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Polyacetylenes from Bidens Marchant, Yu Yoke 1985

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POLYACETYLENES FROM BIDENS by YU YOKE B. Sc., University of M. Sc., University of MARCHANT B r i t i s h Columbia, 1972 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 thesis as conforming .ii^the regu-i*r?ed standard TH&"UNIVERSITY OF BRITISH COLUMBIA August, 1985 © Yu Yoke Marchant, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 E - 6 (3/81) ABSTRACT The Hawaiian species of Bidens are morphologically and e c o l o g i c a l l y diverse taxa which have evolved from a single ancestral species. Adaptive radiation has occurred without the evolution of physiological or genetic i n t e r s p e c i f i c i s o l a t i n g mechanisms since a l l species are i n t e r f e r t i l e and genetic distances among populations, based on isozyme l o c i , show l i t t l e c o r r e l a t i o n with morphological differences or taxonomic c l a s s i f i c a t i o n . This d i s p a r i t y between the evolution of morphological and biochemical characters makes i t of interest to determine whether or not there has been divergence in secondary metabolites in these species. Leaves and roots of 19 species and six subspecies of Hawaiian Bidens were examined for polyacetylenes. Eleven C 1 3 hydrocarbons, aromatic and thiophenyl derivatives, one C,k tetrahydropyran and three C 1 7 hydrocarbons were isolated and i d e n t i f i e d . A l l can be derived from o l e i c a c i d . Polyacetylenes were not detected in the leaves of 13 taxa although they are found in the roots of a l l species. The occurrence of 2-(2-phenylethyne-1-yl)-5 acetoxymethyl thiophene in Bidens has not been previously reported. Most taxa could be distinguished by t h e i r complement of leaf and root acetylenes and no v a r i a t i o n was found within taxa except in B. tort a. There appears to be no taxonomically s i g n i f i c a n t pattern to the d i s t r i b u t i o n of polyacetylenes above the species l e v e l in t h i s group. i i The complexity of polyacetylene inheritance was assessed using experimentally produced i n t e r s p e c i f i c hybrids. Crosses between species which do not produce leaf acetylenes resulted in F, individuals without acetylenes. Crosses between species which produce leaf acetylenes and those which do not yielded hybrids with acetylenes not always i d e n t i c a l to parental arrays. Progeny from parents with d i f f e r e n t sets of acetylenes expressed a combination of the major compounds found in both parents. In a l l cases, nonparental acetylenes in the F, generation were bio s y n t h e t i c a l l y closely related to compounds found in the parents. Polyacetylene synthesis was not segregated in the F 2 individuals from Type B crosses. De novo biosynthesis of polyacetylenes in Bidens leaves was investigated in pulse-chase studies. 1 0 C - l a b e l l e d acetylenes were recovered from three species of Bidens administered 1"C0 2 and subsequently allowed to metabolize in 1 2 C 0 2 for 12, 24 and 168 hours. Radioactive C 1 3 ene-tetrayne-ene was also i s o l a t e d from the roots of a l l plants, indicating that .translocation of 1 " C - l a b e l l e d precursors from a e r i a l tissues occurred. Phenylheptatriyne (PHT) was detected in two day old seedlings of B. alba, suggesting that polyacetylene biosynthesis begins during germination or soon thereafter. Quantities in the leaves continue to increase up to and beyond 24 days while amounts in the hypocotyls peak at seven days. Relative PHT values in the roots are 100 times higher than those in the a e r i a l tissues for the f i r s t 24 days, but there i s also a gradual decline in these l e v e l s beginning at two weeks and continuing beyond the experimental period. Phenylheptatriyne i s absent from the roots of mature B. alba. Many polyacetylenes are toxic to b i o l o g i c a l systems in the presence of UV-A radiation. These in vitro effects have led to speculation about the putative functions of polyacetylenes in' the organisms which produce them. Nineteen species of phylloplane yeasts and yea s t - l i k e fungi were isolated from species of Hawaiian Bidens with and without leaf acetylenes. Although a l l these organisms, members of the Sporobolomycetaceae, Cryptococcaceae and Fungi Imperfecti, were photosensitive to some polyacetylenes and resistant to others, there was no co r r e l a t i o n between the presence or absence of leaf polyacetylenes and the di s t r i b u t i o n of these saprophytes among species of Bidens. Nevertheless, i t i s s i g n i f i c a n t that the only pathogenic species isolated in this study, Col Ietol ri chum gloeospori odes , did not colonize Bidens leaves containing C 1 3 aromatic acetylenes to which i t i s extremely photosensitive in vitro. iv Table of Contents Chapter Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES xi ACKNOWLEDGEMENTS x i i I. GENERAL INTRODUCTION 1 A. POLYACETYLENES 1 B. BIBLIOGRAPHY 11 II . POLYACETYLENES IN HAWAIIAN BIDENS 17 A. INTRODUCTION 17 B. MATERIALS AND METHODS 22 PLANT MATERIAL 22 ISOLATION AND IDENTIFICATION OF POLYACETYLENES 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 C 0 2 74 A. INTRODUCTION 74 B. MATERIALS AND METHODS 77 BIOSYNTHESIS OF POLYACETYLENES FROM 1 *C02 IN BIDENS 77 v PLANT MATERIAL 77 ADMINISTRATION OF 1 aC0 2 77 MEASUREMENT OF 1*C UPTAKE INTO POLYACETYLENES 80 KINETIC STUDIES 83 STATISTICAL ANALYSIS '..83 ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS ..84 C. RESULTS 86 BIOSYNTHESIS OF POLYACETYLENES FROM 1"C0 2 IN BIDENS LEAVES 86 ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS .101 D. DISCUSSION 106 BIOSYNTHESIS OF POLYACETYLENES FROM 1 t tC0 2 IN BIDENS LEAVES 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 128 COMPARISON OF PHOTOTOXICITY OF SELECTED POLYACETYLENES TO CRYPTOCOCCUS LAURENT11 ... 131 TEST COMPOUNDS 132 C. RESULTS 134 v i ISOLATION, IDENTIFICATION AND DISTRIBUTION OF PHYLLOPLANE ORGANISMS 134 PHOTOSENSITIVITY OF MICROORGANISMS TO ACETYLENES 146 COMPARISON OF PHOTOTOXICITY OF SELECTED POLYACETYLENES TO CRYPTOCOCCUS LAURENT11 ...155 D. DISCUSSION 163 ISOLATION, IDENTIFICATION AND DISTRIBUTION OF PHYLLOPLANE MICROORGANISMS 163 PHOTOSENSITIVITY OF PHYLLOPLANE MICRORGANISMS TO POLYACETYLENES 166 E. CONCLUSION 173 F. BIBLIOGRAPHY 174 GENERAL CONCLUSION 179 A. BIBLIOGRAPHY 180 LIST OF TABLES I. Naturally occurring acetylenes 3 II. Bidens taxa examined for polyacetylenes 23 II I . Polyacetylenes from Hawaiian Bidens 37 IV. Polyacetylenes from Hawaiian Bidens 38 V. Polyacetylenes in the leaves of Hawaiian Bi dens 39 VI. Polyacetylenes in the roots of Hawaiian Bi dens 40 VII. Bidens hybrids examined for polyacetylenes... 46 VIII. • Polyacetylenes from Bidens hybrids 47 IX. Polyacetylenes in B. hawaiensis hybrids 48 X. Polyacetylenes in B. cosmpides hybrids 49 XI. Polyacetylenes in b. macrocarpa hybrids 50 XII. Polyacetylenes in F 2 plants 52 XIII. Percent quenching of r a d i o a c t i v i t y by polyacetylenes 1,4 and 5 82 XIV. 1 < tC0 2 uptake during 60 minutes pu l s e - l a b e l l i n g of B. alba 87 XV. E f f i c i e n c y of 1 f tC uptake 88 XVI. Twelve hour uptake of 1"C into PHT by B. al ba leaves 90 XVII. Twelve hour uptake of '*C into ene-tetrayne-ene (1) of B. hi 11 ebr andi ana leaves 91 XVIII. Twelve hour uptake of 1*C into acetylenes 1 and 5 of B. cosmoi des leaves 92 XIX. Twelve hour uptake of 1 ,C into MeOH and PE fractions by B. molokaiensis leaves 93 XX. One week uptake of 1*C into PHT by B. alba leaves 94 XXI. One week uptake of 1*C into ene-tetrayne-ene (1) of B. hi 11 ebr andi ana leaves 95 XXII. One week uptake of 1 ,C into acetylenes 1 and 5 of B. cosmoi des leaves 96 v i i i XXIII. One week uptake of 1 0C into MeOH and PE fractions of B. molokaiensis 97 XXIV. Twenty-four hour uptake of 1*C into PHT by B. al ba leaves 98 XXV. 1ftC uptake into MeOH and PE fractio n s of B. alba leaves in 24 hours 99 XXVI. 1flC uptake into PHT {4):B.alba leaves in 24 hours 100 XXVII. " ^ - l a b e l l e d ene-tetrayne-ene (1) in roots of Bidens given 1*C0 2 102 XXVIII. Accumulation and d i s t r i b u t i o n of phenylheptatriyne (4) in B. alba seedlings..103 XXIX. Bidens taxa sampled for phylloplane fungi...121 XXX. Plants associated with Bidens sampled for phylloplane fungi 124 XXXI. Composition of malt extract (MYPT) culture medium 126 XXXII. Yeasts and yeast-like fungi i s o l a t e d from Hawaiian plants 129 XXXIII. Polyacetylenes used for phototoxicity assays 133 XXXIV. Di s t r i b u t i o n of fungi isolated by the spore f a l l method 135 XXXV. Di s t r i b u t i o n of fungi isolated with the leaf impression method 139 XXXVI. D i s t r i b u t i o n of fungi isolated with the leaf disc method 143 XXXVII. D i s t r i b u t i o n of yeasts and yeas t - l i k e fungi among Hawaiian Bidens 147 XXXVIII. Photosensitivity of microorganisms to extracts of Hawaiian Bidens leaves .149 XXXIX. Photosensitivity of microorganisms to extracts of Hawaiian Bidens leaves 150 XL. Photosensitivity of C. laurentii from di f f e r e n t host plants to polyacetylenes 152 ix XLI. Photosensitivity of phylloplane fungi to polyacetylenes 153 XLII. E f f e c t s of changes in polyacetylene concentration and length of UV exposure on percent survival of C. laurentii 156 XLIII. Survival curves for C. laurentii exposed to polyacetylenes in UV l i g h t 161 x LIST OF FIGURES 1. The Hawaiian Islands 18 2. L o c a l i t i e s of Kauai Bidens populations sampled 24 3. L o c a l i t i e s of Oahu Bidens populations sampled 26 4. L o c a l i t i e s of Maui Bidens populations sampled 28 5. L o c a l i t i e s of Molokai Bidens populations sampled 30 6. L o c a l i t i e s of Hawaii Bidens populations sampled 32 7. Biogenetic relationships of polyacetylenes from Hawaiian Bidens 42 8. Phenylthiophenes from Cor eops i s 44 9. Dendogram of taxa based on s i m i l a r i t y of polyacetylenes 55 10. 1 f tC0 2 - feeding apparatus 78 11. 1*C - E f f i c i e n c y curve 81 12. Accumulation and d i s t r i b u t i o n of PHT in Bidens alba seedlings 104 13. Accumulation and d i s t r i b u t i o n of PHT in Bidens alba seedlings 105 14. E f f e c t of a-terthienyl (21) and UV-A on the 24 hour survival of Crypt ococcus laurentii 157 15. E f f e c t of phenylheptatriyne (4) and UV-A on the 24 hour survival of C. laurentii 158 16. E f f e c t of phenylheptadiyne-ene ( 5 ) and UV-A on the 24 hour survival of C. laurentii 159 17. E f f e c t of heptadeca-tetraene-triyne (8) and UV-A on the 24 hour su r v i v a l of C. laurentii 160 x i ACKNOWLEDGEMENT S I wish to express my appreciation to the people who have contributed their time, e f f o r t and expertise on my behalf throughout the past fiv e years. I thank the members of my research committee, Drs. Fred Ganders, Jack Maze and Anthony Glass, and esp e c i a l l y , Neil Towers, my inimitable supervisor, for the i r individual and c o l l e c t i v e advice and c r i t i c a l support. Dr. L i l y Wat and Zyta Abramovski were always encouraging as well as generous with technical assistance and suggestions. I am also grateful to Dr. R.J. Bandoni for help in matters mycological, to Dr. Jjrfrgen Lam for advice on polyacetylene chemistry, to Dr. Kermit Ritland for his Fortran programmes, to Vince Grant and Ken J e f f r i e s for construction of the 1 t tC0 2 feeding apparatus, to Bob Kantymir for care of greenhouse plants, to James Bjerring for his assistance with s t a t i s t i c a l analysis and to Len Marchant, who helped prepare t h i s manuscript. Finally., the f i n a n c i a l support provided by Mr. Marchant and by the University of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. xi i I . GENERAL INTRODUCTION A. POLYACETYLENES The majority of natural acetylenes known today are polyacetylenes. The name encompasses what now appears to be a biogenetically uniform group of secondary metabolites, usually not s t r i c t l y poly-ynes (Jones and Thaller, 1978), which originate from ole ic - acid (Bu'Lock, 1966) and are found in the roots and a e r i a l parts of plants, and in fungi (Bohlmann et al., 1973), algae (de Napoli et al., 1981), sponges (Cimino et al., 1981), nudibranchs (Walker and Faulkner, 1981), sea hares (Schulte et al., 1981) and insects (Moore and Brown, 1978). H i s t o r i c a l l y , the occurrence of a t r i p l e bond in a natural product was f i r s t c l e a r l y established by Arnaud (1902) in his study of"the monoacetylenic acid, t a r i r i c acid (Table I ) , a component of the seed fat of Picramnia tariri DC. (Simaroubaceae). The f i r s t aromatic compound, c a r l i n a oxide (Table I ) , was isolated and studied by Semmler (1906), who, considering the natural occurrence of a t r i p l e bond unli k e l y , proposed an a l l e n i c formula for the compound. The correct structure was given by Gilman et al. (1933). The str u c t u r a l elucidation of a naturally-occurring pol ^ acetylene was f i r s t achieved by Vil'yams et al. (1935) who recognized the lachnophyllum ester isolated from LachnophylI um gossypinum Bge. as the methyl ester of dec-2-ene-4,6-diynoic acid (Table I ) . These f i r s t compounds 1 2 were found accidentally because they were present in reasonable amounts and e a s i l y p u r i f i e d . Seven acetylenes were described between 1902 and 1950. since then, over 700 more have been i d e n t i f i e d (Thaller, 1976) primarily because a l i p h a t i c polyacetylenes were discovered to show very c h a r a c t e r i s t i c UV spectra with high extinct i o n c o e f f i c i e n t s , thus allowing detection of small quantities of substance (Jones, 1959; 1966; Bohlmann el al . , 1973). Around 1950, a n t i b i o t i c substances produced by Basidiomycetes were characterized by UV spectra showing fine structure, which, by comparison with polyacetylenes from Compositae, t e n t a t i v e l y established their acetylene structures (Anchel et al , 1950; Anchel, 1953; Kavanagh et al; 1950). Serious attention was focussed on t h i s area when Celmer and Solomon (1952a; 1952b; 1953) iso l a t e d and i d e n t i f i e d the a n t i b i o t i c mycomycin as one containing allen e , diacetylene and diene groupings (Table I ) . When Jones and his co-workers started t h e i r broad investigations into acetylenes from fungi, they changed the screening technique from an a n t i b i o t i c test to one of determining the UV spectra of culture f l u i d s (Jones, 1959). It i s largely through the concurrent e f f o r t s of Jones, Sjrfrensen, Bohlmann, Anchel and t h e i r associates in the l a s t 30 years that so many polyacetylenes are known today. About 85% of these were isolated from higher plants. They are f a i r l y widespread amongst the Campanulaceae and Araliaceae 3 TABLE I. NATURALLY OCCURING ACETYLENES CH. 3-(CH 2)^ C=C-(CH 2) ACOOH t a r i r i c acid Picramnia tariri DC. ca r l i n a oxide Car Ii na a caulis L. CH 3-(CH 2) 2-(C=C) 2-CH=CH-COOCH 3 lachnophyllum ester LachnophylI urn gossypinum Bge. H(C=C) 2-CH=C=CH-(CH=CH) 2CH 2COOH mycomycin Nocardia acidophilus 5 - ( 3 - b u t e n - 1 - y n y l ) - 2 , 2 ' - b i t h i e n y l Taget es pat ula L. p h e n y l h e p t a t r i y n e Bi dens alba L. i c h t h y o t h e r e o l lchthyothere terminal is Spreng. H O - C H 2 - ( C H 2 ) 2 - ( C = C ) 2 - ( C H = C H ) 3 - C H (OH)- (CH 2 > 2 ~CH 3 c i c u t o x i n Ci cut a vi r osa L. C H 3 - C H = C H ( C = C ) A - C H = C H 2 C , j-ene-tetrayne-ene Heliantheae: Coreopsidinae C H 3 - ( C = C ) - C H = C H 2 Cij-pentayne-ene Heliantheae: Coreopsidinae C H - C O - ( C = C ) 2 - C H - C H = C H - ( C H 2 ) - C H dehydrofalcarinone Heliantheae: Galinsoginae " ^ - C - ( C = C ) 2 - C H 3 0 c a p i l l i n Artemesia capi 11 or i s Thnb. a w o - t e r t h i e n y l Tageies pat ula L. 6 and have been found sporadically in several other plant families (Bohlmann et al., 1973). They are most frequently found in members of the Umbellifereae and the Compositae, in which they occur in a l l 13 t r i b e s , e s p e c i a l l y the Heliantheae, Anthemideae and Cynareae (S^rensen, 1977; Swain and Williams, 1977) . As d i s t i n c t from the mainly a l i p h a t i c acetylenes of related plant fa m i l i e s , acetylenes of the Compositae are characterized by c y c l i c , aromatic or heterocyclic end groups. Some of these complex structures are r e s t r i c t e d to a single t r i b e while some heterocyclic compounds, such as thiophenes, have been found in the majority of t r i b e s , their occurrence seemingly unrelated to morphological characters (S^rensen, 1977). In fact, the d i s t r i b u t i o n of acetylenes in the Compositae does not often correlate well with botanical c l a s s i f i c a t i o n . Bohlmann et al. (1973) have made an extensive study of the polyacetylenes from th i s family and i t appears that, although acetylene d i s t r i b u t i o n i s discrete, p a r t i c u l a r features are mostly r e s t r i c t e d to some subtribes, some genera or some sections, so that polyacetylenes may be useful taxonomically at d i f f e r e n t l e v e l s below the t r i b e . For example, members of the subtribe Heliantheae: Coreopsidinae are characterized by C 1 3 ene-tetrayne-ene (Table I) and i t s aromatic derivatives, together with less unsaturated compounds. The subtribe H: Galinsoginae contains dehydrofalcarinone and other C 1 7 acetylenes (Table I) 7 (Bohlmann et al., 1973). Within Coreopsidinae, Coreopsis, Bidens and Dahlia, c l o s e l y related genera, have similar acetylene arrays (Bohlmann and Zdero, 1968; Bohlmann and Bornowski, 1966; Bohlmann et al . , 1967; 1966; 1964; S^rensen and Scrfrensen, 1966; 1958a, 1958b, 1958c; Sjrfrensen et al . , 1961), except that Dahlia also has C 1 7 acetylenes (Lam 1971; 1973; Lam and Kaufmann, 1971; Chin et al., 1970) which are c h a r a c t e r i s t i c of two other related genera Glossocardia and Isostigma (Bohlmann et al., 1973). The f i r s t fungal polyacetylenes, l i k e mycomycin (Celmer and Solomon, 1953) and agrocybin (Bohlmann et al., 1969), were detected and isolated because of thei r a n t i b i o t i c properties. In the same way, the discovery of a n t i b i o t i c properties of plants or of plant extracts has led to the i d e n t i f i c a t i o n of polyacetylenes as the active compounds. Thus the antifungal compounds from Artemesia capillar is Thunb. were i d e n t i f i e d as conjugated acetylenic ketones such as c a p i l l i n (Table I) which i s highly active against dermal mycoses (Jones and Thaller, 1978; Wagner, 1977). Reisch et al. (1967) investigated the. ba c t e r i o s t a t i c and f u n g i s t a t i c e f f e c t s of a large number of simple synthetic acetylenes, including hydrocarbons, acids, alcohols, aldehydes and ketones with one or two t r i p l e bonds, as well as the C, 3-ene-tetrayne-ene and pentayne-ene compounds. In general their findings suggest that acetylenes with aromatic substituents were most active and that fungicidal e f f e c t s increase with p o l a r i z a t i o n of the t r i p l e 8 bond and degree of unsaturation in the molecule while compounds which were more hydrophilic tend to be ba c t e r i o c i d a l agents. Several polyacetylenes are known to be generally t o x i c . The plant extract used by natives of the Lower Amazon Basin as f i s h poison on their arrowheads contains the tetrahydropyran ichthyothereol and i t s acetate (Table I) as i t s active p r i n c i p l e s (Cascon et al . , 1965) and the potent t o x i c i t y of Cicut a virosa L. i s due to cicutoxin (Anet et al., 1953). In 1973, Gommers and Geerligs reported that the nematocidal a c t i v i t i e s of a-terthienyl and 5-(3-buten-1-ynyl)-2,2'-bithienyl (Table I) were s i g n i f i c a n t l y enhanced by UV l i g h t . These compounds were subsequently isolated from Tagetes patula L. and found to be phototoxic to Candida albicans (Robin) Berkh. (Daniels, 1965; Chan et al., 1975). This discovery led to a systematic investigation of the phototoxic properties of polyacetylenes and t h e i r thiophene derivatives from the Compositae by Towers and his associates (e.g., Camm et al,, 1975; Towers, 1980; Towers and Wat, 1978; Towers et al., 1977; Wat et a/., 1980). It i s now well established that many polyacetylenes, notably a-terthienyl and phenylheptatriyne (Table I ) , are toxic to b i o l o g i c a l systems in the presence of UV-A (320-400nm) radiation. In addition to bacteria and fungi (Arnason et al . , 1980; DiCosmo et al., 1982), these compounds 9 k i l l human fib r o b l a s t s and erythrocytes (Wat et al . , 1977; MacRae et al., 1980b; Towers et al., 1979), cercaria (Graham et al . , 1980), adult nematodes, insect larvae and eggs (Arnason et al., 1981; Kagan and Chan, 1983; Wat et al., 1981) and deactivate viruses (Warren et al., 1980; Hudson et al., 1982), but they are not genotoxic (MacRae et al., 1980a). Unlike the linear furanocoumarins, whose phototoxic e f f e c t s can be explained by the photo-induced modification of DNA (Song and Tapley, 1979), polyacetylenes act on c e l l membranes. S p e c i f i c a l l y , a-terthienyl acts as a t y p i c a l Type II photodynamic s e n s i t i z e r , requiring oxygen for i t s a c t i v i t y while the photosensitization of E. coli c e l l s and erythrocytes by phenylheptatriyne occurred under both aerobic and anaerobic conditions (Wat el al ., 1980; Arnason et al., 1981; McLachlan et at., 1984). Many d e t a i l s are known about polyacetylenes and th e i r in vitro effects and, although there i s considerable speculation about their putative in vivo functions, at present, no obvious physiological role can be allo c a t e d to polyacetylenes in the organisms which produce them. This does not preclude p r a c t i c a l application of their potent b i o c i d a l properties. Polyacetylenes are notoriously unstable and decompose rapidly in aqueous solution and in l i g h t (e.g., Anchel et a/., 1950; Celmer and Solomon, 1953; Bohlmann et al., 1973; Towers, 1980). This rapid biodegradability may cer t a i n l y be exploited to advantage in 1 0 the search for e f f e c t i v e and environmentally nontoxic b i o l o g i c a l control agents. The purpose of t h i s study is to explore various aspects of polyacetylenes in one group of plants, Hawaiian Bidens, in order to es t a b l i s h preliminary information on their 1 . occurrence and evolutionary significance, 2 . biosynthetic pathways, and 3. a n t i b i o t i c properties. Such preliminary data are necessary in order to id e n t i f y those hypotheses which w i l l lead to a rapid growth of knowledge concerning these chemicals. As well, t h i s information w i l l be useful in determining their potential usefulness and define the l i m i t s of manipulation for human u t i l i z a t i o n . 11 B. BIBLIOGRAPHY Anchel, M. 1953. I d e n t i f i c a t i o n of an a n t i b i o t i c polyacetylene from Cliiocybe di at ret a as a suberamic acid ene-diyne. Am. Chem. Soc. J . 75: 421 - 462. Anchel, M., J . Polatnick and F. Kavanagh. 1950. Isolation of a pair of c l o s e l y related a n t i b i o t i c substances produced by three species of Basidiomycetes. Arch. Biochem. 25: 208 - 220. Anet, E., B. Lythgoe, M.H. S i l k and S. Trippett. 1953 Oenanthotoxin and cicutoxin. Isolation and structures. J. Chem. Soc. 309 - 322. Arnason, T., J.R. Stein, E. Graham, C.K. Wat, G.H.N. Towers and J . Lam. 1981. Phototoxicity to selected marine and freshwater algae of polyacetylenes from species in the Aseteraceae. Can. J . Botany 59: 54 - 58. Arnason, T., C.K. Wat, K. Downum, E. Yamamoto, E. Graham and G.H.N. Towers. 1980. Photosensitization of E. col i and S. cerevisiae by phenylheptatriyne from Bidens pilosa. Can. J . Mi c r o b i o l . 26: 698 - 705. Arnason, T., T. Swain, C.K. Wat, E.A. Graham, S. Partington 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 polyacetylenes from species in the Asteraceae. Biochem. Syst. Ecol. 9: 63 - 68. Arnaud, A. 1902. Sur l a constitution de l'acide t a r r i r i q u e . CR. Hebd. Seanc. Acad. S c i . , Paris 134:473 - 482. Bohlmann, F., T. Burkhardt and C. Zdero. 1973. Naturally Occurring Acetylenes. Academic Press, London. Bohlmann, F., R. Jente and R. Reinecke. 1969. Polyacetylenverbindungen. Uber die biogenese der naturlichen C 1 3 - und C 1 0- acetylenverbindungen. Chem. Ber. 102: 3283 - 3292. Bohlmann, F. and C. Zdero. 1968. Polyacetylenverbindungen, CLVII. Uber die i n h a l t s s t o f f e von Coreopsis nuecensis A. Hel 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 s u b s t i t u i e r t e naturliche acetylenverbindungen. Chem. 12 Ber. 99:1223 - 1228. Bohlmann, F., K.M. Kleine and H. Bornowski. 1966. Polyacetylenverbindungen XCI. 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Isola t i o n , . c r y s t a l l i z a t i o n and chemical characterization. Amer. Chem. Soc. J. 7 4 : 2245 - 2248. Celmer, W.D. and I.A. Solomon. 1953. Mycomycin. I II. The structure of mycomycin, an a n t i b i o t i c containing allene, diacetylene 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 a n t i b i o t i c a c t i v i t y of thiophene compounds of Tagetes. Phytochemistry 14 : 2295 - 2296. Chin, C , M.C. Cutler, S i r E.R.H.Jones, J . Lee, S. Safe and V. Th a l l e r . 1970. Natural acetylenes. Part XXXI. C! a-tetrahydropyranye and other polyacetylenes from the Compositae Dahlia coccinea Cav. var. cocci nea. J. Chem. Soc. (C): 314 - 322. Cimino, G., A. Crispino, S. DeRosa, S. DeStefano and G. Sodano. 1981. Polyacetylenes from the sponge Petrosia ficiformis found in dark caves. Experientia 3 7 : 924 926. Daniels, F. 1965. A simple microbiological method for 13 demonstrating phototoxic compounds. J . Invest. Dermatol. 44: 259 - 263. De Napoli, L., E. Fatturusso, S. Magno and L. Mayol. 1981. Furocaulerpin, a new acetylenic sesquiterpeniod from the green algae Caulerpa prolifera. Experientia 37: 1132. DiCosmo, F., G.H.N. Towers and J. Lam. 1982. Photoinduced fungicidal a c t i v i t y e l i c i t e d by naturally occurring thiophene derivatives. Pestic. S c i . 13: 589 - 594. Gilman, H., P.R. Van Ess and R.R. Burtner. 1933. The constitut i o n of Carlina-oxide. Amer. Chem. Soc. J. 55: 3461 - 3466. Gommers, F.J. and J.W.G. Geerlings. 1973. Lethal 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. Ce r c a r i c i d a l a c t i v i t y of phenylheptatriyne and a-ter t h i e n y l , naturally-occurring compounds in species of Asteraceae (Compositae). Can. J. Zool . - 5 8 : 1955 -1958. Hudson, J.B., E.A. Graham and G.H.N. Towers. 1982. The nature of the interaction between photoactive compound phenylheptatriyne (PHT) and animal viruses. Photochem. Photobiol. 36: 181 - 186. Jones, Sir E.R.H. 1966. Natural polyacetylenes and their precursors. Chem. B r i t . 1966: 6 - 13. Jones, Sir E.R.H. 1959 Polyacetylenes. Pedlar Lecture, 12 February 1959, Chemical Society, London. Jones, S i r . E.R.H. and V. Th a l l e r . 1978. Natural acetylenes, pp 621-633 in The Chemistry of the Carbon-Carbon T r i p l e Bond Part 2. S. Patai (Ed.). J. Wiley and Sons, New York. Kagan, J . and G. Chan. 1983. The photoovicidal a c t i v i t y of plant components towards Drosophila melanogasl er . Experientia 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. Polyacetylenes of Dahlia. Bioch. Systematics 1: 83 - 86. Lam, J . 1971. Polyacetylenes in Dahlia imperial is and Dahlia l enuicaulis . Phytochemistry 10: 2227 - 2228. 14 Lam, J. and F. Kaufmann. 1971. Polyacetylenic C 1 l t-epoxide and C 1 ( ( tetrahydropyranyl compounds from Dahlia scapigera. Phytochemistry 10: 1877 - 1880. 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-terthienyl for thei r a b i l i t y to induce genetic damage. Experientia 36: 1096 - 1097. MacRae, W.D., D.A.J. Irwin, T. Bisalputra and G.H.N. Towers. 1980. Membrane lesions in human erythrocytes induced by the naturally occurring compounds a-terthienyl and phenylheptatriyne. Photobiochem. Photobiophys. 1: 309 318. McLachlan, D., T. Arnason and J. Lam. 1984. The role of oxygen in photosensitizations with polyacetylenes and thiophene derivatives. Photochem. Photobiol. 39: 177 182. Moore, B.P. and W.V. Brown. 1978. Precoccinelline and related alkaloids in the Australian soldier beetle Chaul iognat hus pulchellus (Coleoptera: Cantharidae). Insect Biochem. 8: 393 - 395. Reisch, J . , Spitzner, W. and K.E. Schulte. 1967. Zur frage der mikrobiologischen wirk samkeit einfacher acetylenverbingdungen. Arzneim. Forsch. (Drug Res.) 17: 816 - 840. Schulte, G.R., M.C.H. Chung and P.J. Scheuer. 1981 Two b i c y c l i c C 1 5-enynes from the seahare Aplysia oculifera. J . Org. Chem. 46: 3870 - 3873. Scrimgeour, CM. 1980. Natural acetylenic and o l e f i n i c compounds. Royal Soc. Chem. Aliph. Nat. Prod. Chem. 3: 1 - 25. Scrimgeour, CM. 1981. Natural acetylenic and o l e f i n i c compounds, excluding marine natural products. Royal Soc. Chem. Aliph. Nat. Prod. Chem. 2: 1 - 19. Semmler, L.T. 1906. Zusammensetzung des atherischen oels der eberwurzewl Carlina acauli s L. Ber. dt. Chem. Ges. 39: 726 - 730. Song, P.S. and K.J. Tapley. 1979. Photochemistry and photobiology of psoralens. Photochem. Photobiol. 29: 1177 - 1197. S^rensen, J.S. and N.A. Sf^rensen. 1966. Studies related to naturally occurring acetylene compounds, XXXIII. A preliminary investigation of Coreopsis gigantea (Kell.) H.M.Hall. Acta Chem. Scand. 20: 992 - 1002 1 5 S^rensen, J.S. and N.A. S^rensen. 1958. Studies related to naturally occurring acetylene copounds, XXIV. 2-phenyl-5(a-propynyl)-thiophene from the essential 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. Studies related to naturally occurring acetylene compounds, XXII. Correctional studies on the constitution of the polyacetylenes of some annual Coreopsis species. Acta Chem. Scand. 12: 756 - 764. S^rensen J.S. and N.A. Sfrfrensen. 1958. Studies 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. Polyacetylenes and conservatism of chemical characters in the Compositae. pp. 385-409 in The Biology and Chemistry of the Compositae. Vol. I. V.H. Heywood, J.B. Harborne and B.L. Turner (Eds.). Academic Press, New York. S^rensen, S.L. and N.A. S^rensen. 1961. Studies related to naturally occurring acetylene compounds, XXIX. Preliminary investigations in 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. Williams. 1977. Heliantheae - chemical review. pp. 673-697 in The Biology and Chemistry of the Compositae. Vol. I. V.H. Heywood, J.B. Harborne and B.L. Turner, (Eds.). Academic Press, New York. Thaller, V. 1976-77. Natural acetylenic and o l e f i n i c compounds, excluding marine natural products. Royal Soc. Chem. Aliph. Nat. Prod. Chem. 1: 1 - 19. Towers, G.H.N. 1980. Photosensitizers in plants and their photodynamic action, pp. 183-202 in Progress in Phytochemistry Vol.6. L. Reinhold, J.B. Harborne and T. Swain, (Eds.). Pergamon Press. Towers, G.H.N, and CK. Wat. 1978. B i o l o g i c a l a c t i v i t y of polyacetylenes. Rev. Latinoamer. Quim. 9: 162 - 170. Towers, G.H.N., CK. Wat, E.A. Graham, R.J. Bandoni, G.F.Q. Chan, J.C. Mi t c h e l l and J . Lam. 1977. Ult r a violet-mediated a n t i b i o t i c a c t i v i t y of species of Compositae caused by polyacetylenic compounds. Lloydia 40: 487 - 496. Towers, G.H.N., T. Arnason, CK. Wat, E.A. Graham, J. Lam and J.C. M i t c h e l l . 1979. Phototoxic polyacetylenes and 16 their thiophene derivatives - effect on human skin. Contact Dermatitis 5: 140 - 144. 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 in the essen t i a l o i l of Lac hnophyl I urn gossypinum Bge. Zhur. Obschei Khim. 5: 1195 - 1203; Chem. Abstr. 30: 1176 - 1177. Wagner, H. 1977. Pharmaceutical and economic uses of the Compositae. pp. 412-433 in The B i o l o g y and C h e m i s t r y of the Compositae Vol. I. V.H. Heywood, J.B. Harborne and B.L.Turner, (Eds.). Academic Press, New York. Walker, R. P. and D.J. Faulkner. 1981. Chlorinated acetylenes from the nudibranch Diaul ul a sandi egensis. J . Org. Chem. 46 : 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. Partington, T. Arnason, G.H.N. Towers, and J. Lam. 1981. Photosensitization of invertebrates by natural polyacetylenes. Biochem. Syst. Ecol. 9: 59 - 62. Wat, C.K., T. Johns and G.H.N. Towers. 1980. Phototoxic and an t i b i o t i c a c t i v i t i e s of plants of the Asteraceae used in folk 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. Ultraviolet-mediated cytotoxic a c t i v i t y of phenylheptatriyne from Bidens pilosa L. J . Nat. Products 4 2 : 103 - 111. II . POLYACETYLENES IN HAWAIIAN BIDENS A. INTRODUCTION The Hawaiian Islands are usually considered to be the most isola t e d archipelago on earth. They l i e v i r t u a l l y alone in the North P a c i f i c , separated from the nearest high oceanic islands such as the Marquesas by 3200km, and from the North American coast by 4000km of ocean (Carlquist, 1970). A l l are giant submarine volcanoes that arose from an apparently stationary hot spot beneath the P a c i f i c Plate which has been moving in a northwesterly d i r e c t i o n for tens of m i l l i o n s of years. The island chain stretches 2500km from northwest to southeast across the P a c i f i c , the older Western Leeward Islands having been reduced by erosion to shoals, i s l e t s and a t o l l s , while the main eastern group, the Windward Islands, are mountainous and geologically young. The major islands range in age from 5.6 m i l l i o n years for Kauai to less than 1.0 m i l l i o n years for Hawaii (Figure 1). Geological evidence indicates that the Hawaiian chain has never been connected to a continental land mass (Stearns, 1966). The ancestors of a l l indigenous Hawaiian plants and animals arrived by accidental long-distance d i s p e r s a l . The r a i n f a l l , s o i l and temperature conditions of the Hawaiian Islands make them exceptionally i n v i t i n g f i e l d s for occupation by many groups of organisms. In addition, there i s considerable variation in microclimate on each island 17 KAUAI NIHAU (P v>OAHU LANAI THE MAJOR HAWAIIAN ISLANDS 19 because of the mountainous topography. Upon a r r i v a l , immigrant species faced a new, physically diverse environment with a r e l a t i v e absence of competition, conditions ideal for adaptive r a d i a t i o n (Carlquist, 1966a; 1966b; 1967; 1970). Adaptive radiation i s the evolutionary d i v e r s i f i c a t i o n of one ancestral lineage into numerous species adapted to l i f e in a variety of habitats. Many groups of organisms such as the honeycreepers, f l i e s (Drosophila F a l l e n ) , rutaceous plants (Pelea A. Gray) and composites ( Lipochaeta DC.) have undergone spectacular radiation in the Hawaiian Islands. The genus Bidens L. (Asteraceae), commonly known as beggarticks or Spanish needles in North America and ko'oko'olau in Hawaii, has evolved from a single ancestral species into numerous taxa which exhibit greater morphological and ecological d i v e r s i t y than species of Bidens found elsewhere in the world (Ganders and Nagata, 1983a; 1984). The Hawaiian species of Bidens occur in habitats that range from a r i d to semi-arid lava flows with less than 0.3m of r a i n f a l l per year, to dense r a i n f o r e s t and montane bogs with annual p r e c i p i t a t i o n exceeding 7.0m, and through elevations extending from sea l e v e l to over 2200m. They have d i v e r s i f i e d in growth habit (from small trees with woody trunks over 2m t a l l to t a l l shrubs to erect and prostrate herbaceous forms), leaf shape (from simple to compound to highly dissected), flower head s i z e and shape, achene size and shape (from f l a t and st r a i g h t to t i g h t l y c o i l e d ) , 20 presence and type of dispersal mechanism (awns of various lengths and shapes, pubescence, and presence or absence of wings), as well as in ecological tolerances. Differences between species in a l l these characters are maintained under standard growing conditions, indicating that they are under strong genetic control (Helenurm and Ganders, .1985). Surprisingly, however, a l l Hawaiian Bidens are completely i n t e r f e r t i l e (Ganders and Nagata, 1984). This suggests that adaptive radiation in morphology and ecological tolerance has occurred without the evolution of physiological or genetic i n t e r s p e c i f i c i s o l a t i n g mechanisms ( G i l l e t t and Lim, 1970; G i l l e t t , 1975; Ganders and Nagata, 1983a; 1984). The d i f f e r e n t species of Hawaiian Bidens are as similar genetically at isozyme l o c i as are populations of a single species in most plants. They exhibit l i t t l e d i f f e r e n t i a t i o n in isozymes of primary metabolic processess, and genetic distances among populations based on isozyme l o c i show l i t t l e c o r r e l a t i o n with morphological differences or taxonomic c l a s s i f i c a t i o n (Helenurm and Ganders, 1985). This d i s p a r i t y between the evolution of morphological and biochemical characters makes i t of interest to determine whether or not there has been evolutionary divergence in secondary metabolites in these species. In t h i s study, the polyacetylenes from leaves and roots of a l l the endemic Hawaiian species of Bidens and a l l but two of the subspecies were isolated and i d e n t i f i e d . The primary objectives were to determine the extent of 21 evolutionary d i f f e r e n t i a t i o n of polyacetylenes in Hawaiian Bidens and to see whether they are useful taxonomic characters in the group. The possible rel a t i o n s h i p of polyacetylene d i s t r i b u t i o n to the biology of Hawaiian Bidens i s also considered. Sherff (1943) produced a worldwide taxonomic revision of the genus Bidens based on a study of herbarium material. In t h i s and subsequent publications he recognized 43 species and more than 20 i n f r a s p e c i f i c taxa endemic to the Hawaiian Islands. He had no information, however, on the extent of environmentally determined variation in these plants. Ganders and Nagata (1983a; 1984) have reduced these to 19 species and 8 subspecies. Their c l a s s i f i c a t i o n i s followed in t h i s t h e s i s . Since a l l the species of Hawaiian Bidens are i n t e r f e r t i l e , i n t e r s p e c i f i c hybrids were r e l a t i v e l y easy to obtain experimentally. Several of these hybrids, as well as their F 2 o f f s p r i n g , were examined for their polyacetylenes. The purpose of t h i s portion of the study was to determine the degree of complexity of polyacetylene inheritance. 22 B. MATERIALS AND METHODS PLANT MATERIAL Plants from 54 populations representing 19 species and six subspecies of endemic Hawaiian Bidens were examined for polyacetylenes in leaves and roots (Table I I ) . This includes a l l endemic taxa recognized by Ganders and Nagata (1984) except B. campyl ot heca Schz. Bip. ssp. waihoiensis St.John and B. hi 11 ebrandi ana (Drake del Cast.) Deg. ex Sherff ssp. hi 11 ebrandi ana . L o c a l i t i e s for a l l populations are shown in Figures 1 to 6. Voucher specimens are deposited at the University of B r i t i s h Columbia (UBC), and duplicates of most are also at the Harold Lyon Arboretum, Honolulu (HLA). F, and F 2 hybrids were synthesized by F.R.Ganders and a l l plants were grown from seeds or cuttings in greenhouses at the University of B r i t i s h Columbia under natural l i g h t , and leaves and roots of greenhouse plants harvested for analys i s . ISOLATION AND IDENTIFICATION OF POLYACETYLENES Fresh leaves and roots were extracted with methanol (MeOH) (1g to 10ml r a t i o ) , ground and f i l t e r e d . The f i l t r a t e was d i l u t e d 1:1 with d i s t i l l e d water and extracted twice with equal volumes of l i g h t petroleum ether(PE) (30-60°C). The combined PE fractions were dried with anhydrous Na 2SOi,. Solvent volume was reduced to 3ml for spectral analysis. UV spectra were recorded in spectral grade PE using either a TABLE I I . niCENS TAXA EXAMINED FOR POLYACETYLENES 1. 8fdens amplect ens S h e r f f 12 . ft. menziesii s s p . menzlesi I (Gray) S h e r f f 2. 8. asymmet^ica ( L e v i . ) S h e r f f 12a . P. menzfesii ssp. flliformis ( S h e r f f ) Ganders ft Nagata 3 . 8. campy 1 otheca Schz . BIp. ssp. r.impy 1 ofhec.a 13 . P. micrantha Gaud. ssp. micrantha 3 a . B. campylotheca ssp. pentamera ( S h e r f f ) 13b P. micrantha s s p . ctenophylla ( S h e r f f ) Nagata ft Ganders Nagata ft Ganders 4 . B. cerv i cat a S h e r f f 13a . P. mi crantha ssp. kalealaha Ganders & Nagata 5. 8. conjunct a S h e r f f 14 . 0. molokalensis I H H I e b r . ) S h e r f f 6. 8. cosmoides (Gray) S h e r f f 15. B. popul i f o l i a S h e r f f 7 . 8. f o r b e s i r S h e r f f ssp. f o r b e s M 16. P. sandvIcens<s L e s s . s s p . sandvicensis 7b. 8. forbesiI ssp. kahf1fensis Ganders ft 16a. (i. sandvicensis ssp. confusa Nagata ft Nagata Ganders 8 . 8. hawaiensis Gray * 17 . P. t o r t a 'Sherff 9. 8. hi 11ebrandiana ssp. polycephaij Nagata ft 18. P. valIda S h e r f f Ganders 10. 8. macrocarpa (Gray) S h e r f f 19. • P. itiebkel S h e r f f 11 . 8. maul ensi r, (Gray) S h e r f f 17A (B18, B19): 17B (B36-B41); 17C (B55. B56) : 170 (B110) K) FIGURE 2. LOCALITIES OF KAUAI BIDENS POPULATIONS SAMPLED B. cer vi cat a: BB, BB3; B. cosmoides: B9; B. forbesii s s p . forbesii: B12, B13, B14, B74, B101, B124: B. forbesii s s p . kahiliensis: B71, B134: B. sandvi censi s s s p . sandvi censis: B112; B. sandvi censi s s s p . confuse. B33, B34; B. val i da: B54, B131, B132. KAUAI B12,124 X B U ,B13 B 74 \ B101 \ B 83 B 9 B 33 B 112 B134 B 54,71,132 cn FIGURE 3. LOCALITIES OF OAHU BIDENS POPULATIONS SAMPLED B. amplectens: BI; B. asymmetrical, B4; B211; B. campy I ot heca s s p . campylot hecax B195; B. cervicata: B88; B. macrocarpax B22; B23; B. mol okai e ns i s: B1 1 ; B. populifolia: B42; B. sandvi censis ssp. sandvicensis: B5; B 6 ; B7; B20; B 3 5 ; B43-B47; B. lorla: B18, B 1 9 (A), B36-B41 (B), B55, B56 (C), B110 ( D ) . OAHU B 5t6 B 2 ° . « ' 4 6 FIGURE 4. LOCALITIES OF MAUI BIDENS POPULATIONS SAMPLED B. campyl ot heca s s p . pentamero: B114; B. conjunct ax B60-B63; B. hi 11ebrandi ana s s p . polycephala: B67, B66; B. maui ensi s : BIO, B 2 7 ( c u l t i v a t e d ) , B28, B12B; B. menziesii s s p . menziesii: B31, 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 ssp. fiiiformis: B30, B32, B130, B163; B. micrantha ssp. cl enophylI a: B149, B150. 33 HAWAII 34 Pye-Unicam SP8-100 UV/VIS or a Unicam SP800A UV/VIS spectrophotometer. Samples were evaporated to dryness under nitrogen and resuspended in 1ml spectral grade MeOH for storage at -20°C. Concentrated samples were injected into a Finnigan 1020 Automated GC/MS and the mass spectra of compounds in the PE f r a c t i o n , including polyacetylenes, recorded. Chromatographic separation of compounds was car 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 gradient of 10°/min from 150°C to 250°C, and helium as a c a r r i e r gas. Polyacetylenes were separated on a n a l y t i c a l s i l i c a gel sheets containing a fluorescent indicator (SG-UV254 and SG N-UV254). PE (30-60°C) with increasing percentages of di e t h y l ether(DE), was used to e f f e c t separation of the compounds in each extract according to the methods decribed by Lam et al . ( 1968) and Wrang and Lam (1975). Column chromatography on s i l i c a gel 60 and a chromatotron (Harrison Research) using s i l i c a gel PF-254 with CaSO««l/2H 20 plates were used for larger scale separations. Columns and plates were developed with PE (30-60°C) followed by increasing amounts of DE to elute more polar compounds. The solvent systems used for a l l separations were of the following proportions of PE to DE :19:1; 9:1; 17:3; 8:2; 7:3; 13:7; 5:5. A small aliquot of concentrated acetic acid was added to the developing tank for thin layer chromatography. In a l l cases, compounds 1 to 18 were eluted by PE/DE 13:7. 35 Individual acetylenes were i d e n t i f i e d by comparison of UV and mass spectra with those of known compounds. Extraction, i s o l a t i o n and i d e n t i f i c a t i o n procedures were car r i e d out in dim l i g h t at 0°C or lower. Individuals from one to five populations of each taxon were examined over a period of 18 months. Leaves from individual plants were analyzed for polyacetylenes three to six times throughout t h i s period. Roots were analyzed twice. F, individuals were analyzed three times over 12 months and F 2 populations sampled once. This precluded large scale extractions for s p e c i f i c compounds. 36 C. RESULTS POLYACETYLENES IN BIDENS TAXA Leaves and roots from 19 species and six subspecies of Bidens from Kauai, Oahu, Maui, Molokai and Hawaii were analyzed for polyacetylenes (Table II, Figures 2 to 6). Two taxa (B. campylot heca Schz. Bip. ssp. waihoiensis St. John and B. hi I1ebrandi ana (Drake del Cast.) Deg. ex Sherff ssp. hi I1 ebrandiana) were not available for analysis. Compounds 1 to 18 were isolated chromatographically and i d e n t i f i e d on the basis of UV and mass spectra (Tables I I I , IV) and their d i s t r i b u t i o n among the species recorded (Tables V, VI). Although acetylenes were found in a l l the root samples examined, they were absent from the leaves of 13 of the taxa (Table V). Repeated sampling of greenhouse populations over a period of 18 months revealed no q u a l i t a t i v e v a r i a t i o n in polyacetylene production with changes in season or reproductive state of the plants. This i s in contrast to Dahlia , where considerable v a r i a t i o n in polyacetylene content and composition were encountered within the species in consecutive seasons, and in the same season in plants growing in di f f e r e n t locations (Chin et al. , 1970; Lam et al. ,1968). Many acetylenes are known to be photoactive (Towers et al., 1977; Towers, 1980), and the crude l i g h t petroleum fractions of leaf and root extracts were tested for phototoxicity against nine species of fungi and bacteria using the method of Daniels (1965). While the root samples TABLE I I I . POLYACETYLENES FROM HAWAIIAN BIDENS CHjSCH (CSC) 4CH=CHCH 2 R R • H 1 R « OCOCH3 2 CH 2=CH (C = C ) 5 C H 3 3 <Q(CSC,2CH=CHCHjR . . . R • OH 6 R « OCOCH3 7 CH 3CH=CH (C=C) 2(CH=CH) 2(CH 2> 4CH=CH 2 B CH 3(C=C) 3(CH=CH) 2(CH 2) 4CH=CH 2 9 CH 3CH=CH (C=C) 2CH 2CH = CH(CH 2) 5CH=CH 2 10 CH 2=CHCH=CH (C = C) 3CH = CHCH2R R • H H R « OCOCH3 12 CH 3CH = CH(C=C) 2CH=CH—L ^ ^ J 1 3 ^^C=C0^- R R - CH3 I* R • CH2OH 15 R » CHO 16 R • CH2OCOCH3 1 7 CH,CH=CH (CSC) ,CH = CHCHOHCH_OH 18 38 TABLE I V . POLYACETYLENES FROM HAWAIIAN BIDENS I , 2 t r i d e c a - 1 , 1 1 d i e n e - 3 , 5 , 7 , 9 t e t r a y n e 3 t r i d e c a - 1 ene - 3 , 5 , 7 , 9 ,11 pentayne 4 1-phenylhepta-1 , 3 , 5 t r i y n e 5,6,7 1 - p h e n y l h e p t a - l , 3 d i y n e - 5 ene B h e p t a d e c a - 2 , 7 , 9 , 1 6 t e t r a e n e - 4 , 6 t r i y n e 9 heptadeca - 8 ,10,16 t r i e n e - 2 , 4 , 6 t r i y n e 10 h e p t a d e c a - 2 , 9 , 1 6 t r i e n e -4,6 d i y n e I I , 12 t r i d e c a - 1 , 3 , 1 1 t r i e n e - 5 , 7 , 9 t r i y n e 13 t e t r a h y d r o - 2 1,7 d i e n e - 3 , 5 d i y n y l p y r a n - 3 - o l 14-17 2- 2-phenylethyne-1 y l -5 m e t h y l t h i o p h e n e 18 t r i d e c a - 3 , 1 1 d i e n e - 5 , 7 , 9 t r i y n e - 1 , 2 d i o l 39 TABLE V . POLYACETYLENES IN THE LEAVES OF HAWAIIAN BIDENS Compounds Bidens 1 2 3 4 5 7 8 9 10 11 12 18 1 - - - - - - - - - - -2 _ _ _ _ _ _ _ _ -3 + _ _ _ _ _ + _ + - - + 3a + - - - + + + - + - - -4 + _ _ _ + + _ _ _ _ _ _ 6 + - + - + - - - _ _ _ _ 7 - - - -7a - - - - - _ - _ _ _ _ _ 8 _ _ _ _ _ _ _ _ _ _ _ + 9 + + _ _ _ _ _ _ - + + -10 + - _ _ _ _ + _ + _ _ + "11 - - - - - - - _ _ _ _ 12 _ _ _ _ _ _ + _ • + _ _ _ 12a 1 3 13a 1 3b 14 15 16 16a _ - _ _ + _ - - _ _ _ 17A _ _ _ + - _ - - _ _ _ 17B - - _ - + - - - _ _ _ 17C _ _ - - - - + + _ _ _ 17D _ - - _ + _ - - _ _ _ 18 + - _ _ + _ + _ - + + 19 - _ _ _ 40 TABLE VI. POLYACETYLENES IN THE ROOTS OF HAWAIIAN BIDENS Compounds / de ns 1 2 5 6 7 8 10 11 12 13 14 15 16 1* 1 + + 2 + + + • - - - + 3 + - - - - - - - - + 3a + - - - - - - - -4 + + - - - + - - - - + - + 5 + - + + - + - - + - - - + 6 + + + + + 7 + - - - - - - + - - - + 7a + + + - - - + + - - + + 8 + - - - - + - + - - + -9 - - - - - - - - + + 10 + - - + + + 1 1 + + - - - - + 12 - - - - - -12a - - - + - - - - - + 13 + + - - - - - -13a + - - - + - + - + 13b + + 14 + + 15 • • - - - • - - - - - - + 16 + + - + - - - - - - - -16a - - - - - - - - - -17A - - - - + - - - - - - - + 17B - - + - - - + 17C - - - - + - - - + - * 17D • - - - - - - - - - + IB • - - - - • - - - - - -19 + + 41 were found to be consistently phototoxic, only leaf extracts containing acetylenes were l e t h a l to the microorganisms in the presence of near UV radiation (320-400nm) (see Chapter IV). The absence of polyacetylenes in the leaves of at least two other Bidens species has been previously noted (Bohlmann et a/.,1973), so thi s feature i s not unique to Hawaiian Bidens. However, i t does lead to interesting speculation about the possible b i o l o g i c a l significance of the compounds in question. The polyacetylenes of Hawaiian Bidens include eleven C 1 3 hydrocarbons, aromatic and thiophenic derivatives, a C, „ tetrahydropyran and three C 1 7 hydrocarbons (Table I I I , IV). Compounds 1 to 18 can be derived from o l e i c acid by a series of dehydrogenations, oxidations and reductions (Figure 7). Except for 14 to 18, they have previously been reported in North American and European Bidens, Coreopsis and/or Dahlia (Bohlmann et c/,,1973). Compound 16 occurs in the leaves of C. grandifl or a Hogg ex Sweet (Bohlmann et al. ,1966) while 18 ('safynol') has been reported in Carthamus tinctorius L. (Bohlmann et al. , 1966) and two species of Centaurea (Anderson et al. ,1977; Bohlmann et al .,1958). Naturally-occurring 14, 15 and 17 have not been isolated before although the isomers 19 and 20 (Figure 8), are found in Coreopsis grandiflora and C.nuecensis F. Heller, respectively (Bohlmann and Zdero,1968; Lam et a/.,1968; S^rensen and Sjrfrensen, 1958). Although the presence of the acetate (17) presumes the existence of the precursors (14, FIGURE 7. BIOGENETIC RELATIONSHIPS OF POLYACETYLENES FROM HAWAIIAN BIDENS Compounds 1 to IB can be derived from o l e i c a c i d v i a the intermediate dehydrocrepenynic ac id by a ser ies of transformations which include dehydrogenations (*), a-oxidat ions (a), 0-oxidations (0), and the addi t ion of H 2S (adapted from Bohlmann et al. , 1978). 43 o l e i c a c i d — S — l i n o l e i e acid —CH 3(CH 2> 4CSCCH 2CHssCH(CH 2) 7COOH crcpenynic acid 1-CH 3 (CH2> 2CH=CHC~ CCH^CHasCH (CH2> ?COOH dehydroerepenynie acid C, .-COOH- C, . - C 0 O H- CH,(CH.) CH=CHC = CCH.CH=CH(CHj.CHO IB J 2 2 2 2 b CH,CH=CH (CSC) _CH.CH=CH (CH.) ,CH^ COOH 2'3 2 • . 9 , 10 CH,CH==CH (CSC) ,CH,CH=CH (CH^) 3CH2OH ' 2 2 13 CH3CH =CH (CS C) ^ CH^CHsCH (CH2) jCOOH CHjCH =CH (CSC) (CH = CH) 2CH2CH2CH2OH CH 3CH=CH(CSC 3CH= CHCH2CH2CH2OH / 11. 12 I--CH3CH = CH (CSC) 3CH=CHCH2CH2OH IB CH,CH=CH (CSC).CH,CH,OH 3 4 2 2 1, 2 5. t. 7 I-U 1« . 15 . lfc . 17 3 44 GURE 8. PHENYLTHIOPHENES FROM COREOPSIS 45 15 and 16), these were not detected in many of the root extracts. Compound 17 i s ubiquitous in Bidens roots, and although i t may not serve as a taxonomic marker, i t s unusual structure suggests that i t may have phototoxic and other inter e s t i n g b i o l o g i c a l properties (Towers, 1979). Compound 3('pentayne-ene'), which i s common in the Asteraceae (Bohlmann et al. ,1973), was found in trace quantities in the leaves of only one of the species, B. cosmoi des . This may be due to i t s extreme i n s t a b i l i t y or to low concentrations. Since stringent precautions were taken to prevent polyacetylene degradation during laboratory workup however, and since the compound has a r e l a t i v e l y high extinction c o e f f i c i e n t (e=l0 5), i t would appear that the pentayne-ene does not accumulate in most species of Hawaiian Bidens. The other highly conjugated acetylene hydrocarbon 1 ('ene-tetrayne-ene'), was detected spectrophotometrically in many leaves and in a l l crude root extracts except that of B. tort a A (1 7A) . POLYACETYLENES IN BIDENS HYBRIDS Leaves from 21 Bidens hybrids were analyzed for polyacetylenes. Compounds were isola t e d chromatographically and i d e n t i f i e d on the basis of UV and mass spectra, and their d i s t r i b u t i o n recorded in Tables VII to XI. The leaves of F 2 populations from seeds of two selfed individuals were also examined (five individuals of selfed B. TABLE VII BIDENS HYBRIDS EXAMINED FOR POLYACETYLENES 1. 8. sandvfcensis s s p . sandvicensis X S. 2. B. sandvicensis s s p . sandvfcensis X 8. molokaiensls mlcrantha ssp. micrantha 3. 8. v a l f d a X B. molokalensis 4. 8. molokalensls X 8. macrocarpa 5. 8. mofok a f e n s f s X 8. cosroofdes 6. 8. forbesii ssp. forbesii X 8. cosmofdes 7. 8. menzfesff ssp. flllformls X 8. cosmofdes 8. 8. mlcrantha ssp. mlcrantha X 8. valfda 9. 8. s a n d v f c e n s f s s s p . s a n d v f c e n s i s X 8. 10. 8. menziesii ssp. flllformls X 8. hawalensls valIda 11. 8. mlcrantha ssp. mlcrantha X 8. hawafensfs 12. 8. s a n d v f c 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 ssp. confusa X 8. maufensfs 14. 8. forbesii ssp. forbesii X 8. t o r r a 17A 15. 8. t o r t a 17A X 8. mfcrantha ssp. mlcrantha 16. 8. populi foli a X 8. t o r t a 17C 17. 8. t o r t a 17B X 8. hawalensls 18. 8. t o r t a 17A X 8. v a l / d a 19. 8. valIda X 8. macrocarpa 20. 8. c a r v f e a t a X 8. cosmoides 21. 8. c e r v f c a t a X 8. macrocarpa TABLE VIII. POLYACETYLENES FROM BIDENS HYBRIDS Compounds Bidens F, 1 2 3 4 5 7 8 9 10 11 12 18 Type A Cross 1 _ _ _ _ _ _ _ _ _ _ _ _ 2 _ _ _ _ _ _ _ _ _ _ _ _ Type B Cross 3 + _ _ _ + _ _ _ _ _ + _ 4 + - - + + - + - + - - -5 + - + + + - + - + - - -6 + - + + + - + - + - - -7 + - + + + - + - + - - -8 - _ - _ + _ _ - _ _ _ _ 10 - + - + + - - - - - + + 11 _ _ _ _ + _ _ _ _ _ + + 12 _ + _ _ + _ _ _ _ _ _ _ 13 - + _ _ + _ _ _ _ _ _ _ 14- - - + -15 _ _ _ _ + + _ _ _ _ _ _ 16 _ _ _ _ _ _ + + _ _ _ _ Type C Cross TABLE IX. POLYACETYLENES IN B.HAH'AIENSIS HYBRIDS Compounds Bidens F, 1 2 3 4 5 7 11 12 Type B Cross 10 _ * « . - * « . « . * + 11 - - - - _ *«. Type C Cross 17 • - _ * + bsent in parents \ 49 TABLE X . POLYACETYLENES IN B.COSMO IDES HYBRIDS Bidens F t Type B Cross 5 6 7 Type C Cross 20 + Compounds 2 3 4 • 7 8 - *• - + 10 •absent in parents TABLE X I . POLYACETYLENES IN B. MACROCARPA HYBRIDS Compounds Bidens F} 1 2 3 4 5 7 8 9 10 11 12 Type B Cross 4 + - - * + • * • - • * • - + - -Type C Cross 19 • - - A- - + 2 i + - *+ - + _ + + •absent in parents A present in parents but absent in F , 51 sandvicensis ssp. confusa X B. mauiensis, B 1 8 5 S , and 22 individuals of B. sandvicensis ssp. confusa X B. sandvicensis ssp. sandvicensis, B 1 9 4 S , (Table XII). Type A crosses between two species which do not produce leaf acetylenes result in F, individuals without acetylenes (Table VIII). Crosses between species which produce leaf acetylenes and those which do not (Type B ) result in hybrids which synthesize leaf acetylenes although the compounds are not always . i d e n t i c a l to the arrays present in the parents (Table VIII to XI). In most crosses of this category, the F, progeny did not produce any compounds absent from the leaves of the parents. Nevertheless, in crosses with B. hawaiensis , compound 12 i s expressed in the leaves of the hybrids (Table IX). Bidens 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 , IV), one which i s clos e l y related to compound 12 in the proposed biogenetic scheme shown in Figure 7. Compound 12 also occurs in the roots of B. hawaiensis. Compound 4, phenylheptatriyne, was found in several hybrids from parents which did not contain i t but which did have the closely related compound 5, phenylhepta-diyne-ene. In the three Type B crosses involving B.cosmoides, the F! produced the parental array of C 1 3 acetylenes (compounds 1, 3 and 5) and three novel compounds - compound 4 , and compounds 8 and 10, C 1 7 hydrocarbons which are biosy n t h e t i c a l l y several steps removed from the C 1 3 acetylenes (Table X). Bidens mol okai ensis X B. macrocarpa TABLE XII. POLYACETYLENE IN F 2 PLANTS Compounds Bidens 1 2 Cross 12 (B194) + B194S (F 2) + Cross 13 (B185) + B185S (F 2) + 53 individuals synthesize C V 3 aromatic acetylenes 4 and 5 not found in B. macrocarpa which i s characterized by C 1 7 compounds 8 and 10 (Table X). Type C crosses between two species of Bidens which produce leaf acetylenes result in F, individuals which express a combination of the major acetylenes in both parents (Table VIII). In two cases. B. cervicat a X B. cosmoides and B. tort a MB X B. hawaiensis , compounds not found in either parent were expressed. In the cross B. valida X B. macrocarpa, the C 1 7 acetylenes from B. macrocarpa were absent from the F, whereas there was ad d i t i v i t y of parental arrays in B. cervi cat a X B. macrocarpa (Table XI). F 2 individuals from the progeny of two Type B crosses ( B. sandvicensis ssp. confusa X B. mauiensis - B 1 8 5 S , and B. sandvicensis ssp. confusa X B. sandvicensi s ssp. sandvicensis - B 1 9 4 S ) were analyzed for acetylenes. Every individual examined contained compounds 1 and 5, the same compounds found in a l l the F, and in the leaves of B. sandvicensis ssp. confusa (Table XII). r 54 D. DISCUSSION POLYACETYLENES IN BIDENS TAXA As can be seen in Tables V and V I , there i s no obvious ov e r a l l pattern in the d i s t r i b u t i o n of polyacetylenes in Hawaiian Bidens. 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 s i m i l a r i t y c o e f f i c i e n t and a single linkage (nearest neighbour) cl u s t e r i n g algorithm shows l i t t l e h i e r a r c h i c a l pattern above the species l e v e l (Figure 9 ) . Many taxa cluster at the same l e v e l , and most levels added sequentially. Morphologically similar taxa do not often cluster together, e.g., B.mauiensis and B. mol okai e ns i s , B. cervical a and B. forbesii, B.conjuncta and B.micrantha ssp. micrantha, or the subspecies of B.menzi esi i , B.micrantha, B. campyl ot he ca and B. sandvi censis. The r e l a t i v e constancy of polyacetylenes within taxa and the absence of hi e r a r c h i c a l structure in the c l a s s i f i c a t i o n above the species level provides l i t t l e evidence of relationships among taxa but may be expected in a case of multiple divergences from a common ancestor. Moreover, the r e l a t i v e l y small array of compounds can be derived from a common unsaturated fatty acid precursor, o l e i c acid (Figure 7 ) . Certainly this means that the Hawaiian Bidens share the enzyme system capable of dehydrogenating the o l e f i n i c bond, a capacity widespread in the Asteraceae, as well as the means to synthesize the aromatic acetylenic and thiophenic systems, already notable 55 FIGURE 9. DENDROGRAM OF TAXA BASED ON SIMILARITY OF POLYACETYLENES ro to o ro O 14 h i l l 6 cosm IB v a l i 11 Bieme 9 macr 3 caca 17a t o r i 11a mefi 3a cape 17c tor3 17b tor2 12 mimi 6 hawa 5 conj 4 cerv 12a mict 7a foka 16b saco 7 fofo 10 naui 2 asym I7d tor4 16 sasa 15 popu 13 nolo fc 12b nika 19 weib I 1 ampl 56 in the Tribe Heliantheae (Heywood et al . , 1977). A special feature which separates the Hawaiian group from i t s re l a t i v e s i s the consistent presence of a phenylthiophene derivative. This i s s i g n i f i c a n t in that i t supports the biosystematic evidence indicating that a l l Hawaiian Bidens evolved from a single ancestral species. It would appear, however, that-there has been less evolutionary d i v e r s i f i c a t i o n in polyacetylenes than in morphological characters although more d i f f e r e n t i a t i o n than has occurred in isozymes. A l l except four Hawaiian taxa can be distinguished by unique arrays of leaf and root acetylenes. B.mi cr ant ha ssp. 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 I I I , IV). These four taxa produce the smallest number of acetylenes of any of the Bidens, and since they are not very similar morphologically, their i d e n t i c a l polyacetylene p r o f i l e s probably do not r e f l e c t taxonomic relationships but are, rather, a result of the paucity of compounds. S i m i l a r i t i e s in polyacetylenes among taxa do not correlate well with morphological s i m i l a r i t i e s , and the recognition of related species groups on the basis of polyacetylenes does not appear to be possible. The various compounds seem to be randomly assorted among the taxa, as are many of the morphological differences noted by G i l l e t (1975). Distributions of p a r t i c u l a r acetylenes do show patterns of taxonomic si g n i f i c a n c e , however. Qualitative 57 va r i a t i o n in polyacetylenes was found within only one taxon. Although only one population of some taxa was available for analysis, two to fiv e populations were analyzed for 16 of the taxa. A l l populations of B. sandvicensis ssp. sandvicensis have i d e n t i c a l compounds even though this taxon as interpreted here includes seven species, two v a r i e t i e s and one forma according to Sherff (1937). Bidens molokaiensis was treated as two species by Sherff, B.mol okai ensis on Molokai (Figure 5), and B.cuneata, r e s t r i c t e d to the top of Diamond Head on Oahu (Figure 3). They are decumbent, low spreading herbs whose supposed differences in the shape of their leaf bases and the number of teeth on the leaves disappeared when the plants were cu l t i v a t e d under the same conditions (Ganders and Nagata, 1983b) No acetylenes were found in the leaves and root acetylenes are i d e n t i c a l . Bidens mauiensis is very similar to B.mol okai ens i s except that i t has winged achenes unique in Hawaiian Bidens, and highly variable leaf shape. Sherff recognized several v a r i e t i e s based on leaf shape differences, but they are now considered untenable (Ganders and Nagata, 1983b). A l l B. mauiensis specimens examined were uniform in the occurrence and di s t r i b u t i o n of polyacetylenes. Bidens mauiensis does d i f f e r consistently from B.mol okai ensi s by the presence of compounds 10, 12 and 13 in i t s roots. Var i a t i o n within taxa was found only in B. tort a, a morphologically variable taxon endemic to Oahu and 58 widespread in d i s t r i b u t i o n . Although i t s most d i s t i n c t i v e feature i s a twisted or c o i l e d aohene, plants can vary in the degree of c o i l i n g of achenes even within the same population. They can also vary in the amount and coloration of leaf pubescence and in the number of l e a f l e t s . Variants do not appear to have d i s t i n c t geographical ranges. Individuals from four d i f f e r e n t populations of B. tori a were found to possess d i f f e r e n t combinations of compounds. Populations 17A, 17B, and 17C are from the Waianae Range and 17D from the Koolau Range (Figure 3). Of these, 17A individuals appear to be unique among Hawaiian Bidens being the only plants in which phenylheptatriyne (PHT) i s found. Their roots are also highly unusual in that no ene-tetrayne-ene (1) was detected, although 5 was present. Both 17B and 17D accumulate only 5 in the leaves; 17C did not have any aromatic acetylenes in the leaves, only the C 1 7 hydrocarbons. The combinations of acetylenes in the roots were d i s t i n c t i v e for a l l four populations. These differences remained consistent throughout the period of the study. Although B. tort a i s as variable in i t s polyacetylenes as i t is in i t s morphology, the polyacetylenes appear to be ch a r a c t e r i s t i c of s p e c i f i c populations while morphological v a r i a t i o n i s not. In contrast, no other taxon showed i n t r a - or interpopulation differences. For example, B. hawai ensis, endemic to Hawaii, i s a morphologically uniform and d i s t i n c t i v e species although i t occurs in ec o l o g i c a l l y 59 variable s i t e s . It may rea d i l y be recognized on the basis of leaves or flowers alone. A l l individuals from three populations examined were consistent in the pattern of polyacetylenes accumulated in the leaves and roots. The leaves contained only compound 18 and the roots produced a l l classes except the aromatic acetylenes. In a l l cases examined the subspecies of a species d i f f e r e d in polyacetylene d i s t r i b u t i o n . In some cases, subspecies which have rather subtle d i s t i n c t i o n s in morphological characters can eas i l y be separated on the basis of polyacetylene differences. Bidens sandvicensis i s an extremely variable taxon which inhabits a wide range of habitats on Kauai and Oahu. Leaf shapes can be t r i f o l i o l a t e to bipinnately compound or divided into narrow ultimate segments, and a l l forms may be found in the same population. Many segregates recognized by Sherff (1937) on the basis of leaf forms are i n v a l i d according to Ganders and Nagata (1983b; 1984) and B. sandvi censi s is presently considered to consist of ssp. sandvi censi s and ssp. confusa (Ganders and Nagata, 1983b; 1984). These two taxa are often d i f f i c u l t to t e l l apart morphologically - subspecies confusa i s a high elevation taxon r e s t r i c t e d to the edge of Waimea Canyon in Kauai and tends to have larger ray flowers and flower heads, and narrower l e a f l e t s than ssp. sandvi censi s . They may, however, be distinguished by the presence of phenyl-diyne-ene (5) in the leaves of ssp. confusa and the t o t a l absence of 60 acetylenes in the leaves of ssp. sandvicensis. Five populations from Oahu and one population from Kauai of ssp. sandvicensis, and two populations of ssp. confusa were examined. The root acetylenes of the two subspecies are ident i c a l . Bidens menziesii ssp. menziesii and ssp. filiformis are obviously more cl o s e l y related to each other than to any other Hawaiian Bidens (Ganders and Nagata,1983b). They have c h a r a c t e r i s t i c leaves which vary in size but which are bipinnately divided into long l i n e a r segments less than 5mm wide. Both favour r e l a t i v e l y a r i d , sunny and windswept areas, including the cinder cones and lava flows of Hawaii (Figures 4 to 6). The two subspecies are quite d i s t i n c t morphologically however, and are a l l o p a t r i c on Maui and Molokai (ssp. menziesii) and Hawaii (ssp. filiformis). They are further distinguished by the absence of leaf acetylenes in ssp. filiformis and the presence of C 1 7 compounds 8 and 9 in ssp. menziesii. There are no differences in root acetylenes. Another example where leaf acetylenes separate two subspecies while root acetylenes are i d e n t i c a l can be found in B. campyl ol heca. Bidens campylot heca consists of three subspecies, only two of which were examined in thi s study. Subspecies campylot heca and ssp. pentamera both had a similar range of acetylenes in the leaves except that ssp. pentamera also accumulates 5 and 7, aromatic acetylenes. The l a t t e r subspecies i s r e s t r i c t e d to the foggy rainforests 61 above 1500m on East Maui (Figure 4) and d i f f e r s morphologically from ssp. campylot heca primarily - in leaf shape. Bidens forbesii consists of two subspecies, ssp. forbesii, which occurs primarily along the north coast of Kauai, and ssp. kahiliensis, a montane wet forest form r e s t r i c t e d to the v i c i n i t y of Mt. K a h i l i (Figure 2). Acetylenes are absent from the leaves of a l l six B.forbesii populations although two compounds, 5 and 12 , are found in the roots of ssp. kahiliensis but not in the roots of ssp. for be sii . Most species of Hawaiian Bidens are separable on 'the basis of polyacetylene differences. For example, B.forbesii ssp. kahiliensis i s sympatric with B. val ida on Mt.Kahili, Kauai. They are very similar vegetatively although d i s t i n c t in f l o r a l and achene characters. The two species are i n t e r f e r t i l e but they remain discrete taxa because they flower at di f f e r e n t times of the year. While B.forbesii leaves lack polyacetylenes, those of B.valida contain several compounds, notably 5, the 'phenyl-diyne-ene'. Plants grown from a mixed c o l l e c t i o n of cuttings from th i s l o c a l i t y were distinguished by analysis of leaf acetylenes and their i d e n t i t i e s later confirmed when the plants flowered. The roots of B.valida did not contain compound 5 and may be distinguished from those of B.forbesii ssp. kahiliensis on th i s basis. 62 Bidens cervicata is c l o s e l y related to B.forbesii ssp. forbesii. Both taxa have i d e n t i c a l achenes and prominently quadrangular stems, although B. cervi cat a has s l i g h t l y larger flower heads, terminal inflorescences, and smaller, less succulent and more numerous l e a f l e t s which are narrower and more deeply serrate than those of B.forbesii. Some populations of B.forbesii ssp. forbesii from the north coast of Kauai (B12, B74, B101 and B124) have individuals which appear somewhat intermediate between B.forbesii and B. cervi cat a in morphology. The two species, however, can be separated on the basis of polyacetylene differences. Bidens forbesii does not accumulate acetylenes in the leaves but B. cervi cat a leaves contain compounds 5 and 7. The roots of a l l B.forbesii populations sampled contain 12, the C,n -tetrahydropyran, a compound not found in B. cervicat a. Analysis of morphologically intermediate plants showed that they contained the acetylenes of ty p i c a l B.forbesii ssp. forbesii , suggesting that the two species do not intergrade. Other examples where morphologically similar species can be separated on the basis of polyacetylenes include B. conjunct a and B. mi crantha ssp. mi crantha. Both occur on West Maui and d i f f e r mainly in quantitative characters, but possess d i f f e r e n t polyacetylenes in their roots. B.conjuncta also produces compound 8 in i t s leaves while polyacetylenes are absent in the leaves of B.micrantha. Morphologically, B. asymmel ri ca i s a rather poorly defined taxon and i s sometimes d i f f i c u l t to dist i n g u i s h from 63 B. sandyicensis ssp. sandvi censi s where t h e i r ranges are contiguous in the southern Koolau Range on Oahu. It is also rather similar to B. tort a , which occurs in the northwestern Koolau Range. Although B. tort a exhibits great interplant v a r i a t i o n in polyacetylenes, these three species can be separated on the basis of their polyacetylenes. F i n a l l y , B. cosmoides i s a morphologically unique species endemic to Kauai. It has large flower heads with exserted styles which extend 20 - 25mm beyond the anthers, and achenes which are permanently enveloped by their subtending chaffy bracts, both unique features in the genus (Ganders and Nagata,1983a). It i s s u f f i c i e n t l y d i f f e r e n t from a l l other Bidens species that Sherff (1937) placed i t in the monotypic section Degeneria. G i l l e t t (1975) la t e r proposed that there had been two separate introductions of Bidens to the Hawaiian Islands, one which gave ri s e to B. cosmoides, and the other to a l l the other Bidens species. This hypothesis was considered unlike l y by Ganders and Nagata (1983b; 1984), because B. cosmoides can be crossed with a l l other Hawaiian Bidens. Therefore they most l i k e l y evolved from a single ancestor. The polyacetylenes of B. cosmoides indicate a close r e l a t i o n s h i p with other Hawaiian taxa. Of the nine polyacetylenes found in leaves and roots of B. cosmoi des, only compound 3 was not found in other Hawaiian taxa. Each of the other compounds was found in at least seven other taxa. The polyacetylene data supports a monophyletic o r i g i n for the Hawaiian species of 64 Bi dens . POLYACETYLENES IN BIDENS HYBRIDS Few studies on the inheritance of polyacetylene production have been reported. B i s t i s and Anchel (1966) and Carey et al . (1974) examined the basidiomycete Clitocybe truncicolor which synthesizes trans-dehydromatricarianol, CH 3-(C=C) 3CH CHCH2OH, and i t s methyl ether. Individual homokaryons exhibited d e f i n i t e and reproducible differences in polyacetylene production. These differences were evident in the progeny of crosses between d i s t i n c t i v e homokaryons and the le v e l s of polyacetylenes produced were correlated with s p e c i f i c mating types. The study in 1966 was the f i r s t to provide experimental evidence for genetic control of polyacetylene synthesis. Norton (1984) performed a similar investigation using Bidens alba L. var. radial a (Schz. Bip. ) Ballard and B. pilosa var. minor (Blume) Sherff. Bidens alba synthesizes phenylheptatriyne (PHT or compound 4 in thi s paper) in i t s leaves whereas acetylenes are absent from B. pilosa leaves. PHT was found in the leaves of a l l F, individuals r e s u l t i n g from B. alba X B. pilosa but at level s which were less than half of that in B. alba. PHT synthesis segregated in the F 2 generation although the ra t i o s of segregants did not agree with expected values and individual values were much lower than anticipated i f PHT lev e l s are a function of gene 65 dosage. In the present study, the inheritance of polyacetylene biosynthesis in Hawaiian Bidens was examined. A l l Hawaiian Bidens produce acetylenes in their roots, but only 15 taxa express t h i s a b i l i t y in the leaves. Biosynthesis in leaves and roots appear to be independent (Van Fleet, 1970) and occurs de novo in the leaves (see Chapter I I I ) . Most of the Hawaiian species can be separated on the basis of thei r leaf acetylene arrays and selected hybrids were used for this a n a l y s i s . Quantitative l e v e l s of acetylenes produced were not measured. The only other study of th i s nature was reported by Van F l e e t ( l 970). He worked with Cor eopsi s and examined the genetics of polyacetylene formation by the endodermis of roots, stems and leaves of C. saxicola Alexander, C. grandiflora Hogg ex. Sweet and their a r t i f i c i a l and natural hybrids. The stems and leaves of the two parent species produced mainly PHT but in some forms of C. grandiflora and in most of the a r t i f i c i a l hybrids, a mixture of the tri d e c a - t r i e n e - t r i y n e (compounds 11 and 12) and the phenylhepta-diyne-ene (compounds 5 and 6) was produced. An ent i t y Van Fleet c a l l s a 'general ecotype', which i s interpreted here as natural hybrids in general, contains mixtures of compounds 4, 5 and 6 in i t s stems and leaves. The roots of C. saxicola and C. gr andi flora produce predominantly the trideca-ene-tetrayne-ene (compounds 1 and 2) and "....compounds produced in the roots of the hybrids 66 are predominantly the same as the parents." Biosynthesis in the a e r i a l tissues seems to be more variable than in the roots. Coreopsis 'hybrid ecotypes' could not be distinguished or separated on the basis of acetylenes produced in the roots but could be distinguished from parental types on the basis of leaf acetylenes. The v a l i d i t y of Van Fleet's data may be questioned because the information provided i s descriptive and s t a t i s t i c a l l y nonspecific. Nevertheless, i t does indicate that the genetics of polyacetylene biosynthesis in higher plants i s a challenging problem. Data from a preliminary investigation of Hawaiian Bidens hybrids suggests that acetylene biosynthesis per se in leaves i s a heritable and dominant phenotype. Polyacetylenes were found in the leaves of progeny from Type B and Type C crosses but not from Type A crosses (Tables VII to XI). Acetylene synthesis was not segregated in the small number of F 2 individuals examined from Type B crosses. Instead, a l l plants produced the acetylenes found in B. sandvicensis ssp. confusa (Table XII). Whether there was si g n i f i c a n t v a r i a t i o n in the quantitative lev e l s of compounds produced i s not known. In general, Hawaiian Bidens produce a limited array of acetylenes, consisting of C 1 7 and C 1 3 compounds. According to the scheme proposed here, these compounds may be commonly derived from o l e i c acid but are subsequently elaborated along bi o s y n t h e t i c a l l y divergent pathways (Figure I I - 7 ) . In 67 the leaves, most species tend to produce predominantly C 1 3 or C 1 7 compounds. With the exception of crosses involving B. cosmoi des and B. macrocarpa, Type B progeny produced only compounds in the parental c l a s s . In B. cosmoi des hybrids, C 1 7 compounds not found in the parents were synthesized, and in B. macrocarpa hybrids, C 1 3 aromatic compounds absent from the parental array were observed. Data from Type C crosses is s i m i l a r . There was a d d i t i v i t y of parental polyacetylenes in F, progeny but there was also synthesis of C 1 7 compounds when only C 1 3 arrays were expected and vice versa. This i s not surprising since one would'expect that the complete set of instructions for de novo acetylene synthesis exists in the leaf genome. Moreover, regulation and control of genetic expression i s further complicated by the polyploid condition of Hawaiian Bidens (2N=72; X=12) (Mears, 1980; Fedorov, 1974; G i l l e t t and Lim, 1970; Skottsberg, 1953). That only c e r t a i n compounds are expressed in si g n i f i c a n t amounts in each species could be due to any number of factors. Certain enzymes along the sequence may have depressed a c t i v i t y or may be absent, the pool size of key precursors and intermediates, and the turnover rates of the end products would aff e c t the di r e c t i o n of equilibrium in the synthetic sequence. In addition, the sequence does not exist in 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 be influenced by the state of primary processes such as fatty acid metabolism. Whatever the governing factors, i t i s clear that the status quo i s altered when Bidens are h y b r i d i z e d . 69 E. CONCLUSION The leaves and roots of Hawaiian species of Bidens accumulate a moderate d i v e r s i t y of polyacetylenes which may a l l be biosy n t h e t i c a l l y related. Of these compounds, the phenylthiophenes 14 to 17 appear to be ubiquitous and unique to the species. This i s consistent with other evidence that the Hawaiian species are a l l derived from a single ancestral immigrant to the Hawaiian Islands. There has been less evolutionary d i v e r s i f i c a t i o n in polyacetylenes than in morphology and ecology in Hawaiian Bidens, but greater d i f f e r e n t i a t i o n than i s found in isozymes. Polyacetylenes are usually constant within a given taxon. Only B.torta exhibited interpopulational variation in compounds accumulated. Nearly a l l taxa can.be distinguished by the array of acetylenes in roots and leaves although species s p e c i f i c compounds are rare. Even subspecies which are d i f f i c u l t to dist i n g u i s h morphologically can be unequivocally i d e n t i f i e d on the basis of their polyacetylenes. The d i s t r i b u t i o n of polyacetylenes in the populations studied strongly supports the species concepts of Ganders and Nagata (1983b,1984) based on morphological and ecogeographical data. Subspecies of the same species exhibited as many differences in polyacetylenes as did di f f e r e n t species. Above the l e v e l of subspecies and species, polyacetylenes were not correlated with relationships based on morphology. Therefore, i t is not yet possible to define species groups 70 within Hawaiian Bidens based on correlated morphological and chemical characters. Adaptive radiation in Hawaiian Bidens has produced a group of species that combine an assortment of morphological and chemical characters which occur in a large number of combinations. The de novo synthesis of polyacetylenes in Bidens leaves i s a heritable and dominant t r a i t . Acetylenes were expressed in the leaves of hybrids with at least one leaf acetylene-producing parent. Synthesis, however, was not segregated in the small number of F 2 individuals examined, a l l of which produced the parental arrays. 71 F. BIBLIOGRAPHY Andersen, A.B., J . Lam and P.Wrang. 1977. Polyunsaturated compounds of Centaurea scabiosa. Phytochem. 16: 1829-1831. B i s t i s , G. and M. Anchel. 1966. Evidence for genetic control of polyacetylene production in a Basidiomycete. Mycologia 58: 270-274. Bohlmann, F., F.T. Burkhardt and C. Zdero. 1973. Naturally Occurring Acetylenes . Academic Press, London. Bohlmann, F. and C. Zdero. 1968. Uber die inhalsstoffe von Coreopsis nuecensis A. He l l e r . 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 polyine der gattung Carthamus L. Chem. Ber. 99: 3433-3436. Bohlmann, F., S. Postulka and J . Ruhnke. 1958. Polyacetylenverbindungen, XXIV. Die polyine der gattung Centaurea, L. Chem. Ber. 91: 1642-1656. Carey, S., M. Anchel and G. B i s t i s . 1974. Polyacetylene production in Clilocybe truncicola : Effect of mating-type combination. Mycologia 66 : 327-332. Carlquist, S. 1970. Hawaii: A Natural History. Natural History Press, New York. Carlquist, S. 1967. The biota of long-distance Dispersal. V. Plant dispersal to P a c i f i c islands. B u l l . Torrey Bot. Club 94 : 129-162. Carlquist, S. 1966a. The biota of long-distance d i s p e r s a l . I. P r i n c i p l e s of dispersal and evolution. Quart. Rev. B i o l . 41 : 247-270. Carlquist, S. 1966b. The Biota of long-distance d i s p e r s a l . I I . Loss of d i s p e r s i b i l i t y in P a c i f i c Compositae. Evolution 20 : 30-48. Chin,C, M. C. Cutler, E. R. H. Jones, J. Lee, S. Safe and V. Th a l l e r . 1970. Natural Acetylenes. Part XXXI. C, i,-tetrahydropyranyl and other polyacetylenes from the Composite Dahlia cocci nea Cav. var. cocci nea. J . Chem. 72 Soc. (C): 314-322. Daniels, F. 1965. A simple microbiological method for demonstrating phototoxic compounds. J . Invest. Dermat. 44 : 259-263. Fedorov, A. A. 1974. Chromosome Numbers of F l o w e r i n g P l a n t s . Otto Koeltz Science Publishers, Koenigstein. Ganders, F. R. and K. M. Nagata. 1984. The role of hybridization in the evolution of Bidens on the Hawaiian Islands. P.179-194 in 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. Relationships and f l o r a l biology of Bidens cosmoides (Asteraceae). Lyonia 2: 23-31. Ganders, F. R. and K. M. Nagata. 1983b. New taxa and new combinations in Hawaiian Bidens (Asteraceae). 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 history of Polynesian Bidens . Harold L. Lyon Arboretum Lecture No. 6. University 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) in the Hawaiian Islands. Univer. C a l i f . Publ. Bot. 56 : 1-63. Helenurm, K. and F.R. Ganders. 1985. Adaptive radiation and genetic d i f f e r e n t i a t i o n in Hawaiian Bidens . Evolution (in press). Heywood, V. H., J . B. Harborne and B. L. Turner (Eds.) 1977. The B i o l o g y and Chemistry of the Compositae . Volumes I and I I . Academic Press, New York. Lam, J ., F. Kaufmann and 0. Bendixen. 1968. Chemical Constituents of the genus Dahlia . III . 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 polyploids: A summary with comments on Parthenium (Asteraceae-Ambrosiinae). Pages 77-101 in W. H. Lewis (Ed.) P o l y p l o i d y : B i o l o g i c a l R e l e v a n c e . Plenum Press, New York. Norton, R. A. 1984. Studies of Polyacetylene production in normal and transformed tissue cultures of Bidens alba . Ph.D. Dissertation, University of B.C. 73 Sherff, E. E. 1937. The genus Bidens F i e l d Mus. Nat. Hist. Bot. Ser. 16 : (I and I I ) . Skottsberg, C. 1953. Chromosome Numbers in Hawaiian Flowering Plants. Preliminary Report. Arkiv. for Bot. 3: 63-70. SpVensen, J.S. and N. A. S^rensen. 1958. Studies related to naturally-occurring Acetylene compounds. XXIV. 2-Phenyl-5(a-propynyl)-thiophene from the essential o i l s of Coreopsis grandiflora Hogg Ex Sweet. Acta. Chem. Scand. 12 : 771-776. Stearns, H. T. 1966. Geology of the State of Hawaii P a c i f i c House Inc. Palo Alto, C a l i f . Sturtervant, A. H. 1965. A History of Genetics . Harper and Row, New York. Towers, G. H. N. 1980. Photosynthesizers from plants and their photodynamic action. Prog. Phytochem. 6 : 183-202. Towers, G.H.N., C.K. Wat, E.A. Graham, R.J. Bandoni, G.F.Q. Chan, J . Mit c h e l l and J. Lam. 1977. Ultraviolet-mediated a n t i b i o t i c a c t i v i t y of species of Compositae caused by polyacetylenic compounds. Lloydia 40 : 487-498. Van Fleet, D.S. 1970. Enzyme l o c a l i z a t i o n and the genetics of polyenes and polyacetylenes in the endodermis. Advancing Frontiers of Plant Science 26: 109-143. Wrang, P.A. and J . Lam. 1975. Polyacetylenes from Chrysanthemum I eucanl hemum. Phytochem. 14 : 1027-1035. III. BIOSYNTHESIS OF POLYACETYLENES FROM 1*C0 2 A. INTRODUCTION Natural polyacetylenes comprise a wide range of combinations of d i f f e r i n g chain lengths (C 6 - C 1 8 ) , degrees of. unsaturation, and a considerable number of functional groups and c y c l i c systems in varying relationship to the chromophores (Jones and Thaller, 1978). The almost exclusive occurrence of straight carbon chains from C 1 8 down suggests that the biosynthesis of polyacetylenes i s a variant of that of fatty acid synthesis from acetate, and there i s abundant experimental evidence to support t h i s assumption (e.g.,Bu'Lock and Gregory, 1959; Bu'Lock et al., 1961; Bu'Lock and Smith, 1962; 1963; Jones , 1966; Bohlmann and Jente, 1966; Fairbrother et al., 1967). The currently accepted hypothesis for the biogenesis of polyacetylenes in plants was f i r s t proposed by Bu'Lock (1966). This primarily involves the desaturation of the d i s t a l half ( C 1 0 ~ C 1 8) of o l e i c acid via the a-en-6-yne system of crepenynic ac i d . Subsequent transformations include chain-shortening (usually by the c l a s s i c a l a- or ^-oxidations of fatty acids at the carboxyl end), rearrangement and/or oxidation of the conjugated system, extension of the chromophore, chain-shortening at the d i s t a l end by deformylation or decarboxylation, f u n c t i o n a l i z a t i o n and c y c l i z a t i o n (Jones and Thaller, 1978). The exact sequence of reactions would be 74 75 c h a r a c t e r i s t i c of the organism and i t s physiology, producing a variety of acetylenes depending on the type and a v a i l a b i l i t y of enzymatic substrates. Hawaiian Bidens species synthesize a li m i t e d array of C 1 3 and C 1 7 polyacetylenes in t h e i r leaves and roots (Tables III, IV, V, VI). A l l these compounds may be th e o r e t i c a l l y derived from o l e i c acid in the sequence of reactions outlined in Figure 7 . The presence of non-parental acetylenes in the leaves of F, progeny from Bidens crosses may be explained using this biogenetic scheme (Chapter I I ) , thus demonstrating i t s u t i l i t y . Although only half the taxa of Hawaiian Bidens synthesize leaf acetylenes, t h i s a b i l i t y has been shown here as a dominant and heritable phenotype. Since a l l the native species are believed to have evolved from a common ancestor (Ganders and Nagata, 1983a; 1983b; 1984; Helenurm and Ganders, 1985; Marchant et al., 1984), a l l presumably had the genetic information for acetylene biosynthesis which some taxa do not express in the leaves. One of the objectives of t h i s study was to establish that de novo polyacetylene synthesis occurs in the leaves of Bidens independently of the system in the roots. This was followed by investigation of the kinetics of acetylene accumulation in intact plants with comparisons of species producing d i f f e r e n t leaf acetylenes and an assessment of the re l a t i v e e f f i c i e n c y of acetylene synthesis from 1*C0 2. This would provide some indication of the p r a c t i c a l i t y of synthesizing 1 " C - l a b e l l e d acetylenes using whole plants. 76 F i n a l l y , the accumulation and d i s t r i b u t i o n of polyacetylenes in Bidens seedlings was determined using B. alba as a representative species. 77 B. MATERIALS AND METHODS BIOSYNTHESIS OF POLYACETYLENES FROM 1 *C0 2 IN BIDENS PLANT MATERIAL Bidens cosmoides, B. hi I I ebr andi ana ssp. polycephala, B. molokaiensis and B. alba var. radiata were grown in UBC greenhouses under standard conditions. With the exception of B. molokaiensis, plants used for 1 * C 0 2 feeding experiments were approximately six months old. ADMINISTRATION OF 1 " C 0 2 Whole, freshly watered plants were placed inside the photosynthetic chamber i l l u s t r a t e d in Figure 10. The pots and s o i l surfaces were f i r s t covered with aluminum f o i l . Plants were allowed to equilibrate in the closed chamber for 30 minutes to one hour before each experiment. The entire chamber was set up in a fume hood with the overhead fluorescent l i g h t s switched on. A tungsten lamp emitting white l i g h t with an intensity of 303.57 ± 48 joules sec" 1 cm - 2 in the chamber was the major source of rad i a t i o n . A tray of water, 50 cm in depth, was kept between the l i g h t source and the plants in order to minimize temperature fluctuations within the box. A small fan c i r c u l a t e d the a i r within the chamber and temperature during 1 * C 0 2 administration.remained constant at 23°C. FIGURE 10. ' 'COi - FEEDING APPARATUS 79 A l l valves were closed at the beginning of each experiment and the chamber then p a r t i a l l y evacuated through stopcock 3 for 1.5 minutes. Stopcock 3 was then closed for the rest of the feeding. Three hundred m i c r o l i t r e s or 500ML of aqueous NaH 1"C0 3 (53.0 j/Ci/umole, New England Nuclear) was pipetted into flask A equipped with a small magnetic s t i r bar. Equimolar quantities of concentrated HC1 were added to the NaH 1*C0 3 solution and generation of i a C 0 2 according to the equation: NaH 1 4C0 3 + HCl(conc) —>-H20 + 1 *C02 + NaCl allowed to proceed. The reaction flask was kept in a warm water bath (35-40°C). Stopcocks 1 and 2 were opened and 1*C0 2 flushed into the photosynthetic chamber by allowing atmospheric a i r in v i a the 1*C0 2-generating apparatus. When chamber pressure was equalized with atmospheric pressure, stopcock 1 was closed. Plants were allowed to metabolize for one hour in the 1 aC0 2-enriched environment, at the end of which the 1*C0 2 generator was disconnected and the unused 1*C0 2 evacuated through stopcock 3 into 1.0M NaOH, where i,t was trapped as NaH 1 8C0 3. The rate of 1"C0 2 u t i 1 i z a t i o n during each feeding period was measured by taking three 0.2 mL samples of chamber a i r through a rubber septum at 15 minute i n t e r v a l s . 1*C0 2 was dissolved in Oxifluor-C0 2 (New England Nuclear) and subsequently counted for r a d i o a c t i v i t y . 80 MEASUREMENT OF 1"C UPTAKE INTO POLYACETYLENES In a l l experiments a 60-minute pulse of 1 f lC0 2 was given to plants in the chamber. Leaf samples were then taken from the plants immediately aft e r 1 f lC0 2 administration and at predetermined time intervals thereafter. Plants were allowed to photosyhthesize in atmospheric a i r for 12, 24 and 168 hours after the radioactive pulse. Roots were sampled p e r i o d i c a l l y as plants became available. A l l samples were extracted for polyacetylenes according to ,the method described in Chapter I I . Aliquots of MeOH and PE fractions were counted for ra d i o a c t i v i t y in 10 mL of Aquasol 2 (New England Nuclear). The l i g h t petroleum (PE) extracts were fractionated on preparative TLC plates (Merck, SG 60 F-254 0.25mm, 0.5mm and 2.0mm thick, 20 X 20cm) and respective polyacetylenes eluted off the s i l i c a gel with PE (30-60°C). A l l samples were run with p u r i f i e d acetylenes as reference compounds. The concentrations (C) of polyacetylenes were calculated from the absorbance (A) at the wavelength of maximum absorbance and the molar extinction c o e f f i c i e n t of the compounds according to the formula e = AX/C.l where '1' i s the length of the sample c e l l and equals 1cm (Parikh, 1974). Samples were dried and resuspended in 1OmL of Aquasol 2 and placed in a PDS/3-ISOCAP/300 (Searle) l i q u i d s c i n t i l l a t i o n counter. Radioactivity, measured as counts per 18 XIII. PERCENT QUENCHING OF RADIOACTIVITY POLYACETYLENES 1, 4 AND 5. Compound Percent decrease in dpm. ug 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 0.00 500 • 0.00 0.00 0.00 800 0.00 0.02 0.07 1000 4.54 0.00 0.36 83 minute (cpm) and the disintegrations per minute (dpm) of each sample, corrected for background, was calculated from a standard 1 4 C - e f f i c i e n c y curve (Figure 11). Quenching effects of the three acetylenes examined here (1,4 and 5) were also prepared and are shown in Table XIII. KINETIC STUDIES The uptake of 1*C into the methanol and l i g h t petroleum fractions and polyacetylenes in leaves of the four Bidens species was determined for 12 hours and 168 hours (one week) after 1*C0 2 administration. In the 12 hour studies, samples were taken at two hour inter v a l s and in the week long studies, samples taken once a day. Spec i f i c a c t i v i t y for the MeOH and PE fractions was expressed per unit fresh weight and for acetylenes as dpm/mg compound. Total amounts of acetylenes extracted were calculated for each time period and values expressed per gram of leaves. Bidens alba was used in 24 hour time course studies where samples were taken at two hour i n t e r v a l s . STATISTICAL ANALYSIS Raw data from the 24 hour time course tracer experiments using B.alba were transformed and subjected to analysis of variance using the programme UBC Anovar (1978). In each experiment, 3 plants and 3 rep l i c a t e s of MeOH and PE 84 fractions per plant were available for 13 time i n t e r v a l s . Samples for each plant subsequently had to be combined for i s o l a t i o n of PHT because of the low l e v e l of r a d i o a c t i v i t y present. Acetylene cal c u l a t i o n s are based on readings from a l l plants at each time i n t e r v a l sampled. Data analysis was thus complicated by uneven c e l l sizes and two separate s t a t i s t i c a l comparisons were made. Run 1: Three-level nested anova design for 1 BC uptake into MeOH and PE fracti o n s . Run 2: Two-level nested anova design for 1 4C uptake into PHT. Four species of Bidens, B. hi I I e b r andi ana, B. cosmoides, B. alba and B. molokaiensis were used in the 12 hour and 168 hour studies. Only one plant was available per experiment and 3 r e p l i c a t e s for MeOH and PE fractions had to be combined into one aliquot for polyacetylene p u r i f i c a t i o n . Subsequently, only one set of data was available for acetylenes at each time sampled and was not analyzed for var iance. A C C U M U L A T I O N A N D D I S T R I B U T I O N O F P H E N Y L H E P T A T R I Y N E I N BIDENS ALBA SEEDLINGS Seeds of B.alba c o l l e c t e d from greenhouse plants were germinated on damp f i l t e r paper in covered s t e r i l e p e t r i dishes at 22 ° C . Seedlings were kept in the dark for one week and then allowed to develop under white l i g h t (15 hour 85 photoperiod/day, "Universal White" fluorescent tubes). Whole seedlings were harvested at days 2, 4 and 5 of germination and leaves, hypocotyls and roots at days 7, 15, 20 and 24. A l l samples were weighed and crude l i g h t petroleum (BP 30-60°C) fractions prepared according to the method described in Chapter I I . Dried PE fractions were resuspended in 1.0mL spectral grade MeOH and 20uL samples fractionated by high performance l i q u i d chromatography (HPLC) on a Varian MCH-10 reverse phase column and Varian Model 5000 HPLC with Varian Series 634 variable wavelength UV detector. The UV detector was set at 250nm which i s the wavelength of maximum absorption (e = 167,000) for phenylheptatriyne (PHT). Separation of the crude fractions was achieved at a flow rate of 1.0mL/min with a solvent gradient proceeding from 70% CH3CN/20% H 20 to 100% CH3CN in 15 minutes. Retention time (R_, for PHT under these conditions was 11.94 mins. (=* 93% CH3CN). Quantitation of eluted peaks was performed by an SP4100 Integrator (Spectra-Physics) and these values expressed per unit fresh weight of tissue. 86 C. RESULTS BIOSYNTHESIS OF POLYACETYLENES FROM 1 *C02 IN BIDENS LEAVES Three species of Hawaiian Bidens, B.cosmoides, B. hi 11 e br andi ana ssp. polycephala, B. molokaiensis and B.alba var. radiat a were used in a series of time course tracer studies of polyacetylene biosynthesis. Choice of Hawaiian species was limited by the a v a i l a b i l i t y of healthy young plants which produce ea s i l y detectable leaf acetylenes in acceptable quantities. Bidens cosmoides leaves contain measurable amounts of compounds 1 and 5 (Table V) and were used in order to assess the pa r t i t i o n i n g of 1*C into these acetylenes. Bidens hi 11 ebrandi ana which produces mainly compound 1 (Table V), and B. molokaiensis, which does not have leaf acetylenes, were used as comparisons. Bidens alba produces PHT (4) in i t s leaves and numerous plants were grown from seeds in UBC greenhouses. Experiments with Hawaiian species were repeated twice and those with B. alba four times. In a l l experiments, plants were allowed to photosynthesize for 60 minutes in a 1 4C0 2-enriched atmosphere within the enclosed chamber (Figure 10). 1 < tC0 2 disappearance during the 60 minute pulse was monitored and the data presented in Table XIV. Conversion of NaH1ftC03 to 1*C0 2 was nearly complete (99.42%) for a l l experiments and to t a l uptake of 1 4C into the plants and percent 1 f tC incorporation into polyacetylenes shown in Table XV. TABLE XIV. '*C02 UPTAKE DURING 60 MINUTE PULSE-LABELLING OF B. ALBA* Time Total 1 a C 0 2 in Chamber (mins) (dpm) 0 5.44 X 10s + 6.2% 1 5 4.54 X 106 + 3.5% 30 2.80 X 10s + 7.2% 45 2.12 X 10 s + 2.2% 60 1 .32 X 106 + 3.8% 0.20mL air/sample (0.032% C0 2) or 0.0064 mL C0 2/sample t o t a l volume 1 S C 0 2 in chamber (80L) = 25.6mL therefore: t o t a l 1<lC02 in chamber at sampling = dpm/0.0064mL X 25.6mL * average of 4 experiments TABLE XV. EFFICIENCY OF 1*C UPTAKE* Percent conversion of NaH^COa to 1 4 C 0 2 99.4% Total 1*C0 2 in chamber: from 300 uCi NaHC03 6.73 X 108 dpm (298 uCi) from 500 uCi NaHC03 11.28 X 108 dpm (499 uCi) Percent t o t a l 1 4C uptake 1.0% (0.1% - 2.4%) Percent incorporation of 1 f lC into 2.9% acetylenes (0.1% - 12.9%) * data from 15 experiments 89 The uptake of 1*C into the MeOH and PE fractions and the polyacetylenes in leaves of the four Bidens species was determined for 12 hours and 168 hours (one week) after i a C 0 2 administration. In the 12 hour studies, samples were taken at two hour intervals and in the week long studies, samples taken once a day. Total amounts of acetylenes extracted were calculated for each time period and values expressed per gram fresh weight of leaves. These results are shown in Tables XVI to XXIII and Figures 12 to 21. In general, 0.1 to 2.0 percent of the administered r a d i o a c t i v i t y was incorporated into the methanol fractions and 0.02 to 12.9 percent of t h i s recovered in the polyacetylenes. Bidens alba was used in 24 hour time course studies where samples were taken at two hour i n t e r v a l s . Uptake of 1 4C into B.alba leaves i s shown in Table XXIV. Less than 0.1 percent of the administered l a b e l was incorporated into the methanol fr a c t i o n , from which 0.22 percent went into PHT. Analysis of variance for th i s data i s reported in Tables XXV and XXVI. The 24 hour accumulation of 1"C-PHT in B. alba leaves was s t a t i s t i c a l l y s i g n i f i c a n t . Differences between samples within each time period for both MeOH and PE components were also s i g n i f i c a n t . This i s c l e a r l y due to technical problems inherent in the extraction procedure. A l l the Bidens species used in these studies produce the ene-tetrayne-ene (1) in the roots. De novo acetylene biosynthesis in roots and leaves seem to occur independently (Van Fleet, 1970) but t h i s does not preclude translocation TABLE XVI. TWELVE HOUR UPTAKE OF 1«C INTO PHT BY B. ALBA LEAVES T I me (h o u r s ) MeOH dpm/gFW PE dpm/gFW PHT dpm/mg PHT ug/gFW 1 3.38 X 10' ± 5.7% 15.26 X 10' 3 3.32 X 10' ± 6.57. 9.79 X 10' 5 1.16 X 10" ± 2.4% 3.96 X 10 1 7 1.31 X 10' 1 4 4/„ 26. B1 X 10' 9 1.17 X 10' ± 5.1/. 5.68 X 10' 11 0.77 X 10* ± 3.27. 5.54 X 10' 13 0.75 X 10' ± 6.27. 30.77 X 10' + 1.9% 400 ± 2.4% 308 : 3.0% 2600 ± 1.9% 230 ± 8.9% 600 ± 8.1% 298 ± 6.5% 6000 ± 2.5% 237 ± 8.4% 38700 ± 6.3% 266 ± 7.4% 4 100 ± 2.9% 156 ± 6.0% 62500 + 4.5% 189 T o t a l p e r c e n t ''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 • 2.02 T o t a l p e r c e n t "'C I n c o r p o r a t i o n i n t o PHT f r a c t i o n =0.38 VO O TABLE XVII. TWELVE HOUR UPTAKE OF ''C INTO ENE-TETRAYNE-ENE (1) OF 3. HILLEORANOtANA LEAVES Time MeOH PE 1 1 (hours) dpm/gFW dpm/gFW dpm/mg 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 1 1 2 . 26 X 10' + 3 OX 229 + 6 97 12600 ± 3.2% 0.93 13 1 . 19 X 10' + 3 4% 197 ± 4 . 27 640O ± 5.5X 1.91 Total percent 1 4C Incorporation into MeOH f r a c t i o n = 0.457= Total percent 1'C incorporation into Compound 1 = 0.023% TABLE XVIII. TWELVE HOUR UPTAKE OF •«C INTO ACETYLENES 1 AND 5 OF B. COSM01DES LEAVES Time MeOH PE (hours) dpm/gFW dpm/gFW 1 4.89 X 10' ± 3.5/ 27 ± 1.5% 3 9.16 X 10' ± 6.1% 565 ± 3.4% 5 4.65 X 10» ± 2.7% 675 ± 2.4% 7 5.41 X 10 s ± 3.2% 19J6 ± 2.2% 9 2.84 X IO' i 3.11% 322 1 + 1.3% 11 1.04 X 10' ± 5.2% 1936 ± 6.7% 13 0.91 X 10' ± 4.8% 2153 ± 8.7% 1 5 t o t a l acetylenes dpm/mg dpm/mg ug/gFW 100 + 4.8% 600 • 4.8% 102 600 ± 3.1% 1600 ± 6.7% 33 400 ± 2.9% 800 • 2.8% 128 1900 i 6.9% 2100 ± 5.2% 28 5600 ± 7.2% 6200 +5.9% 19 1800 ± 7.1% 2600 ± 6.5% 47 4700 ± 5.8% 9000 ± 4.5% 20 Total percent '4C incorporation into MeOH f r a c t i o n = 1.01 Total percent ''C incorporation into Compound 1 = 0.089 Total percent '*C Incorporation into Compound 5 =0.044 Total percent ''C incorporation into t o t a l acetylenes = 0.13 TABLE XIX. TWELVE HOUR UPTAKE OF 1"C INTO MEOH AND FRACTIONS OF B. MOLOKAIENSES LEAVES Time MeOH PE (hours) dpm/gFW dpm/gFW 1 4.64 X 105 ± 5.9% 496 ± 2.6% 3 2.73 X 105 ± 2.4% 933 + 4.8% 5 3.81 X 105 ± 5.8% 240 ± 9.2% 7 1.06 X 105 ± 4.1% 1115 ± 5.8% 9 1.79 X 105 ± 5.9% 393 ± 9.7% 11 3.27 X 105 ± 3.1% 2633 ± 8.9% 13 2.09 X 105 ± 4.4% 3090 ± 6.3% Total percent '"C incorporation into MeOH fraction= 0.41 TABLE XX. ONE WEEK UPTAKE OF 1«C INTO PHT BY B. ALBA LEAVES Time MeOH PE PHT PHT (hours) dpm/gFW dpm/gFW dpm/mg ug/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 Total percent ''C Incorporation Into MeOH f r a c t i o n =0.15 Total percent 1*C Incorporation Into PHT = 12.8 VJD TABLE XXJ. ONE WEEK UPTAKE OF •*C INTO ENE-TETRAYNE-ENE M ) OF P. HILLfPKANOiANA LEAVES Time W=OH PE 1 1 (hours) dpm/gFW dpm/gFW dpm/mg ug/gFW 25 1 28 X 10' 4 . 1% 464 + 6.97. 4 1300 6 .2% 0 .37 49 1 5B X 10' + J .5/, 2595 t 8.57- 92100 • 3 .57- 0 80 73 O. 89 X 10' * 3 .97, 1 105 • 5 . 37- 40600 • 9 .2% 0 97 145 2 49 X 10' * 9 8"/- 128 i 7 .07 25800 • 6. .27. 0. 94 169 O 79 X 10' * 3 5/. 794 * 7 37. 46100 3 o 38 Total percent ''C Incorporation Into MeOH f r a c t i o n « 0.17 Total percent 1'C Incorporation Into Compound 1 =0.20 TABLE XXII. ONE WEEK UPTAKE OF '«C INTO ACETvLENES 1 AND 5 OF 8. COSMOWtS LEAVES Time MeOH PE (hours) dpm/gFW dpm/gFW 25 a. 59 X 10' + 4.97. 2.25 x 10* ± 4.4y. 49 6.66 x 10* ± 8 0" 1.26 x IO' ± 6.87 73 3 10 x 10" ± 5 27. 0.91 x 10' ± 6.8% 145 0.42 X 10" ± 1.8% 0.18 x 10' ± 1.1% 169 3.10 X 10* 1 9.2% 1.4 1 X 10* ± 2.4% 1 5 Total dpm/mg dpm/mg ug/gFW 1.6 X 10' ± 2.3% 2.6 X 10' ± 1.8% 35 6.7 X 10" ± 5.2% 4.52 X 10' ±3.9% 9 3.54 x 10* ± 8.1% 2.88 X 10' ± 4.5% 13 3 15 X 10' ± 6.6% 0.49 X 10' ± 4.8% 47 3.48 X 10' • 6.1% 0.72 X 10' ± 6.2% 24 Total percent • -c incorporation Total percent • 'C Incorporation Total percent • 'C Incorporation Total percent ' 'C Incorporation Into MeOH f r a c t i o n » 0.55 into Compound 1 = 0.59 into Compound 5 = 0.33 into t o t a l acetylenes = 0.93 97 TABLE XXIII. ONE WEEK UPTAKE OF 1 BC INTO MEOH AND PE FRACTIONS OF B. MOLOKAJENSIS LEAVES Time MeOH PE (hours) dpm/gFW dpm/gFW 25 1.15 X 10 5 ± 4.4% 1042 ± 6.2% 49 1.49 X 105 ± 8.5% 547 ± 7.5% 73 0.51 X 10s ± 9.1% 324 + 1.9% 97 7.38 X 105 ± 9.0% 907 ± 5.4% 169 0.86 X 10s ± 2.8% 8686 ± 1.0% T o t a l P e r c e n t '"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 = 0.07% TABLE XXIV. TWENTY-FOUR HOUR UPTAKE OF "C INTO PHT BY S. ALBA LEAVES T Ime (hours) MeOH dpm/gFW PE dpm/gFW PHT dpm/mg PHT ug/gFW 1 2 .94 X 10' 7 .8% 1224 ± 5.2% 11.23 ± 6 .4% 373 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 Total percent 1"C incorporation Into MeOH f r a c t i o n = 0.09 Total percent ''C incorporation Into PHT » 0.22 vo co TABLE XXV. "C UPTAKE INTO MEOH AND PE FRACTIONS OF P. ALBA LEAVES IN 24 HOURS* ANOVA: for dpm MeOH/gFW Source TIME SAMPLE AL I ERROR TOTAL D F 12 26 78 0 1 16 s.s. 8 34 x 10' ' 9 57 x 10'' 121 x 10" 0 1.80 X lO'' M.S. 6 95 X 10'" 3.68 X 10'' 1.44 X 10" F value 1.8873 256.0845 F prob. O.0851 O.OOOO Tested against 2 3 4 ANOVA for dpm PE/gFW: SOURCE D. F . TIME 12 SAMPLE 26 AL I 78 ERROR 0 TOTAL 116 S.S. 1.45 X 10' 1.07 X 10' 5.05 x 10' 0 2.57 x 10* M.S. 1.21 X 10 4 . 12 X 10" 6.47 X 10* F value 2 9323 63.6992 F prob. 0.0106 0.0000 Tested against 2 3 4 •Three-level nested design. Run *1. VO vo 100 TABLE XXVI. '*C UPTAKE INTO PHT(3): B. ALBA LEAVES IN 24 HOURS* ANOVA for dpm/mg (3): Source D.F. S.S. TIME SAMPLE ERROR TOTAL 12 26 0 38 138012 116337 0 254349 M.S. F value F prob. Tested against 11501 2.5704 0.0214 2 .4474 3 Two-level nested design, Run# 2. 101 of precursor molecules from leaf to root. Roots were harvested and extracted for compound 1 at the end of the 12 hour and week long experiment and the data in Table XXVII shows substantial incorporation of 1*C into the acetylene. ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS Two-day to 24-day seedlings of B. alba were extracted for PHT in the leaves, hypocotyls and roots. The r e l a t i v e amounts of PHT in each sample was determined using HPLC and the results shown in Table XXVIII and Figures 12 and 13. Phenylheptatriyne was present in two day old seedlings and lev e l s in leaves increased throughout the experimental period. Concentrations in the hypocotyls decreased after one week. The roots contained 100 times higher amounts of PHT than the a e r i a l tissues i n i t i a l l y although quantities began to decline after two weeks. TABLE XXVII. 1•OLABELLED ENE-TETRAYNE-ENE (1) IN ROOTS BIDENS GIVEN 1 SC0 2. Time (hours) P l a n t 1 dpm/mg ug/gFW 1 3 1 3 1 3 1 3 169 1 6 9 1 6 9 1 6 9 B. alba B. molokai ensi s B. cosmoi des B. hi 11 ebr andi ana B. alba B. molokai e ns i s B. cosmoi des B. hi I I ebr andi a na 1 2 0 7 8 7 0 0 0 0 2 6 8 2 5 0 0 0 2 2 3 5 6 6 5 0 2 6 9 2 4 6 8 9 9 . 0 0 . 1 1 1 . 3 1 . 0 1 4 . 7 7 . 1 2 . 7 1 . 0 1 03 TABLE XXVIII. ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE (4) IN B. ALBA SEEDLINGS Days FW mg/20uL mg/lOOOuL sample % area PHT/sample Rel.amt. PHT/mg sample Seedlings: 2 122.4 4 125.3 5 100.8 Leaves: 7 152.4 15 140.8 20 156.5 24 159.8 Hypocotyls: 7 150.1 15 180.0 20 199.3 24 169.9 Roots: 7 0.560 15 0.625 20 0.231 24 0.821 2.45 2.51 2.02 3.05 2.82 3.13 3.20 3.00 3.60 3.99 3.79 0.0112 0.0125 0.0046 0.0164 49.31 77.05 76.88 65.59 76.86 95.59 96.54 81.43 58.78 63.42 64. 15 26.37 46. 17 12.46 30.64 20 31 39 22 28 31 30 27 16 16 17 2355 3694 2709 1666 901 106 D. DISCUSSION BIOSYNTHESIS OF POLYACETYLENES FROM 1*C0 2 IN BIDENS LEAVES The term biosynthesis i s defined by Swain (1965) as the in vivo endothermic production of more complex molecules from simpler ones. Natural polyacetylenes are thought to be derived from fatty acid precursors which are systematically transformed into a dazzling array of compounds, some of which have potent photobiocidal effects (Bu'Lock, 1966; Bohlmann et al. , 1973; Jones and Thaller, 1978; Towers, 1979). At present, no real evidence exists concerning the in vivo formation of the carbon-carbon t r i p l e bond although dire c t dehydrogenation via cis double bonds was favoured speculatively (Bu'Lock and Smith, 1967), and appears probable (Haigh et al., 1968; Jones et al., 1975; Jones and Thaller, 1978). Nevertheless, the various biosynthetic i n t e r r e l a t i o n s h i p s of acetylenes such as modifications of the chain lengths, introduction of t r i p l e bonds, rearrangements, introduction of oxygen- and sulphur-containing groups and c y c l i z a t i o n s have been studied in great d e t a i l in the laboratories of F. Bohlmann and S i r Ewart R.H. Jones, mostly with the use of s p e c i f i c a l l y l a b e l l e d precursors obtained by t o t a l synthesis. These precursors include 1 4C- and/or 3H-labelled acetate, o l e i c and l i n o l e i c acids, crepenynic acid and dehydromatricaria 107 ester variously administered v i a the intact root or leaf surface or the fungal culture medium (Bohlmann et al . , 1968; Bohlmann and Schulz, 1968; Fairbrother et al., 1967; Jente and Richter, 1976; Jones, 1966). Unlike the situation with microorganisms where putative precursors can be ea s i l y and naturally fed to a defined system, the uptake of l a b e l l e d substances into plants i s often d i f f i c u l t and unsatisfactory, the results not necessarily a true r e f l e c t i o n of the in vivo situation (Swain, 1965; Brown and Wetter, 1972; Floss, 1977). The only way in which 1"C can be administered to plants in a physiological fashion i s as 1 t tC0 2 even though rates of incorporation into complex secondary metabolites may be rather low. This has not been previously demonstrated for polyacetylenes. In the present study, the de novo biosynthesis of polyacetylenes in Bidens leaves was investigated in time course studies. 1 " C - l a b e l l e d polyacetylenes were recovered from three species of Bidens f i r s t administered 1 0CO 2 and subsequently allowed to metabolize in 1 2C0 2 for 12, 24 and 168 hours (Tables XVI to XXIV). In general, the plants incorporated 0.1 to 2.4 percent (mean = 0.9%) of the administered r a d i o a c t i v i t y into the methanol fractio n from which 0.1 to 12.9 percent (mean - 2.9%) went into the polyacetylenes examined (Table XV). The range in values may be due to individual and/or i n t e r s p e c i f i c variations in photosynthetic rates since environmental factors (C0 2 108 concentrations, H20, l i g h t and temperature) were e s s e n t i a l l y uniform for a l l experiments. These differences are most pronounced between B. alba and B. mol okai enses in the 12 hour experiments. The B. molokaiensis plants available were older and evidently less vigorous than B. alba plants. In a l l experiments, peak s p e c i f i c a c t i v i t y in the methanol fractions preceded that in the l i g h t petroleum fractions where most of the l i p o p h i l i c compounds, including polyacetylenes, are found. Peak a c t i v i t y of polyacetylenes generally coincided with that of the l i g h t petroleum fr a c t i o n s . Specific a c t i v i t i e s of these components would be expected to r i s e i n i t i a l l y with increasing formation from 1*C0 2-derived intermediates and then f a l l as flushing with 1 2 C 0 2 proceeded. Total amounts of polyacetylenes isolated at each time period are expressed as micrograms per gram fresh weight of leaves. This information suggests that PHT (4) and the ene-tetrayne-ene (1) are synthesized at a r e l a t i v e l y constant rate in B. alba and B. hi 11ebrandiana leaves, respectively (Tables XVI, XVII and XX, XXI). Levels of acetylenes remain e s s e n t i a l l y uniform while s p e c i f i c a c t i v i t y fluctuates. Bidens cosmoides leaves synthesize compounds 1, 3 and 5 but only 1 and 5 were e a s i l y detected in the system used here (TLC p u r i f i c a t i o n ) . The data for acetylene lev e l s in B. cosmoides i s inconclusive and may be due to the exclusion of the pentayne-ene from the analysis. 109 According to the scheme in Figure 7, compounds 1 and 5 are synthesized in p a r a l l e l but separate pathways; one does not precede the other along the same sequence of reactions. In B. cosmoides, maximum s p e c i f i c a c t i v i t i e s of the two compounds occurs at the same time and decreases s i m i l a r l y . Either conversion from one acetylene to the other i s extremely rapid or both are synthesized concurrently. Compound 3 was not investigated in these experiments but a comparison of i t s 1*C-uptake rate with that of 1 and 5 may have been more enlightening. The highest s p e c i f i c a c t i v i t y of 1UC-PHT synthesized by B. alba was 0.0275 /zCi/mg PHT (6.25 X 10" dpm/mg), 12 hours afte r 300 MCi of 1 f tC0 2 was administered (Table XVI and Figure 14). In the 24 hour experiments, 500 uCi of 1 4 C 0 2 was fed to the plants and peak a c t i v i t y was even lower (0.0001 nCi/mg PHT or 234 dpm/mg) (Table XXIV) while that for the one week experiments was 0.0105 nCi/mg PHT or 2.38 X 10* dpm/mg (Table XX). These results r e f l e c t the differences in t o t a l 1*C incorporated into the plants during each of the experiments (2.0%, 0.1% and 0.2%) and i l l u s t r a t e the d i f f i c u l t y of co n t r o l l i n g the actual dose of 1*C fixed photosynthetically by whole plants. Other factors contributing to the variance may include r e l a t i v e differences in the metabolic a c t i v i t y of the biosynthetic s i t e s as well as differences in the pool sizes of the acetylene precursors, postulated intermediates and those of the f i n a l products. In any case i t appears that 1 10 the natural synthesis of 1 * 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 amount of r a d i o a c t i v i t y may not be possible using t h i s method. At the end of the 12 hour and 168 hour experiments, roots were extracted for polyacetylenes. 1 " C - l a b e l l e d ene-tetrayne-ene (1) was detected in a l l plants (Table XXVII), including B. mol okai ensi s , which incorporated 1"C into i t s M e O H and P E fractions (Tables XIX, XXIII), in spite of the fact that i t does not synthesize leaf acetylenes. This indicates that 1 " C - l a b e l l e d precursors were translocated from a e r i a l tissues to si t e s in the roots where de novo synthesis of root compounds takes place. ACCUMULATION AND DISTRIBUTION OF PHENYLHEPTATRIYNE IN BIDENS ALBA SEEDLINGS In mature B. alba plants, leaves contain mainly P H T (4) while the stems have comparable amounts of P H T and phenylhepta-diyne-ene (5) and the roots 5 as well as the ene-tetrayne-ene (1) (Norton, 1984). The accumulation of polyacetylenes in developing B. al ba plants has not been previously reported. Detectable levels of P H T were found in two-day old seedlings of B. alba, suggesting that polyacetylene biosynthesis begins during germination or soon thereafter (Table XXVII and Figures 12, 13). Quantities in the leaves continue to increase up to 24 days and presumably beyond 111 that to adult levels while amounts in the hypocotyls peak at seven days and subsequently decline to a lower concentration. This i s probably accompanied by a concomitant increase in the level s of compound 5 (Figure 12). PHT i s absent from the roots of mature Bidens alba plants (Towers, 1980; Norton, 1984) but is present in the seedlings. Relative PHT levels in the roots are 100 times higher than those in the a e r i a l tissues for the f i r s t 24 days. Nevertheless, there i s also a gradual decline in these levels beginning at two weeks and continuing beyond the experimental time period (Table XXVII and Figure 13). This is accompanied by a concomitant increase in levels of compounds 1 and 5 (data not shown). It appears that the di s t r i b u t i o n of PHT in B. alba reaches i t s adult proportions by one month after the onset of germination. 1 12 E. CONCLUSION The complete elucidation of a biosynthetic pathway requires the application of several d i f f e r e n t techniques. According to Adelberg (1953), these include isotopic l a b e l l i n g with precursors, in vitro enzyme studies and the use of microorganisms with blocked synthetic pathways. The main source of current knowledge of the pathways of acetylene biosynthesis are experiments of the f i r s t category (e.g., Bu'Lock, and Smith, 1963; Jones, 1966; Bohlmann et al., 1968). In fungal cultures, biosynthetic experiments are easier and give higher incorporations than those with plants, although a variety of alte r n a t i v e sequences may be available for both types of organisms (Jones and Thaller, 1978). In t h i s study, the de novo biosynthesis of polyacetylenes in the leaves of selected species of Hawaiian Bidens and Bidens alba was demonstrated using 1*C0 2. Levels of 1 < tC incorporated into the f i n a l products were minimal but a l l three acetylenes isolated were s i g n i f i c a n t l y l a b e l l e d . The v a l i d i t y of postulated biosynthetic sequences must be tested by more than one method, and i d e a l l y should be confirmed by the detection and i s o l a t i o n of enzymes catalyzing key steps in the pathway. Hawaiian species of Bidens should be useful organisms for in vitro studies for several reasons: they produce a limited array of acetylenes which are close l y related, d i f f e r e n t species produce d i f f e r e n t arrays in leaves and roots and may be selected for 1 13 p a r t i c u l a r compounds, i n t e r s p e c i f i c hybrids are ea s i l y produced and the plants are r e l a t i v e l y easy to propagate and maintain under standard greenhouse conditions. 1 1 4 F. BIBLIOGRAPHY Adelberg, E.A. 1953. The use of metabolically blocked organisms for the analysis of biosynthetic pathways. B a c t e r i o l . Rev., 17: 253 - 267. Bohlmann, F., H. Bonnet and R. Jente. 1968. Uber die biogenese des phenylheptatriins. Chem. Ber. 101: 855 -860. Bohlmann, F. and R. Jente. 1966. Zur biogenese der phenylpolyine. Chem. Ber. 99: 995 - 1001. Bohlmann, F. and H. Schulz. 1968, Uber die bildung von polyinen mit z e l l f r e i e n homogenaten. Tetrahedron Letters, 4795 - 4798. Brown, S.A. and L.R. Wetter. 1972. Methods for investigation of biosynthesis in higher plants. pp1-45 in Progress in Phytochemistry: Volume II. L. Reinhold and Y. Liwschitz (Eds.). John Wiley and Sons, London. Bu'Lock, J.D. 1966. The biogenesis of natural acetylenes. pp79-95 in Comparative Phytochemistry. T. Swain (Ed.). Academic Press, London. Bu'Lock, J.D., D.C. Al l p o r t and W.B.Turner. 1961 (Part I I ) . The biosynthesis of Polyacetylenes. Part I I I . Polyacetylenes and triterpenesin in Polyporus ant hracophi I us . J . Chem. Soc. 1654 - 1662. Bu'Lock, J.D. and H. Gregory. 1959. The biosynthesis of polyacetylenes. 2. Origin of the carbon atoms. Biochem. J. 72: 322 - 325. Bu'Lock, J.D. and G.N. Smith. 1963. Acetylenic fatty acids in seeds and seedlings of sweet quandong. Phytochemistry 2: 289 - 296. Bu'Lock, J.D. and G.N. Smith. 1962. Polyacetylenic acids in developing seedlings of sweet quandong. Biochem. J. 85: 35P. Fairbrother, J.R.F., S i r E.R.H. Jones and V. Th a l l e r . 1967. Natural acetylenes. Part XXV. The biosynthesis of benzenoid polyacetylenes. J . Chem. Soc. (C): 1035 1039. Floss, H.G. 1977. Radiotracers in biosynthetic studies, pp. 689-732 in Radiotracer Techniques and Applications. Vol 2. E.A.Evans and M.Muramatsu (Eds.). M. Dekker Inc. Ganders, F.R. and K.M. Nagata. 1984. The role of 115 hybridization in the evolution of Bidens on the Hawaiian Islands. P. 179-194 in 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. Relationship and f l o r a l biology of Bidens cosmoides (Asteraceae). Lyonia 2: 23 - 31. Ganders,F.R. and K.M. Nagata. 1983b. New taxa and new combinations in Hawaiian Bidens (Asteraceae). Lyonia 2: 1 - 16. Haigh, W.G., L.J. Morris and A.T.James. 1968. Acetylenic acid biosynthesis in Crepis rubra. Lipids 3 : 307 - 312. Helenurm, K. and F.R. Ganders. 1985. Adaptive radiation and genetic d i f f e r e n t i a t i o n in Hawaiian Bidens. Evolution (in press). Jente, R. and E. Richter. 1976. Zur biosynthese des dehydromatricanaesters. Phytochem. 15: 1673 - 1679. Jones, S i r E.R.H. 1966. Natural polyacetylenes and their precursors. Chem. in B r i t . 1966: 6 - 13. Jones, Si r E.R.H. and V. Thal l e r . 1978. Natural acetylenes, pp. 621-633 in The C h e m i s t r y of the Carbon-Carbon T r i p l e Bond. P a r t 2. S. Patai (Ed.). John Wiley and Sons, New York. Jones, S i r E.R.H., V. Thaller and J.L. Turner. 1975. Natural acetylene. Part XLVII. Biosynthetic experiments with the fungus Lepista di emi i (Singer). Biogenesis of the C 8 acetylenic cyanoacid diatretyne 2. J. Chem. S o c , Perkin Trans. I : 424 - 428. Marchant, Y.Y., F.R. Ganders, CK. Wat and G.H.N. Towers. 1984. Polyacetylenes in Hawaiian Bidens. Biochem. Syst. E c o l . 12: 167 - 178. Norton, R.N. 1984. Studies of Polyacetylene production in normal and transformed tissue cultures of Bidens alba. Ph.D. Dissertation, University of B.C. Parikh,V.M. 1974. A b s o r p t i o n S p e c t r o s c o p y of O r g a n i c M o l e c u l e s . Addison-Wesley, Ontario. Swain, T. 1965. Methods used in the study of biosynthesis, pp. 9-36 in B i o s y n t h e t i c Pathways i n Hig h e r P l a n t s . J.B.Pridham and.T. Swain (Eds.). Academic Press, New York. Towers, G.H.N. 1980. Photosynthesizers from plants and their photodynamic action. Prog. Phytochem. 6:183 - 202. 116 UBC ANOVAR. 1978. Analysis of variance and covariance. Adapted by M.Greig and D. O s t e r l i n . UBC Computing Centre. Van Fleet, D.S. 1970. Enzyme l o c a l i z a t i o n and the genetics of polyenes and polyacetylenes in the endodermis. Adv. Frontiers P i . S c i . 26 : 109 - 143. Wiermann, R. 1981. Secondary plant products and c e l l and tissue d i f f e r e n t i a t i o n , pp. 86-116 in The Biochemistry of Plants. A Comprehensive Treatise. Vol.7. P.K. Stumpf and E.E.Conn (Eds.). Academic Press, New York. Weiss, U. and J.M. Edwards. 1980. The Biosynthesis of Aromatic Compounds. J . Wiley and Sons. Toronto. IV. PHOTOTOXICITY OF POLYACETYLENES TO PHYLLOPLANE FUNGI A. INTRODUCTION A l l Hawaiian Bidens possess acetylenes in the roots but only 15 taxa synthesize them in the leaves (Marchant et al .,1984). There appears to be no s i g n i f i c a n t correlation between the presence or absence of leaf acetylenes and any other feature of Bidens, including habitat (F.R.Ganders, pers. comm.). Nearly a l l rainforest species (e.g., B. cosmoides and B. macrocarpa) have leaf acetylenes, but species with and without leaf acetylenes occur in drier habitats and lower elevations. Since morphological, genetic and biochemical data suggest that a l l native Hawaiian Bidens have evolved from a common ancestor (Ganders and Nagata, 1983a; 1983b; 1984; Helenurm and Ganders, 1985; Marchant et al.,1984), the genetic information for de novo acetylene synthesis in leaves and roots i s probably present in Bidens but i s not expressed in the leaves of ce r t a i n taxa. This phenomenon i s not unique to Hawaiian Bidens species or even to Bidens in general. Bidens cernua L. (Bohlmann el al.,1973), B. tripartita L. (Bohlmann et a/.,1962), B. pilosa var. minor (Blume) Sherff (Norton, 1984), as well as other species of Compositae (e.g., Artemesia vulgaris L., Chrysanthemum douglasii Hulten) (Bohlmann et al.,1973) do not synthesize leaf acetylenes, although they do so in the roots. This may be merely fortu i t o u s . 117 1 18 Nevertheless many polyacetylenes are powerful photosensitizers (Towers, 1980) and are toxic to a wide range of organisms, including bacteria and fungi (Arnason et a/., 1980; Camm et al . , 1975; Chan et al., 1975; DiCosmo el a/.,1982; Towers et a/.,1977; Wat el al .,1977). This information has led to speculation about the possible b i o l o g i c a l significance of polyacetylenes in plants. Do these compounds have a s p e c i f i c function or set of functions in t h e i r parent plants? Does their presence s i g n i f y a defense strategy against s p e c i f i c enemies or against a l l po t e n t i a l l y threatening organisms? If so, why are they absent from the leaves of some species of Bidens? Do bioassays ca r r i e d out on t y p i c a l laboratory organisms such as Escherichia coli (Migula) Cast. et Chalm. and Candida albicans (Robin) Berkh. have relevance to the ecological conditions faced by Bidens cosmoi des or other species growing in the wet jungle of Kauai? The present study i s a preliminary attempt to answer some of these questions. The a e r i a l surfaces of higher plants growing under natural conditions are usually colonized by large and varied populations of microorganisms. Such populations colonizing leaf surfaces form an ecological niche which i s termed the phylloplane (Last and Price, 1969; Davenport, 1976; Dickinson, 1976). Comparisons of the fungal populations on the leaves of d i f f e r e n t plants have led some authors to consider that one surface i s very l i k e another (DiMenna, 1971; Ruscoe, 1971), and although the majority of 119 phylloplane studies have been carr i e d out in temperate regions and on a g r i c u l t u r a l crops, the few studies which have hitherto been published on t r o p i c a l plants suggest that many phylloplane fungi are cosmopolitan in d i s t r i b u t i o n (Dickinson, 1976). These fungi grow on a wide range of host plants and populations, and species of fungi, p a r t i c u l a r l y in temperate regions, are very similar (Last and Deighton, 1965). Nevertheless, Lamb and Brown (1971) examined Pas pal um dilatatum L. (Poaceae), Salix babylonica L. (Salicaceae) and Eucalyptus stellulata Sieb. ex DC. (Myrtaceae) growing together on a creek bank and.found that the leaves of these plants support d i s t i n c t i v e microbial populations. They suggest that the phylloplane i s a niche which i s unfavourable for many of the organisms present in the airborne f l o r a and that the leaf exerts sel e c t i v e pressure which determines the nature of the resident phylloplane microflora af t e r the inoculum has come into contact with i t . One of the objectives of th i s study was to isol a t e and id e n t i f y the phylloplane yeasts and yeast-like fungi on species of Hawaiian Bidens and on selected sympatric plants. The second purpose was to fi n d out whether the occurrence and d i s t r i b u t i o n of phylloplane fungi was correlated with the presence or absence of leaf acetylenes in Bidens . F i n a l l y , the s e n s i t i v i t y of these organisms to polyacetylenes was assessed and the b i o l o g i c a l role of acetylenes in leaves was evaluated. 120 B. MATERIALS AND METHODS PLANT MATERIAL Healthy green leaves from 12 taxa of Hawaiian Bidens and 23 sympatric species of other plants were c o l l e c t e d in August, 1983 and February, 1984, placed in paper envelopes, sealed and allowed to dry during a i r transfer from Hawaii to Vancouver, Canada (Tables XXIX and XXX). ISOLATION AND IDENTIFICATION OF FUNGI Three methods were used to is o l a t e phylloplane yeasts and yeast-like fungi from a l l the leaves: the spore f a l l method (Last, 1955), the leaf impression method (Potter, 1910; Lamb and Brown, 1970) and the leaf disc method (Petrini et al., 1982; C a r r o l l and C a r r o l l , 1978). A l l isol a t e s were cultured in a malt extract medium with te t r a c y c l i n e (MYPT), (Bandoni, 1972), (Table XXXI). In the spore f a l l method (A), the dried leaves were f i r s t rehydrated by soaking in s t e r i l e water for 30 minutes, then placed in a p l a s t i c bag with a wad of moistened tissue paper, sealed and kept thus for 24 hours. Whole leaves were then rinsed in s t e r i l e water, dried and attached to the l i d s of s t e r i l e p e t r i dishes, abaxial or adaxial surfaces exposed to the MYPT agar below. This method i s a selective one which favours the i s o l a t i o n of members of the Sporobolomycetaceae by allowing their ballistospores to drop from leaf samples onto the nutrient medium. TABLE XXIX. ft I DENS TAXA SAMPLED FOR PHYLLOPLANE FUNGI Taxa LocalI t l e s S i t e d e s c r i p t i o n s 3. amp tec tens Man i n ! P a l l . Oahu 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 , north end of the Waianae Range, In scrub vegetation of Lucaena Ieucocephala. Canthium odoratum. Myoporlum sandwlcense and Slda r al I ax 8. cervicata Makaha Ridge. Kauai On roadside and open areas on dry ridge i n planted pine f o r e s t : area r a i n f a l l about 40" per year: with 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 State Park near Rainforest of Acacia koa. Metros Ideros with Cyanea Waimea Canyon overlook. and other p l a n t s : about 1000— e l e v a t i o n . kaua 1 8. forbesii ssp. f o r b e s i i Lumahai Beach overlook, 20-30m e l e v a t i o n on steep co a s t a l b l u f f s above K.ma i beach; in wet (75-iOO" r a i n per year) but rather scrubby vegetation with t'anrtantis and Met ros i deros . —» to B. nmra'ens's P. M I IetsrsndI ana ssp. potycephaia B. maul ens Is Graveyard near Kalmu. Hawa11 West side of MaHko Bay Maul Near Chinese cemetery, WaIhee, Mau1 B. menriesil ssp HI Iformls Ahumoa. Hawa11 Open mesle HfefrosIderoa forests end open f i e l d s on ol d a'a lava flows; about 30m elevation; r a i n f a l l about 75" per year. On top of sea c l i f f s . 30-40* elevation; on windward side of island, exposed to ocean spray: bare s o l i between plants on the v e r t i c a l c l i f f face and introduced weeds on top. On windy, exposed, dry l l t h l f i e d sand dunes on the windward coast of west Maul; elevation 50m: with vegetation of native low shrubs and herbs; r a i n f a l l about 30" On leeward slope of Mauna Kea, 1n open scrub and open dry forests of Sophora and Myoporum on the cinder cone of Ahumoa: about 2000m elevation; r a i n f a l l about 30" per year. 0. mlcrantha ssp. etenoohyila Old quarry near Kallua, Kona, Hawa11 8. popu11f olI a B. sandvicensls ssp. confusa B. sandvfcensfs ssp. sandv fcensIs South Mdge bordering Kahana Valley. Oahu IHau nature t r a i l , Walmea Canyon. Kauai Waahlla Ridge, Oahu Watmea Canyon, Kauai At about 30m elevation on ancient a'a lava flow; In arid, open scrub of Schfnus. tfalfherfa. sida and grasses; very low ra i n f a l l , about 10-15" per yaar. In clearings on ridge about 70-100m elevation; wat Wef/rosfderos forest with Pandanus, Schfnus and Srhef flera tn open scrubland with WlIkesla, Oodonaea and StypheMa: elevation about 700m: In open meslc forests of Acacia koa and Metrosfderos at about 900m elevat Ion. On exposed crest of ridge in mas 1c scrub vegetation; with Slda. Osteomeres and Acacia koa: about 400m elevation. About 350m elevation; in dry scrub of Oodonaea and introduced shrubs and herbs. oo 124 TABLE XXX. PLANTS ASSOCIATED WITH BIDENS SAMPLED FOR PHYLLOPLANE FUNGI Bidens Associated Plant Species B. amplectens Cant hi urn odor alum Forst. f. 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 (Forst) Gray Stachyl arpheta jamaicensis (L.)Vahl. Bidens pi Iosa L. B. hi 11 ebrandiana hi coliana glauca Grah. SSp. polycephala Emilia sp. Trifoliurn sp. V 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 A.Gray B. populifol ia St achytarphela jamaicensis (L.)Vahl. Pas si fI or a sp. Ost eomel es ant hylI idifol ia L i n d l . Euphorbia sp. yfi ks t r oemi a Sp. B. sandvicensis ssp. Styphelia lamehameha (Cham.) F. confusa Muell. Dodonae a Sp. Yi'ilkesia gymnoxi phi urn A.Gray Walt heria americana L. Lant ana camara L. B. sandvicensis Passiflora sp. ssp. sandvicensis Acacia koa A. Gray St achyt arphela jamaicensis (L.)Vahl. Si da f alI ax Walp. Ost eomeles ant hylIidifolia L i n d l . 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 ) C U L T U R E MEDIUM Ingredients grams / l i t re malt extract 7 . 0 yeast extract 0.5 soytone 1 0 . 0 bacto agar 1 5 . 0 t e t r a c y c l i n e HC1 0 . 0 5 1 27 The leaf impression method (B) was f i r s t described by Potter (1910) who used t h i s technique to demonstrate the presence of fungi and bacteria on Solanum and Helianthus leaves. Since then, t h i s method has been regularly used to provide data on the readily detachable component of the phylloplane saprophyte population. It provides an indication of the frequencies of both resident and transient fungal populations. Whole leaves were washed in s t e r i l e water and dried. Each leaf was placed, adaxial or abaxial surface up, on the agar surface, l i g h t l y pressed on the medium and subsequently removed. The leaf disc method (C) was used to is o l a t e endophytic organisms. It was modified from the methods described for conifer needles by P e t r i n i et al. (1982) and C a r r o l l and C a r r o l l (1978). Leaves were washed in s t e r i l e water, then dipped b r i e f l y in 50% EtOH and transferred to a solution of 3.5% NaOCl:H20 (1:9) for one minute. After a f i n a l rinse in s t e r i l e water, several discs of 10 mm diameter were cut from each leaf and transferred to the agar surface, covered and allowed to incubate. In a l l three methods, prepared plates were incubated at 23°C (12 hour light/dark cycle) for up to 48 hours. Yeast and y e a s t - l i k e fungal colonies were s e l e c t i v e l y transferred to new agar plates u n t i l pure cultures were obtained. Cultures are kept refrigerated at 4°C and transferred every two months. 1 28 The fungal i s o l a t e s were i d e n t i f i e d by the Centraalbureau voor Schimmelcultures, P.O. Box 273, Oosterstraat 1, 3470 AG Baarn, Netherlands. PHOTOTOXICITY ASSAYS The photosensitivity of selected test organisms to the crude l i g h t petroleum extracts of Hawaiian Bidens leaves and that of isolated Hawaiian phylloplane fungi to several polyacetylenes was assayed using the method of Daniels (1965). Phylloplane fungi tested are l i s t e d in Table XXXII. Other test organisms included the yeasts Saccharomyces cerevisiae and Candida albicans , the gram p o s i t i v e bacteria Staphylococcus al bus , Streptococcus faecalis and Bacillus subtil is ,• and the gram negative bacteria Escherischia col i , , Pseudomonas fluorescens and P. aeuroginosa. Cultures of bacteria and yeasts were obtained from the UBC culture c o l l e c t i o n . Sabouraud's medium was used for the yeasts and nutrient agar plates for the bacteria. A l l phylloplane fungi were cultured on MYPT agar plates. Forty-eight hour l i q u i d cultures in MYPT were streaked on agar plates with s t e r i l e cotton swabs. Light petroleum fractions and test compounds were dissolved in 95% EtOH at concentrations of 0.2 mg/mL, 1.0 mg/mL and/or 2 mg/mL. Five m i c r o l i t r e s of each solution ( i . e . , 1 jig/disc, 5 Mg/disc and 10 Mg/disc) were applied to paper discs, 7mm in diameter, prepared from Whatman No.1 f i l t e r paper and the solvent allowed to dry in the dark. 8-Methoxypsoralen was used as a 1 29 TABLE XXXII. YEASTS AND YEAST-LIKE FUNGI ISOLATED FROM HAWAIIAN PLANTS Class Basidiomycetes Sporobolomycetaceae Cryptococcaceae Class Fungi Imperfecti But(era sp. Rhodoiorula gr ami ni s di Menna R. mucilaginosa (Joerg.) Harrison R. pal 11da Lodder Sporobol omyces salmoni col or (Fischer et Brebeck) Kluyver et van Ni e l S. shi bat anus (Okunuki) Verona et C i f e r r i S. roseus Kluyver et van Niel Tilleliopsis pallescens Gokhale Cryptococcus albidus (Saito) Skinner C. laurentii (Kuff) Skinner C. luteolus (Saito) Skinner Alternaria tenuissima (Kze:Fr.) Wiltsch Aureobasidi urn put Iulans (de Bary) Arnaud Cladosporium cladosporiodes (Fres.) de Vries C. c f . cladosporiodes (yellow pigment) 1 30 Col I el otrichum gloeosporiodes (Pe'nzig) Penzig et Sacc. Epi coccum pur purescens Ehrenb. Hyphozyma variabilis de Hoog et M. Th. Smith Phoma sorghina (Sacc.) Boerema et a l 131 reference photoactive compound (Fowlks et al., 1958). The discs were placed on the inoculated plates which were prepared in duplicate. Test plates were exposed to longwave UV-A l i g h t (320-400nm) in a Psycrotherm incubator for two hours. Four Sylvannia black l i g h t s , F20T12-BLB, with irradiance of 10 watts/m2 at 10 cm from source, measured with a Y S I-Kettering Model 65 radiometer, were used. The controls were kept in the dark. A l l phylloplane organisms were subsequently incubated at 23°C for 48 hours. Saccharomyces, Candida and b a c t e r i a l species were incubated at 37°C. Compounds which produced zones of i n h i b i t i o n of microbial growth only upon i r r a d i a t i o n are phototoxic. Those samples which gave similar sizes of zones of i n h i b i t i o n both in l i g h t and dark are a n t i b i o t i c . A l l assays were repeated three to fi v e times and the resu l t s combined. COMPARISON OF PHOTOTOXICITY OF SELECTED POLYACETYLENES TO CRYPTOCOCCUS LAURENTII A 48 hour stationary culture of Crypt ococcus laurentii grown in Malt Yeast Peptone (MYP) broth at 23°C was diluted to approximately 1.69 X 10* cells/mL in fresh medium. One hundred m i c r o l i t r e aliquots of t h i s suspension were added to wells of s t e r i l e microtitre plates (Nunc-96FB with l i d s ) using a Titertek Multichannel pipette. A series of nine two-fold d i l u t i o n s of phenylheptatriyne (PHT, compound 4), phenyl-diyne-ene (compound 5) , heptadeca-tetraene-diyne 1 32 (compound 8) and a-terthienyl (compound 21) were made with growth medium (MYP). Six replicates of 100UL of each d i l u t i o n were added to the test wells containing yeasts. The s t a r t i n g concentrations were 5 nq/mh or 10 uq/mL with a maximum of 1% EtOH to avoid solvent t o x i c i t y . One row in each plate was set up as a control, with growth medium only. For each compound, one plate served as the dark control (0'), and test plates were i r r a d i a t e d with long wave UV (320-400nm) for 5,10 and 20 minutes. A l l plates were subsequently incubated at 23°C. The o p t i c a l density (O.D.), at 492 nm, of each solution in each of the test wells was read before i r r a d i a t i o n ( T 0 ) , and at 19 hours ( T 1 9 ) , 24 hours (T 2«), 40 hours (T f t 0) and 48 hours ( T a 8 ) l a t e r . TEST COMPOUNDS Crude l i g h t petroleum (BP 30-60°C) extracts and compounds 1,4, 5,7,8 and 9 were prepared and isolated from selected species of Hawaiian Bidens according to the methods described in Chapter II (Table XXXIII). Compound 21, a-terthienyl was a g i f t from Thor Arnason, University of Ottawa, compound 22 thiarubine A was i s o l a t e d by Alex Finlayson from Eriophyllum lanatum (Pursh) Forbes (Norton et al. , 1985). 8-Methoxypsoralen was obtained from Sigma Chemical. A l l compounds were dissolved in 95% EtOH at 0.2, 1.0 or 2.0 mg/ml. 1 33 TABLE XXXIII. POLYACETYLENES USED FOR PHOTOTOXICITY ASSAYS CE= CH(C=C) ACH=CH-CH3 o -= C ) 3 -CH3 C)_-CH=CH-CH. C ) o-CH=CH-CH20C0CH3 CH3-CH=CH ( C = C ) 2(CH=CH) 2 (CHj) 4>CH=CH2 Q CH 3-(C=C) 3(CH=CH) 2(CH 2) 4-CH=CH 2 9 CH,-C=C-(/ \)-(C=C) 2-CH=CH 2 22 134 C. RESULTS ISOLATION, IDENTIFICATION AND DISTRIBUTION OF PH7LL0PLANE ORGANISMS Leaves from 12 species of Bidens and 23 species of associated t r o p i c a l plants were coll e c t e d from 13 d i f f e r e n t l o c a l i t i e s in Hawaii in August, 1983 and February, 1984 (Tables XXIX and XXX). Nineteen taxa of yeasts and yeast-like fungi belonging to the Sporobolomycetaceae, the Cryptococcaceae and the Fungi Imperfecti were s e l e c t i v e l y i s o l a t e d and samples of isolat e s i d e n t i f i e d by the Centraalbureau voor Schimmelcultures (Table XXXII) (Kreger-van R i j , 1984; 1965; Barnett et al., 1979). Three methods were used for the i s o l a t i o n of microorganisms, each one with i t s pa r t i c u l a r advantages and li m i t a t i o n s (Potter, 1910; Last, 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 isolated by each method i s presented in Tables XXXIV to XXXVI. On agar plates inoculated with d i l u t i o n s or leaf washings, yeasts such as Sporobol omyces are usually overgrown by other fungal species. Separation can be effected by allowing these organisms to discharge their spores so that they f a l l on the culture medium (Last, 1955). The spore f a l l method also provides an indication of the ac t i v e l y growing and sporulating fungi on the surfaces of leaves. This 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 0. sandvfcensfs ssp. confusa Styphel I a tamehameha Oodonaea sp. Wllkesla gymnoxlphlum Waltheria amerlcana Lant ana camara Site 6: Waahila Ridge, Oahu B. sandvfcensfs ssp. sandvfcensfs Pass Ifiora sp. Acad a koa sp. Stachytarpheta Jamalcensls SI da rail ax Osteomeies anthylIIdlfolI a' 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 odoratum 11f ma sp. Myoporum sandttlcen»9 S i t e 10: Ahumoa. Hawaii 8. menzleslI ssp. f i l i f o r m i s * less 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 *•** greater than 8 c o l o n i e s / p l a t e ++•+ covers 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 Isolated r f f f e t f o p s f s pallescens 138 by a c t i v e l y growing fungi are more l i k e l y to be liberated and f a l l to the n u t r i t i v e agar surface under the action of gravity than spores which are present by chance and merely adhere to the leaf surface (Lamb and Brown, 1970). Several species of Sporobol omyces and Crypt ococcus were isolated with this method (Table XXXIV). Bui I era and Tilletiopsis pallescens Golkhale were detected once, from the leaves of B. cosmoides , whereas Cladosporium cI adospori odes (Fres.) deVries was frequently i s o l a t e d this way, probably because i t s spore discharge i s affected by humidity changes (Dickinson, 1971). In general, Cladosporium appears to be the most commonly occurring species found in thi s study, being present on leaves of a l l plants from a l l l o c a l i t i e s sampled by each method (Tables XXXIV to XXXVI). Tilletiopsis, a mycelial genus, i s reported to be regularly isolated from leaves (Last, 1955; 1970; Ruscoe, 1971; Pady, 1974; Dickinson, 1976). Its rare occurrence in t h i s study may be because of two reasons. It appears to require a higher growth temperature than Sporobol omyces and i t grows more slowly on laboratory culture media so that i t may be eas i l y overlooked in spore f a l l i s o l a t i o n plates (Dickinson, 1976; Last and Price, 1969). If a species i s detected by the spore f a l l method, i t is l i k e l y to be an active saprophyte and therefore sporulating on the leaf surface. It therefore should also be isolated by the leaf impression method. This c o r r e l a t i o n i s demonstrated by the data in Table XXXV where the presence of TABLE XXXV. DISTRIBUTION OF FUNGI ISOLATED WITH THE LEAF IMPRESSION METHOD Plants Sp Rh Cry Clad Epl Aureo Coll S i t e 1: Mallko Bay. Maul 8. hiI Iebrandlana ssp. pofycephafa NlcotI ana glauca EmfII a sp. Trlfollum sp. S i t e 2: Kokee State 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 S i t e 4 : Kohana Valley, Oahu B. popu11folI a St achyt arphet a Jamalcensls Pass!fI or a sp. Osteomeles anthyI 11dlfolla Euphorbia sp. WIkstroemla sp. + 4 •f +++••• 2++ + + +++ +++ 4-4-• + + GO Site S: Walmea Canyon, Kauai B. sandvfcensfs ssp. confusa Styphella tamehameha Oodonaea sp. Wllkeala gymnoxlphlum Walthena amerlcana Lantana camera 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 Site 8: Cemetery, Waihee, Maul 8. maufensfs Stachytarpheta jamalcensls LIpochaeta ssp. 11Ima ssp. Scaveola taccada Waltheria amerlcana 4.4.4. 4-4.4. 4-4-4-4-4. 4-4-4-4-+ 4.44. 4.44.4-24.4.4. 4-4-4. 4.+. + 4-4-4-4. 24.4.4. O Site 9: Manlnl Pall. Oahu P. ampfeetens Canthlum odoratum 11fma 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 +++ - •+ ++* ++ *• 2*++ - +* 2+*+ - +• + 4.4-142 Sporobol omyces, Crypt ococcus and CI adosporium species i s recorded. Rhodotorula species and Aur eobasi di um pulI ul ans (de Bary) Arnaud are also important primary leaf colonizers which adhere to the phylloplane surface (Last and Price, 1969; Pugh and Buckley, 1971), whereas Epicoccum pur purescens i s believed to be a phylloplane invader which exhibits a pattern of r e s t r i c t e d development u n t i l conditions on the leaf surface become p a r t i c u l a r l y favourable (Dickinson, 1976). A l l three were isolated by the leaf impression method. Col I et ol ri chum gl oeos por i odes (Penzig) Penzig et Sacc. is a pathogenic species which invades leaf tissue (Blakeman et a l . , 1971; Marks et a l . , 1965). It was isolated mainly from s u r f a c e - s t e r i l i z e d leaves using the leaf disc method, (Table XXXVI) but was also found using the leaf impression method, presumably because the organism exists on the leaf before i t enters the tissue (Blakeman, 1971; Marks et al., 1965). Species of CI adospori um, Epicoccum and, rarely, Crypt ococcus and Sporobolomyces were also isolated from surface s t e r i l i z e d leaves (Table XXXVI). The f i r s t two genera have been reported to grow ac t i v e l y within the leaf and C. cladospori odes forms microsclerotia which are able to withstand desiccation and probably other adverse environmental factors (Pugh and Buckley, 1971; Ruscoe, 1971; Dickinson, 1976). One of the objectives of t h i s study was to establish whether the occurrence and d i s t r i b u t i o n of phylloplane fungi Plants TABLE XXXVI. DISTRIBUTION OF FUNGI ISOLATED WITH THE LEAF DISC METHOD Sp Rh Cry Clad Ep1 Aureo Coll Site 1: Maliko Bay. Maul B. hi IIebrandlana ssp. polycephala Nlcotlana glauca Eml11a sp. T r l f o l I u m 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 ssp. f o r b e s i i Ageratum sp. Metroslderos c o l l l n a Stachytarpheta Jamalcensls 0. p i l o s a Site 4: Kohana Valley, Oahu 0. p o p u l I f o l l a Stachytarpheta Jamalcensls P a s s l f I o r a sp. Osfeomeles anthylI IdlfolI a Euphorbla sp. Wlkstroemla sp. + +++ Site S: Walmea Canyon, Kauai B. sandvicensis ssp. confusa Styphella tamehameha Dodonaea sp. Wlikesla sp. Waltherla amerIcana Lantana camara 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 sp. Waft her ia americana S<t( 9: Martini Pall. 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 i s correlated with the presence or absence of leaf acetylenes in Bidens. The data shown in Table XXXVII indicates that at least non filamentous saprophytes do not seem to distinguish among Bidens species. Nevertheless, Col I etr otTi chum was found in only two of the f i v e (40%) leaf acetylene producing Bidens species, and i s absent from Bidens which produce the C 1 3 aromatic compounds 5 and 7 (Tables I I I , IV). It was detected in five of seven (71.4%) Bidens species without acetylenes. In addition, Aureobasi di um pullulans, a common phylloplane organism, was isolat e d from only one of the f i r s t group of Bidens (20%) and from three out of four species of the second. A l l except three Bidens taxa were hosts to at least f i v e of the seven fungi l i s t e d . Bidens hawaiensis and esp e c i a l l y B. menziesii ssp. filiformis and B. mi crantha ssp. ct enophylI a were a l l co l l e c t e d from a r i d to semiarid exposed s i t e s on the island of Hawaii (Table XXIX) which would be expected to have a lower d i v e r s i t y of fungal species than a t y p i c a l rainforest l o c a l i t y such as that of B. cosmoides (Dickinson, 1967; 1976; Dickinson and O'Donnell, 1977; Ruinen, 1961). PHOTOSENSITIVITY OF MICROORGANISMS TO ACETYLENES The l i g h t petroleum fractions of leaf and root extracts from species of Hawaiian Bidens were tested for phototoxicity against nine species of fungi and bacteria 147 TABLE XXXVII. DISTRIBUTION OF YEASTS AND YEAST-LIKE FUNGI AMONG HAWAIIAN BIDENS Bidens with leaf Sp Rh Cry Clad Epi Aureo C o l l acetylenes B. hi 11 ebr andi ana + + + + - + B. cosmoi des + B. cervical a • - + + + B. sandvi ce ns i s ssp. + • + + + + confusa B. hawaiensis + + - - - + Bidens w i thout l e a f a c e t y l e n e s B. forbesii ssp. + + + + + forbesii B. mauiensis • + - + + - • 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 148 using the method of Daniels (1965). While the root samples were found to be consistently phototoxic as expected, only leaf extracts containing acetylenes were l e t h a l to the test organisms in the presence of long wave UV l i g h t (Tables XXXVIII, XXXIX). The yeasts S. cer evi s i ae and C. albicans were sensitive to most of the Bidens containing acetylenes, as were the gram-positive b a c t e r i a . The gram-negative bacteria were generally unaffected. This i s in agreement with the data of Towers et al ., ( 1977). The most photoactive extracts were those from Bidens species which produce the C 1 7 hydrocarbon compounds 8, 9, and 10 (B. campylotheca, ssp. pent amera, B. conjuncta, B. macrocarpa, B. menziesii ssp. menziesii, B. hi I I ebr andi ana ssp. polycephala, B. torta 17C) and/or C 1 3 hydrocarbon and aromatic compounds 1, 4, 5 and 7 ( B. campyl ot heca ssp. pentamera, B. cosmoides, B. sandvicensis S S p . confusa, B. torta 17A and 17B). Bidens hawaiensis with leaves containing compound 18 ("safynol") also exhibited phototoxicity against several of the test organisms. 8-Methoxypsoralen (8-MOP) was used as a reference test compound. It i s a well known phototoxic furanocoumarin (Towers, 1980; Warren et al., 1980; Averbeck, 1982; Ashwood-Smith et al . , 1980) and i s l e t h a l to the organisms used in th i s study except Pseudomonas sp. (Fowlks et al . , 1958. Relative differences in the phototoxicity of test extracts can be qua n t i f i e d by measurement of the 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 Saecfi. cerevlsiae *** - +• - * - * ++* - •+- *- - - --Candida albicans *** - - - * - *** - -- *•- *- -- - -Staphylococcus aureus *** - -- -- *- -- **- * - - - - -S. a I bus *•+ - **- • - ** - -- *** - - - **•* - - - - -Streptococcus raecaiis *** - *•• - * - - - - - *** - - - - - - - - -Bacillus subtil Is *•* - * - **- -- **++ - -- -- - -Escherichia col' **+ - - - - - • - - - *- - - - - - - - -Pseudomonas fluorescens - - - - -- - - -- -- -- --P. aevroglnosa - - - - - - - - - - - - - - . - - - - - -• 10 ug/dlsc of light petroleum extracts: Bidens key, In Table II * clear zone diameter 7-10mm ** clear zone diameter io-l4mm *+• clear zone diameter 14-I8mm *•** clear zone diameter >> 18mm 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 ug/dlsc of light petroleum extracts: Ridcns key In Table II * 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 151 of the clear zones around the impregnated f i l t e r paper discs. The minimum measurable zone of i n h i b i t i o n i s 7mm, the diameter of the disc. Clear zone diameters were assigned values on a scale of + (7-1Omm) to ++++ (greater than 18mm) in order to demonstrate r e l a t i v e p h o t o t o x i c i t i e s . Irradiation i t s e l f does not affect the growth of the organisms. Thirteen species of yeasts and yeast-like fungi isolated from Hawaiian Bidens leaves were tested for photosensitivity to polyacetylenes from Bidens as well as to a-terthienyl (compound 21) and thiarubrine A (compound 22) from Tagetes and Eriophyllum species (Compositae) respectively (Tables XXXIII, XL and XLI). Concentrations of 1,5 and 10 nq/xtiL were used because leaf acetylenes in Bidens do not exceed 10 jxg/mL. The C 1 3 'ene-tetrayne-ene' compound 1 was in e f f e c t i v e against a l l organisms except Tilletiopsis pallescens and Col I el ot ri chum gl oeospori odes . The C 1 7 compounds 8 and 9, which were strongly photoactive against bacteria, Saccharomyces and Candida (Tables XXXVIII, XXXIX) were s l i g h t l y toxic to Crypt ococcus species but not at a l l deleterious to their 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 well as 8-methoxypsoralen were phototoxic to most of the phylloplane fungi. The thiophenes 21 and 22 were used in t h i s study because they are known to be powerful photosensitizers (Towers, 1979; Downum et al., 1982). They TABLE XL. PHOTOSENSITIVITY OF CR. LAURENTII FROM DIFFERENT HOST PLANTS TO POLYACETYLENES Host Plants 8 MOP UV DK UV DK 5 UV DK 7 UV DK 8 UV DK 9 UV DK 4 UV DK 21 UV DK 22 UV DK 0. cosmoides 0. hllIIbrandlana ssp. polycephala 0. maul ens Is 0. sandvfcensfs ssp. sandvIcensIs 0. sandvfcensfs ssp. confusa + -+ -+++ 4 * 4 . -4- + + -4-4- -4.4- -4-4- -4-4- •» 4-4- 4-+4- 4-• 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 UV DK 5 uv OK 7 UV DK 8 UV DK 9 UV DK 4 UV DK 21 UV DK 22 UV DK 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 • -* 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 ++++ ++ +4-4-4- • • *+ +* • + 4- -++ + + -4- -4-+ -NT NT 4- -NT NT + 4- -4- -NT NT *++ + -• 4.4- -NT NT ++ -4. -4-4- -+ + + -4- -• *++ -4- -NT NT + 4- -+ + + -4-4- -+ + + + + 4- . 4-4- 4 + + + + • + • • * + + NT NT + 4-4-4- + + • + + + + + -+4- 4-+ +4- 4-NT NT *•• •+++ 4-*4- +4-4- + * + NT NT co 1 5 4 were e f f e c t i v e against a l l the organisms tested except for Rhodoiorula muciIagi nosa (Joerg.) Harrison which was resista n t to a-terthienyl and not inhibited by thiarubrine A in the dark. Except for one population (17A) of B. t o r t a , compound 4 (PHT) does not occur in Hawaiian Bidens. It i s also deleterious to the majority of fungi tested. 8-Methoxypsoralen, used as a reference photoactive compound, was e f f e c t i v e against a l l but Spor obol omyces roseus Kluyver et van N i e l . This organism i s also resistant to fi v e other test acetylenes and r e l a t i v e l y insensitive to a-terthienyl and thiarubrine A. The pathogenic endophyte Col Iet ot richum gloeospori odes i s very sensitive to compound 1 both in the l i g h t and in the dark. In addition, i t i s e a s i l y k i l l e d by compound 5. This organism was absent from leaves of Bidens containing aromatic acetylene 5 (Table XXXVII) although present in Bidens containing other acetylenes. It i s resista n t to compounds 8 and 9. Aureobasidium pulIulans, i s o l a t e d from only one of f i v e Bidens species producing leaf acetylenes, B. sandvi censi s ssp. confusa (Table XXXVII), i s only s l i g h t l y photosensitive to compounds 5 and 7 which occur in the host plant and to the c l o s e l y related compound 4. It i s also least sensitive of a l l organisms tested against thiarubrine A. Epicoccum pur purescens i s insensitive to a l l Bidens compounds except compound 5. CI adosporium cladosporiodes, the most commonly is o l a t e d organism in thi s study, i s sensitive to the aromatic acetylenes and the thiophenes but not to the 155 straight chain compounds 1, 8 and 9. COMPARISON OF PHOTOTOXICITY OF SELECTED POLYACETYLENES TO CRYPTOCOCCUS LAURENTII Two-fold s e r i a l d i l u t i o n s of 5 nq/mL and 10 nq/mh of compounds 4,5,8 and 21 were added to approximately 1.69 X 10" cells/mL of C. laurentii in MYP. Control plates were kept in the dark and test plates were irradiated for 5,10 and 20 minutes. The op t i c a l density (O.D.) at 492 nm of each sample was read before i r r a d i a t i o n (T 0) and at T 2 0 h , T 2„h, T 0 0 h and T„ Bh l a t e r , and the differences expressed as a percentage of control O.D. (Tables XLII and XLIII). Percent survival of c e l l s , 24 hours after exposure to varying concentrations of compounds was plotted against the time of UV-A i r r a d i a t i o n (Figures 14 to 17). The synergistic action of a l l test compounds and UV-A i r r a d i a t i o n on the v i a b i l i t y of C. laurentii i s demonstrated in these graphs. The Minimum Inhibitory Concentration (MIC), causing complete growth i n h i b i t i o n , was 0.078 uq/mL for a-terthienyl after 10 minutes exposure to UV-A. No other compound was as l e t h a l . Alpha-terthienyl also had a dark effect and k i l l e d some 20-30% of c e l l s in the control plates at concentrations between 5 uq/mL and 0.039 uq/mL. None of compounds 4, 5 and 8 (Figures 15 to 17) was completely l e t h a l to C. laurentii even at 10 nq/mL. Higher concentrations were not used for two reasons: polyacetylenes p r e c i p i t a t e in aqueous solutions 1 TABLE XLII. EFFECTS OF CONCENTRATION AND LENGTH OF. OF Cr. Cone.(vq/mL) T 0 Compound 4** 0.00 100% 1.25 100% 5.00 B3% 10.00 47% Compound 5 0.00 100% 1.25 98% 5.00 77% 10.00 46% Compound 8 0.00 100% 5.00 100% 10.00 75% Compound 21 0.00 100% 0.0195 81% 0.039 83% 0.078 75% 5.00 73% CHANGES IN POLYACETYLENE UV EXPOSURE ON PERCENT SURVIVAL laurentii* Time of I r r a d i a t i o n (mins) T 5 T ,o T 2 o 100% 100% 100% 7 9 % 62% 41% 67% 59% 39% 53% 33% 19% 100% 100% 100% 69% 57% 83% 61% 72% 62% 44% 52% 59% 100% 100% 100% 61% 96% 95% 60% 68% 62% 100% 100% 100% 68% 53% NT*** 17% 14% NT 22% 3% NT 17% 5 % NT * A l l sampled incubated for 24 hours ** See Table IV-5 f o r compound names *** NT not t e s t e d 157 FIGURE 14. EFFECT OF a-TERTHIENYL (21) AND UV-A ON THE 24 HOUR SURVIVAL OF CRYPTOCOCCUS LAURENTII 0 ug/mL 00195 ug/mL o.o T 1 r 2.4 4.B 7.2 TIME OF 1RRRD1RT10N I 9.6 (MIN5) Q039 ug/mL 50ug/mL 0.078 ug/mL 12 158 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 - j — 1 1 r 4 4 6.6 13.2 17 TIME OF 1RRRD1R710N (MIN5) 22.0 159 16. EFFECT OF PHENYLHEPTADIYNE-ENE (5) AND UV-A ON THE 24HOUR SURVIVAL OF CRYPTOCOCCUS LAURENTII 0 ug/mL 1.25 ug/mL • 5.0ug/mL 0 10.0ug/mL ^ i 1 — r 4 4 _ ^ B.B 13.2 17. 7 ME OF 1RRRD1RT10N (M1NS) 22.0 160 17. E F F E C T O F H E P T A D E C A - T E T R A E N E - T R I Y N E (8) A N D U V - A ON T H E 2 4 H O U R S U R V I V A L O F CRYPTOCOCCUS LAURENTII -° 0 ug/mL •° 5L0 ug/mL 10.0 ug/mL • T 1 1 1 4-4 8.B 13 7 \i c O-J TIME OF IRRADIATION (MINSJ 161 TABLE X L I I I . SURVIVAL CURVES FOR C. LAURENTII EXPOSED TO POLYACETYLENES IN UV LIGHT* Time of I n c u b a t i o n Compounds** T 2 0. T 2 , T, 0 C o n t r o l 100% 100% 100% Compound 3 44% 59% 79% Compound 5 50% 72% 88% Compound 8 84% 96% 96% Compound 21 9% 5% 4% * 5 ug/mL exposed t o 10 minutes UV l i g h t ** See t a b l e IV-5 f o r names of compounds 162 at such concentrations and values of polyacetylenes in Bidens leaves do not exceed 10 ug/mL. Compounds 4, 5 and 8 had an a n t i b i o t i c effect at 10 ug/mL and compounds 4 and 5 also at 5 Mg/mL. Irradiation of greater than 5 minutes duration seemed to cause breakdown of compounds 5 and 8. This i s not unexpected as polyacetylenes are known to be unstable in l i g h t and aqueous solutions (Towers, 1979). Figure 31 i s a direct comparison of the phototoxic effects of the four test compounds against Cr. laurentii. At 5 xig/mL and afte r 10 minutes UV a-terthienyl i s the most powerful photosensitizer, followed by PHT (4) phenyl-diyne-ene (5) and heptadeca-tetraene-triyne (8). 163 D. DISCUSSION ISOLATION, IDENTIFICATION AND DISTRIBUTION OF PHYLLOPLANE MICROORGANI SMS Yeasts are defined by Kreger-van R i j (1984) as un i c e l l u l a r fungi reproducing by budding or f i s s i o n , which may or may not be a stage in the l i f e cycle of m u l t i c e l l u l a r fungi. They are characterized by morphological and physiological c r i t e r i a and are taxonomically diverse, including ascomycetes, basidiomycetes and imperfect fungi with both ascomycetous and basidiomycetous a f f i n i t i e s . In t h i s study, 19 taxa of yeasts and yeast-like fungi were s e l e c t i v e l y isolated from 12 species of Bidens and 23 species of Hawaiian plants and their d i s t r i b u t i o n recorded (Tables XXXII, XXXIV, XXXV, XXXVI). Isolation of filamentous fungi per se was li m i t e d by the nature of imminent phototoxicity assays which were o r i g i n a l l y developed for bacteria and yeasts (Fowlks, 1958; Daniels, 1965). The data shows that, in Hawaii, just as in Indonesia (Ruinen, 1963) and temperate regions (Voznyakovskaya, 1962; Last and Deighton, 1965; Dickinson, 1976), healthy green leaves are inhabited by members of the Sporobolomycetaceae and Cryptococceae together with Aureobasi di um and a few imperfect fungi such as CI adosporium and Epicoccum species. Using a leaf washing technique, Voznyakovskaya (1962) isola t e d a range of epiphytic microorganisms including Spor obol omyces roseus, Rhodotorula rubra (Demme) Lodder, R. 1 64 muci I agi nosa, R. aurantiaca (Saito) Lodder, Crypiococcus laurentii and C. albidus from a diverse selection of hosts. Her survey served to stress the ubiquity of most leaf yeasts and also indicated the lack of host s p e c i f i c i t y among them. Phylloplane species also tend to be found more frequently on leaves than in s o i l , probably because they are well adapted to the microenvironment of the le a f . Many species are pigmented, which may be an adaptation enabling them to survive the high l i g h t intensity at the leaf surface (Last and Deighton, 1965; Last and Price, 1969; Pugh and Buckley, 1971; Ruscoe, 1971; Dickinson, 1976; McCauley and Waid, 1981). Unlike those colonizing f r u i t s and flowers, leaf yeasts are unable to ferment sugars (Last and Price, 1969), although some species have been shown to possess lipase a c t i v i t y which would enable them to become embedded into the wax layers of the c u t i c l e . When Crypt ococcus laurentii (Kuff.) Skinner and Rhodotorula gl ut i ni s (Fres.) Harrison were cultured on the stripped epidermis of Aloe sp. and the c u t i c l e fragments of Sansevi eria sp., the c u t i c l e s were seriously eroded within f i v e days (Ruinen, 1966). Yeasts colonizing leaves above the cut in-reinforced a n t i c l i n a l c e l l walls, may, as a result of the i r enzymic a c t i v i t y , detrimentally affect the intact c u t i c l e and i t s functions. The phytopathogen, Col Iet otrichum gl oeospori odes enters the leaf by direct penetration without hyphal growth on the surface of the leaf (Blakeman, 1971; Marks et a/.,1965) and 165 is usually isolated from s u r f a c e - s t e r i l i z e d leaves ( P e t r i n i et al., 1982; C a r r o l l and C a r r o l l , 1978). Despite the cosmopolitan d i s t r i b u t i o n of phylloplane yeasts, the comparative study of Lamb and Brown (1971) suggests that the leaf i t s e l f may exert sele c t i v e pressure which p a r t i a l l y determines the nature of the resident phylloplane microflora on any given host. The s p e c i f i c nature of the pressure was not investigated or discussed. One of the objectives of t h i s study was to determine whether the presence or absence of polyacetylenes in the leaves of Hawaiian Bidens exerts such an e f f e c t . Numerous reports that polyacetylenes are photoactive against bacteria and fungi and other organisms (eg. Arnason et al., 1980; Camm et al., 1975; Chan et al., 1975; DiCosmo et al., 1982; Towers et al., 1977; Wat et al., 1977) have raised speculation about the putative b i o l o g i c a l functions of polyacetylenes in plants. Certainly the raison d'etre of secondary compounds in general may never be f u l l y c l a r i f i e d because a concerted, m u l t i d i s c i p l i n a r y , long-term approach is required for each class of chemicals, using appropriate source and test organisms (Janzen, 1979). Nevertheless, progress toward an answer may be made by small d e f i n i t i v e steps. In th i s study, two s p e c i f i c questions were asked: does the presence or absence of polyacetylenes in Hawaiian Bidens leaves have any co r r e l a t i o n with the d i s t r i b u t i o n of selected phylloplane inhabitants and are these organisms sensitive to polyacetylenes? 166 In general, the nonfilamentous saprophytes i s o l a t e d in thi s study occurred on both Bidens with leaf acetylenes and Bidens without acetylenes, as well as on most of the other plants sampled at any p a r t i c u l a r s i t e (Tables XXXVI, XXXVII). It appears that the presence or absence of polyacetylenes within the leaves of Bidens i s not correlated with the nature of the phylloplane yeasts and yeast-like fungi. Only Col Ietol ri chum gl oeospori odes demonstrates discrete d i s t r i b u t i o n . It was consistently absent from Bidens taxa which produce the C 1 3 aromatic compounds 4 ,5 and/or 7 (Table XXXVII). PHOTOSENSITIVITY OF PHYLLOPLANE MICRORGANISMS TO POLYACETYLENES If polyacetylenes in leaves are photoactive against the microorganisms which dwell within or on the leaves, one might expect resident organisms to be unaffected by the compounds found in the i r host plants. Furthermore, phylloplane organisms from plants without leaf acetylenes would have no such resistance. Since most of the organisms were found on a l l plants sampled (Table XXXIV to XXXVI), one representative species from several hosts was checked for possible d i f f e r e n t i a l photosensitivity to acetylenes. Crypt ococcus laurentii from B, cosmoides (compounds 1,4 and 5), B. hi 11 ebrandiana ssp. polycephala (compounds 1,8, and 9), B. sandvicensis ssp. confusa (compound 5), B. 167 sandvi censi s ssp. sandvi censi s (no leaf acetylenes) and B. mauiensis (no leaf acetylenes) were a l l tested for photosensitivity to the polyacetylenes l i s t e d in Tables XXXIII. No differences were detected (Table XL), which implies that the presence or absence of leaf acetylenes bears no rel a t i o n s h i p to the responses of C. laurentii to polyacetylenes. Crypt ococcus species, notably C. laurentii, have been isolated from northern and southern temperate regions, from the tropics and even from the Antarctic (diMenna, 1960; Last and Deighton, 1965), and are the only non-pigmented fungi found in th i s study. The incidence of pigmentation among phylloplane fungi and bacteria is high (70% among bacteria) (Ruinen, 1961; 1963a; Last and Deighton, 1965) and i s thought to be an adaptation in response to UV radiation on exposed leaf surfaces. Ruscoe (1971), in a detailed dir e c t examination study of Nothofagus leaves, found that hyaline species generally showed a hypophyllous d i s t r i b u t i o n . Phragmites leaves, which are displayed in a nearly v e r t i c a l p o s i t i o n , had similar populations on both surfaces (Apinis et a/., 1972). Although the significance of radiation as a determining factor may be debated, pigmentation i t s e l f may aff e c t the degree of photosensitivity of organisms to sp e c i f i c acetylenes. Compounds 8 and 9 were found to be very toxic to bacteria and to S. cerevisiae and C. albicans (Tables XXXVIII and XXXIX), none of which are pigmented. These C 1 7 compounds did not k i l l any of the pigmented 1 68 phylloplane fungi but were phototoxic against a l l Crypt ococcus species tested (Tables XL, XLI). Rhodotorula spp., pigmented r e l a t i v e s of Crypt ococcus was resistant to compound 8 and 9. Epi coccum pur purescens, A. pullulans, S. roseus and R. pallida, a l l pigmented species, seem to be resistant to most acetylenes occurring in Bidens (Table XLI). In addition, S. roseus i s unaffected by 8-methoxypsoralen suggesting that i t may have a metabolic mechanism for disabling this toxic furanocoumarin. Dose response curves for compounds 4,5, 8 and 21 were determined using C. laurentii as a representative phylloplane yeast because of i t s s e n s i t i v i t y to a wider range of polyacetylenes, which may or may not be because of i t s lack of pigmentation (Figures 14 to.17). The data in these graphs are generally in agreement with those obtained using the disc test (Tables XL, XLI), except that the Titertek method seems to be more sensitive because i t revealed previously undetected a n t i b i o t i c e f f e c t s of these compounds. The two methods are not d i r e c t l y comparable because one uses l i q u i d suspensions of c e l l s , unstable aqueous solutions of polyacetylenes of known concentrations and short i r r a d i a t i o n times while the other uses s o l i d medium, solvent-free acetylenes which d i f f u s e in unknown quantities across agar and i r r a d i a t i o n periods of up to two hours. These differences may account for discrepancies between data sets. For example, in 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 even though the data in Tables XLII and XLIII and Figures 14 and 15 indicate that the opposite should be true. This may be a r e f l e c t i o n of the d i f f e r e n t modes of action of these two photosensitizers. The photodynamic action of a-terthienyl i s oxygen-dependent whereas that of PHT i s not (Arnason et al . , 1980; Downum et a l . , 1982; McRae et a l . , 1985), and the two hour i r r a d i a t i o n period in the disc test may favour the mechanism of action of PHT and enhance i t s toxic effect on the organisms. With the exception of C. l u t e a l us, a l l Sporobol omyces, Rhodotorula and Crypl ococcus yeasts in t h i s study were more sensitive to PHT than a-terthienyl in the disc tests (Table XLI). It remains to be seen whether the opposite i s true using the Titertek method. Although the phototoxicity assays performed in t h i s study provide an indication of the s e n s i t i v i t y of selected phylloplane organisms to polyacetylenes in v i t r o , such data cannot be unequivocally extrapolated to the situation in v i v o . T o x i c i t y in v i t r o does not prove t o x i c i t y in vivo although resistance in v i t r o implies resistance in any s i t u a t i o n . A l l the fungi tested, with one exception (Col I e t o t r i c h u m ) , exhibit d i f f e r e n t i a l s e n s i t i v i t y to the test compounds. These responses are not related to the physical d i s t r i b u t i o n of the organisms among Bidens (Tables XXXVII, XLI). Unlike B. alba and Coreopsis species (Towers and Wat, 1978), in Hawaiian Bidens taxa there is no evidence for the 170 presence of polyacetylenes in the leaf c u t i c l e or within leaf surface structures such as trichomes. Resin canals exi s t but their minute diameters preclude sampling the contents for analysis. Whether acetylenes occur in resin canals, within c e l l s or e x t r a c e l l u l a r l y , any contact between surface microorganisms and leaf acetylenes must occur within leaf tissue. Yeasts such as Crypt ococcus and Rhodotorula spp., which degrade leaf c u t i c l e to some extent (Ruinen, 1963b; 1966), may or may not encounter acetylenes, but species of Col I et ol ri chum, as well as Aureobasidium, which penetrate leaf tissue and dwell within as endophytic pathogens (Blakeman, 1971), would be exposed to int r a and ex t r a c e l l u l a r constituents. Col I etotrichum gloeospori odes was isolat e d from numerous species of Hawaiian Bidens and other plants sampled in t h i s study. Its occurrence appears to be s i t e related (Table XXXV, XXXVI). It was not found in B. cosmoides, B. cervi cat a or B. sandvicensis ssp. confusa and in only one other plant at the f i r s t f i v e l o c a l i t i e s , in no other plant sampled at the t h i r d s i t e . In six other l o c a l i t e s where Col I et ot r i chum was isolat e d from Bidens, two to four of the sympatric species were also hosts to the endophyte. Nevertheless, C. gl oeospori odes did not occur in Bidens which produce the C 1 3 acetylenes (1,4,5 and/or 7) . It was found to be very sensitive to 1 and 5 in the presence of UV radiation (Table XLI). Its s e n s i t i v i t y to a-terthienyl (Compound 21) has been previously reported (di Cosmo et al ., 1982). It was 171 not affected by compounds 8 and 9. Although in vitro responses may not be a true r e f l e c t i o n of the s i t u a t i o n in vivo, t h i s data suggests that the presence of polyacetylenes 1 and/or 5 in B. cosmoi des (or B. cervi cat a or B. sandvicenses ssp. confusa) leaves precludes colonization of i t s tissues by Col Iet otri chum gloeospori odes. The organism would presumably come into contact with polyacetylenes, which may be located e x t r a c e l l u l a r l y or within c e l l s , as i t invades the leaf and subsequently becomes i n h i b i t e d by the photoactive compounds. There i s also the p o s s i b i l i t y that some hitherto unknown phytoalexin(s) may be produced in response to fungal invasion. The selective photosensitivity may be caused by inherent morphological, physiological and/or biochemical characters s p e c i f i c to C. gloeosporiodes which causes i t to react more strongly with some acetylenes than others and af f e c t i n g i t s a b i l i t y to grow within some plants. Certainly this information i s limited and cannot be interpreted as evidence for polyacetylene function in vivo. Nevertheless, i t indicates that further research using phylloplane microorganisms on s p e c i f i c host plants may y i e l d interesting information. Hawaiian Bidens not sampled in t h i s study must be checked for Col Iet ot ri chum, e s p e c i a l l y , B. campylotheca ssp. pentamera, B. torta 17B and 17D and B. valida, a l l containing compounds 1,5 and/or 7 (Table V). Future investigations should focus on leaf-invading pathogens in order to determine whether there are other 172 species excluded from Bidens leaves with s p e c i f i c polyacetylenes to which the organisms are sensitive, and whether there are pathogens which dwell within leaves which are resistant to the host acetylenes. Current bioassays for phototoxicity must be suitably refined and modified for filamentous fungi (Daniels, 1965; DiCosmo et al., 1982). If such organisms can be found, a tentative argument for the case against the f o r t u i t y of polyacetylene phototoxicity to microorganisms may be made. As for the answer(s) to the central question of the putative role of polyacetylenes in nature, the complexity of the pot e n t i a l research problems to be surmounted cannot be overemphasized. These problems need to be c a r e f u l l y dissected into a methodical series of hypotheses which can be tested by experimentation. P a r a l l e l studies using d i f f e r e n t organisms and polyacetylene producing plants must be c a r r i e d out in the laboratory as well as in the f i e l d . The present study has established that the phylloplane microflora/5/dens system i s a useful model for further investigation. 173 E . CONCLUSION Yeasts and yeast-like fungi were isolated from the leaves of Hawaiian Bidens and other plants and i d e n t i f i e d as members of the Sporobolomycetaceae, the Cryptococcaceae and the Fungi Imperfecti. A l l are common phylloplane inhabitants of worldwide occurrence. 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Ultraviolet-mediated a n t i b i o t i c a c t i v i t y of species of compositae caused by polyacetylenic compounds. Lloydia 40: 487 - 498. Voznyakovskaya, Yu. M. 1962. Epiphytic yeast organisms. Mikrobiologiya 31: 616 - 622. Warren, R.A.J., J.B. Hudson, K. 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, CK., R.K. Biswas, E.A. Graham, L. Bohm and G.H.N. Towers. 1977. Ultraviolet-mediated cytotoxic a c t i v i t y of phenylheptatriyne from Bidens pilosa L. J. Nat. Products 42: 103 - 1 1 1 . V . GENERAL CONCLUSION In t h i s dissertation several aspects of the biology and chemistry of polyacetylenes synthesized by the native Hawaiian species of Bidens were examined. The occurrence and d i s t r i b u t i o n of acetylenes in these plants was consistent with the species concepts of Ganders and Nagata (1983) in their r e v i s i o n of the group, and with other evidence that the Hawaiian species are derived from a single ancestral lineage (Ganders and Nagata, 1984). Some species have lost the a b i l i t y to produce leaf acetylenes although de novo synthesis was c l e a r l y established in others. This t r a i t i s apparently dominant although acetylene inheritance seems complex and may be affected by the polyploid condition of the plants. An endophytic fungus, C. gloeospori odes , was discovered to be highly photosensitive to C 1 3 aromatic acetylenes which occur in the leaves of Bidens species not inhabited by the organism. This seems to suggest that the presence of acetylenes in leaves may be a deterrent to colonization by certain fungal pathogens. This however, does not explain why some species no longer synthesize such leaf compounds. F i n a l l y , an unusual aromatic thiophene was isola t e d from the roots of Hawaiian Bidens species. It has a unique combination of a phenyl ring and a thiophene ring bridged by a carbon-carbon t r i p l e bond. This compound may have interesting b i o l o g i c a l properties and merits future study. 179 180 A . BIBLIOGRAPHY G a n d e r s , 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 in the e v o l u t i o n of Bidens on the Hawai ian I s l a n d s , pp 179-194 in P l a n t B i o s y s t e m a t i c s . W . F . Grant ( E d . ) . Academic P r e s s , Canada. G a n d e r s , F . R . and K . M . Nagata . 1983. New taxa and new c o m b i n a t i o n s in Hawai ian Bidens ( A s t e r a c e a e ) . L y o n i a 2: 1 - 16. 

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