  i s not expressed This  et  (e.g.,  that a l l n a t i v e Hawaiian  s y n t h e s i s i n leaves and r o o t s i s probably but  (F.R.Ganders,  and lower e l e v a t i o n s . Since m o r p h o l o g i c a l ,  biochemical data suggest  have  et  including habitat  and any  (Norton, (e.g., Hulten)  acetylenes, may be merely  1 18 Nevertheless photosensitizers range et  of  Camm et  a/.,1982;  information  al . , 1975; et  l e d to  speculation of  parent  plants?  defense s t r a t e g y a g a i n s t  Does  absent from  the  of  albicans  growing  coli  some  (Migula) Berkh.  faced  in  the in  If  or so,  species  by  Cast.  have  Bidens  et  possible  p l a n t s . Do  signify a  why  are they  Bidens?  of  Do  relevance cosmoi des  surfaces  of  higher  Candida  and  t o the e c o l o g i c a l or  other  the wet jungle of Kauai? The present  aerial  This  against a l l  Chalm.  p r e l i m i n a r y attempt to answer some of these The  (Arnason  out on t y p i c a l l a b o r a t o r y organisms such  (Robin)  conditions  wide  DiCosmo  presence  enemies  organisms?  as Escherichia  a  al .,1977).  about  their  threatening  carried  to  f u n c t i o n or s e t of f u n c t i o n s  potentially  bioassays  powerful  1975;  polyacetylenes  specific  leaves  al.,  Wat el  a/.,1977;  significance  their  toxic  et  Chan  these compounds have a s p e c i f i c in  are  are  i n c l u d i n g b a c t e r i a and fungi  Towers has  biological  polyacetylenes  (Towers, 1980) and  organisms,  a/., 1980;  el  many  species  study  is a  questions.  p l a n t s growing under  n a t u r a l c o n d i t i o n s a r e u s u a l l y c o l o n i z e d by l a r g e and v a r i e d populations leaf  Dickinson, leaves  consider 1971;  microorganisms. Such p o p u l a t i o n s c o l o n i z i n g  s u r f a c e s form an e c o l o g i c a l niche which i s  phylloplane  the  of  (Last  and  Price,  1969;  Davenport,  1976). Comparisons of the fungal of  different  plants  that one s u r f a c e i s very Ruscoe,  1971),  and  termed  the 1976;  populations  on  have l e d some authors to like  although  another the  (DiMenna,  majority  of  119 phylloplane  studies  have  been  r e g i o n s and on a g r i c u l t u r a l  carried  crops,  out  the few  i n temperate  studies  which  have h i t h e r t o been p u b l i s h e d on t r o p i c a l p l a n t s suggest that many p h y l l o p l a n e (Dickinson,  fungi  a r e cosmopolitan  temperate  regions, a r e very  1965). N e v e r t h e l e s s ,  L. (Poaceae),  Salix  Eucalyptus  stellulata  Sieb.  plants  support  babylonica  distinctive  that  unfavourable airborne  the  ex  DC. (Myrtaceae)  phylloplane  f o r many  flora  microflora after  growing  of  populations.  is a  the organisms  pressure  of  the r e s i d e n t  Finally,  the  polyacetylenes acetylenes  phylloplane  the inoculum has come i n t o c o n t a c t with i t .  purpose  presence  is  and that the l e a f e x e r t s s e l e c t i v e  s p e c i e s of Hawaiian Bidens  and d i s t r i b u t i o n  which  i n the  the p h y l l o p l a n e  second  niche  They  present  of the o b j e c t i v e s of t h i s study  identify  the  (Last and Deighton,  L. ( S a l i c a c e a e ) and  microbial  which determines the nature  The  similar  particularly  on a creek bank and.found that the leaves of these  suggest  One  fungi,  Lamb and Brown (1971) examined Pas pal um  dilatatum  together  distribution  1976). These fungi grow on a wide range of host  p l a n t s and p o p u l a t i o n s , and s p e c i e s of in  in  yeasts  and  plants.  was t o f i n d out whether the occurrence  absence  of  sensitivity was  and  y e a s t - l i k e fungi on  and on s e l e c t e d sympatric  of p h y l l o p l a n e fungi  or  was t o i s o l a t e  assessed  leaf  was  correlated  acetylenes  i n Bidens  of  these  and  the b i o l o g i c a l  i n leaves was e v a l u a t e d .  with  organisms  . to  r o l e of  120 B. MATERIALS AND METHODS  PLANT MATERIAL Healthy green leaves from 12 taxa and  23  August,  of  Bidens  Hawaiian  sympatric s p e c i e s of other p l a n t s were c o l l e c t e d i n 1983 and February, 1984, p l a c e d i n paper  envelopes,  s e a l e d and allowed t o dry during a i r t r a n s f e r from Hawaii t o Vancouver,  Canada (Tables XXIX and XXX).  ISOLATION AND IDENTIFICATION OF FUNGI Three methods were used t o i s o l a t e and  yeast-like  method (Last, 1910;  Lamb  (Petrini isolates  et  In  from  and al.,  Brown,  impression  1970) and  1982; C a r r o l l cultured  in a  and malt  (MYPT), (Bandoni, 1972),  the spore  fall  yeasts  a l l the leaves: the spore  1955), the l e a f  were  tetracycline  fungi  phylloplane  method  fall  (Potter,  the l e a f  d i s c method  Carroll,  1978). A l l  extract  medium  with  (Table XXXI).  method (A), the d r i e d l e a v e s were  first  rehydrated by soaking i n s t e r i l e water f o r 30 minutes,  then  p l a c e d i n a p l a s t i c bag with a wad of moistened  tissue  paper, s e a l e d and kept thus f o r 24 hours. Whole l e a v e s then r i n s e d  were  i n s t e r i l e water, d r i e d and a t t a c h e d t o the l i d s  of  s t e r i l e p e t r i d i s h e s , a b a x i a l or a d a x i a l s u r f a c e s  to  the MYPT agar below. T h i s method i s a s e l e c t i v e one which  f a v o u r s the i s o l a t i o n of members of the by  allowing  Sporobolomycetaceae  t h e i r b a l l i s t o s p o r e s t o drop from l e a f  onto the n u t r i e n t medium.  exposed  samples  TABLE XXIX. Taxa  ftI DENS  LocalI t l e s Man i n ! P a l l . Oahu  3. amp tec tens  TAXA SAMPLED FOR PHYLLOPLANE FUNGI Site descriptions Sunny ledges on steep c l i f f s about 200m e l e v a t i o n on windward s i d e , n o r t h end of the Waianae Range,  In scrub v e g e t a t i o n of  Lucaena Ieucocephala.  Canthium odoratum. Myoporlum sandwlcense r al I ax  Makaha Ridge. Kauai  8. cervicata  On r o a d s i d e and open areas on d r y r i d g e i n p l a n t e d p i n e f o r e s t : area r a i n f a l l about 40" per year: w i t h  n a t i v e shrubs such as  Dodonaea  and  Styphelia:  e l e v a t i o n 500-700—•  B. cosmoides  Kokee S t a t e Park near Waimea Canyon o v e r l o o k .  R a i n f o r e s t of  Acacia koa. Metros Ideros  with  Cyan  and other p l a n t s : about 1000— e l e v a t i o n .  kaua 1 8. forbesii ssp.  forbesii  Lumahai Beach overlook, K.ma i  20-30m e l e v a t i o n on steep c o a s t a l b l u f f s above beach; i n wet (75-iOO" r a i n per y e a r ) but r a t h e r  scrubby v e g e t a t i o n w i t h  t'anrtantis  and  Met ros i deros to  Graveyard near Kalmu. B. nmra'ens's Hawa11  Open mesle  old  HfefrosIderoa  a'a lava flows;  f o r e s t s end open f i e l d s on  about 30m e l e v a t i o n ;  rainfall  about 75" per year.  P. M I IetsrsndI ana ssp. potycephaia  West s i d e of MaHko Bay  Maul  On top of sea c l i f f s .  30-40* e l e v a t i o n ; on windward  s i d e of i s l a n d , exposed t o ocean spray: b a r e s o l i  between p l a n t s on the v e r t i c a l c l i f f  introduced  B. maul ens Is  Near Chinese cemetery,  WaIhee, Mau1  f a c e and  weeds on top.  On windy, exposed, d r y l l t h l f i e d sand dunes on the  windward coast  vegetation  of west Maul; e l e v a t i o n 50m:  of n a t i v e  low shrubs and herbs;  with  rainfall  about 30"  B. menriesil ssp HI Iformls  Ahumoa. Hawa11  On leeward s l o p e of Mauna Kea, 1n open s c r u b and  open dry f o r e s t s of Sophora and Myoporum on the c i n d e r cone of Ahumoa: about 2000m e l e v a t i o n ; rainfall  about 30" per year.  0. mlcrantha ssp. etenoohyila  Old quarry near Kallua, Kona, Hawa11  At about 30m elevation on ancient a'a lava flow; In arid, open scrub of Schfnus. tfalfherfa.  sida  and  grasses; very low r a i n f a l l , about 10-15" per yaar.  8. popu11f olI a  South Mdge bordering Kahana Valley. Oahu  In clearings on ridge about 70-100m elevation; wat Wef/rosfderos forest with Srhef  B. sandvicensls ssp. confusa  IHau nature t r a i l , Walmea Canyon. Kauai  Pandanus,  Schfnus and  flera  tn open scrubland with WlIkesla, Oodonaea StypheMa: elevation about 700m: In open meslc forests of  Acacia koa  and Metrosfderos at about  900m elevat Ion. B. sandvfcensfs ssp.  Waahlla Ridge, Oahu  On exposed crest of ridge in mas 1c scrub vegetation; with  sandv fcensIs  Slda.  Osteomeres and  Acacia koa:  about 400m elevation. Watmea Canyon, Kauai  About 350m elevation; in dry scrub of  Oodonaea  introduced shrubs and herbs.  oo  and  124  TABLE  XXX.  PLANTS  ASSOCIATED  WITH  BIDENS SAMPLED  FOR  PHYLLOPLANE FUNGI Bidens  A s s o c i a t e d Plant  B. amplectens  Cant hi urn odor alum F o r s t . f .  Species  Si da falI ax Walp. 11 ima sp. Myoporum sandwicense A. Gray B. cervicata  Styphelia  lamehameha (Cham.)  F.Muell.  Dodonaea sp. Lani ana camara B. cosmoides  Psychol ri a sp. Pas s i fI or a sp. Acacia koa A. Gray  B. forbesii  ssp.  Ageralum sp.  forbesii Met rosi deros col Iina  ( F o r s t ) Gray  Stachyl arpheta jamaicensis Bidens pi Iosa L. B. hi 11 ebrandiana  hi coliana  glauca Grah.  SSp. polycephala  Emilia sp. Trifoliurn sp.  V  (L.)Vahl.  125  B. mauiensis  Lipochaeta  sp.  Stdchytarphela  jamaicensis  (L.)Vahl.  11ima sp. Si da f alI ax Walp. Scaveola  taccada  (Gaertn.) Roxb.  Volt heri a amer i cana L. Nama sandwicensis B.  populifol  ia  St achytarphela  A.Gray  jamaicensis  (L.)Vahl.  Pas si fI or a sp. Ost eomel es ant hylI idifol  ia  Lindl.  Euphorbia sp. yfi ks t r oemi a Sp. B.  sandvicensis  confusa  ssp.  Styphelia  lamehameha (Cham.) F.  Muell. Dodonae a Sp. gymnoxi phi urn A.Gray  Yi'ilkesia Walt heria  americana L.  Lant ana camara L. B.  sandvicensis  ssp. sandvicensis  Passiflora Acacia  sp.  koa A. Gray  St achyt arphela  jamaicensis  (L.)Vahl.  Si da f alI ax Walp. Ost eomeles ant hylIidifolia  Lindl.  126  T A B L E  X X X I .  C O M P O S I T I O N  OF  M A L T  E X T R A C T  ( M Y P T )  MEDIUM  Ingredients  grams/litre  malt e x t r a c t  7.0  yeast e x t r a c t  0.5  soytone  10.0  bacto agar  15.0  tetracycline  HC1  0.05  C U L T U R E  1 27  The Potter  leaf  impression  (1910)  who  method (B) was f i r s t  used  t h i s technique  presence of f u n g i and b a c t e r i a leaves.  Since  then,  by  t o demonstrate the  Solanum  on  described  Helianthus  and  t h i s method has been r e g u l a r l y used t o  p r o v i d e data on the r e a d i l y  detachable  component  of the  p h y l l o p l a n e saprophyte p o p u l a t i o n . I t provides an i n d i c a t i o n of the f r e q u e n c i e s of both populations.  Whole  resident  and  transient  fungal  leaves were washed i n s t e r i l e water and  d r i e d . Each l e a f was p l a c e d , a d a x i a l or a b a x i a l s u r f a c e on  the agar  subsequently The  surface,  lightly  on the medium and  removed.  l e a f d i s c method (C) was used t o i s o l a t e  organisms. I t was modified conifer  pressed  up,  needles  by  from the methods  Petrini  et  al.  endophytic  described f o r  (1982) and C a r r o l l and  Carroll  (1978). Leaves were washed i n s t e r i l e  dipped  briefly  water,  then  i n 50% EtOH and t r a n s f e r r e d t o a s o l u t i o n of  3.5% NaOCl:H 0 (1:9) f o r one minute. A f t e r a f i n a l 2  rinse in  s t e r i l e water, s e v e r a l d i s c s of 10 mm diameter were c u t from each l e a f and t r a n s f e r r e d t o the agar s u r f a c e , allowed  (12 hour  p l a t e s were incubated a t  l i g h t / d a r k c y c l e ) f o r up t o 48 hours.  and y e a s t - l i k e fungal c o l o n i e s were s e l e c t i v e l y to  new  and  t o incubate.  In a l l three methods, prepared 23°C  covered  agar  plates  until  pure  cultures  transferred  were  obtained.  C u l t u r e s a r e kept r e f r i g e r a t e d a t 4°C and t r a n s f e r r e d two months.  Yeast  every  1 28 The  fungal  Centraalbureau Oosterstraat  isolates  voor  were  identified  Schimmelcultures,  P.O.  by Box  the 273,  1, 3470 AG Baarn, Netherlands.  PHOTOTOXICITY ASSAYS The  p h o t o s e n s i t i v i t y of s e l e c t e d t e s t organisms t o the  crude l i g h t petroleum e x t r a c t s of Hawaiian Bidens  leaves and  that  to several  of  isolated  polyacetylenes (1965). Other  was  Phylloplane test  cerevisiae  is  assayed  phylloplane using  and Candida  included albicans  the gram  bacteria  and y e a s t s  of  Daniels  i n Table XXXII.  the yeasts  Saccharomyces  , the gram p o s i t i v e b a c t e r i a faecalis  negative  col i , , Pseudomonas fluorescens of  the method  al bus , Streptococcus  ,• and  fungi  fungi tested are l i s t e d  organisms  Staphylococcus subtil  Hawaiian  and  Escherischia  bacteria  and P. aeuroginosa.  were obtained  Bacillus  Cultures  from the UBC c u l t u r e  c o l l e c t i o n . Sabouraud's medium was used f o r the yeasts n u t r i e n t agar p l a t e s f o r the b a c t e r i a . A l l p h y l l o p l a n e  and fungi  were c u l t u r e d on MYPT agar p l a t e s . Forty-eight  hour l i q u i d c u l t u r e s i n MYPT were  on agar p l a t e s with s t e r i l e c o t t o n fractions  and  concentrations  swabs.  of 0.2 mg/mL, 1.0 mg/mL and/or 2 mg/mL.  Whatman  No.1  Five  1 jig/disc, 5 Mg/disc and  10 Mg/disc) were a p p l i e d t o paper d i s c s , from  petroleum  t e s t compounds were d i s s o l v e d i n 95% EtOH a t  m i c r o l i t r e s of each s o l u t i o n ( i . e . ,  prepared  Light  streaked  filter  7mm  i n diameter,  paper and the solvent  allowed t o dry i n the d a r k . 8-Methoxypsoralen was used as a  1 29  TABLE XXXII.  YEASTS  AND  YEAST-LIKE  FUNGI  ISOLATED  FROM  HAWAIIAN PLANTS  Class  Basidiomycetes  Sporobolomycetaceae  But(era  sp.  Rhodoiorula  gr ami ni s d i Menna  R. mucilaginosa R.  (Joerg.) H a r r i s o n  pal 11da Lodder  Sporobol omyces salmoni col or  (Fischer  et Brebeck) Kluyver e t van N i e l S.  shi bat anus  (Okunuki)  Verona  et  Ciferri S.  Cryptococcaceae  roseus  Kluyver e t van N i e l  Tilleliopsis  pallescens  Cryptococcus  albidus  C. laurentii  ( K u f f ) Skinner  C. luteolus C l a s s Fungi  Alternaria  Imperfecti  Wiltsch Aureobasidi  Gokhale ( S a i t o ) Skinner  ( S a i t o ) Skinner tenuissima  urn  put Iulans  (Kze:Fr.)  (de Bary)  Arnaud Cladosporium  cladosporiodes  (Fres.)  de V r i e s C.  cf.  pigment)  cladosporiodes  (yellow  1 30  Col I el otrichum  gloeosporiodes  (Pe'nzig) Penzig e t Sacc. Epi coccum pur purescens  Ehrenb.  Hyphozyma  de Hoog e t M.  Th.  variabilis  Smith  Phoma sorghina  (Sacc.) Boerema e t a l  131 et  r e f e r e n c e p h o t o a c t i v e compound (Fowlks discs  were  prepared UV-A  placed  the  inoculated  (320-400nm)  Four  irradiance  Sylvannia of  10  i n a Psycrotherm black  watts/m  2  lights,  plates  controls  were  kept  subsequently  Saccharomyces,  The  which were longwave  i n c u b a t o r f o r two F20T12-BLB,  with  a t 10 cm from source, measured  with a Y S I - K e t t e r i n g Model 65  were  1958).  i n d u p l i c a t e . Test p l a t e s were exposed t o  light  hours.  on  al.,  radiometer,  were  used.  The  i n the dark. A l l p h y l l o p l a n e organisms incubated  at  23°C  Candida and b a c t e r i a l  for  48  hours.  s p e c i e s were incubated  at 37°C. Compounds  which  produced  zones  of  inhibition  of  m i c r o b i a l growth only upon i r r a d i a t i o n are p h o t o t o x i c . Those samples which gave s i m i l a r s i z e s of zones of i n h i b i t i o n in l i g h t and dark are a n t i b i o t i c . A l l assays  were  both  repeated  three t o f i v e times and the r e s u l t s combined.  COMPARISON OF PHOTOTOXICITY OF  SELECTED  POLYACETYLENES  TO  CRYPTOCOCCUS LAURENTII A 48 hour s t a t i o n a r y c u l t u r e of Crypt ococcus grown  laurentii  i n Malt Yeast Peptone (MYP) broth at 23°C was d i l u t e d  to approximately  1.69 X 10* c e l l s / m L i n f r e s h  medium.  One  hundred m i c r o l i t r e a l i q u o t s of t h i s suspension were added t o w e l l s of s t e r i l e m i c r o t i t r e using  a  Titertek  plates  Multichannel  (Nunc-96FB  pipette.  with  lids)  A s e r i e s of nine  t w o - f o l d d i l u t i o n s of p h e n y l h e p t a t r i y n e (PHT, compound 4 ) , phenyl-diyne-ene  (compound  5),  heptadeca-tetraene-diyne  1 32  (compound growth  8)  and a - t e r t h i e n y l  medium  dilution  (MYP).  Six  (compound 21) were made with  replicates  of  100UL  were added t o the t e s t w e l l s c o n t a i n i n g s t a r t i n g c o n c e n t r a t i o n s were 5 nq/mh  The  of  each  yeasts.  or  uq/mL  10  with a maximum of 1% EtOH t o a v o i d s o l v e n t t o x i c i t y . One row in each p l a t e was set up as a c o n t r o l , only.  For  control UV  each  (320-400nm)  492  for  5,10  plate  served  medium  as the dark  and  20 minutes. A l l p l a t e s were  incubated at 23°C. The o p t i c a l d e n s i t y  nm,  of each s o l u t i o n  read b e f o r e i r r a d i a t i o n hours  one  growth  ( 0 ' ) , and t e s t p l a t e s were i r r a d i a t e d with long wave  subsequently at  compound,  with  ( T « ) , 40 hours  i n each of the t e s t w e l l s was  ( T ) , and  at  0  (T  2  ft0  (O.D.),  19  ) and 48 hours  hours (T  a 8  (T  1 9  ),  24  ) later.  TEST COMPOUNDS  Crude  light  petroleum  (BP  30-60°C)  compounds 1,4, 5,7,8 and 9 were prepared and  extracts isolated  and from  s e l e c t e d s p e c i e s of Hawaiian Bidens  a c c o r d i n g t o the methods  described  XXXIII).  in  a-terthienyl  Chapter was  a  Ottawa, compound Finlayson et  al.  from  , 1985).  Chemical.  22  II gift  (Table  A  lanatum  8-Methoxypsoralen  was  was  isolated  by  Alex  (Pursh) Forbes (Norton obtained  A l l compounds were d i s s o l v e d  1.0 or 2.0 mg/ml.  21,  from Thor Arnason, U n i v e r s i t y of  thiarubine  Eriophyllum  Compound  from  Sigma  i n 95% EtOH at 0.2,  1 33  TABLE XXXIII. POLYACETYLENES USED FOR PHOTOTOXICITY ASSAYS  CE= CH(C=C) CH=CH-CH A  =C)  3  -CH  3  3  C)_-CH=CH-CH.  C ) -CH=CH-CH 0C0CH  o -  2  o  3  CH -CH=CH ( C = C ) (CH=CH) (CHj) >CH=CH 3  2  2  4  CH -(C=C) (CH=CH) (CH ) -CH=CH 3  3  CH,-C=C-(/  2  2  4  \)-(C=C) -CH=CH 2  2  2  2  Q  9  22  134 C.  RESULTS  ISOLATION,  IDENTIFICATION  AND  DISTRIBUTION  OF  PH7LL0PLANE ORGANISMS Bidens  Leaves from 12 s p e c i e s of associated  i n Hawaii i n August, XXIX  yeast-like  species  February,  of  taxa  of  1984  y e a s t s and  fungi belonging t o the Sporobolomycetaceae, and  and  samples  Centraalbureau  voor  the  the Fungi Imperfecti were s e l e c t i v e l y of  isolates  identified  Schimmelcultures  1984; 1965; Barnett et  (Kreger-van R i j , methods  1983 and  and XXX). Nineteen  Cryptococcaceae isolated  23  t r o p i c a l p l a n t s were c o l l e c t e d from 13 d i f f e r e n t  localities (Tables  and  by  (Table al.,  the  XXXII)  1979).  Three  were used f o r the i s o l a t i o n of microorganisms, each  one with i t s p a r t i c u l a r advantages and l i m i t a t i o n s 1910; L a s t ,  (Potter,  1955; Lamb and Brown, 1970; C a r r o l l and C a r r o l l ,  1978). A comparison of the d i s t r i b u t i o n of  the seven  most  common genera i s o l a t e d by each method i s presented i n T a b l e s XXXIV t o XXXVI. On washings, overgrown  agar  plates  yeasts by  inoculated  such  other  e f f e c t e d by a l l o w i n g  as fungal  these  spores so that they f a l l The spore f a l l actively  with  Sporobol omyces species. organisms  and  or  an  leaf  are  usually  Separation  can be  to discharge  on the c u l t u r e medium ( L a s t ,  method a l s o p r o v i d e s  growing  dilutions  indication  their 1955).  of the  s p o r u l a t i n g fungi on the s u r f a c e s of  l e a v e s . T h i s i s based on the assumption that spores produced  TABLE XXXIV. DISTRIBUTION OF FUNGI ISOLATED BY THE SPORE FALL METHOD Plants  Sp  Rh  Cry  Clad  Ep1  Aureo  Coll  Site 1: Mallko Bay. Maul  B. hi I lebrandlana ssp. polycephala NlcotI ana glauca Eml11a ap. Trlfollum sp. Site 2: Kokee State Park. Kauai  B. cosmoides* Psychotrta sp. Pass/flora ap.  Acacia koa Site 3: Lumahai Beach, Kauai  0. forbesii ssp. forbesii Ageratum sp. Metros Ideros colllna Stachytarpheta Jamalcensls B. pi lose Site 4: Kohana Valley. Oahu  0. popullfoila Stachytarpheta jamalcensls Pass!fI or a sp. Osteomefes anthyllldlfolla Euphorbia sp. Wlkstroemla sp.  CO  cn  Site 5: Walmea Canyon, Kauai  confusa Styphel I a tamehameha Oodonaea sp. Wllkesla gymnoxlphlum Waltheria amerlcana Lant ana camara 0. sandvfcensfs ssp.  Site 6: Waahila Ridge, Oahu B. sandvfcensfs ssp. sandvfcensfs  Ifiora sp. Acad a koa sp. Stachytarpheta Jamalcensls SI da rail ax Osteomeies anthylIIdlfolI a' Pass  Site 7: Makaha Ridge. Kauai  0. cerv'cata** Styphella tamehameha Oodonea sp. Lantana camara Site 8: Cemetery. Walnee. Maul  0. maulensis Stachytarpheta Jamalcensis Llpochaeta ssp. 11Ima ssp. Scaveola taccada Waltheri a amerlcana  S i t e 9: Manlnl P a l l . Oahu 0.  amp)*cfens  Canthlum  11f ma  odoratum  sp.  Myoporum  sandttlcen»9  S i t e 10: Ahumoa. Hawaii 8.  menzleslI  ssp.  f i l i f o r m i s  * l e s s than 4 c o l o n i e s / p l a t e ++ 4-8 c o l o n i e s / p l a t e *•** g r e a t e r than 8 c o l o n i e s / p l a t e ++•+ c o v e r s e n t i r e p l a t e  * a l s o i s o l a t e d B u f f e r * sp., Wyphomycefes sp. ** a l s o I s o l a t e d r f f f e t f o p s f s  pallescens  138 by  actively  and  fall  gravity  growing  fungi are more l i k e l y  t o be l i b e r a t e d  t o the n u t r i t i v e agar s u r f a c e under the than  spores which are present  adhere t o the l e a f s u r f a c e  (Lamb and  a c t i o n of  by chance and  merely  Brown, 1970).  S e v e r a l species of Sporobol omyces and Crypt ococcus were isolated  with  Tilletiopsis the  this  pallescens of B.  leaves  cI adospori odes way,  probably  humidity  method  (Table  Golkhale  cosmoides  Bui I era  XXXIV).  were detected ,  Cladosporium  whereas  discharge  1971). In g e n e r a l , Cladosporium  changes ( D i c k i n s o n ,  study,  being present  isolated 1974; may  t o XXXVI).  a m y c e l i a l genus, i s reported t o be r e g u l a r l y  from leaves  Dickinson,  ( L a s t , 1955;  1976).  growth  1970;  Ruscoe, 1971;  I t s r a r e occurrence  be because of two reasons.  higher  found i n  on leaves of a l l p l a n t s from a l l  l o c a l i t i e s sampled by each method (Tables XXXIV Tilletiopsis,  this  i s a f f e c t e d by  appears t o be the most commonly o c c u r r i n g s p e c i e s this  once, from  (Fres.) d e V r i e s was f r e q u e n t l y i s o l a t e d  because i t s spore  and  I t appears  in this  Pady, study  to require  a  temperature than Sporobol omyces and i t grows  more slowly on l a b o r a t o r y c u l t u r e media so that  i t may be  e a s i l y overlooked  isolation plates  (Dickinson,  species i s detected  by the spore f a l l  method, i t  t o be an  saprophyte  1976;  Last and P r i c e , 1969). If  is  in spore f a l l  a  likely  active  and  therefore  s p o r u l a t i n g on the l e a f s u r f a c e . I t t h e r e f o r e should a l s o be i s o l a t e d by the l e a f  impression  demonstrated by the data  method. T h i s c o r r e l a t i o n i s  i n Table XXXV where the presence of  TABLE XXXV. DISTRIBUTION OF FUNGI ISOLATED WITH THE LEAF IMPRESSION METHOD Plants  Site  Sp  Rh  Cry  Clad  Epl  Aureo  Coll  1: Mallko Bay. Maul  8.  hiI Iebrandlana NlcotI ana glauca  ssp. pofycephafa  +++•••  EmfII a sp.  Trlfollum sp.  2++ + +  S i t e 2: Kokee S t a t e Park. Kauai S. cosmofdes  Psychot rla sp. Pass IfI or a sp. Acacia koa S i t e 3:  Lumahal Beach, Kaua!  B. forbesii ssp. forbesiI Ageratum sp. Metrosideros colllna Stachytarpheta Jamalcensls B. pilosa  +++ + 4  •f  +++  S i t e 4 : Kohana V a l l e y , Oahu  B. popu11folI a St achyt arphet a Jamalcensls Pass!fI or a sp. Osteomeles anthyI 11dlfolla Euphorbia sp. WIkstroemla sp.  4-4-  •++  GO  Site S: Walmea Canyon, Kauai  confusa Styphella tamehameha Oodonaea sp. Wllkeala gymnoxlphlum Walthena amerlcana Lantana camera B. sandvfcensfs ssp.  4.4.4. 4-4.4.  4-4.  4-4-4-  4-  Site 6 : Wnahlla Ridge. Oahu fl. sandvfcensfs ssp. sandvfcensfs  Passlflora sp. Acacia koa sp. Stachytarpheta jamalcensls Slda falI ax Osteomeiea anthylIIdlfol I a Site 7: Makaha Ridge, Kauai  8. cervlcata Styphella tamehameha Oodonaea sp. Lantana camara  4-4-4-+ 4.44. 4.44.4-  Site 8: Cemetery, Waihee, Maul 8. maufensfs  Stachytarpheta jamalcensls LIpochaeta ssp. 11Ima ssp. Scaveola taccada Waltheria amerlcana  24.4.4.  4-4-4. 4.+. + 44-4-4. 24.4.4.  O  Site 9: Manlnl P a l l . Oahu P. ampfeetens  +++  -  Canthlum odoratum  ++*  ++  11fma  2*++  -  +*  2+*+  -  +•  sp.  Myoporum sandwlcense Site 10: Ahumoa. Hawaii  0. menrlesll ssp. flllformls Site 11: Kallua. Kona. Hawaii  0. mlcrantha ssp. ctenophylla Site 12: Kalmu, Hawaii  0. hawalensls  * less than 4 colonies/plate ++ 4-8 colonies/plate ++«• greater than 8 colonies/plate •+•• covers entire plate  + 4.4-  •+ *•  142  Sporobol  omyces,  recorded.  Crypt ococcus  Rhodotorula  and  species  CI adosporium and  (de Bary) Arnaud are a l s o important which  adhere  to  1969;  Pugh  and  pur purescens exhibits  is  a  Buckley,  of  the  1971),  from  Marks et a l . , 1965). I t  (Table XXXVI) but was  before  presumably  Species  Crypt ococcus surface  of  and  cladospori  was  a l s o found using the  Penzig  One  et  Sacc.  isolated  mainly  leaf  impression  because the organism e x i s t s on the  CI adospori  leaves  um,  Marks  Epicoccum were  (Table  and,  also  leaf  et  al.,  rarely,  isolated  XXXVI).  The  from  first the  two leaf  odes forms m i c r o s c l e r o t i a which are able to  desiccation  and  environmental f a c t o r s (Pugh and Dickinson,  the  leaves using the l e a f d i s c method,  Sporobolomyces  sterilized  withstand  until  particularly  genera have been reported to grow a c t i v e l y w i t h i n and C.  which  l e a f t i s s u e (Blakeman  i t e n t e r s the t i s s u e (Blakeman, 1971;  1965).  invader  become  (Penzig)  s p e c i e s which invades  surface-sterilized  method,  Epicoccum  method.  a pathogenic  et a l . , 1971;  (Last and P r i c e ,  1976). A l l three were i s o l a t e d by  Col I et ol ri chum gl oeos por i odes is  colonizers  development  surface  is  pulI ul ans  whereas  restricted  leaf  (Dickinson,  impression  primary l e a f  b e l i e v e d to be a p h y l l o p l a n e  on  favourable  Aur eobasi di um  p h y l l o p l a n e surface  pattern  conditions  leaf  the  species  probably  other  Buckley, 1971;  adverse  Ruscoe,  1971;  1976). of  the  o b j e c t i v e s of t h i s study  whether the occurrence  was  to e s t a b l i s h  and d i s t r i b u t i o n of p h y l l o p l a n e  fungi  TABLE XXXVI. DISTRIBUTION OF FUNGI ISOLATED WITH THE LEAF DISC METHOD Sp  Plants  Rh  Cry  Clad  Ep1  Aureo  Coll  Site 1: Maliko Bay. Maul B.  hi IIebrandlana  Nlcotlana  ssp.  polycephala  glauca  Eml11a sp. TrlfolIum  sp.  Site 2: Kokee State Park. Kauai 0.  cosmoides  Psychotrla  sp.  P a s s l f I or a  sp.  Acacia  koa  Site 3: Lumahal Beach, Kauai 0. forbesM Ageratum  sp.  Metroslderos  colllna  Stachytarpheta 0.  +  ssp. f o r b e s i i  Jamalcensls  pilosa  Site 4: Kohana Valley, Oahu 0.  populIfolla  Stachytarpheta  Jamalcensls  PasslfIora  sp.  Osfeomeles  anthylI  Euphorbla Wlkstroemla  sp. sp.  IdlfolI  a  +++  Site S: Walmea Canyon, Kauai  sandvicensis ssp. confusa Styphella tamehameha Dodonaea sp. Wlikesla sp. Waltherla amerIcana Lantana camara B.  Site 6: Waahlla Ridge. Oahu  B. sandvicensis ssp. sandV'eensfs Pasal(lora sp. Acad* koa Stachytarpheta jamaicensis SI da fall ax Osteomeles anthylIIdlfolI a Site 7: Makaha Ridge, Kauai  B. cervI cat a Styphella tamehameha Dodonaea sp. Lantana camara Site 8: Cemetery. Walhee, Maul  B. mauI ens Is St achyt arphet a Jamaicensis Llpochaeta ssp. 11 I ma ssp. Scaveola  Waft h e r i a  sp. americana  S<t( 9: Martini P a l l . Oahu 8. ampfeetens  Canthlum odoratum tI fma sp. Myoporum sandwlcense Site 12: Kalmu, Hawaii  B. hawalensls  * less than 4 colonies/plate 4-8 colonies/plate greater than 8 colonies/plate ++** covers entire plate  +  +++  +  -  -  ++++  +  146 is  correlated  acetylenes indicates seem t o  with  the presence  i n Bidens. that  The  data  or  shown  Bidens  among  Bidens  Bidens  producing  species.  which  produce  species,  the C  (Tables I I I , I V ) . I t was detected Bidens  species  without  Aureobasi di um pullulans, isolated and  from  only  XXXVII  Nevertheless, (40%) l e a f  i s absent  from  compounds 5 and 7  i n f i v e of  seven  acetylenes.  In  (71.4%) addition,  a common p h y l l o p l a n e organism, one of the f i r s t  was  group of Bidens (20%)  from three out of four s p e c i e s of the second. All  f i v e of  except  B.  Bidens  three  the seven  especially  fungi  menziesii  taxa were hosts t o a t l e a s t  exposed  sites  on  would be expected species  than  cosmoides  Bidens  listed.  s s p . filiformis  ssp. ct enophylI a were a l l c o l l e c t e d  B.  and  aromatic  1 3  leaf  saprophytes do not  Col I etr otTi chum was found i n only two of the f i v e acetylene  of  i n Table  at l e a s t non filamentous  distinguish  absence  from  the i s l a n d of Hawaii to  have  a  lower  a typical rainforest (Dickinson,  hawaiensis  and  and B. mi crantha arid  to  semiarid  (Table XXIX) which  diversity  of  fungal  l o c a l i t y such as t h a t of  1967;  1976;  Dickinson  and  O'Donnell, 1977; Ruinen, 1961).  PHOTOSENSITIVITY OF MICROORGANISMS TO ACETYLENES The from  l i g h t petroleum f r a c t i o n s of l e a f and root e x t r a c t s  species  phototoxicity  of  Hawaiian  against  nine  Bidens species  were  tested  for  of fungi and b a c t e r i a  147 TABLE  XXXVII.  DISTRIBUTION  OF YEASTS AND YEAST-LIKE FUNGI  AMONG HAWAIIAN BIDENS  Bidens  with l e a f  Sp  Rh  Cry  C l a d E p i Aureo  + •  + + + +  + +  +  -  -  +  +  +  +  +  •+  -  +  +  -  B. ampl eel ens  +  +  +  +  B. menziesii ssp. filiformis B. popul i folia B. sandvicensis ssp. sandvi ce ns i s B. mi crantha ssp. cl enophylI a  -  -  +  Coll  acetylenes  B. hi 11 ebr andi ana + B. cosmoi des B. cervical a • B. sandvi ce ns i s ssp. + confusa B. hawaiensis + Bidens  without  +  -  +  + +  + -  leaf  acetylenes  B. forbesii ssp. forbesii B. mauiensis  + + -  +  +  + +  +  +  -  -  •  + +  + + +  +  148 using the method of D a n i e l s were  found  (1965). While the root  samples  t o be c o n s i s t e n t l y p h o t o t o x i c as expected,  only  l e a f e x t r a c t s c o n t a i n i n g a c e t y l e n e s were l e t h a l t o the organisms  i n t h e presence  of  long wave UV l i g h t  test  (Tables  XXXVIII, XXXIX). y e a s t s S.  The  cer evi s i ae and C. albicans  to most of the Bidens gram-positive  containing  bacteria.  The  were s e n s i t i v e  acetylenes,  as  gram-negative  were the  bacteria  were  g e n e r a l l y u n a f f e c t e d . T h i s i s i n agreement with the data et  Towers  al .,  ( 1977). The most p h o t o a c t i v e e x t r a c t s were  from Bidens  those  s p e c i e s which produce the C  compounds  8,  conjuncta,  B. macrocarpa,  9, and 10 (B. campylotheca,  hi I I ebr andi ana  B. menziesii  s s p . polycephala,  hydrocarbon and aromatic  s s p . pentamera,  SSp.  B. torta  leaves  containing  1,  4,  compound. I t (Towers,  is a  1980;  Ashwood-Smith et used  in this  and  B.  ( B.  7  sandvicensis  hawaiensis  with  organisms.  (8-MOP) was used as a r e f e r e n c e  well  Warren  known et  phototoxic  al.,  test  furanocoumarin  1980; Averbeck,  al . , 1980) and i s l e t h a l t o study except  1 3  ("safynol") a l s o e x h i b i t e d  p h o t o t o x i c i t y a g a i n s t s e v e r a l of the t e s t 8-Methoxypsoralen  B.  17C) and/or C  5  B. cosmoides,  18  hydrocarbon  s s p . menziesii,  17A and 17B). Bidens  compound  1 7  ssp. pent amera, B.  B. torta  compounds  campyl ot heca confusa,  of  1982;  the organisms  Pseudomonas sp. (Fowlks et  al . ,  1958. Relative  differences  i n the p h o t o t o x i c i t y  e x t r a c t s can be q u a n t i f i e d by measurement of  the  of  test  diameters  TABLE XXXVttl. PHOTOSENSITIVITY OF MICROORGANISMS TO EXTRACTS OF HAWAIIAN BIDtNS LEAVES*  Microorganisms  8 MOP  3a  5  6  7  10  11  12  12a  13  UV DK  UV DK  UV DK  UV OK  UV OK  UV DK  UV DK  UV OK  UV DK  UV DK  cerevlsiae *** - +• * Candida albicans *** - - * Staphylococcus aureus *** - --S. a I bus *•+ - **- • Streptococcus raecaiis *** - *•• - * Bacillus subtil Is *•* * Saecfi.  Escherichia col'  **+ -  Pseudomonas fluorescens P. aevroglnosa - -  - -  - - -  • 10 ug/dlsc of light petroleum extracts: * clear zone diameter 7-10mm ** clear zone diameter io-l4mm *+• clear zone diameter 14-I8mm *•** clear zone diameter >> 18mm  Bidens  - -  - - -  • -  * ++* •+*- *** - -- *•*-*-- *** - ** -- *** - - - **•* - - - - *** - - - **- -- **++ --- -  *-  - -  -- -- - - - -  key, In Table II  - -  - -  --- -. - -  -  - -  -- -  -  TABLE XXXIX. PHOTOSENSITIVITY OF MICROORGANISMS TO EXTRACTS OF HAWAIIAN BtOENS LEAVES  Microorganisms  8 MOP  9  16  16a  17A  17B  17C  18  8  15  UV OK  UV OK  UV OK  UV OK  UV OK  UV OK  UV OK  UV OK  UV OK  UV OK  Sacchfomycea t»r*visMf *** **-* * • ** *. • Candida albicans **+ - *• -* * - +* *- St aphylococeua aureus + + * - +*- -- » * - + + -S. gibus + + * - +++ - - * * - '* - • - *• Str»ot ococcus faecal is **+ - ++- -- .• -- - * Bacillus subtil is *** - -- *•» + - *• ** -€scn*rlehla coll •*• - *--- ---- *- -Pseudomonas fluorescens - - **- -- + -- --- - - r. aeuroginoaa - - -- -- -- ---- - -  * 10 u g / d l s c of l i g h t petroleum e x t r a c t s : Ridcns * c l e a r zone diameter of 7-iomm •* c l e a r zone diameter of 1 0 - I 4 m m ••• c l e a r zone diameter of i d - i 8 m m •*** c l e a r zone diameter >> 19 mm  key In Table II  151 of the c l e a r  zones  around  the  impregnated  filter  d i s c s . The minimum measurable zone of i n h i b i t i o n diameter of the d i s c . C l e a r values in  zone  diameters  paper  i s 7mm, the  were  assigned  on a s c a l e of + (7-1Omm) t o ++++ (greater than 18mm)  order  to  Irradiation  demonstrate  itself  does  relative  not a f f e c t  phototoxicities. the growth  of the  organisms. Thirteen isolated  from  species  of  yeasts  Hawaiian  Bidens  and  leaves  yeast-like were  tested  p h o t o s e n s i t i v i t y to p o l y a c e t y l e n e s from Bidens (compound 21) and t h i a r u b r i n e A  a-terthienyl  Tagetes  from  respectively  and  Eriophyllum  fungi for  as w e l l as t o (compound  species  22)  (Compositae)  (Tables XXXIII, XL and X L I ) . C o n c e n t r a t i o n s  of  1,5 and 10 nq/xtiL were used because l e a f a c e t y l e n e s i n Bidens do not exceed 10 jxg/mL. The C 1  1 3  'ene-tetrayne-ene'  Tilletiopsis  was i n e f f e c t i v e a g a i n s t a l l organisms except  pallescens compounds  and Col I el ot ri chum 8  gl oeospori odes .  The  C  1 7  and 9, which were s t r o n g l y p h o t o a c t i v e a g a i n s t  b a c t e r i a , Saccharomyces were  compound  slightly  and Candida  t o x i c t o Crypt ococcus  (Tables XXXVIII,  XXXIX)  s p e c i e s but not at a l l  d e l e t e r i o u s t o t h e i r pigmented r e l a t i v e s Rhodotorula  sp. or  to members of the Sporobolomycetaceae and Fungi Imperfeci. The C  1 3  aromatic compounds 4,5 and 7, the thiophenes 21  and 22, as w e l l as 8-methoxypsoralen were p h o t o t o x i c t o most of the p h y l l o p l a n e f u n g i . The thiophenes 21 and 22 were used in  this  study  photosensitizers  because  they  a r e known  (Towers, 1979; Downum et  to al.,  be  powerful  1982).  They  TABLE XL. PHOTOSENSITIVITY OF CR. LAURENTII FROM DIFFERENT HOST PLANTS TO POLYACETYLENES  Host Plants  8 MOP UV DK  0. cosmoides 0. hllIIbrandlana ssp. polycephala 0. maul ens Is  UV DK  5  7  8  9  4  UV DK  UV DK  UV DK  UV DK  UV DK  21  +++  22  UV DK  UV DK  4-4-  -  4-4- 4-  4.4-  -  +4-  + -  4*4.  -  4-4-  -  + -  4- + +  -  4-4-  •»  4-  0. sandvfcensfs ssp.  sandvIcensIs 0. sandvfcensfs ssp.  confusa  • 1-5 ug/dlsc: see Tables III and XXXIII + clear rone diameter of 7-10mm clear zone diameter of 10-14mm 4.4.4. clear zone diameter of 14-18mm +*++ clear zone diameter >>18 mm  cn  TABLE XLI. PHOTOSENSITIVITY OF PHYLLOPLANE FUNGI TO POLYACETYLENES*  M1croorgan1sms  8 MOP UV  DK  7  5  UV  DK  uv  UV  OK  SporoboIomyces roseus - S. shlbat anus *** 5. salmonlcol or +• Tilletiopsis pallescens *•*• Phodotorula pallida •+ ++++ ++ P. mucllaglnosa * Cryptococcus albldus + C. laurentii •+ C. luteolus • CIadosporI urn cladosporI odes ** -*+ +* • + Eplcoccum purpurescens Aureobasldlum putlulans * C. gloeosporlodes • 4-  +4-4-4-  • •  4-  8  DK  -  4-+  -  DK  UV DK  UV  21  DK  *++ + -  NT NT  • 4.4-  -  NT NT ++ 4.  4-  -  NT NT  -  4-4-  -  ++ +  -  4-  + 4-  -  -  -  DK  UV  DK  + 4-  -  4-4-  4  +++  -  ++  +  4-4-  -  +++ + + 4-  +• +•  • * + +  NT NT .  + 4-4-4-  +4-  ++• +  -  NT NT  -  4-+  +4-  4-  NT NT *•• •+++ 4-*4-  44-  UV  22  ++++  • *++ -  -  ++ + +  UV  4  9  *  +4-4- + +  NT NT  NT NT  * 1-5 ug/dlsc: see Tables III and XXXIII • clear zone diameter of 7-10mm +• clear zone diameter of 10-14 mm +++ clear zone diameter of 14-18mm •+•»•+ clear zone diameter >> 18 mm NT not tested  co  154  were e f f e c t i v e a g a i n s t a l l the organisms t e s t e d Rhodoiorula  muciIagi  (Joerg.)  nosa  except f o r  Harrison  which  was  r e s i s t a n t t o a - t e r t h i e n y l and not i n h i b i t e d by t h i a r u b r i n e A in  the dark.  Except f o r one p o p u l a t i o n  (17A) of B.  compound 4 (PHT) does not occur  i n Hawaiian  also  majority  deleterious  to  the  It i s  Bidens.  of  fungi  8-Methoxypsoralen, used as a r e f e r e n c e p h o t o a c t i v e was  effective  a l l but Spor obol omyces  against  et van N i e l . T h i s organism i s a l s o r e s i s t a n t test and  acetylenes  and r e l a t i v e l y  tested. compound,  roseus  to  Kluyver  five  other  insensitive to a-terthienyl  t h i a r u b r i n e A. The pathogenic  gloeospori  torta,  Col Iet ot  endophyte  i s very s e n s i t i v e t o compound 1 both i n the  odes  l i g h t and i n the dark. In a d d i t i o n , i t i s e a s i l y  killed  5. T h i s organism was absent from l e a v e s of  compound  c o n t a i n i n g aromatic present  richum  acetylene  i n Bidens  5  containing  (Table other  XXXVII) acetylenes.  by  Bidens  although It i s  r e s i s t a n t t o compounds 8 and 9. Aureobasidium Bidens  s p e c i e s producing  ssp. confusa  i s o l a t e d from only one of f i v e  pulIulans,  leaf  acetylenes,  (Table XXXVII), i s only s l i g h t l y  to compounds 5 and 7 which occur the  closely  r e l a t e d compound 4.  of a l l organisms t e s t e d pur purescens  B.  against  i n the host  isolated  organism  aromatic  acetylenes  and  s  photosensitive plant  thiarubrine  study,  A.  and t o  i s sensitive  the thiophenes  Epicoccum  compounds except  the most  cladosporiodes,  in this  censi  It i s also least sensitive  i s i n s e n s i t i v e t o a l l Bidens  compound 5. CI adosporium  sandvi  commonly t o the  but not t o the  155 s t r a i g h t c h a i n compounds 1, 8 and 9.  COMPARISON  OF  PHOTOTOXICITY  OF SELECTED POLYACETYLENES TO  CRYPTOCOCCUS LAURENTII Two-fold  serial  of 5 nq/mL and 10 nq/mh of  dilutions  compounds 4,5,8 and 21 were added t o approximately laurentii  cells/mL  kept  i n the dark and t e s t p l a t e s were  and  of  C.  10"  i n MYP. C o n t r o l p l a t e s were  0 0  irradiated  f o r 5,10  20 minutes. The o p t i c a l d e n s i t y (O.D.) a t 492 nm of each  sample was read before i r r a d i a t i o n T  1.69 X  h  and  percentage survival  T„ h  later,  B  and  ( T ) and at 0  T  2 0  h,  cells,  24  2  the d i f f e r e n c e s expressed  of c o n t r o l O.D. (Tables XLII and X L I I I ) . of  T „h,  hours  after  exposure  as a  Percent  to varying  c o n c e n t r a t i o n s of compounds was p l o t t e d a g a i n s t the time UV-A  irradiation  ( F i g u r e s 14 t o 17). The s y n e r g i s t i c a c t i o n  of a l l t e s t compounds and UV-A i r r a d i a t i o n on the of C. laurentii  i s demonstrated i n these  The Minimum  of  Inhibitory  viability  graphs.  Concentration  (MIC),  causing  complete growth i n h i b i t i o n , was 0.078 uq/mL f o r a - t e r t h i e n y l after  10 minutes exposure t o UV-A. No other compound was as  lethal.  Alpha-terthienyl  some 20-30% of c e l l s between  a l s o had a dark e f f e c t and k i l l e d  i n the c o n t r o l p l a t e s at c o n c e n t r a t i o n s  5 uq/mL and 0.039 uq/mL.  None of compounds 4,  8 ( F i g u r e s 15 t o 17) was completely even  at  10  nq/mL.  Higher  l e t h a l t o C.  5 and  laurentii  c o n c e n t r a t i o n s were not used f o r  two reasons: p o l y a c e t y l e n e s p r e c i p i t a t e i n aqueous s o l u t i o n s  1  TABLE  XLII.  CONCENTRATION  EFFECTS  OF  CHANGES  IN  POLYACETYLENE  AND LENGTH OF. UV EXPOSURE ON PERCENT SURVIVAL OF  Cr.  laurentii* Time o f I r r a d i a t i o n  Cone.(vq/mL)  Compound  T  0  T  5  (mins)  T,o  T o 2  4**  0.00  100%  100%  100%  100%  1.25  100%  79%  62%  41%  5.00  B3%  67%  59%  39%  10.00  47%  53%  33%  19%  0.00  100%  100%  100%  100%  1.25  98%  69%  57%  83%  5.00  77%  61%  72%  62%  10.00  46%  44%  52%  59%  0.00  100%  100%  100%  100%  5.00  100%  61%  96%  95%  10.00  75%  60%  68%  62%  100%  100%  100%  100%  0.0195  81%  68%  53%  0.039  83%  17%  14%  NT  0.078  75%  22%  3%  NT  5.00  73%  17%  5 %  NT  Compound 5  Compound 8  Compound 21 0.00  * A l l sampled  i n c u b a t e d f o r 24 h o u r s  ** See T a b l e IV-5 f o r compound names *** NT not t e s t e d  NT***  157 FIGURE 14. EFFECT OF a-TERTHIENYL (21) AND UV-A HOUR SURVIVAL OF CRYPTOCOCCUS  ON  THE  LAURENTII  0 ug/mL  00195 ug/mL  Q039 ug/mL  T  o.o  1  r  50ug/mL 0.078 ug/mL I  7.2 9.6 TIME OF 1RRRD1RT10N (MIN5)  2.4  4.B  12  24  158 EFFECT OF PHENYLHEPTATRIYNE (3) AND UV-A  ON  THE  24HOUR SURVIVAL OF CRYPTOCOCCUS LAURENTII  X> 0  ug/mL  ,o 125 ug/mL ° 5.0 ug/mL  •° 10.0 ug/mL  -  —  1  1  4 4  6.6  13.2  j  r  17  TIME OF 1RRRD1R710N (MIN5)  22.0  159  16.  E F F E C T OF PHENYLHEPTADIYNE-ENE  (5)  AND  U V - A ON  THE 24HOUR SURVIVAL OF CRYPTOCOCCUS LAURENTII  0 ug/mL  1.25 ug/mL  • 5.0ug/mL 10.0ug/mL 0  ^ 44  _  ^  i  1  B.B  13.2  — r 17.  7 ME OF 1RRRD1RT10N (M1NS)  22.0  160  17. ON  EFFECT  OF HEPTADECA-TETRAENE-TRIYNE  T H E 24HOUR  SURVIVAL  (8)  AND  UV-A  CRYPTOCOCCUS LAURENTII  OF  -° 0 ug/mL •° 5L0 ug/mL  10.0 ug/mL  •  T  4-4  8.B  1  13 7  1  \i c  TIME OF IRRADIATION (MINSJ  1 O-J  161  TABLE  XLIII.  SURVIVAL  CURVES  FOR C.  LAURENTII EXPOSED TO  POLYACETYLENES I N UV L I G H T *  Time of I n c u b a t i o n Compounds**  T .  T ,  T,  100%  100%  100%  Compound 3  44%  59%  79%  Compound 5  50%  72%  88%  Compound 8  84%  96%  96%  9%  5%  4%  Control  Compound 21  20  * 5 ug/mL e x p o s e d t o 10 m i n u t e s UV ** See t a b l e  2  light  I V - 5 f o r names o f compounds  0  162 at such  concentrations leaves  Bidens  and  values  do not exceed 10  had an a n t i b i o t i c e f f e c t a t 10 also  at  5  Mg/mL.  of  and compounds 4  ug/mL  of  i s not  unstable Figure  unexpected  in light 31  and  after  5  compounds  5  and  8.  as p o l y a c e t y l e n e s are known to be  aqueous  solutions  (Towers,  1979).  i s a d i r e c t comparison of the p h o t o t o x i c  effects  of the four t e s t compounds a g a i n s t and  and  g r e a t e r than 5 minutes  d u r a t i o n seemed to cause breakdown of This  in  Compounds 4, 5 and 8  ug/mL.  Irradiation  polyacetylenes  10  photosensitizer,  Cr.  minutes UV a - t e r t h i e n y l f o l l o w e d by PHT  and h e p t a d e c a - t e t r a e n e - t r i y n e  (8).  laurentii.  At 5 xig/mL  i s the most  powerful  ( 4 ) phenyl-diyne-ene  (5)  163 D. DISCUSSION  ISOLATION,  IDENTIFICATION  AND  DISTRIBUTION  OF  PHYLLOPLANE MICROORGANI SMS Yeasts  are defined  unicellular may  fungi  by  Kreger-van  Rij  reproducing by budding  (1984)  as  or f i s s i o n , which  or may not be a stage i n the l i f e c y c l e of m u l t i c e l l u l a r  fungi.  They  are  physiological including  characterized  criteria  and  ascomycetes,  by  are  morphological  taxonomically  basidiomycetes  this  study,  of  affinities.  19 taxa of yeasts and y e a s t - l i k e fungi 12 s p e c i e s of Bidens  and 23  Hawaiian p l a n t s and t h e i r d i s t r i b u t i o n  recorded  were s e l e c t i v e l y species  diverse,  and imperfect fungi  with both ascomycetous and basidiomycetous In  and  isolated  from  (Tables XXXII, XXXIV, XXXV, XXXVI). I s o l a t i o n of filamentous per  fungi  se  was  limited  p h o t o t o x i c i t y assays which b a c t e r i a and y e a s t s (Fowlks,  by  were  the nature originally  are  imperfect a  isolated  for  just  as  in  Indonesia  1965; D i c k i n s o n ,  together  with  healthy  Aureobasi di um  f u n g i such as CI adosporium leaf a  1976),  green  i n h a b i t e d by members of the Sporobolomycetaceae  and Cryptococceae  Using  developed  1963) and temperate regions (Voznyakovskaya, 1962;  L a s t and Deighton, leaves  imminent  1958; D a n i e l s , 1965).  The data shows t h a t , i n Hawaii, (Ruinen,  of  washing  range  of  Spor obol omyces roseus,  technique, epiphytic Rhodotorula  and  a few  and Epicoccum s p e c i e s . Voznyakovskaya  microorganisms rubra  (1962) including  (Demme) Lodder,  R.  1 64 muci I agi nosa,  R.  aurantiaca  (Saito)  Crypiococcus  Lodder,  laurentii  and C. albidus  Her  served t o s t r e s s the u b i q u i t y of most l e a f  survey  and  a l s o i n d i c a t e d the l a c k of host  Phylloplane leaves to  from a d i v e r s e s e l e c t i o n of hosts.  probably  the microenvironment which  may  s u r v i v e the high l i g h t and  s p e c i f i c i t y among them.  species a l s o tend t o be found more f r e q u e n t l y on  than i n s o i l ,  pigmented,  yeasts  of be  because they a r e w e l l adapted the l e a f .  an  Many  adaptation  species  are  e n a b l i n g them to  i n t e n s i t y a t the l e a f  surface  (Last  Deighton, 1965; Last and P r i c e , 1969; Pugh and Buckley,  1971;  Ruscoe, 1971; D i c k i n s o n ,  1976; McCauley  and  Waid,  1981).  are  U n l i k e those c o l o n i z i n g f r u i t s and f l o w e r s , l e a f  yeasts  unable  although  to  ferment  sugars  (Last  and  Price,  1969),  been  shown  t o possess  lipase  some species have  a c t i v i t y which would enable them t o become embedded i n t o the wax  layers  (Kuff.)  of  the c u t i c l e .  Skinner  and  When  Rhodotorula  Crypt ococcus  gl ut i ni s (Fres.) H a r r i s o n  were c u l t u r e d on the s t r i p p e d epidermis cuticle  fragments  of  Sansevi eria  s e r i o u s l y eroded w i t h i n f i v e  laurentii  days  of Aloe  sp. and the  sp., the c u t i c l e s were (Ruinen,  1966).  Yeasts  c o l o n i z i n g leaves above the cut i n - r e i n f o r c e d a n t i c l i n a l walls,  may,  detrimentally The leaf  as  a  result  affect  of  direct  penetration  s u r f a c e of the l e a f  enzymic  activity,  the i n t a c t c u t i c l e and i t s f u n c t i o n s .  phytopathogen, Col Iet otrichum by  their  cell  gl oeospori odes enters  without  the  hyphal growth on the  (Blakeman, 1971; Marks et  a/.,1965) and  165  is usually isolated et  al.,  from s u r f a c e - s t e r i l i z e d  1982; C a r r o l l and C a r r o l l ,  D e s p i t e the cosmopolitan yeasts,  the comparative  suggests  that the l e a f  which  partially  nature One  of  of  Lamb  i t s e l f may exert  phylloplane microflora  any  of  phylloplane  and Brown (1971)  selective  the nature  on  (Petrini  1978).  distribution  study  determines  leaves  given  of  host.  pressure  the r e s i d e n t The  specific  the pressure was not i n v e s t i g a t e d or d i s c u s s e d .  of the o b j e c t i v e s of t h i s study was t o determine whether  the  presence  or absence of p o l y a c e t y l e n e s i n the leaves of  Hawaiian Bidens Numerous  e x e r t s such an e f f e c t . reports  that  polyacetylenes are photoactive  a g a i n s t b a c t e r i a and f u n g i and other organisms (eg. Arnason et  al., 1980; Camm et  et  al., 1982; Towers et  raised  speculation  al., 1975; Chan et al.,  1977; Wat et  al., 1975; DiCosmo al.,  1977) have  about the p u t a t i v e b i o l o g i c a l  functions  of p o l y a c e t y l e n e s i n p l a n t s . C e r t a i n l y the r a i s o n d'etre secondary  compounds i n g e n e r a l may never be f u l l y  because a concerted, m u l t i d i s c i p l i n a r y , is  required  source and progress  f o r each c l a s s test  toward  organisms an  s t e p s . In t h i s study, does Bidens  the presence  of  clarified  long-term  approach  of chemicals, u s i n g a p p r o p r i a t e (Janzen,  1979).  Nevertheless,  answer may be made by small d e f i n i t i v e two  specific  questions  were  asked:  or absence of p o l y a c e t y l e n e s i n Hawaiian  l e a v e s have any c o r r e l a t i o n  selected  phylloplane  inhabitants  sensitive  to polyacetylenes?  with the d i s t r i b u t i o n and  of  a r e these organisms  166  In g e n e r a l , the nonfilamentous  saprophytes  study occurred on both Bidens  this Bidens  without  plants  at  any  I t appears  particular  that  site  p o l y a c e t y l e n e s w i t h i n the l e a v e s of Bidens of the p h y l l o p l a n e  fungi.  Col Ietol ri chum  Only  discrete Bidens  distribution. taxa  which  produce  or  and  of  yeast-like  demonstrates  consistently 1 3  XXXVI,  i s not c o r r e l a t e d  yeasts  the C  other  absence  gl oeospori odes  I t was  the  (Tables  the presence  with the nature  in  with l e a f a c e t y l e n e s and  a c e t y l e n e s , as w e l l as on most of  sampled  XXXVII).  isolated  absent  from  compounds 4 , 5  aromatic  and/or 7 (Table XXXVII).  PHOTOSENSITIVITY TO  OF  PHYLLOPLANE  MICRORGANISMS  POLYACETYLENES If p o l y a c e t y l e n e s i n l e a v e s a r e p h o t o a c t i v e a g a i n s t the  microorganisms which dwell w i t h i n might  expect  compounds  resident  found  phylloplane  in  organisms  or  organisms their from  on  to  host  the l e a v e s ,  be u n a f f e c t e d by the  plants.  p l a n t s without  would have no such r e s i s t a n c e . S i n c e most of were found  on a l l p l a n t s sampled  differential  Crypt ococcus 5), 9),  B.  laurentii  hi 11 ebrandiana  B. sandvicensis  leaf  acetylenes  the  was  photosensitivity from B, cosmoides s s p . polycephala ssp.  Furthermore,  organisms  (Table XXXIV t o XXXVI), one  r e p r e s e n t a t i v e species from s e v e r a l hosts possible  one  confusa  checked f o r  to  acetylenes.  (compounds 1,4 and (compounds 1,8, and (compound  5),  B.  167 sandvi censi s ssp. sandvi censi s (no l e a f a c e t y l e n e s ) mauiensis  (no  leaf  photosensitivity to XXXIII.  No  acetylenes)  the p o l y a c e t y l e n e s  differences  were  i m p l i e s that the presence bears  no  were  relationship  detected  or  absence  and B.  a l l tested for listed  in  (Table  XL), which  of  leaf  Tables  acetylenes  t o the responses of C. laurentii  to  polyacetylenes. Crypt ococcus isolated  notably C. laurentii,  species,  from northern  and southern  have been  temperate r e g i o n s ,  from  the t r o p i c s and even from the A n t a r c t i c (diMenna, 1960; and Deighton, found  1965), and are the only  in this  phylloplane (Ruinen,  study.  non-pigmented  fungi  The i n c i d e n c e of pigmentation  among  fungi and b a c t e r i a i s high  1961; 1963a;  Last  thought t o be an a d a p t a t i o n exposed  leaf  (70% among  and Deighton,  i n response t o UV  1965) and i s radiation  of Nothofagus  generally  showed  a  l e a v e s , found hypophyllous  that  l e a v e s , which are d i s p l a y e d i n a n e a r l y  position,  had  s i m i l a r populations  hyaline  vertical  on both s u r f a c e s  a/., 1972). Although the s i g n i f i c a n c e of r a d i a t i o n  determining affect  on  distribution.  Phragmites  et  bacteria)  s u r f a c e s . Ruscoe (1971), i n a d e t a i l e d d i r e c t  examination study species  Last  factor  the degree  may be debated, pigmentation of  photosensitivity  of  (Apinis as a  i t s e l f may  organisms  to  s p e c i f i c a c e t y l e n e s . Compounds 8 and 9 were found t o be very t o x i c t o b a c t e r i a and (Tables These C  XXXVIII 1 7  t o S.  and XXXIX),  compounds  cerevisiae  and C.  albicans  none of which are pigmented.  d i d not k i l l  any  of  the pigmented  1 68 phylloplane  fungi  Crypt ococcus  but  species  were  tested  phototoxic  compound roseus  8  resistant XLI).  to  In  a l l pigmented  most  acetylenes S.  addition,  8-methoxypsoralen  roseus  suggesting  that  response  determined  C.  using  phylloplane  curves  yeast  species,  seem  S.  t o be  i n Bidens  is  (Table  unaffected  by  furanocoumarin.  f o r compounds 4,5, 8 and 21 were as  of  a  representative  i t s s e n s i t i v i t y t o a wider  range of p o l y a c e t y l e n e s , which may or may not be because its  lack  of  pigmentation  to  i t may have a metabolic  laurentii  because  resistant  A. pullulans,  occurring  mechanism f o r d i s a b l i n g t h i s t o x i c Dose  was  and 9. Epi coccum pur purescens,  and R. pallida,  Rhodotorula  (Tables XL, X L I ) .  pigmented r e l a t i v e s of Crypt ococcus  spp.,  against a l l  of  ( F i g u r e s 14 t o . 1 7 ) . The data i n  these graphs a r e g e n e r a l l y i n agreement with those  obtained  using  that the  the d i s c  T i t e r t e k method revealed  test  (Tables  seems  t o be  previously  XL, more  undetected  X L I ) , except sensitive  because i t  a n t i b i o t i c e f f e c t s of these  compounds. The  two methods a r e not d i r e c t l y comparable because one  uses l i q u i d suspensions of  polyacetylenes  irradiation solvent-free  times  of c e l l s ,  of while  known  unstable aqueous s o l u t i o n s concentrations  the other  uses  and  solid  short  medium,  a c e t y l e n e s which d i f f u s e i n unknown q u a n t i t i e s  across agar and i r r a d i a t i o n p e r i o d s  of  up  to  two  hours.  These d i f f e r e n c e s may account f o r d i s c r e p a n c i e s between data s e t s . For example, i n Tables XL and XLI, compound 4 causes a  169 wider zone of i n h i b i t i o n than compound 21 data i n Tables XLII and that the opposite of  the  XLIII and  even  Figures  14 and  should be t r u e . T h i s may  different  p h o t o s e n s i t i z e r s . The  modes  of  though  be  action  15  a  reflection  of  these  i s not  Downum et a l . , 1982;  McRae et a l . , 1985), and  hour  irradiation  in  the  and  period  mechanism of a c t i o n of PHT  Sporobol  omyces,  study  were  With  the  two  d i s c t e s t may  favour  the  enhance i t s t o x i c  effect  on  and  i s true using  provide  an  ococcus  assays  i n d i c a t i o n of the  unequivocally  although  vitro  resistance  situation.  All  (Col I e t o t r i c h u m ) ,  the  physical XXXVII,  in  this  whether  in  performed  in v i t r o ,  extrapolated  does  not  vitro  fungi  exhibit  t e s t compounds. These  seen  to the  prove implies  tested,  the  are  this  selected  such  data  s i t u a t i o n in  toxicity resistance  with  differential  responses  in  s e n s i t i v i t y of  cannot  T o x i c i t y in  yeasts  the T i t e r t e k method.  organisms to p o l y a c e t y l e n e s  vivo.  l u t e a l us, a l l  than a - t e r t h i e n y l in the  phylloplane be  C.  It remains to be  Although the p h o t o t o x i c i t y study  of  Crypl  s e n s i t i v e to PHT  d i s c t e s t s (Table X L I ) . opposite  the  exception  Rhodotorula  more  two  (Arnason et al . ,  1980;  organisms.  indicate  photodynamic a c t i o n of a - t e r t h i e n y l i s  oxygen-dependent whereas that of PHT  the  the  one  in  vivo in  exception  s e n s i t i v i t y to  not  related  d i s t r i b u t i o n of the organisms among Bidens  any  to  the the  (Tables  XLI).  Unlike  B.  alba  and Coreopsis  1978), i n Hawaiian Bidens  taxa there  species  (Towers and  Wat,  i s no evidence f o r  the  170 presence leaf  of p o l y a c e t y l e n e s i n the l e a f  surface  structures  e x i s t but t h e i r contents  minute  for analysis.  such  as  or w i t h i n  trichomes. Resin  diameters Whether  cuticle  preclude  canals  sampling  a c e t y l e n e s occur  the  in resin  c a n a l s , w i t h i n c e l l s or e x t r a c e l l u l a r l y , any c o n t a c t between s u r f a c e microorganisms and l e a f a c e t y l e n e s must occur w i t h i n l e a f t i s s u e . Yeasts such spp.,  which  degrade  1963b; 1966), may  as Crypt ococcus  leaf  or may  and  Rhodotorula  c u t i c l e t o some extent not encounter  (Ruinen,  a c e t y l e n e s , but  of Col I et ol ri chum, as w e l l as Aureobasidium,  species penetrate  leaf  tissue  pathogens  (Blakeman,  and dwell 1971),  within  as  which  endophytic  would be exposed t o i n t r a and  extracellular constituents. Col I etotrichum  gloeospori  odes  numerous s p e c i e s of Hawaiian Bidens in  this  study.  I t s occurrence appears t o be s i t e  or B.  sandvicensis  i n B.  s s p . confusa  other p l a n t a t the f i r s t  five l o c a l i t i e s ,  sampled  site.  a t the t h i r d  species  were  also  1 3  acetylenes  cosmoides,  B.  and i n only one plant  hosts  two t o four of the t o the endophyte. i n Bidens  which  ( 1 , 4 , 5 and/or 7 ) . I t was found t o  be very s e n s i t i v e t o 1 and 5 i n the presence (Table  related  i n no other  N e v e r t h e l e s s , C. gl oeospori odes d i d not occur produce the C  from  In s i x other l o c a l i t e s where  Col I et ot r i chum was i s o l a t e d from Bidens, sympatric  isolated  and other p l a n t s sampled  (Table XXXV, XXXVI). I t was not found cervi cat a  was  of UV r a d i a t i o n  XLI). Its s e n s i t i v i t y to a-terthienyl  (Compound 21)  has been p r e v i o u s l y r e p o r t e d ( d i Cosmo et al ., 1982). I t was  171 not a f f e c t e d by compounds 8 and 9. Although  in  responses  vitro  r e f l e c t i o n of the s i t u a t i o n the (or  presence B.  cervi  of  cat a  precludes  odes.  contact  with  not  or  B.  The  ssp.  sandvicenses  of  i t s tissues  organism  be  a  true  t h i s data suggests that  vivo,  p o l y a c e t y l e n e s 1 and/or 5 i n  colonization  gloeospori  in  may  would  polyacetylenes,  B.  des  leaves  confusa)  by  Col Iet otri  presumably  which  cosmoi  may  chum  come be  into  located  e x t r a c e l l u l a r l y or w i t h i n c e l l s , as i t invades the l e a f subsequently  becomes i n h i b i t e d by the p h o t o a c t i v e compounds.  There i s a l s o the p o s s i b i l i t y phytoalexin(s)  may  be  that  some  produced  in  hitherto  response  i n v a s i o n . The s e l e c t i v e p h o t o s e n s i t i v i t y may inherent  morphological,  c h a r a c t e r s s p e c i f i c t o C. react  more  affecting  strongly  its ability  Certainly  this  Nevertheless,  it  be  unknown  to  fungal  caused  and/or  gloeosporiodes  which causes i t t o  biochemical  some a c e t y l e n e s than others and  t o grow w i t h i n some p l a n t s . information  is  l i m i t e d and cannot be  f o r polyacetylene function  indicates  that  further  in  vivo.  research  using  p h y l l o p l a n e microorganisms on s p e c i f i c host p l a n t s may i n f o r m a t i o n . Hawaiian  study must be checked campylotheca valida,  Future  ssp.  for  B.  torta  a l l c o n t a i n i n g compounds 1,5 investigations  should  determine  yield  not sampled i n t h i s  Bidens  especially,  Col Iet ot ri chum,  pentamera,  pathogens i n order t o  by  physiological  with  i n t e r p r e t e d as evidence  interesting  and  17B  and/or  focus whether  on  and  7  17D  and  (Table  B. B.  V).  leaf-invading  there  are  other  172  species  excluded  polyacetylenes  to  from  Bidens  which  the  leaves  with  specific  organisms are s e n s i t i v e ,  whether there are pathogens which dwell w i t h i n leaves are  resistant  suitably  refined  and  DiCosmo et  fungi ( D a n i e l s , 1965;  such organisms can be found, a t e n t a t i v e case  which  to the host a c e t y l e n e s . Current bioassays f o r  p h o t o t o x i c i t y must be filamentous  and  modified al.,  for  1982). If  argument  for  the  a g a i n s t the f o r t u i t y of p o l y a c e t y l e n e p h o t o t o x i c i t y to  microorganisms may  be made. As  central  of the p u t a t i v e r o l e of p o l y a c e t y l e n e s i n  nature, be  question  the complexity  the  answer(s)  to  the  of the p o t e n t i a l r e s e a r c h problems to  surmounted cannot be overemphasized. These problems need  to be  carefully  hypotheses studies producing  dissected  into  a  methodical  series  which can be t e s t e d by experimentation.  using plants  different must  phylloplane  organisms  be c a r r i e d out  w e l l as i n the f i e l d . The the  for  present  study  microflora/5/dens  for further i n v e s t i g a t i o n .  and  of  Parallel  polyacetylene  in the l a b o r a t o r y as has e s t a b l i s h e d that  system i s a u s e f u l model  173 E.  CONCLUSION  Yeasts  and y e a s t - l i k e  leaves of Hawaiian Bidens members  nonfilamentous generally  correlated  with  among  pathogenic  a l l of  them are  the d i s t r i b u t i o n of  Hawaiian  the presence  that  Bidens  i s not  or absence  Col Iet otri chum  species  endophyte which does not grow  found  of  acetylenes  in vitro.  T h i s suggests that f u r t h e r  gloeos por i odes,  i n t h i s study,  i n Bidens  aromatic  relationship  inhabitants  i n the l e a v e s .  i s significant  only  Although  polyacetylenes,  saprophytes  polyacetylenes  leaves  i s an  with  C  1 3  t o which i t i s extremely p h o t o s e n s i t i v e investigation  of the  between l e a f - i n v a d i n g pathogens, fungal and/or  b a c t e r i a l , and Hawaiian Bidens those  from the  and other p l a n t s and i d e n t i f i e d as  occurrence.  p h o t o s e n s i t i v e t o Bidens  the  isolated  I m p e r f e c t i . A l l a r e common p h y l l o p l a n e  worldwide  It  were  of the Sporobolomycetaceae, the Cryptococcaceae and  the Fungi of  fungi  curious  polyacetylenes.  about  w i l l be of great  the p u t a t i v e  interest  biological  role  to of  174 F. BIBLIOGRAPHY  Apinis, A.E., C.G.C.Chesters C o l o n i z a t i o n of Phragmites Nova Hedwigia 23: 113-124.  and H.K. T a l i g o o l a . communis l e a v e s by  1972. fungi.  Arnason, T., C K . Wat, K. Downum, E. Yamamoto, E. Graham and G.H.N.Towers. 1980. P h o t o s e n s i t i z a t i o n of Escherichia col i and Saccharmomyces cerevisiae by p h e n y l h e p t a t r i y n e from Bidens pilosa. Can. J . M i c r o b i o l . 26: 698-705. Ashwood-Smith, M.J., G.A. Poulton, M. Barker and M. Mildenberger. 1980. 5-Methoxypsoralen, an i n g r e d i e n t i n s e v e r a l suntan p r e p a r a t i o n s , has l e t h a l , mutagenic and c l a s t o g e n i c p r o p e r t i e s . Nature 285: 407 - 409. Averbeck, D. 1982. Photobiology of furocoumarins. pp. 295-307 i n Trends i n P h o t o b i o l o g y , C. Helene, M. Charlier, T. Montenay-Garestier and G. Laustriast, (Eds.). Plenum Press, New York. Bandoni, R.J. 1972. T e r r e s t r i a l occurrence of some hyphomycetes. Can. J . Bot. 50: 2283 - 2288.  aquatic  B a r n e t t , J.A., R.W. Payne and D. Yarrow. 1979. A Guide t o I d e n t i f y i n g and C l a s s i f y i n g Y e a s t s . Cambridge U n i v e r s i t y Press. Blakeman, J.P. 1971. The chemical environment of the l e a f surface i n r e l a t i o n t o growth of pathogenic fungi. pp256-268 i n Ecology of Leaf Surface Microorganisms, T.F.Preece and C H . D i c k i n s o n , ( E d s . ) . Academic P r e s s , London. Bohlmannn, F., T. Burkhardt and C. Zdero. 1973. N a t u r a l l y O c c u r r i n g A c e t y l e n e s . Academic P r e s s , London. Bohlmann, F., C. Arndt, H. Bornowski and K.M. K l e i n e . 1962. Uber d i e P o l y i n e der gattung Bidens L. Chem. Ber. 95: 1315-1319. Camm, E.L., G.H.N. Towers and J.C. M i t c h e l l . 1975. UV-mediated a n t i b i o t i c activity of some Compositae s p e c i e s . Phytochemistry 14: 2007 - 2011. Carroll, G.C and F.E. C a r r o l l . 1978. S t u d i e s on the i n c i d e n c e of c o n i f e r o u s needle endophytes i n the P a c i f i c Northwest. Can. J . Bot. 56: 3034 - 3043. Chan, G.F.D., G.H.N. Towers and J.C. M i t c h e l l . 1975. Ultraviolet-mediated a n t i b i o t i c activity of thiophene compounds of Tagetes. Phytochemistry 14: 2295 - 2296.  175 Daniels, F. 1965. A simple m i c r o b i o l o g i c a l method f o r demonstrating phototoxic compounds. J . I n v e s t . Dermat. 44: 259 - 263. Davenport, R.Ri 1980. An i n t r o d u c t i o n t o yeasts and yeast-like organisms, pp 1-27 i n B i o l o g y and A c t i v i t i e s of Yeasts. F.A. Skinner, S.M. Passmore and R.R. Davenport, (Eds.). Academic Press, London. Davenport, R.R. 1976. E c o l o g i c a l concepts i n s t u d i e s of microorganisms on a e r i a l p l a n t s u r f a c e s . ppl99-215 i n Microbiology o f A e r i a l Plant Surfaces. C.H. D i c k i n s o n and T.F. Preece, (Eds.). Academic Press, New York. Dickinson, C.H. and J . O'Donnell. phylloplane fungi on Phased us Mycol. Soc. 68: 193 - 199.  1977. Behavior of l e a v e s . Trans. B r i t .  D i c k i n s o n , C.H. 1976. Fungi on the a e r i a l s u r f a c e s of higher plants. pp293-324 i n M i c r o b i o l o g y of A e r i a l Plant Surfaces. C.H. Dickinson and T.F. Preece, (Eds.) Academic Press, New York. Dickinson, C.H. 1967. Fungal c o l o n i z a t i o n of Pi sum Can. J . Bot. 45: 915 - 927.  leaves.  DiCosmo, F., G.H.N.Towers and J . Lam. 1982. Photo-induced fungicidal activity elicited by n a t u r a l l y o c c u r r i n g thiophene d e r i v a t i v e s . P e s t i c . S c i . 13: 589 - 594. DiMenna, M.E. 1960. Yeasts from M i c r o b i o l . 23: 295 - 300.  Antarctica.  J.  Gen  DiMenna, M.E. 1971. The m i c r o f l o r a of leaves of pasture plants i n New Zealand. ppl59-174 i n Ecology o f Leaf Surface Microorganisms. T.F. Preece and C.H. D i c k i n s o n , ( E d s . ) . Academic Press, London. Downun, K.R., R.E.W.Hancock and G.H.N.Towers. 1982. Mode of a c t i o n of a - t e r t h i e n y l on Escherichia coli : evidence f o r a photodynamic effect on membranes. Photochem. P h o t o b i o l . 36: 517 - 523. Fowlks, W.L., D.G. G r i f f i t h and E.L. Oginsky. 1958. Photosensitization of b a c t e r i a by furocoumarins and r e l a t e d compounds. Nature 181: 571 - 572. Ganders, F.R. and K.M. Nagata. 1984. The r o l e of h y b r i d i z a t i o n i n the e v o l u t i o n of Bidens on the Hawaiian Islands. pp179-l94 i n P l a n t B i o s y s t e m a t i c s . W.F. Grant, (Ed.). Academic Press, Canada. Ganders, F.R. and K.M. Nagata. 1983a. R e l a t i o n s h i p s and f l o r a l b i o l o g y of Bidens cosmoides ( A s t e r a c e a e ) . Lyonia  176  2: 23 - 31 . Ganders, F.R. and K.M. Nagata. combinations i n Hawaiian Bidens 1 - 16.  1983b. New taxa and new ( A s t e r a c e a e ) . Lyonia 2:  Godfrey, B.E.S. 1976. Leachates from a e r i a l p a r t s of p l a n t s and their relation to plant surface microbial p o p u l a t i o n s . pp433-439 i n M i c r o b i o l o g y of A e r i a l Plant Surfaces. C.H. D i c k i n s o n and T.F. Preece. (Eds.). Academic Press, New York. Helenurm, K. and F.R. Ganders. 1985. Adaptive r a d i a t i o n and g e n e t i c d i f f e r e n t i a t i o n i n Hawaiian Bidens. Evolution (in press). Janzen, D.H. 1979. New h o r i z o n s i n the b i o l o g y of p l a n t d e f e n s e s . pp33l-348 i n Herbivores, Their Interaction with Secondary P l a n t M e t a b o l i t e s . G.A. Rosenthal and D.H.Janzen, (Eds.). Academic Press, New York. Kreger-van R i j , N.J.W. (Ed.). 1984. The Yeasts: Study. 3rd E d i t i o n . E l s e v i e r , H o l l a n d .  A  Taxonomic  Kreger-van R i j , N.J.W. 1965. Endomycetales, Basidomycetous y e a s t s and r e l a t e d f u n g i . Ch.2 i n The Fungi. Volume IVA. A Taxonomic Review with Keys: Ascomycetes and Fungi Imperfecti. G.C. Ainsworth, F.K.Sparrow and A.S. Sussman, ( E d s . ) . Academic Press, London. Lamb, R.J. and J.F. Brown. 1970. N o n - p a r a s i t i c m i c r o f l o r a on l e a f s u r f a c e s of Pas pal urn diI at at um, Salix babyl onica and Eucalyptus si el Iulat a. Trans. B r i t . Mycol. Soc. 55: 383 - 390. Last, F.T. 1955. Seasonal i n c i d e n c e of Sporobolomyces on c e r e a l l e a v e s . Trans. B r i t . Mycol. Soc. 38: 221 - 239. L a s t , F.T. and D. P r i c e . 1969. p l a n t s and t h e i r e n v i r o n s . Yeasts. V o l . 1. B i o l o g y H a r r i s o n , (Eds.). Academic  Yeasts a s s o c i a t e d with l i v i n g Ch. 5, pp 182-218 i n The of Yeasts. A.H. Rose and J.S. Press, New York.  Last, F.T. and F.C. Deighton. 1965. The n o n - p a r a s i t i c m i c r o f l o r a on the s u r f a c e s of l i v i n g leaves. Trans. B r i t . Mycol. Soc. 48: 83 - 89. Marchant, Y.Y., F.R. Ganders, C. K. Wat and G.H.N. Towers. 1984. P o l y a c e t y l e n e s i n Hawaiian Bidens. Biochem. S y s t . E c o l . 12: 167 - 178. Marks, G . C , J.G. Barbee and A . J . R i k e r . 1965. D i r e c t penetration of leaves of Populus tremuloides by Col Ietotrichum gloeosporoi des Phytopathology 55: 408 -  177  412. McCauley, B.J. and J.S. Waid. 1981. Fungal p r o d u c t i o n on leaf s u r f a c e s . Ch. 26., pp. 501-532 i n The Fungal Community: I t s O r g a n i z a t i o n and Role i n t h e Ecosystem. D.T. Wicklow and G . C . C a r r o l l , (Eds.). Marcel Dekker Inc., New York. McRae, D., E. Yamamoto and G.H.N. Towers. 1985. The mode of a c t i o n of p o l y a c e t y l e n e s and thiophene photosensitizers on liposome p e r m e a b i l i t y t o g l u c o s e . In p r e s s . Norton, R.A., A . J . F i n l a y s o n and G.H.N. Towers. 1985. Identification of two dithiacyclohexadiene p o l y a c e t y l e n e s from Chaenactis douglasii and Eriophyllum lanatum. Phytochemistry 24: 356 - 357. Norton, R.N. 1984. Studies of P o l y a c e t y l e n e production in normal and transformed t i s s u e c u l t u r e s of Bidens alba. Ph.D. D i s s e r t a t i o n , U n i v e r s i t y of B.C. Pady, S.M. 1974. Sporobolomycetaceae i n Kansas. 66: 333 - 338.  Mycologia  Petrini,0., J . Stone and F. E. C a r r o l l . 1982. Endophytic fungi i n evergreen shrubs in western Oregon: A p r e l i m i n a r y study. Can. J . Bot. 60: 789 - 796. • Phaff, H.J. and W.T. Starmer. 1980. S p e c i f i c i t y of n a t u r a l habitats f o r yeasts and y e a s t - l i k e organisms, pp.79-102 in Biology and A c t i v i t i e s of Yeasts. F.A.Skinner, S.M.Passmore and R.R.Davenport, (Eds.). Academic Press, London. P o t t e r , M.C. pathology.  1910. B a c t e r i a in their r e l a t i o n to plant Trans. B r i t . M y c o l . Soc. 3 : 150 - 168.  Pugh, G.J.F. and N.G. Buckley. 1971. The l e a f s u r f a c e as a s u b s t r a t e f o r c o l o n i z a t i o n by f u n g i , pp. 431-435 i n Ecology of Leaf Surface Microorganisms. T.F. Preece and C H . D i c k i n s o n , ( E d s . ) . Academic Press, N.Y. Ruinen, J. 1956. Occurrence of Beijeri nckia i n the " p h y l l o s p h e r e " . Nature (London) 177: 220 - 221. Ruinen, J . 1961. The p h y l l o s p h e r e I . An n e g l e c t e d m i l i e u . P i . S o i l . 15: 81 - 109.  ecologically  Ruinen, J . 1963. The p h y l l o s p h e r e I I . Yeasts from the p h y l l o s p h e r e of t r o p i c a l f o l i a g e . J . M i c r o b i o l . S e r o l . 29: 425 - 438. Ruinen, J . 1966. The p h y l l o s p h e r e IV. C u t i c l e by microorganisms i n the phyllosphere.  decomposition Anals. Inst.  178 Pasteur 111: 342 - 346. Ruscoe, D. W. 1971. Mycoflora of l i v i n g and dead leaves of Nothofagus truncata. Trans. B r i t . Mycol. Soc. 56: 463 474. Towers, G.H.N. 1980. P h o t o s e n s i t i z e r s i n p l a n t s and t h e i r photodynamic a c t i o n (a review), pp. 183-202 i n Progress in Phytochemistry, V o l . 6. L. Reinhold, J.B. Harborne and T. Swain, (Eds.). Pergamon P r e s s . Towers, G.H.N, and C K . Wat. 1978. B i o l o g i c a l a c t i v i t y of p o l y a c e t y l e n e s . Rev. Latinoamer. Quim. 9: 162 - 170. Towers, G.H.N., C K . Wat, E.A. Graham, R.J. Bandoni, G.F.Q. Chan, J.C. Mitchell and J. Lam. 1977. Ultraviolet-mediated antibiotic a c t i v i t y of species of compositae caused by p o l y a c e t y l e n i c compounds. L l o y d i a 40: 487 - 498. Voznyakovskaya, Yu. M. 1962. E p i p h y t i c M i k r o b i o l o g i y a 31: 616 - 622.  yeast  organisms.  Warren, R.A.J., J.B. Hudson, K. Downum, E.A. Graham, Norton and G.H.N. Towers. 1980. Bacteriophages indicators of the mechanism of action photosensitizing agents. Photobiochem. Photobiophys. 385 - 389. Wat,  R. as of 1:  C K . , R.K. Biswas, E.A. Graham, L. Bohm and G.H.N. Towers. 1977. U l t r a v i o l e t - m e d i a t e d c y t o t o x i c a c t i v i t y of p h e n y l h e p t a t r i y n e from Bidens pilosa L. J . Nat. Products 42: 103 - 1 1 1 .  V.  GENERAL CONCLUSION  In t h i s d i s s e r t a t i o n chemistry  of  s e v e r a l aspects  polyacetylenes  Hawaiian s p e c i e s of Bidens distribution  of  of the b i o l o g y and  synthesized  by  the n a t i v e  were examined. The occurrence and  acetylenes  i n these p l a n t s was c o n s i s t e n t  with the s p e c i e s concepts of Ganders and Nagata their  revision  of  the group, and with other evidence  the Hawaiian s p e c i e s a r e d e r i v e d lineage  (1983) i n  from  a  single  that  ancestral  (Ganders and Nagata, 1984).  Some s p e c i e s have l o s t acetylenes established although  de  although  i n others. This  acetylene  the a b i l i t y  to  produce  novo  synthesis  trait  i s apparently  inheritance  seems  was  complex  leaf  clearly dominant  and may be  a f f e c t e d by the p o l y p l o i d c o n d i t i o n of the p l a n t s . An endophytic  fungus, C. gloeospori  to be h i g h l y p h o t o s e n s i t i v e t o C i n the leaves of Bidens  occur  organism. acetylenes certain  This  seems  to  1 3  odes , was d i s c o v e r e d  aromatic  acetylenes  which  s p e c i e s not i n h a b i t e d by the  suggest  that  the presence  of  i n leaves may be a d e t e r r e n t t o c o l o n i z a t i o n by  f u n g a l pathogens. T h i s however, does not e x p l a i n why  some s p e c i e s no longer s y n t h e s i z e such l e a f compounds. F i n a l l y , an unusual from  aromatic  the r o o t s of Hawaiian Bidens  thiophene  was  isolated  s p e c i e s . I t has a unique  combination of a phenyl r i n g and a thiophene r i n g b r i d g e d by a  carbon-carbon  triple  bond.  This  compound  i n t e r e s t i n g b i o l o g i c a l p r o p e r t i e s and m e r i t s  179  future  may  have  study.  180  A.  BIBLIOGRAPHY  Ganders, F . R . and K . M . N a g a t a . 1984. The role of h y b r i d i z a t i o n i n t h e e v o l u t i o n of Bidens on t h e H a w a i i a n I s l a n d s , pp 179-194 i n P l a n t B i o s y s t e m a t i c s . W . F . G r a n t ( E d . ) . Academic P r e s s , Canada. G a n d e r s , F . R . and K . M . N a g a t a . 1983. New t a x a a n d new combinations i n H a w a i i a n Bidens ( A s t e r a c e a e ) . L y o n i a 2: 1 - 16.  

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