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Comparison of some chemical constituents of the lycopods McMullan, Eleanor Elizabeth 1966

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COMPARISON OF SOME CHEMICAL CONSTITUENTS OF THE LYCOPODS by ELEANOR ELIZABETH McMULLAN B.Sc, University of B r i t i s h Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biology and Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1966 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 avail able f o r reference and study, I further agree that permission-for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. i i ABSTRACT A survey of some chemical c o n s t i t u e n t s of the lycopods was c a r r i e d out in order t o determine whether the chemistry of these p l a n t s i s c o r -r e l a t e d w i t h t h e i r taxonomy. One approach t o t h i s problem was to study the products of photosynthesis of species of Lycopodium. S e l a g i n e l l a and Isoetes. R a d i o a c t i v e C^0£ w a s t o -hese p l a n t s and the d i s t r i b u t i o n of r a d i o a c t i v i t y in sugars and amino acids was examined by means of paper chromatography. The d i s t r i b u t i o n of r a d i o a c t i v i t y in sugars was c h a r a c t e r i s t i c f o r each genus, but the d i s t r i b u t i o n of r a d i o a c t i v i t y in amino acids was not. In S e l a g i n e l l a 80% or more of the r a d i o a c t i v i t y was incorporated i n t o t r e h a l o s e w h i l e most of the r e s t of the r a d i o a c t i v i t y was found i n sucrose. There was one exception t o t h i s : in S. kraussiana 40% of r a d i o a c t i v i t y was incorporated i n t o trehalose w h i l e most of the r e s t was incorporated i n t o an u n i d e n t i f i e d sugar. In Isoetes k% t o 8% of the r a d i o a c t i v i t y was found in t r e h a l o s e w i t h most of the r e s t in sucrose. In Lycopodiurn 95% or more of the r a d i o a c t i v i t y was found in sucrose and none was found in t r e h a l o s e . R a d i o a c t i v e t r e h a l o s e was administered t o species of these genera and i t was shown that they are a l l able t o metabolize t r e h a l o s e to some degree. Species of S e l a g i n e l l a . Isoetes and Phylloglossum were examined to determine whether they c o n t a i n a l k a l o i d s . Phylloglossum e x t r a c t s contained compounds w i t h the chromatographic p r o p e r t i e s of Lycopodiurn a l k a l o i d s , but S e l a g i n e l l a and Isoetes species d i d not c o n t a i n d e t e c t a b l e amounts of these compounds. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF SUBJECT 1 Morphology and Taxonomy 1 Chemistry 8 ( i ) General Considerations 8 ( i i ) Chemistry of the Lycopods 16 METHODS AND MATERIALS 31 1. Sources of Pl a n t M a t e r i a l 31 2. Preparation of Aqueous E x t r a c t s 31 3. Preparation of Neutral C a t i o n , and Anion F r a c t i o n s of Plant E x t r a c t s 31 k. Preparation of A l k a l o i d E x t r a c t s 35 5. Methyl at ion of Sugars 35 6. Vapour Phase Chromatography 36 7. One D i r e c t i o n a l Paper Chromatography of Sugars f o r Q u a l i t a -t i v e A n a l y s i s 36 8. One D i r e c t i o n a l Paper Chromatography of Sugars f o r Prepara-t i v e I s o l a t i o n 37 9. One D i r e c t i o n a l Paper Chromatography of A l k a l o i d s 37 10. Two D i r e c t i o n a l Chromatography of Sugars and Amino Acids 37 11. Thin Layer Chromatography of A l k a l o i d s 38 12. Column Chromatography of Sugars 39 13. Detection of Spots 39 14. Technique f o r Adm i n i s t e r i n g c '^0, 40 iv TABLE OF CONTENTS, cont'd. Page METHODS AND MATERIALS, cont'd. 15. Technique for Administering Trehalose-c'^ hi 16. Measurement of Radioactivity hi EXPERIMENTAL AND RESULTS hh 1. Vapour Phase Chromatography hh 2. Preliminary Survey of the Sugars of Lycopodiurn and Selaginella hh 3. Survey of the Distribution of Radioactivity in Sugars and Amino Acids of Lycopods Fed C'^ O- hh h. Isolation of Radioactive Trehalose 48 1h 5. Administration of Trehalose-C to Plants 55 6. Examination of Selaginella, Isoetes and Phylloglossum for the Presence of Alkaloids 58 DISCUSSION 1. Use of Vapour Phase Chromatography for Quantitative Surveys of Sugars in Plants 60 2. Survey of the Metabolic A c t i v i t y of Sugars and Amino Acids in Lycopods as Indicated by C 1^ Incorporation 62 3. Survey of Lycopods for the Presence of Alkaloids 67 BIBLIOGRAPHY 69 SUMMARY 7h APPENDIX 76 LIST OF TABLES Table Page I Sources of Plant M a t e r i a l 32 II P r e l i m i n a r y Survey of Sugars i n Lycopodiurn and S e l a q i n e l l a 47 III D i s t r i b u t i o n of R a d i o a c t i v i t y i n the Neutral Aqueous E x t r a c t of Plants Fed 49 IV D i s t r i b u t i o n of R a d i o a c t i v i t y in the Cation F r a c t i o n of Aqueous E x t r a c t of Plants Fed C ^ 0 2 51 V D i s t r i b u t i o n of R a d i o a c t i v i t y in Trehalose, Sucrose, Glucose and Fructose from Plants Administered T r e h a l o s e - C l ^ 57 v i LIST OF FIGURES Figure Page 1 Trehalose 17 2 Lycopodine 22 3 Lyconnotine 23 k Annotinine 23 5 Lycodine 2k 6 Selagine 2k 7 Cernuine, Lycocernuine, and Suggested Precursors 25 8 B i o s y n t h e s i s of Annotinine Suggested by Leete 26 9 B i o s y n t h e s i s of Q u i n o l i z i d i n e Skeleton 27 10 B i o s y n t h e s i s of Four Basic Skeletons of Lycopodiurn A l k a l o i d s Suggested by Conroy 28 11 Biosynthesis of Annotinine Suggested by Ayer et al_. 30 12 Apparatus f o r A d m i n i s t e r i n g k\ 13 Vapour Phase Chromatography of Mixtures of Methylated Sucrose and Trehalose kS \k Radioautographs Showing D i s t r i b u t i o n of R a d i o a c t i v i t y in Sugars in Representative Chromatograms 5k 15 1. Infra - r e d Spectrum of C r y s t a l s Obtained from S e l a g i n e l l a w a l l a c e i 2. Infr a - r e d Spectrum of Trehalose 56 16 Chromatogram of A l k a l o i d s from E x t r a c t s of 1 gm of Plant M a t e r i a l 59 17 Chromatogram of A l k a l o i d s from E x t r a c t s of 1 gm of Plant M a t e r i a l 59 18 Chromatogram of A l k a l o i d s from E x t r a c t s of 2 1/2 gm of Plant M a t e r i a l 61 VI I LIST OF FIGURES, cont'd. Figure Page 19 Representative Graph of C0 2 Concentration i n A i r Stream During C^0_ Feedings 64 20 Representative Graph of R a d i o a c t i v i t y i n A i r Stream During C l^0 2 Feedings 64 21 Dioxane Quenched Standards E f f i c i e n c y versus Channels R a t i o 77 i v i i i ACKNOWLEDGMENTS I am very g r a t e f u l t o Dr. G.H.N. Towers f o r h i s a s s i s t a n c e and advice in the p r e p a r a t i o n of t h i s t h e s i s . I would a l s o l i k e t o thank Dr. E.B. Tregunna f o r i n s t r u c t i o n in the use of r a d i o i s o t o p e s , Dr. W.B. S c h o f i e l d f o r h i s c r i t i c i s m of the s e c t i o n d e a l i n g w i t h morphology, Dr. T.M.C. T a y l o r , Dr. V.J. K r a j i n a , and Dr. W.B. S c h o f i e l d f o r t h e i r a s s i s t a n c e in i d e n t i f y i n g c o l l e c t i o n s , and Dr. T. B i s a l p u t r a and Miss Nancy Corfman f o r the use of t h e i r photographic equipment. I wish t o thank Mrs. Aida Tse f o r the i n f r a - r e d s p e c t r a , and Mr. D.E. McMullan, Mr. L.K. Wade, Dr. R.C. Brooke, Mr. T. F l e g e l , Mr. L. Cordes, and Mr. G. Davis f o r t h e i r c o l l e c t i o n s . 1 INTRODUCTION The extant members of the lycopods are remnants of a group of pl a n t s whose h i s t o r y extends back at l e a s t to the Devonian, some 50 m i l l i o n years before the c o n i f e r s appeared and 300 m i l l i o n years before the angio-sperms (60). They have c h a r a c t e r i s t i c s i n common with each other and with f o s s i l i z e d members of the d i v i s i o n which suggest that they have common ancestors. However, there are marked d i s s i m i l a r i t i e s among some of them, implying that they have been e v o l v i n g s e p a r a t e l y f o r a very long time. The f i v e l i v i n g genera are placed i n three d i f f e r e n t orders r e f l e c t i n g these e v o l u t i o n a r y l i n e s . Some chemical c o n s t i t u e n t s of these genera were surveyed i n an attempt to discover whether metabolic p e c u l i a r i t i e s had a r i s e n over t h i s long period of e v o l u t i o n , and t o assess the d i a g n o s t i c value of these changes. REVIEW OF THE SUBJECT Morphology and Taxonomy The lycopods are d i s t i n c t from the f e r n s , w i t h which they were once c l a s s i f i e d , in t h e i r leaves, p o s i t i o n of sporangia, type of branching, the gametophytes. Their leaves are m i c r o p h y l l s , g e n e r a l l y small and a l -ways s u p p l i e d by a s i n g l e v e i n which lacks a l e a f gap in the stem. Even before the lycopods were recognized as a d i v i s i o n separate from the f e r n s , The c l a s s i f i c a t i o n o u t l i n e d i n Scagel et_ a l . (49) w i l l be fo l l o w e d . The p l a n t kingdom i s given twelve d i v i s i o n s , i n c l u d i n g P s i l o p h y t a , Lycopodophyta, Arthrophyta and Pterophyta f o r the former P t e r i d o p h y t a . 2 microphyl ls were thought to have a d i f f e r en t phylogenet ic o r i g i n from the megaphylls of f e r n s , having developed as stem enations whi le megaphylls developed from a spec i a l i z ed branch system (50). The sporangia of lycopods are borne s ing l y on the adaxial s ide of a leaf or l a t e r a l l y on the stem but not in aggregates on the leaf margin or on the underside as they are in the f e rns . Sporophyl ls are of ten l o ca l i z ed at the ends of branches to form s t r o b i l i , s t ructures which do not occur in f e r n s . Branching of the stem is dichotomous with t r ans i t i ons to l a t e ra l branching brought about by unequal growth of the branches. This s i t ua t i on is considered p r im i t i v e to that in the ferns where l a te ra l branches a r i s e in leaf a x i l s as a r u l e , though there may be revers ion to dichotomous branching. The r e l a t i on of a branch to a p a r t i c u l a r leaf does not occur in the lycopods except in some dors i vent ra l S e l ag i ne l l a spec i e s , where i t apparently arose secondar i l y as a resu l t of the fac tors that caused d o r s i -ventral i t y (50) . The gametophytes of the lycopods are s impler than those of f e r n s . In the Se l ag ine l l a l e s and Isoetales the gametophytes develop la rge ly wi th in the spore w a l l , on n u t r i t i v e substances deposited in the spore from the sporophyte (50) . In the Lycopodiales the gametophyte germinates exospora l ly but is smal ler than a fern gametophyte and f requent ly lacks c h l o r o p h y l l . The morphology of these gametophytes seems to be re la ted to t he i r development; in one species i f the spore germinates above ground i t produces a carrot-shaped green gametophyte, but i f the gametophyte develops underground i t is co lour l ess and c y l i n d r i c a l . Apparently a l l gametophytes in the Lycopodiales have a mycorrhizal assoc ia t ion (52). The d i f f e rences 3 between lycopod and fern gametophytes are not as important phylogenetic-a l l y as the sporophytic differences discussed, since reduction and loss of chlorophyll in gametophytes are known in fern genera (e.g. Botrychium. Qphioglossum) and since heterospory has arisen independently in more than one group of plants (20) . The lycopods are separated from the arthrophytes (Equisetum and i t s f o s s i l r e l a t i v e s , which also have microphylls) by differences in thei r leaf p o s i t i o n , branching pattern, and position of sporangia. The leaves of arthrophytes are borne in whorls of three or more and branches arise at the nodes, while the lycopods have dichotomously branched stems with s p i r a l l y arranged leaves. The sporangia of arthrophytes are borne on lat e r a l appendages; in more recently evolved groups these are highly specialized and are ca l l e d sporangiophores. These la t e r a l appendages are thought to represent modified branches. It has been suggested that the microphylls of Equisetum are also very reduced branches, i.e. that they have originated in the same way as the megaphylls of ferns (37). The lycopods are distinguished from the psilophytes (Psilotum. Tmesipteris and f o s s i l a l l i e s ) in that the l a t t e r lack true roots and bear t h e i r sporangia on lat e r a l branches or terminally on shoots. Different authors have placed different degrees of importance on these distinguishing c h a r a c t e r i s t i c s . The way they assess the separateness of plants with microphylls and plants with megaphylls depends on th e i r idea of how these organs arose. Authors who believe a l l leaves arose in the same way consider the evolutionary significance of microphylls versus megaphylls slight (19). S i m i l a r l y , the importance of the a x i l l a r y position of the lycopod sporangium as a c h a r a c t e r i s t i c d i s t i n g u i s h i n g the lycopods from the arthrophytes and p s i l o p h y t e s diminishes i f i t i s be l i e v e d that the lycopod sporangium was once borne on an a x i l l a r y branch which became very much reduced. Bower (13) advanced t h i s theory. Schoute (50) f e l t that t h i s conception was "so improbable that i t hardly needs d e t a i l e d d i s c u s s i o n . " Lam (37), using the same f o s s i l evidence (Baragwanathia) implied that i t was so i n diagrams but d i d not assert i t . Zimmerman (64) derived the lycopod sporangium from an overtopped f e r t i l e telome fused w i t h a s t e r i l e *elome, and put Psil o t u m and Tmesipteris w i t h the lycopods in h i s group "Lycophyta." The question of whether the lycopod sporangium o r i g i n a t e d on a branch i s r e l a t e d t o that of the s t a b i l i t y of branching form. Schoute (50) thought that the f a c t that a x i l l a r y branching i s unknown i n the lycopods provided an argument against the o r i g i n of a sporangium on a branch, but Lam (37) who s t r e s s e d the telome theory, saw no problem in assuming changes i n branching. The d i s t i n c t i o n of the lycopods from the P s i l o p h y t e s on the basis of the absence of roots in the l a t t e r might be c a l l e d a r b i t r a r y s i n c e "the phylogenetic o r i g i n of the root from a s p e c i a l i z e d kind of stem . . . has always been more or l e s s probable" (50). Stem and root c h a r a c t e r i s t i c s intergrade in some organs of lycopods, e.g. the S e l a g i n e l l a rhizomorph and and the Isoetes root base. Associated w i t h the problem of how t o assess the morphological d i f f e r e n c e s between the l i v i n g lycopods and other p l a n t s i s the problem of f o s s i l s in which these d i f f e r e n c e s are less d i s t i n c t . For example, in Protohyenia. an e a r l y Devonian member of the Arthrophyta, the stem i s not 5 j o i n t e d though i t s other characters r e l a t e i t to Carboniferous arthrophytes (49). Baragwanathia possesses features that d e f i n i t e l y r e l a t e i t t o the lycopods (37) but i t s sporangia are attached to the stem by a sporangio-p h o r e - l i k e s t r u c t u r e . There are more c o n t r o v e r s i e s about the e v o l u t i o n a r y s i g n i f i c a n c e of the d i s t i n c t i o n s between these groups of p l a n t s . T h e i r s o l u t i o n requires c o n s i d e r a t i o n of many c h a r a c t e r i s t i c s and i t changes as more becomes known of f o s s i l i z e d and l i v i n g p l a n t s . In any case, f o s s i l evidence shows that the three main d i v i s i o n s of extant lycopods were e s t a b l i s h e d in the Carboniferous and have had a continuous h i s t o r y s i n c e (49). It i s c e r t a i n that these p l a n t s have been e v o l v i n g f o r a long time, so i t i s p o s s i b l e that comparing t h e i r chemical c o n s t i t u e n t s would throw l i g h t on trends in metabolic changes. The three extant orders of the Lycopodophyta are the Lycopodiales (one f a m i l y and two genera, Lycopodiurn and Phylloglossurn). the S e l a g i n a l -l a l e s (one genus, S e l a g i n e l l a ) • and the Isoetales (one f a m i l y and two genera, Isoetes and S t y l i t e s ) . Lycopodiurn and Phylloglossurn are homosporous, the other genera are heterosporous. Isoetes. S t y l i t e s and S e l a g i n e l l a have a l i g u l e , a t r i -angular s t r u c t u r e on the adaxial s i d e of the l e a f w i t h i t s base sunken i n t o the t i s s u e of the l e a f . The sporangia of Isoetes are d i s t i n c t from those of S e l a g i n e l l a because they are crossed by bars of c e l l s c a l l e d t r a b e c u l a e , and because the megasporangia c o n t a i n many megaspores ( S e l a g i n e l l a has four megaspores per megasporangium). S e l a g i n e l l a and Lycopod?um are q u i t e s i m i l a r i n h a b i t . They have erect or creeping branched axes bearing s p i r a l l y arranged leaves; a d v e n t i -6 tous roots arise on the basal or horizontal parts. The leaves associated with sporangia may be compacted into s t r o b i l i . Phylloglossum is apparently very reduced; possibly the short growing season where t h i s plant is found served to select t h i s habit. The stem is a shortened storage organ with i t s leaves crowded together. It bears a single strobilus with sporangia and sporophylls resembling those of Lycopodiurn. Isoetes represents a remnant of a l i n e of plants with t r e e - l i k e members which co-existed in the Carboniferous with Lycopodites and Sel l a g i n e l 1 i t e s . Isoetes consists of a very short erect axis which has been c a l l e d a corm, an erect rhizome, a stock, a stem, and a stem plus rhizophore (25). The nature of t h i s axis is obscured by an anomolous cambium, which occurs outside the phloem and produces storage tissue to the outside and mixed vascular tissue to the inside. The axis produces a rosette of leaves ( a l l f e r t i l e ) on i t s upper surface and roots on i t s lower surface. A change in the shape of the s t e l e forms the demarcation between the , ,stem , , and "rizophore." The lobed rhizophore has been interpreted as a very reduced Stigmarian axis through a series of forms comparable to Pleuromeia, Nathorstiana. and Sty 1ites, though t h i s has been debated (50) . In fact some authors have cal l e d i t a rhizomorph in com-parison to the rhizomorphs of Selaginella, structures produced by aer i a l stems which have a stem-like anatomy but lack appendages and produce true roots when they reach the ground. The fact that the origins of these structures are obscure emphasizes the antiquity of the plants which bear them. The gametophytes of Lycopodiurn and Phylloglossum are unlike those of Selaginella; Selaginella gametophytes are much smaller, a consequence 7 of endosporal gemination. They are unisexual, and the male gametophytes are much smaller than the female gametophytes, since the plant is hetero-sporous. However, Selaginella and Lycopodiurn both produce b i f l a g e l l a t e sperm. Isoetes is heterosporous, but i t produces s p i r a l l y twisted multi-f l a g e l l a t e sperm sim i l a r to those of ferns and Equisetum. The relationships among the orders of lycopods are obscure. On morphological grounds the Lycopodiales and Isoetales are quite d i s t i n c t . The Selaginellales share the presence of a l i g u l e with the Isoetales and b i f l a g e l l a t e sperms with Lycopodiales. Whether either character infers a closer relationship with one group or the other is a matter of opinion (52). The f o s s i l evidence (Selaginel 1ites co-existent with Lepidodendron) implies that Selaginella is not a direct descendent of the lepidodendrid l i n e , whereas Isoetes may be. Within the genus Lycopodiurn there is a great d i v e r s i t y between species, and various subdivisions of the group have been proposed (48, 49, 60). The divisions have been made on the basis of degree of d i f f e r e n t i a -t i o n and compaction of sporophylls, type of branching, and degree of heterophylly, as well as gametophytic c h a r a c t e r i s t i c s . Different taxonomic ranks have been given to these subdivisions. Rothmaler (48) segregates them into two families with one genus in the f i r s t and three genera in the second. As chemical differences might exist between these subdivisions, as many species of Lycopodiurn as possible were examined. Phylloglossum contains a single species. Selaginella species are less heterogenous and Rothmaler has placed these in three genera in one family. In the present study, l i v i n g material was available for only four species of Selaginella. so a survey of the species was not made. The species of Isoetes are quite 8 s i m i l a r , so no attempt was made to survey a large number of spec i es . Chemistry ( i ) General Considerat ions The chemical const i tuents of plants have provided chemical problems fo r many years . The pharmacological proper t ies of p lants have been studied at least s ince the beginnings of h i s to ry (56), and a large body of know-ledge of interest to chemists and pharmacologists has accumulated. This information has aroused the in teres t of workers concerned with b i o l o g i c a l problems, and in some cases has proved useful to them. Chemical cha rac te r -i s t i c s have been used both as ind ica tors of phylogeny and as taxonomic markers. For example, in the algae " the primary c r i t e r i a used in d i s t i n g -u ish ing the d i v i s i ons are biochemical . . . " (49). The phylogeny of these organisms is r e f l e c t ed in t he i r chemical c h a r a c t e r i s t i c s . Examples of chemical c h a r a c t e r i s t i c s used as taxonomic markers are common; the l i chen acids are an example. Spot tes ts fo r these acids are used in keys to l ichens (27). V i s i b l e chemical c h a r a c t e r i s t i c s such as the presence of raphides and the form of s tarch grains have been used as taxonomic markers fo r some t ime. The i r degree of s p e c i f i c i t y and constancy under varying cond i t ions have been es t ab l i shed , and so they are accepted as d iagnost ic characters (26). This is not so fo r most low molecular weight plant con -s t i t u e n t s . But s ince these may be def ined p r e c i s e l y , and s ince they provide an independent l i n e of evidence, they may be va luable addi t ions to morphol-og i ca l c h a r a c t e r i s t i c s in desc r ib ing p l a n t s . However, most chemical c h a r a c t e r i s t i c s are not v i s i b l e , and genera l ly i t is much more p r a c t i c a l to use morphological c h a r a c t e r i s t i c s to ident i f y p l a n t s . Chemical 9 characteristics are of more interest in c lar i fy ing plant relationships. The chemistry of a plant, l ike its morphology is determined by the plant 's genetic complement, so it is to be expected that chemistry wi l l reflect phylogeny. But it is d i f f i cu l t to interpret phylogeny from chemistry for the same reasons that early c lass i f icat ion systems based on morphology did not necessarily reflect phylogeny. It was not known what degree of relationship was implied if two plants shared a certain charac-t e r i s t i c . As more became known of how these characteristics evolved, the phylogeny of the organisms which possessed them became clearer. Fossil evidence showed which structures primitive organisms possessed and how these structures were modified to perform a function which gave the plant a better chance for survival . Analogous structures could be distinguished from homologous structures and primitive structures could be distinguished from reduced structures. Characteristics that are conservative were found, and the phylogenetic implications of morphological s imi lar i ty could be assessed. Information about the evolution of most chemical characteristics is lacking. Therefore, the phylogenetic implications of chemical s imi lar i ty are mostly speculative. There is interest in these problems. The proceedings of several symposia (e.g. those edited by Swain, "Chemical Plant Taxonomy") provide some examples. The qualit ies of chemical characteristics that might prove useful in taxonomy have received some attention. Erdtman (22) suggests that sub-stances which have intermediate distr ibution and "biosynthet?c complexity" would best indicate relationship. Hegnauer (31) stresses the importance of looking at a whole range of products. Sorenson (51) feels a useful 10 compound would have a limited d i s t r i b u t i o n , that i t s d i s t r i b u t i o n would have some correlation with botanical c l a s s i f i c a t i o n , and that i t would be independent of divergent or convergent evolution. This last requirement draws attention to some problems involved in applying biological theories to chemical c h a r a c t e r i s t i c s . The concepts of divergence and convergence can be applied to the evolution of biosynthetic routes, but not enough is known of most biosynthetic routes to know i f the application is reasonable. Erdtman (22) points out that "chemical divergence" is easy since a muta-tion that blocks an early step in a biosynthetic pathway may produce plants with "dormant" synthetic routes and abnormal chemistry. Also, chemical p a r a l l e l evolution is possibly more l i k e l y than morphological p a r a l l e l evolution, although convergence of a large number of chemical characteristics in unrelated plants is u n l i k e l y . Erdtman's theories are plausible but d i f f i c u l t to demonstrate. He gives as an example of diverg-ence a case where apparently s i m i l a r enzyme systems produce d i s s i m i l a r alkaloids. However, i t is possible that these systems have ent i r e l y different enzymes which happen to act s i m i l a r l y on s i m i l a r compounds. As an example of convergence, he c i t e s the p o s s i b i l i t y that the stilbene skeleton may be synthesized by two e n t i r e l y different routes. But the existence of only one of these routes has been supported by tracer studies; the other is hypothetical. The transformations between flavonoids provide another speculative example of divergence. Birch (11) notes that these transformations depend on the stereochemistry of an intermediate when i t is attached to an enzyme surface. Slight changes in the enzyme could result in quite drastic changes in the product. He emphasizes that the products of enzyme systems are not 11 such d i r e c t ind ica tors of phylogeny as are the enzymes themselves. Since many b iosynthet i c routes are interwoven "not much phylogenet ic information can be obtained from a few f o r t u i t o u s l y i so la ted substances . " Sorenson (51) gives an example of p a r a l l e l evo lut ion of s im i l a r ace ty len ic com-pounds in the Basidiomycetes and Compositae. In t h i s case the morphology of the organisms c l e a r l y demonstrates that they are unre la ted . A l s o , the ace ty len ic compounds are "completely d i f f e r en t types of metabol ic product in the two t a x a . " He a lso gives an in te res t ing example of non-convergence of a chemical c h a r a c t e r i s t i c in two composites in which morphological con -vergence had occur red . He examined two species which E. Hulten had placed in d i f f e r en t genera, contrary to most op in ion . The species had the ace ty len ic compounds c h a r a c t e r i s t i c of the two genera appropr iate to Hulten 's c l a s s i f i c a t i o n . When asked what h is botanical d i v i d i n g l i n e was, Hulten rep l i ed that he had known them persona l l y from twenty-five years in the f i e l d . Hegnauer (31) a lso appl ies the concept of convergence to chemical charac te rs . He gives examples of analogous modes of formation of s i ng l e a lka lo ids and of a l ka l o id ske le tons . He a lso gives examples of d ivergence; he c i t e s cases of qu i te d i f f e r en t a lka lo ids that are re la ted b i o s y n t h e t i c a l l y . He notes that the poss ib l e "homology" of a r i s t o l o c h i c ac id and the aporphine bases may prov ide an add i t iona l argument fo r p l ac ing the Aristolochiaceae in the Po lycarp iceae. The ac id and the bases have the same ske le ton , and the conversion of the bases to the ac id is l i k e l y . The problem of analogy of morphological features is better under-stood when the funct ions of analogous s t ructures are cons idered . But the funct ions of p r a c t i c a l l y a l l the secondary metabo l i tes , which are ofi imore l imi ted d i s t r i b u t i o n and so of more taxonomic interest than so-ca l led 12 primary metabolites, are a matter of speculation. Gibbs (26) draws attention to the p o s s i b i l i t y that they may determine the food s p e c i f i c i t y of insects or other animals, acting as repellants or in a few cases as attractants (an advantage to entomophilous plants). Bate-Smith (7) gives two of the few examples of secondary compounds for which there apparently are obvious functions, the anthocyanins and betacyanins. There is a biosynthetic l i n k between the flavonoids (including anthocyanins) and the betacyanins. Both anthocyanins and betacyanins produce flower colour but do not co-exist in the same plant. Anthocyanins are produced in p r a c t i c -a l l y a l l families of Angiosperms except eight of the nine families in the Centrospermae (29) . In these f a m i l i e s , flower colour is produced by betacyanins. The two types of compounds are produced from intermediates synthesized by the shikimic-prephenic pathway, but the further development of these intermediates proceeds along different paths to produce one or the other type of compound (7) . Thus, thei r d i s t r i b u t i o n may i l l u s t r a t e a case in which the loss of a step in a biosynthetic sequence (that to produce anthocyanins) put' the plant at an evolutionary disadvantage and selection of individuals which could compensate for t h i s (by producing betacyanins) occurred. Neish (43) considers that many secondary metabolites accumulate in plant c e l l s simply because the plant has no method of getting r i d of them. In some cases these "wastes" may have conferred an evolutionary advantage on the plant so selection of individuals which produced them occurred. In other cases, secondary metabolites may have no function. Consideration of function leads to consideration of what effect environment may have in modifying the parts responsible for the function. Since p r a c t i c a l l y nothing is known of the function of secondary chemical 13 constituents, i t is hard to take this general view. There are examples in which environment is known to a l t e r chemistry, at least in individuals. These do not show how environment might effect biochemical evolution, but do point out other reasons why caution must be used in applying chemical characteristics to taxonomy. Fluck (2h) points out that i t is necessary to know i f a constituent i s genetically controlled or i f i t s presence is dependent on outside factors before i t can be used in taxonomy. He gives examples of substances which may vary with i n t r i n s i c (diurnal, onto-genetic) changes and with e x t r i n s i c changes. There are many examples of the second case; substances vary especially with s o i l types (different physical, chemical, and microbiological factors) and with climatic types (different combinations of radiation, temperature, and water). He notes that "no one has observed a substance disappearing, or a new substance appearing by change in any of the e x t r i n s i c factors," but that so far most studies have been carried out with compounds present in r e l a t i v e l y large amounts. If environment modifies the characteristics of a population permanently by selecting certain individuals, there is a progression over a period of time from " p r i m i t i v e " to "advanced" organisms. Suggestions about changes in the chemical characteristics of populations as a result of natural selection have been made. It has been postulated that larger molecules and a higher degree of unsaturation in glycerides are advanced c h a r a c t e r i s t i c s , and that the development: carbohydrate - saturated o i l -unsaturated o i l in seeds represents recapitulation. The possible phylogenetic value of degree of unsaturation is supported by the work of Mirov on Pinus (26). However, large molecules need not characterize 14 advanced plants. Birch (11) notes that a mutation is most l i k e l y to result in loss of a stage in a biosynthetic sequence, possibly resulting in a decrease of molecular complexity. Hegnauer (31) questions whether an increase in s i z e or complexity of a molecule represents progression in connection with McNair's c l a s s i f i c a t i o n of alkaloid containing plants. In chemical as well as morphological characteristics there is the problem of whether s i m p l i c i t y represents primitiveness or reduction. Another suggestion about chemical progression was made by Harborne (29) who drew attention to the d i s t r i b u t i o n of pelargonidin and delphinidin in most advanced flowering plants "replacing cyanidin, which was considered to be the most 'primitive' anthocyanidin." It is possible that some metabolic systems undergo l i t t l e change because of natural selection. Their products could be c a l l e d "conserva-t i v e " chemical c h a r a c t e r i s t i c s . Again, though the theory is applicable, examples are not known for c e r t a i n . Sorenson (51) feels that the presence of acetylenic f a t t y acids is "a good chemotaxonomic character" since they occur regularly in the Santalaceae, an o l d , morphologically conservative group. Unfortunately, arguments such as this for the evolutionary s t a b i l -i t y of chemical characteristics are based on t h e i r co-occurrence with conservative morphological characters which could lead to a c i r c u l a r argument. Erdtman (22) sums up that no lines of progression in chemical characteristics are known with certainty. The conclusive evidence for lines of progression is found in the f o s s i l record. Examples of "biochemical f o s s i l s " are known. Eglington and Hamilton (21) point out that Recent marine sediments show a marked alternation in the amount of odd and even numbered n-alkanes, a pattern 15 c h a r a c t e r i s t i c of plants. This pattern becomes less pronounced with i n -creasing age of the sediments. Other " f o s s i l biochemicals" in petroleum deposits include complex molecules probably derived from steroids, triterpenoids, etc., and o p t i c a l l y active compounds (1) . Abelson (1) feels i t w i l l be possible to isolate ancient biochemicals such as amino acids, l i p i d s , and degradation products which can be related to more complex molecules from rocks and f o s s i l s . As an example of the last type , of " f o s s i l " he quotes the work of Triebs who isolated porphyrins from many sediments which he related to chlorophyll. Extensions of his work gave good evidence of the occurrence of chlorophyll in l i v i n g systems for the past 550 m i l l i o n years. He also notes the p o s s i b i l i t y that isotopic studies may give indirect evidence about the biosynthesis of f o s s i l com-1 2 1 3 pounds; for example, low Czir'/C;'! fractionation may imply CO2 f i x a t i o n other than by the pathway involving ribulose diphosphate. Ideas which developed over many years of study of the morphology of plants are beginning to be applied to some of the chemical constituents of plants. Studies so far have been very limited; Erdtman (22) says "the structures of only a few thousand natural products have been established, and these obviously represent only a very small group as compared to those that remain to be discovered." Price (hk) points out that the majority of species have never been examined chemically. Many species which have been examined have been subjected to general tests (e.g. "spot-tests" for alkaloids) which are useful for indicating whether further examination would reveal a high concentration of an economically important chemical but which are not useful for taxonomic purposes. In most instances where a complete chemical i d e n t i f i c a t i o n has been made, factors such- as seasonal 16 v a r i a t i o n or v a r i a t i o n in d i f f e r e n t organs have not been regarded. He concludes that now i t i s not p o s s i b l e to argue on chemical grounds with morphological evidence, but that d e t a i l e d knowledge of the chemistry of p l a n t s would be v a l u a b l e in the study of morphology. Bate-Smith (8) regards the value of chemical s i m i l a r i t y to be t o draw a t t e n t i o n t o otherwise obscure r e l a t i o n s h i p s , provide supplementary evidence f o r debatable r e l a t i o n s h i p s , or to help in s o l v i n g taxonomic problems that have not yet been touched. ( i i ) Chemistry of the Lycopods  S e l a g i n e l l a S e l a g i n e l l a species have been included i n a few chemical surveys and some i n t e r e s t i n g compounds have appeared. Wall et_ aj_. (59) included S e l a g i n e l l a r u p e s t r i s among the thousands of species of p l a n t s they s u r -veyed f o r s t e r o i d a l saponins, a l k a l o i d s , f l a v o n o i d s , and s t e r o i d s of pharmaceutical i n t e r e s t . They had negative r e s u l t s w i t h S e l a g i n e l l a f o r everything except f l a v o n o i d s , but the spot t e s t s they used would give p o s i t i v e r e s u l t s only i f large amounts of these compounds were present. Harada and S a i k i (28) included S e l a g i n e l l a t a m a r i s c i n a and S e l a g i n e l l a  pachystachys in a survey of the f l a v o n o i d s of ferns using one d i r e c t i o n a l paper chromatography. They recorded apigenin from both. Hsu -(3*0 has i s o l a t e d the b i f l a v o n y l s amentoflavone and s o t e t s u f l a v o n e from S e l a g i n e l l a t a m a r i s c i n a . Towers and Gibbs (54) found that the l i g n i n of S e l a g i n e l l a resembles angiosperm l i g n i n in having both s y r i n g y l a l d e h y d e and v a n i l l i n in i t s nitrobenzene o x i d a t i o n mixture. Apparently the a b i l i t y to produce t h i s type of l i g n i n has evolved independently in these two groups. It i s i n t e r e s t i n g that vessel elements have a l s o evolved independently in S e l a -17 g i n e l l a and in angiosperms. Arthur and Cheung (4) surveyed the f o l k medicinal plants of Hong Kong for alkaloids using two extraction techniques and a variety of spot tes t s . They included Selaginella at rover i d i s and Selaginella involvens in t h e i r survey. They only reported strongly p o s i t i v e tests (e.g. Lycopodiurn cernuum. from which two alkaloids have been isolated, was not reported to contain a l k a l o i d s ) . Selaginella was not l i s t e d as an alka l o i d containing plant. An extension of the survey of Selaginella species for alkaloids is .reported in t h i s thesis. In 1913, Anselmo and Gilg (30) isolated the disaccharide trehalose (two D-glucose molecules linked a,a[l - 1]) from Selaginella lepidophylla. Figure 1 Trehalose Hydrogen bonding probably forces one ring to l i e over the other (23). In 1929, Yamishito and Sato (63) isolated trehalose from 21 species of Selaginella from Japan and Southern Manchuria. This sugar has been found in bacteria, a variety of fungi and lichens, crustaceans, and is the 18 main sugar in the c i r c u l a t o r y system of insects (58). As i t is non-reducing and r e l a t i v e l y d i f f i c u l t to hydrolyze i t is hard to detect by means of the common tes ts used fo r chromatographic ana lys is of sugars . It may be more common than is known at present . A l lsopp (2), in surveying sugars and organic acids in Archegoniates, reported that sucrose, glucose and f ruc tose are prominent in the bryophytes and pter idophytes , except in two of the three species of S e l a g i n e l l a he examined. He noted that t reha lose may be an important sugar in these spec i es , but that th i s would not be revealed by h is methods. However, he concluded that there was no notable d i f f e r ence between the carbohydrate metabolism of bryophytes, p ter idophytes , and higher p l an t s . He did not examine any Lycopodiurn species in his survey. Isoetes Isoetes l a c u s t r i s . which was included in Wa l l ' s survey (59), gave negative tes ts for f l a vono ids , a l k a l o i d s , tannins and s t e r o l s . Bate-Smith (8) made a survey of vascular p lants fo r leucoanthocyanins using a spot test (anthocyanidin t e s t ) . He confirmed p o s i t i v e react ions with another spot tes t ( v a n i l l i n t e s t ) . He included Isoetes l a c u s t r i s , f o r which the anthocyanidin tes t was inconc lus ive but which gave a p o s i t i v e v a n i l l i n t e s t . From th i s he in fer red that Isoetes contained leucoanthocyanins. In t h i s respect , Isoetes d i f f e r ed from Lycopodiurn and S e l a g i n e l l a . Bate-Smith found that the presence of leucoanthocyanin is genera l ly associated with woody hab i t , and a lso with the more p r im i t i v e members o f a phy lo -genet ic l i n e . He concludes that they are "par t of a p r im i t i v e metabolic pattern assoc iated wi th , but not essent i a l t o , a t r ee- l i ke or woody h a b i t . " The presence of leucoanthocyanins in Isoetes (for which the evidence is 19 admittedly s l i m ) could p o s s i b l y t i e Isoetes more c l o s e l y t o the t r e e - l i k e Lep idodendrales. Towers and Gibbs (54) examined the a l k a l i n e nitrobenzene o x i d a t i o n products of the l i g n i n of Isoetes muricata and found that i t contained both v a n i l l i n and syringaldehyde. A l l s o p p (2) included Isoetes echinospora i n h i s survey of sugars and organic acids of Archegoniates and found that i t had a comparable a c i d content t o succulents such as Bryophyllum and Sedum. He d i d not report anything unusual about i t s sugars but, as he says, h i s method would not reveal t r e h a l o s e . Examination of Isoetes f o r t r e h a l o s e i s reported in t h i s t h e s i s . Chenery (16) examined three species of Isoetes f o r aluminum content but none of them were found to be accumulators. He re-examined a species which Japanese workers had reported as aluminum accumulating and found i t contained a moderate amount, but f e l t that i t may have r e s u l t e d from d i f -f u s i o n from very aluminous waters. Phylloglossum Chenery (16) included Phylloglossum drummondii in h i s survey, and reported that i t " d i s p l a y e d i t s r e l a t i o n s h i p w i t h Lycopodiurn" by being an aluminum accumulator. Examination of Phylloglossum f o r carbohydrates and a l k a l o i d s i s reported i n t h i s t h e s i s . Lycopod i urn Chenery (16) included 81 species of Lycopodiurn i n h i s survey and found 27 to be aluminum accumulators. His work confirmed that of o t h e r s , who found that the members of the subsection Urostachys were s l i g h t l y 20 aluminum accumulating (5 of 48 species examined contained 0.3 to 2.8% of aluminum on an ash weight basis), while the members of the Rhopalostachya accumulated more (in 22 of 33 species examined, the content was 3.7 to 26.1% aluminum, ash weight). Rothmaler (48) puts the members of these sections in different f a m i l i e s . Differences in t h e i r a b i l i t y to accumulate aluminum may result from a general difference in the physiology of the mem-bers of the two sections. Chenery states that aluminum accumulation is probably related to a complex of physiological c h a r a c t e r i s t i c s . Kratzl and Eibl (36) examined the oxidation products of Lycopod?urn  clavatum l i g n i n . They found v a n i l l i n was present and concluded that large amounts of syringaldehyde were not present. Towers and Maass (55), using chromatographic methods, found that a l k a l i n e nitrobenzene oxidation of Lvcopodiurn l i g n i n did not y i e l d syring-aldehyde. In th i s i t differed from the l i g n i n of Selaginella . Isoetes. and angiosperms, and coincided with gymnosperm l i g n i n . Some species of Lycopodiurn did y i e l d syringic acid, both in the l i g n i n oxidation products and in the ethanol soluble f r a c t i o n . These species were confined to two genera in one of the families described by Rothmaler. Zetsche-Huggler (30) isolated dihydrocaffeic acid after hydrolysis of Lvcopodiurn clavatum with IN NaOH. Achmatowicz and Werner-Zamojska (30) isolated v a n i l l i c and f e r u l i c acids from Lycopodiurn annotinum. Lycopod?urn  clavatum and Lycopodiurn selago. Wall's chemical survey included four species of Lycopod?urn. They a l l gave pos i t i v e alkaloid reactions and negative tests for flavonoids, tannins, and s t e r o l s . However, Muszynski (30) isolated a compound from Lycopodium selago which he named selaginosid and which had the properties 21 of a quercetin glycoside. Harada and Saiki (28) examined eight species of Lycopodiurn in t h e i r survey of flavonoids, and reported apigenin from four species and the p o s s i b i l i t y of a different flavonoid in the other four. Reibsomer and Johnson (47), prompted by c o n f l i c t i n g reports, exam-ined the fat of the spores of Lycopodiurn clavatum. and found i t contained 50-60% o l e i c acid and 30-35% hexadec-9-enoic acid, along with palmitic and 1 i n o l e i c acids. H i l d i t c h (32) notes that hexadec-9-enoic acid is now known to be a constituent of nearly a l l natural f a t s , but that i t is a "major component only in the lower forms of l i f e " ( i t has been isolated from a ba c i l l u s and from Saccharomyces cerevesel). . . . "and the more developed aquatic f l o r a and fauna." He notes that in animal fats "a progressive diminution occurs in the proportion of hexadec-9-enoic acid corresponding with the evolutionary development of the species." Achmatowicz and Werner-Zamojska (30) isolated azelaic acid (C00H-(CH2)7~C00H) from Lycopodiurn annotinum and Lycopodiurn clavatum. Inubushi et. al_. (35) have isolated the triterpenoids a-onocerin and serratenediol from Lycopodiurn clavatum and Lycopodiurn serratum. Sucrose has been found in the spores of Lycopodiurn clavatum (30). It has also been found to be a major component of the ethanol soluble f r a c t i o n of the whole plant in miany species of Lycopod?urn (55). Manske and Marion (40) did a preliminary investigation of the insoluble polysaccharides of Lycopodiurn compIanaturn and found large amounts of galactose in the hydrolysis product. A search for fucose and mannose was negative. Hegnauer (30) reports that Lycopodiurn accumulates starch. An investigation of the soluble carbohydrates of Lycopodiurn is reported in t h i s thesis. 22 The most chemical work done on any lycopod has been the investiga-tion of the alkaloids of Lycopodiurn. Bodecker (30) in 1881 c r y s t a l l i z e d a base from Lycopodiurn complanaturn which he named "lycopodine.' 1 In 1938 Achmatowicz and Uzieblo (30) c r y s t a l l i z e d lycopodine and two other a l k a l -oids from Lvcopod?urn clavatum. In 19^2 Manske and Marion (kO) began a series of structural studies on these alkaloids and isolated many new ones. Lately, with the use of new chemical techniques, great advances have been made in the structural elucidation of Lvcopod?urn alkaloids. The structures of 31 alkaloids have been determined. Most of these are variations of the same basic skeleton (Figure 2). Twenty d i f f e r only in the substituents of rings A and B from lycopodine; Figure 2 Lycopodine 23 In lyconnotine (Figure 3) the methyl-substituted ring is broken, Figure 3 Lyconnotine (3) and annotinine (Figure 4) has a four-membered ring and epoxide and lactone side rings. Figure k Annotinine Seven have the basic structure of lycodine (Figure 5). Figure 5 Lycodine In selagine (Figure 6) the N-substituted ring is opened. 25 The only other skeleton is the one recently proposed (5) for lycocernuine and cernuine: Figure 7 Cernuine, Lycocernuine, and Suggested Precursors (5) Cernuine X = H Lycocernuine X = OH This structure was suggested partly because i t f i t s well with a scheme of biogenesis involving condensation of poly-p-keto acids, as in Figure 7. 26 The formation of Lycopodium alkaloids from condensation of poly-p-keto acids was f i r s t suggested in 1958 by Leete (38) for annotinine as follows: T *0 -h 'CO OH C O O K (XS D a r l " Q~tc_ 'HO coow Figure 8 Biosynthesis of Annotinine Suggested by Leete (38) There is condensation of a s i x carbon polyacetate straight chain (1) with mevalonic ketone (2); nitrogen is introduced as the dialdehyde amine (3) 27 which can be derived from aspartic acid or i t s equivalent. A series of internal condensations, oxidations and reductions produce annotinine. The quinolizidine alkaloid lupinine has been postulated to be formed from lysine v i a i t s lower diamine homologue cadaverine as follows: l y s i n e . c a d a v e r i n e l u p i n i n e Figure 9 Biosynthesis of Quinolizidine Skeleton (42) The more complex quinolizidine alkaloids of Lupinus and the PapiIionaceae have been derived by s i m i l a r schemes. This hypothesis has been borne out by tracer studies. Labelled lysine and cadaverine have been shown to be incorporated extensively into lupinine and the more complex homologues, and some det a i l s of the pathway have been suggested (42). This type of biosynthetic pathway has apparently not been suggested for the Lycopodiurn alkaloids. Conroy (18) proposed that annotinine could be derived by condensa-tion of two eight-carbon polyacetate straight chains, with incorporation of nitrogen by a mechanism analogous to Mannich condensation with ammonia. After c l a r i f i c a t i o n of the structures of other Lycopodiurn alkaloids, he found these could be f i t t e d into the same scheme (Figure 10). 28 Condensation of two eight-carbon polyketo acid chains gives struc-ture 1 from which a l l the others are derived. (Structures 1, 2 and 3 are equivalent.) S e l a q ( n e LjCo_ine Annotinine L^copoe(ir»e Figure 10 Biosynthesis of Four Basic Skeletons of Lycopodiurn Alkaloids Suggested by Conroy (18) 29 In support of t h i s theory, Conroy notes that every oxygenated position of the proposed precursor "either (1) takes a direct part in the formation of the f i n a l skeleton, or (2) can be otherwise accounted for as an oxygenated or unsaturated center in one or the other of the f i n a l a l k aloids." If t h i s is true, then the Lycopodiurn alkaloids are "analogous" to the quinolizidine alkaloids of Lupinus and the Papi1ionaceae, since, although the alkaloids of both genera are s t r u c t u r a l l y s i m i l a r , they are derived from amino acids in one case and from poly-p-keto acids in the other case. According to one d e f i n i t i o n (31) the Lycopodiurn alkaloids should be ca l l e d "pseudo-alkaloids," a term which emphasizes their bio-synthetic difference. However, as Conroy says, "the scheme requires experimental study;" the appropriate tracer studies would be d i f f i c u l t because the poly-p-keto acids are rather unstable. Ayer (6) and co-workers suggest that lycopodine, which occurs in p r a c t i c a l l y a l l species of Lycopodiurn, is a central intermediate in the biosyntheses of the Lycopodiurn alkaloids. They derive the annotinine skeleton more less d i r e c t l y from lycopodine v i a an annofoline type inter-mediate (Figure 11) and have shown the f e a s i b i l i t y of the f i r s t conversion in the laboratory. 30 Figure 11 Biosynthesis of Annotinine Suggested by Ayer et al_. Aside from lycopodine, the d i s t r i b u t i o n of alkaloids within the genus Lycopodiurn may be ch a r a c t e r i s t i c of a species. Frequently the same alk a l o i d appears in different species but in some species a combination of alkaloids is ch a r a c t e r i s t i c of the species. In others there is variation within the species; different alkaloids have been reported in L. annot inurn from Canada, Poland, Germany, and the West Indies (30). These may have been quantitative differences, or seasonal differences, or possibly genetically controlled differences. Determination of the biosynthetic pathway to these alkaloids may c l a r i f y the problem. Manske and Marion (41) proposed that Lycopodiurn annotinum var. acrifolium Fernald be given species rank (Lycopodiurn acrifoliurn (Fern.)) since i t s alkaloids were ent i r e l y different from those they found in other Lycopodium annotinum material. Alkaloid content may prove useful to the taxonomy of Lycopodiurn species. 31 METHODS AND MATERIALS 1. Sources of Plant Material This information is found in Table I. 2 . Preparation of Aqueous Extracts Whole plants were chopped in a Waring Blendor with b o i l i n g 80% ethanol. This material was f i l t e r e d hot, washed with b o i l i n g 80% ethanol, and taken to dryness in a rotary f l a s h evaporator. The residue was sus-pended in b o i l i n g water and f i l t e r e d through " C e l i t e " (diatomite f i l t e r aid, trade mark of Johns-Manvi1le Products). The f i l t r a t e was concen-trated to about 10 ml. The extracts were stored at ; 8*C after 5 ml of ethanol had been added to them. 3 . Preparation of Neutral Cation, and Anion Fractions of Plant Extracts Samples of about 20 gm of plant material were k i l l e d and extracted with b o i l i n g 80% ethanol as described. The residue obtained by evaporating the 80% ethanol extract was suspended in 15 ml chloroform and 25 ml water. The layers were separated by centrifugation. The aqueous layer was passed onto a column of 60 ml of Rexyn 101 (organic strong acid cation exchanger, trade mark of Fisher S c i e n t i f i c Company) arranged above a column of 50 ml of Dowex 3 (weakly basic anion exchanger, trade mark of J.T. Baker Chemical Company). A neutral f r a c t i o n was obtained by eluting the columns with about 250 ml of d i s t i l l e d water, the eluate from the cation exchanger passing onto the anion exchanger. The columns were then eluted separately, the cation exchanger with about 250 ml of 1M NH^ OH and the anion exchanger with about 250 ml of 1M (NH I F ) 2 C0,. The volumes of the neutral, cation, and 32 Table I Sources of Plant Material Plant Source Col lector Selaqinella pallescens "Emmeliana" (Presl.) Sprinq Munich, Botanical Gardens Courtesy of Dr. W.S.G. Maass S. pallescens " n o b i l i s 1 1 II II S. poulteri Hort. Veitch. II II S. martensii Sprinq II II S. usta ii it S. sp. 1 it ii S. caulescens (= " f l a b e l lata") (Wall.) Spring II II S. eleqans it n S. qrandis Moore II II S. sp. 2 II II S. wall acei Heiron II II II Horseshoe Bay, B.C. Sooke, B.C. Oregon Tofino, B.C. E.E. McMullan D. E. McMullan L.K. Wade E. E. McMullan S. densa Rybd. Kootenay Lake, B.C. II S. emmeliana Schmitz U.B.C. Greenhouse S. kraussiana A. Br. n Lycopodium squarrosum Forst. Munich, Botanical Gardens Courtesy of Dr. W.S.G. Maass L. sp. II II L. hippuris Wirzburg, Botanical Gardens II - continued 33 Table I, cont'd. Plant Source Collector L. phlegmaria L. L. pinifolium Blume L. clavatum L. II II it L. selago. L. i i L. s itchense Rupr. i i L. annotinum L. i i II L. complanaturn L. II j . . obscurum L. Isoetes occidental is Hend. l_. bo lander i Engelm. Botryehi urn virginianum ( U ) Kew Gardens 41 P a r k s v i l l e , B.C. bog, Tofino, B.C. steep mountain slope Tofino, B.C. Squamish, B.C. Nanaimo, B.C. Tofino, B.C. Mt. Seymour, B.C. Garibaldi Park, B.C. Bowron Lakes, B.C. Prince George, B.C. White River, B.C. Bowron Lakes, B.C. White River, B.C. Bowron Lakes, B.C. Marion Lake, B.C. Kennedy Lake, B.C. P a r k s v i l l e , B.C. Sw. White River, B.C. Courtesy of Dr. W.S.G. Maass II D.E. McMullan II II Dr. G.H.N. Towers D.E. McMullan R.C. Brooke T. Flegel II D.E. McMullan T. Flegel D.E. McMullan T. Flegel G. Davis L. Cordes D.E. McMullan (1) Horseshoe Bay (2) Sooke (3) Tofino (4) Kootenay Lake (5) U.B.C. Greenhouse (6) P a r k s v i l l e (7) Squamish (8) Nanaimo (9) Mt. Seymour (10) G a r i b a l d i Park (11) Bowron Lakes (12) P r i n c e George (13) White River (14) Marion Lake (15) Kennedy Lake 35 anion fractions obtained were recorded. The neutral and cation fractions were concentrated to about 10 ml in a fla s h evaporator, and stored in the same way as the aqueous extracts. 4. Preparation of Alkaloid Extracts The method of Manske and Marion (40) modified for much smaller amounts of material was used. Ten to 50 gm of whole plant material, chopped with 80% ethanol in a Waring Blendor, were heated under reflux for about two hours and f i l t e r e d hot. The residue was washed well with b o i l i n g absolute ethanol. The f i l t r a t e was taken to dryness in a fla s h evaporator, and re-suspended in a mixture of 30 ml d i s t i l l e d water and 10 ml chloroform. The pH was adjusted to 3 or less with HCl, and the layers were separated by centrifugation. Unless otherwise stated, the aqueous layer was adjusted to pH 8 with NH^ OH and placed with 30 ml chloro-form in a 125 ml Erlenmeyer f l a s k on a culture shaker for s i x hours. At the end of t h i s time the layers were separated by centrifugation and the chloroform layer containing the alkaloids was concentrated to 1 or 2 ml. In a few cases, attempts were made to fractionate the alkaloids by adding base to the acidic aqueous extract stepwise, extracting with diethyl ether after each addition and f i n a l l y extracting with chloroform. 5. Methyl at ion of Sugars Sugars were methylated p r i o r to t h e i r separation and estimation using vapour phase chromatography. Methyl at ion was carried out according to the method of Kuhn as used by Rast (46). S i l v e r oxide was prepared by pre c i p i t a t i o n from AgNO^ solution with either Ba(0H)2 or K0H. S u f f i c i e n t AgNO, and base were weighed out to give approximately 25 gm Ag,0, and a 36 concentrated solution of base was prepared and added to a d i l u t e solution of the AgNOy Adding excess base was avoided by testing the pH of the mixture after most of the base was added. The precipitated AgNO^ was thoroughly washed with water, absolute methanol and acetone, and dried in  vacuo over P 20,j. The Ag 20 was used after at least 12 hours drying and before 72 hours. Two grams of Ag 20 were added with each addition of 1 ml of methyl iodide so that the sequence of additions to the reaction mixture was: i n i t i a l l y , 10 ml Mel + 16 gm Ag 20; after 24 hours, 1 ml Mel + 2 gm Ag 20; after 48 hours, 1 ml Mel and 2 gm Ag 20. 6. Vapour Phase Chromatography A Perkin-Elmer 8]0 Gas Chromatograph with a thermal conductivity detector was used to separate the methylated sugars. The column was s i x feet long, 1/4 inch in diameter, packed with SE-30 Silicone Gum Rubber, coating weight 5%, on Chromosorb W.A.W. support material of 60/80 mesh si z e (Perkin-Elmer Packed Column 2). Helium c a r r i e r gas was used at a flow rate of 20 ml/min. The oven temperature was programmed from 150 to 290*C at a rate of 8°/min. 7. One Directional Paper Chromatography of Sugars for Qualitative Analysis Aliquots (about 50 MO of the aqueous extract were spotted onto s t r i p s of Whatman #1 f i l t e r paper, 7" x 22", and chromatographed by the descending method for 8 to 10 hours with a solvent mixture of ethyl acetate/ pyridine/water (12:5:4). Spots of a 0.1% solution of known sugars were placed beside the aqueous extract spots. The chromatography chamber was a c y l i n d r i c a l glass tank, diameter 1', height 2', with a glass plate l i d which was sealed with stop-cock grease. Solvent trays were glass troughs 37 8 1/2" x 2 1/2"; the paper was held in place as described in Block, Durrum and Zweig (12). 8. One Directional Paper Chromatography of Sugars for Preparative Isola- tion The neutral fraction of 100 gm of material was concentrated to about 5 ml and aliquots were streaked in a l i n e 3 1/2" from the long edge of 18" x 22" Whatman #1 f i l t e r paper. Spots of a 0.1% solution of tr e h a l -ose were placed at each end of the l i n e . The chromatograms were made as described above, except that rectangular chromatography tanks, 27" x f x 23" deep, f i t t e d with hinged l i d s and 26" glass solvent troughs were used. The solvent used was n-propanol/ethyl acetate/water (7:1:2). The chrom-atograms were run for 15 to 24 hours. About s i x t y chromatograms were required to separate the neutral fraction obtained from about 100 gm of material. 9. One Directional Paper Chromatography of Alkaloids Aliquots (about 50 pi) of the alkal o i d extract were spotted on 18" x 22" sheets of Whatman #1 f i l t e r paper beside known Lvcopodiurn a l k a l -oids. Chromatograms were made as described above, except that the paper was equilibrated in the chamber saturated with fumes from the lower layer of the solvent mixture. After e q u i l i b r a t i o n for one hour, the solvent trays were f i l l e d . The solvent used was the upper layer of a mixture of tert-amyl alcohol/acetic acid/water (70:10:40). 10. Two Directional Chromatography of Sugars and Amino Acids Aliquots of the neutral f r a c t i o n and of the cation fraction were spotted in one corner, 1 1/4" from each edge, of 8" x 8" sheets of Whatman 38 #1 f i l t e r paper. The chromatograms were made by the ascending method, by suspending the sheets so that one edge just touched the solvent. To do t h i s , glass rods were run through holes punched in the edges opposite the o r i g i n . Aquarium tanks with glass plate l i d s sealed with stop-cock grease and f i t t e d to hold the glass rods were used as solvent tanks. The bottom of the tanks acted as solvent trays. When the chromatograms had been run in one d i r e c t i o n , they were dried thoroughly, the glass rods slipped through the holes punched in the second side, and the chromatograms were run in the second direction in a tank containing the second solvent. Chromatograms were run u n t i l the solvent front rose to about 1/2 inch of the edge of the paper. Using t h i s method, up to twelve chromatograms could easily be run simultaneously. The solvent used for the f i r s t d i r e c-tio n was phenol/water/conc. NH^ OH (80:19.7:0.3); for the second direction n-propanol/ethyl acetate/water (7:1:2) was used. Both sugars and amino acids could be separated in these solvents. 11. Thin Layer Chromatography of Alkaloids Plates were prepared as described by Stahl (53), using a Desaga thin layer spreader. Layers were made 0.25 mm thick, of Aluminum Oxide G (with gypsum binder; Warner-Chilcott Laboratories Instrument Division). When th i s was not available, S i l i c a Gel G (E. Merck Ag.) was used. Adsorbents were applied as a 33% w/v aqueous s l u r r y . When S i l i c a Gel G was used the slu r r y was made up with 0.1N NaOH. Aliquots (about 50 MO of the alkaloid extract were applied one inch from the edge of the plate, beside known Lycopodiurn alkaloids. Chrom-atography chambers were glass tanks, 9" x 8 1/2" x k", f i t t e d with glass l i d s which were sealed with stop-cock grease. The bottom of the tanks 39 acted as solvent trays. Chromatograms were made by the ascending method by standing the plates in the tanks so the edge nearest the o r i g i n was immersed in the solvent. The solvents used with Aluminum Oxide G were chloroform, chloroform/ethanol (95:5), or benzene/ethyl ether (1:1). With S i l i c a Gel G benzene/chloroform/ethanol (5:3:2) was used. 12. Column Chromatography of Sugars A column was prepared by packing Whatman standard grade c e l l u l o s e powder into a 2 1/2" x 30" chromatography column as described by Hirst and Jones (33), and the column was tested with cresol red. The neutral f r a c t i o n from a large amount of material was applied to the column and eluted with one l i t r e of the top layer of a mixture of n-butanol/acetic acid/water (40:10:18). Fifteen ml fractions were collected using a mechanical fr a c t i o n c o l l e c t o r . Each fraction was concentrated to about 2 ml, and aliquots were chromatographed as described under "One Direc-tiona l Chromatography" to determine which sugars were in each f r a c t i o n . 13. Detection of Spots After paper chromatography, sugar spots were located by the method of Trevelyan, Procter and Harrison (57). This method was tedious and the background was rather dark, but i t consistently produced a very d i s t i n c t spot withtrehalose after any of the solvent systems described. The only solvent which interfered with t h i s method of detection was 80% phenol, but as i t was always used as the f i r s t solvent in two directional chrom-atography i t only obscured the solvent front. A more even background was produced i f the f i n a l NH^ OH wash was made up in 20% ethanol. The per-manganate/per iodate reagent described by Lemieux and Bauer (39), the 40 benzidine/periodate reagent described by Bean and Porter (9) and the permanganate/periodate/benzidine reagent described by Wolfrom and M i l l e r (62) were tested but they gave no or only a very f a i n t spot with trehalose after the solvents described had been used. Amino acid spots were revealed with a commercial ninhydrin spray (Sigma Chemical Company). Alkaloid spots on paper chromatograms were revealed by spraying with Dragendorff's Reagent, as described by Block, Durrum and Zweig (12). Alkaloid spots on thin layer plates were detected by spraying with Dragendorff's Reagent, or with the iodoplatinate reagent described by Randerath (45). 14. Technique for Administering C^OV, The apparatus (Figure 12) was essenti a l l y the one used by Bidwell (10). A i r was circulated over the plants in a glass chamber. A pump, a 14 Geiger tube, a manometer, and a trap containing Na2C Oj into which 2N h^SO^ could be injected were connected into the a i r stream. The window end of the Geiger tube was sealed onto the bottom end of a p l a s t i c bottle through which the a i r passed. The Geiger tube was connected to a ratemeter, to which a chart recorder was attached. The manometer was connected to warn of pressure changes that might break the Geiger tube window, but the pump (Thiborg Suction Pump, Model 1) maintained an even flow. The chamber was illuminated with about 1000 foot-candles from two sunlamps. An aquar-ium tank lined with f i l t e r paper on one side and f u l l of cold water was placed between the chamber and the li g h t source to diffuse the light and avoid over-heating the chamber. Whole plants were placed with t h e i r roots in water in a beaker, and H B ra D G IT I 4 r Figure 12 Apparatus for Administering COV, A = Reflector B = Pump C = Chamber D = Geiger Tube, connected to Recorder I = Light Source E = Manometer F = CO2 Analyzer G = Na2C 1 i f0 3 Trap H = Aquarium Tank with Paper 42 the beaker was put in the chamber, in the case of plants with extensive creeping axes, a section of the horizontal axis was coi l e d and the roots and the cut end were kept in water. The l i d of the chamber was sealed 14 with stop-cock grease, and C 0 2 was released by injecting acid through 14 the rubber septum sealing the Na2C 0_ trap. The change in the amount of rad i o a c t i v i t y in the a i r stream during feeding was recorded on the chart connected to the ratemeter. In some cases, an infrared CO- analyzer was also connected into the a i r stream and the change in C0 2 concentration was recorded. When the feeding was fini s h e d , the plant material was immed-iat e l y k i l l e d as described under "Preparation of Aqueous Extracts." 15. Technique for Administering Trehalose-C^ 14 Trehalose-C was administered to cut shoots in the dark. Plants with erect axes were used. Shoots were cut at the base and placed in water. The shoots were blotted, weighed, and replaced in water where another 1/8 inch was cut off the base under water with a sharp blade; then they were rapidly transferred to 10 dram v i a l s containing 7 mgm of trehalose-c '^ (1 He c'^ ) and about 10 cc water. In the case of Isoetes. the stem was cut as close as possible to the outermost leaves, the roots and most of the lobes were removed. After this plant was placed in the v i a l a t i n f o i l cone was dropped over the leaf t i p s to reduce evaporation. The v i a l s holding the plant material were set into holes in a board. Over t h i s was placed a rectangular metal box with screens at each end. Light was excluded by draping a black p l a s t i c sheet loosely over the ends. 16. Measurement of Radioactivity A l l measurements were made with a Nuclear-Chicago Liquid S c i n t i l -43 l a t i o n Counter, 750 Series. Measurements were corrected for background and e f f i c i e n c y . To correct for factors which decrease e f f i c i e n c y , the channels r a t i o method was used. Quenched standards with toluene s c i n t i l -l a t i o n solvent were provided with the instrument, so a quench correction curve was prepared and the effici e n c y calculated for any sample made with the toluene solvent. For the dioxane solvent quenched standards had to be prepared. The method for doing t h i s and the results are given in Appendix 1. To measure the rad i o a c t i v i t y of aqueous solutions the s c i n t i l l a t i o n solvent described by Bray (14) (naphthalene 60 gm, PPO 4 gm, P0P0P 200 mgm, methanol 100 ml, ethylene glycol 20 ml, dioxane to make 1 l i t r e ) was used. Duplicate aliquots of the neutral, cat ion and anion fractions were taken for r a d i o a c t i v i t y determination before the fractions were concentrated. The aliquots varied from 10 pi to 100 pi depending on the expected radio-a c t i v i t y of the solution. To f a c i l i t a t e comparison between different sized aliquots d i s t i l l e d water was added to each to make a to t a l volume of 2 ml before the s c i n t i l l a t i o n solvent was added. To measure the rad i o a c t i v i t y of spots excised from paper chromato-grams toluene solvent was used, prepared from commercial concentrate ("Liquiflour," P i l o t Chemicals Inc., f i n a l solution containing 4 gm PPO, 50 mg P0P0P, toluene to make 1 l i t r e ) . The chromatogram spot was placed in the bottle and solvent added u n t i l i t was two-thirds f u l l . 44 EXPERIMENTAL AND RESULTS 1. Vapour Phase Chromatography Standard mixtures cf sucrose and trehalose were methylated and separated by vapour phase chromatography as described. The mixtures con-tained 100% trehalose, 75% trehalose + 25% sucrose, 50% trehalose + 50% sucrose, 25% trehalose + 75% sucrose, and 100% sucrose. Each mixture contained a tota l of 250 mgm of sugar. The results of vapour phase chromatography are shown in Figure 13. Samples containing about 2 mgm methylated sugar were applied to the column. In some cases samples con-taining up to 10 mgm tota l methylated sugar were applied in an attempt to improve resolution of the peaks. 2. Preliminary Survey of the Sugars of Lycopodium and Selaginella The sugars of ten species of Selaginella and f i v e species of Lycopodium were surveyed by the method described for the one directional paper chromatography of sugars for q u a l i t a t i v e analysis. A small amount (0.4 gm) of Phylloglossum drummondii was available for analysis. An aqueous extract of this sample was prepared and de-ionized with a small amount of mixed ion exchange resin. The neutral fraction obtained was chromatographed in the same way as the Lycopodiurn and Selaginella extracts, except that the n-propanol/ethyl acetate/water solvent was used. The results are shown in Table I I . 3. Survey of the Distribution of Radioactivity in Sugars and Amino Acids 14 of Lycopods Fed C 0-Four species of Selaginella. two species of Isoetes. and s i x species 45 "I l«t«!Hjfllli»|lll!'l!'l ' >*JI « j iiniiiwiimlmlllll^ ^ 2 3 Figure 13 ( cont 'd , next page) 46 1 "1 4 \7\ A :A " 5 Figure 13 (cont'd.) Vapour Phase Chromatography of Mixtures of Methylated Sucrose and Trehalose 1. 100% Trehalose 2. 75% Trehalose, 25% Sucrose 3. 50% Trehalose, 50% Sucrose 4. 25% Trehalose, 75% Sucrose 5. 100% Sucrose 47 Table II Preliminary Survey of Sugars in Lycopodiurn and Selaginella No. of Chromato Name grams Trehalose Sucrose Glucose Fructose Other Selaginella pallescens "Emmeliana" 8 5 4 5 0 5 S. pallescens " n o b i l i s " 2 2 2 2 0 2 S. poulteri 6 6 1 03) 6 0 6 S. martensii 7 7 (3.?) 5(1?) 0 5 S. usta 8 3(4'.?) 1 4 5 7 S. (undetermined " sp., 1) 2 2 0 2 0 2 S. canlescens 4 3 (1-J) 3 2 3 S. elegans 3 3 3 3 3 3 S. grand is 2 2 2 2 0 2 S. (undetermined sp., 2) 3 3 3 3 3 3 S. wallacei 1 1 1 1 03) 1 Lycopodium squarrosum 4 era) 4 • 4 4 4 JL. (undetermined sp.) 1 0 1 1 1 1 L. hippuris 2 (2?) 2 2 (2,?) 2 L. phleqmata 2 <n) 2 2 2 2 L. pinifolium 2 0 2 2 OS) 2 Phylloqlossurn drummondii 1 0 1 1 1 1 The number of chromatograms which contained a spot which co-chromato-graphed with the appropriate sugar are indicated by the number in the column. 48 of Lycopodium of which l i v i n g material were available were administered for two hours as described. Individuals of a species from different locations were tested to assess v a r i a b i l i t y within a species. The fern Botryehium virginianum was included to provide a comparison with a plant in another d i v i s i o n . The two species of Isoetes took up l i t t l e radio-a c t i v i t y in two hours so they were also fed for f i v e hours. Neutral and cation fractions were prepared and chromatographed two d i r e c t i o n a l l y as described. Radioautographs were made for each chromatogram, the radio-active spots were located, excised, and measured with the l i q u i d s c i n t i l -l a t i o n counter. The results of this survey are shown in Tables III and IV. Representative radioautographs of sugar chromatograms are shown in Figure 14. 4. Isolation of Radioactive Trehalose Positive i d e n t i f i c a t i o n of trehalose in Selaginella wallacei was carried out by i s o l a t i n g the compound which co-chromatographed with trehalose from this plant and comparing i t s infra-red spectrum to that of commercial trehalose. At the same time, radioactive trehalose for use in 14 other experiments was obtained by feeding the plant C 0 2 before iso l a t i n g the trehalose. Small samples (about 10 gm) of Selaginella Wallace? were 14 fed 30 He of C 0 2 as described u n t i l a t o t a l of about 100 gm of plant material had been fed. Preliminary attempts to isol a t e trehalose by the method of Yamashito and Sato (63) and by the method of Clark (17) were not successful, even after the sugar fr a c t i o n had been separated from amino acids and organic acids by passing the aqueous extract through ion exchange resins. An attempt to isolate trehalose from the neutral aqueous extract of 60 gm of Table I 11 Distribution of Radioactivity in the Neutral Aqueous Extract of Plants Fed Total radio- % Radioactivity a c t i v i t y in ~_ the fraction Plant From (uc) Trehalose Sucrose Glucose Fructose Origin Other Selaginella wallacei Horseshoe Bay 12.1 " Sooke 21.4 " Oregon 19.3 " Tofino 21.9 S. kraussiana UBC:Greenhouse 18.8 S_. densa Kootenay Lake-2 hr 21.2 " " " -3/4 hr 19.6 II II II _3//+ n r + 3/4 hr 15.3 isoetes bolanderi Kennedy Lake-2 hr 0.6 II II II -5 h r _» occidental is Marion Lake-2 hr 1.2 I I it II _ 5 H R Lvcopod i urn clavatum P a r k s v i l l e 19.5 11 Tofino, bog 23.5 «' " , mountain 19.8 " Squamish 16.2 91 8 0.5 0.5 - -88 10 0.2 0.5 0.3 1 81 18 0.3 - 0.2 0.5 98 1 0.2 - 0.3 0.5 42 1 0.2 0.2 0.6 56** 96 3 0.4 0.1 - 0.5 95 1 0.2 1.7 1.8 0.3 96 3 0.3 0.1 - 0.6 4 77 3 3 4 9 7 63 2 2 16 10 8 64 2 2 11 13 17 40 2 2 21 18 96 1 1 _ 2 - 96 2 1 - 1 - 97 1 0.4 - 1.6 - 98 0.6 0.7 - 0.7 - continued Table M l , cont'd. Total radio- % Radioactivity a c t i v i t y in the fraction Plant From (uc) Trehalose Sucrose Glucose Fructose Origin Other L. selaqo Nanaimo 18.8 - 97 0.5 0.5 2 II Tof i no 19.1 — 96 0.5 0.5 3 L. sitchense Garibaldi 6.6 - 98 0.8 0.8 0.3 II Seymour 15.4 - 98 0.5 0.5 1 L. annotinum Bowron Lakes 18.6 - 96 1 1 2 II Prince George 19.0 - 98 0.5 0.5 1 II White River 20.1 — 98 — 0.5 1.5 L. complanatum I I II 21.9 - 98 0.5 0.5 1 L. obscurum Bowron Lakes 18.2 - 95 0.7 0.3 4 Botrychium virqinianum Parksvi1le 17.8 - 82 8 0.5 9.5 II White River 19.5 11 63 10 1 15 * As per cent of ra d i o a c t i v i t y in the compounds excised from the chromatogram. These were a l l but the faintest spots that darkened the chromatograms, and represented at least 99.5% of the t o t a l radio-a c t i v i t y on the chromatograms. * F i f t y - f i v e % of the r a d i o a c t i v i t y in the neutral fraction of S. krauss iana occurred in a spot which chromatographed nearer to the o r i g i n than trehalose (see Figure 14). Indicates the compound was not detectable. Table IV Distribution of Radioactivity in the Cation Fraction of Aqueous Extract of Plants Fed C 0 2 it % Radioactivity* Total radio- o o c " a c t i v i t y in * J % ^ ^ f % n c c the f ract ion 1O+J*O m +J ^ o — Plant From (pc) g- = g- .2 ' » J a) a> c d) 0) c c c c c o 0) .n c c c +J o (D _c t — 0) • o +J u c 1 _ <£ L. 0) 4) u x: 1 .>» (/> o K- 00. Selaginella wallacei Horseshoe Bay 1.1 11 14 60 - 5 10 II Sooke 1.0 6 52 15 8 4 - 5 S. emmeliana UBC Greenhouse 0.7 k 17 30 (0 13 7 4 - 1 20 - - 2 2 S. kraussiana ii II 1.2 9 5 22 18 19 9 4 - 1 7 - 3 1 S. densa Kootenay Lake-2 hr 1.2 10 12 28 1 22 6 - 0.2 0.6 0.8 ii » " 3A hr 2.2 11 6 64 0.5 5 11 1.5 - 0.2 ii it II -3//+ hr + - 1.8 0.4 3 A hr 0.9 13 13 36 0.7 24 9 0.1 -Isoetes 54 bolanderi Kennedy Lake-2 hr 0.2 31 10 - — 5 *• ii II II -5 hr 0.3 3 4 17 13 14 5 - 4 1. occidentalis Marion Lake-2 hr 0.5 11 17 30 1 20 6 - - 4 - 8 -it •i II _5 HR 0.5 10 14 24 1 35 3 4 8 - continued oo r-O • < rt —• 0 < cr n o in —• _ O _ —• c —• c -1 Ol 3 c _ 3 c 3 — o • • vn vn — Co VO V-ON\_ CO fO -vj ON VO I- 00 o 4 "^ I tO I N3 I I O • I ON I-o t§ cu 3 OJ Pt c 3 | - |r-w rt O rr -(A (D _ K o o at r~ oa. c 3 00 s: -j ro c> H _ _• at O 3T -1 O at at o at _ o a> — -i s -< n -h 3 c _ -h 1 Pt TT ~i Pt 3 — 3 —• -. at at — TT (D W O fl> o o o 3 -• 3 3 01 < 3 c O 3 o < o cn _ *# —• —« 1— — o r - a. 3" —• < — at < <D at 3 cr — fl) O 7? at o TT o O (D — O -» n fl> _ unta CO o o o o o — — o — O O O — Jr rs> vo vn ro 4>0 oovnv~jr-ON o vn ho ro — ro — — ro co vD oo vo vo VO V*> O N row N) — — NJ — — vo — Ul N)s| to vn ts) W - VO O v~ — jr-jr-ON oovj vn ON £- Jr- to Jr-VD ON M O M — VO to VD 00 CO to 00 4r- 4r ro — oo to to v~ ro -vo i v» ro .p- - N N ON .p- jr- — f j r i cr. ro V™ ON vn vo — ON «vj —• ON vo ro ON vn vo ro — i i i Vi-Ct CO ro o • • ro i i i • ro —. vo ro i i i — o i i • • VA) — — VA) • W N < I I I i ro ro vn -«j • vo i i i I I i i i i a> 3 rt a H —TOO at pt pt — at —. -t, < — x -i — o at pt n o ~< at rt a. o" -" o 3 I Aspartic Glutamic Glycine Asparagine Glutamine a-Alanine Leucine Proline Methionine Ser ine Ornithine Threonine p-Alanine Tyrosine at _ o" cH rt <* rt •< Footnote to Table IV As per cent of the ra d i o a c t i v i t y in the compounds excised from the chromatograms. These were a l l but the faintest spots that darkened the radioautographs, and represented at least 98% of the t o t a l r a d i o a c t i v i t y on the chromatograms. 2 Figure 14 Radioautographs Showing D i s t r i b u t i o n of R a d i o a c t i v i t y in Sugars in Representative Chromatograms 1. S e l a g i n e l l a w a l l ace i 2. S_. krauss i ana 3. Isoetes bolander? 4. Lvcopod?urn clavatum 55 Selaginella martensii using column chromatography as described was not successful. Isolation of trehalose from the plants fed C was possible using one directional paper chromatography as described. The band of trehalose on each chromatogram was located by developing s t r i p s cut from the edges of the chromatogram. The trehalose bands were excised and cut into pieces. Trehalose was eluted from the paper with about 2 l i t r e s of water, the eluate was concentrated, decolourized with charcoal and mixed ion exchange resins, and taken to dryness in a f l a s h evaporator. The residue was dissolved in b o i l i n g ethanol and the minimum amount of hot water. After the solution stood at room temperature for 12 hours, large crystals appeared. About one gram of crystals with an a c t i v i t y of 0.143 uc/mgm (1 uc in 7 mgm) was obtained. Infra-red spectra of t h i s material and of commercial trehalose c r y s t a l l i z e d from ethanol/water were obtained. The spectra are shown in Figure 15. 14 5. Administration of Trehalose-C to Plants 14 One uc of trehalose-C was fed to 10 gm of each of Lycopodiurn  selago. L. obscurum. Selaginella emmeliana, Isoetes bolander?. and Tmesipteris tannensis as described. Large amounts (about 500 ul) of the concentrated neutral f r a c t i o n were banded on 18" x 22", 3 mm paper and chromatographed one d i r e c t i o n a l l y . The n-propanol/ethyl acetate/water solvent was used. Standard spots of glucose, fructose, sucrose, and trehalose were run at the side of the chromatogram. The bands correspond-ing to these sugars were excised, cut in small pieces, and measured with the l i q u i d s c i n t i l l a t i o n counter. The results are shown in Table V. Figure 15 1. Infra-red spectrum of c r y s t a l s obtained from Selaginel l a Wallace? 2. Infra-red spectrum of trehalose ON 57 Table V Distribution of Radioactivity in Trehalose, Sucrose, Glucose and Fructose from Plants Administered Trehalose-C'** Total radio- % Radioactivity ' a c t i v i t y in aqueous neutral Plant f r a c t i o n Trehalose Sucrose Glucose Fructose Origin (He) Lycopod i urn  selago L. obscurum Selaginella emmeliana Isoetes bolanderi Tmesipteris tannensi s 0.057 0.078 0.180 0.129 0.070 32 14 23 79 32 38 48 58 9 37 22 29 14 9 24 6 7 2 3 3 1 3 As per cent of ra d i o a c t i v i t y in the compounds excised from the chrom-atogram. 58 6. Examination of Selaginella. Isoetes and Phylloglossum for the Presence  of Alkaloids A preliminary examination of 20 gm of Selaginella wallacei for alkaloids was made using paper chromatography. The aqueous extract was partitioned with ether at increasing pH as described. Alkaloids appeared to be present in th i s species. However, the resolution of standards was poor, and there was a tendency for the compounds to form double spots. Extracts of 30 gm of S. martens? i and 50 gm each of S_. emmel iana and S_. kraussiana were made by the same method and chromatographed using aluminum oxide thin layer plates. Spots were located with Dradendorff's reagent. Small amounts of alkaloids appeared to be present, but the presence of chlorophyll interfered with the extracts made at low pH. About 100 gm of each of S. wallacei. S. kraussiana and L. clavatum were extracted by the method described in d e t a i l , chlorophyll being removed before the alkaloids were extracted. Aliquots containing about 1% of the chloroform extracts were chromatographed on aluminum oxide plates. Spots were located with Dragendorff's reagent. The results are shown in Figure 16. A small amount (1.6 gm) of Phylloglossum drummondii was extracted by the same method except that the alkaloids were extracted from the aqueous layer in a l i q u i d / l i q u i d extractor rather than on a culture shaker. About 60% of the chloroform extract was chromatographed on s i l i c a gel plates made up with 0.1N NaOH. Spots were located with the iodoplatinate reagent. The results are shown in Figure 17. About 5 gm each of Selaginel l a w a l l a c e i , S_. kraussiana. S. emmeliana. Isoetes bolanderi. Tmesipteris tannensis and Lycopod?urn sitchense were extracted and about half the chloroform extract was chromatographed in the same way. Spots were located with the iodo-59 Figure 16 Chromatogram of A l k a l o i d s from E x t r a c t s of 1 gm of Plant M a t e r i a l 1. Annotinine 2. Lycodine 3. Acetyldihydrolycopodine 4. S. kraussiana 5. L. clavatum 6. S. emmeliana Figure 17 Chromatogram of Alkaloids from Extracts of 1 gm of Plant Material 1. Lycoclavine 2. Clavolonine 3. Dihydrolycopodine 4. a-Obscurine 5. Di-N-methyl-a-obscurine 6. Phylloglossum drummondi i 7» Fawcettidine 60 platinate reagent. The results are shown in Figure 18a. The same plate was then sprayed with cone. h^SO^ and placed in an oven for an hour to char any organic compounds present in the alkaloid extract. The results are shown in Figure 18b. DISCUSSION 1. Use of Vapour Phase Chromatography for Quantitative Surveys of Sugars  in Plants Since trehalose is non-reducing and d i f f i c u l t to hydrolyze quan-t i t a t i v e analysis of t h i s compound by means of the colourimetric methods generally used for sugars is d i f f i c u l t . Quantitative analysis of the sugars found in l i v i n g organisms is time consuming since i t involves separation of the sugars before they can be analyzed. It was hoped that the use of vapour phase chromatography would solve both these problems since separation and quantitative estimation of the sugars could be carried out at the same time. Vapour phase chromatography of sugars can be carried out i f they are methylated to increase the i r v o l a t i l i t y . Before the tech-nique could be applied to quantitative analysis of unknown mixtures i t would be necessary to determine exactly what y i e l d of completely methylated derivative could be expected for each sugar to be assayed. As can be seen in Figure 13, the methylated sugars prepared were poorly resolved by t h i s technique. Programming the temperature of the columns to improve the resolution resulted in an uneven base l i n e ; in an unknown mixture poorly resolved peaks could have been mistaken for such unevenness. Increasing the sample applied to the column to about 10 mgm 61 L L. 2 3_ 4^ 5 6 7 8 9 (b) Figure 18 Chromatogram of Alkaloids from Extracts of 2 1/2 gm of Plant Material 1. Clavolonine 2. L. sitchense 3. T. tannensis 4. JL bolanderi 5. S. emmeliana 6. S. krauss iana 7. S. wal1 ace? 8. Annotinine 9. Lycopodine (a) Sprayed with iodoplatinate reagent (b) Charred 62 to t a l methylated sugar did not improve the resolution. This might have been improved by decreasing the chart speed. By greatly increasing the sample size the detector could be used at a lower s e n s i t i v i t y which would decrease the background unevenness. However, a large number of samples must be surveyed to establish a "chemical p r o f i l e " for a plant, and usually only small samples are available. This technique was not suitable for such a survey since the methylation procedure is time consuming ( i t requires three days) and the amount of sample required (at least 20 grams) is rather large. It would be useful i f quantitative data were absolutely necessary, and large samples were available. 2. Survey of the Metabolic A c t i v i t y of Sugars and Amino Acids in Lycopods  as Indicated by Incorporation The preliminary survey of the sugars of Lycopodiurn and Selaginella indicated that in addition to trehalose, glucose, and fructose, a small amount of sucrose and other sugars were present in Selaginella. A very small amount of trehalose was possibly present in Lycopodiurn. in addition to sucrose, glucose and fructose, the main sugars. (These questionable trehalose spots in the Lycopodiurn extracts were possibly due to sugar acids since these extracts had not been de-ionized.) It seemed that the d i f f e r -ence between the sugars of Lycopodiurn and Selaginella was quantitative; so the p o s s i b i l i t i e s of gas chromatography were examined. Later i t was found that radioisotope techniques provided a much more pra c t i c a l technique for getting quantitative data. Quantitative analysis of the metabolic a c t i v i t y of different sugars and amino acids was obtained by feeding C ^ 0 2 to the plants for about two hours before extracting them. Provided was incorporated into a l l com-63 pounds at the same rate, the radioactivity in each compound would indicate i t s r e l a t i v e amount, since after a long period of photosynthesis, the amount of ra d i o a c t i v i t y would be proportional to the amount of the chemical. Overall CO2 uptake ceased after about half an hour as shown in Figure 19. Figure 20 shows that c'^ uptake continued over two hours, though i t became s l i g h t after about one hour. Carbon'** saturation of the most actively metabolized compounds might have occurred. A rough estimation of the amount of an amino acid present was obtained from the intensity of the spot i t produced with the ninhydrin spray. Glycine, though i t contained appreciable amounts of ra d i o a c t i v i t y (l6%-80%), produced a very f a i n t spot with the spray reagent. Apparently c'^ was not incorporated into a l l amino acids at the same rate. There-fore, the amount of rad i o a c t i v i t y in these compounds indicated their metabolic a c t i v i t y but not thei r r e l a t i v e amounts. The d i s t r i b u t i o n of rad i o a c t i v i t y in the amino acids of these plants was not correlated with the i r taxonomy. The amount of rad i o a c t i v i t y in the common amino acids was not cha r a c t e r i s t i c of a species, and there were no amino acids which occurred only in one species,consistently. This was not the case with the sugars. The d i s t r i b u t i o n of radio-a c t i v i t y in sugars was very ch a r a c t e r i s t i c of a genus. A l l except one species of Selaginella examined had 80% or more ra d i o a c t i v i t y in trehalose and 10%-18% ra d i o a c t i v i t y in sucrose. The one exception was Selaginella  kraussiana. in which 40% was in trehalose and most of the rest in an uniden-t i f i e d sugar. Both species of Isoetes had 4%-8% in trehalose and 40%-80% in sucrose. A l l species of Lycopodium had no rad i o a c t i v i t y in trehalose, and had 95% or more ra d i o a c t i v i t y in sucrose. Apparently Phylloglossum Figure 19 Representative Graph of C0_ Concentration in A i r Stream During Feedings Figure 20 Representative Graph of Radioactivity in A i r Stream During C 1 ^ Feedings 65 was similar to Lycopodium; the one directional chromatograms of i t s neutral aqueous extract indicated that i t did not contain trehalose. A l l these genera had small amounts of r a d i o a c t i v i t y in glucose and fructose. The d i s t r i b u t i o n of r a d i o a c t i v i t y in the sugars of these plants provided a very good "metabolic marker" at the family l e v e l . Since these families are easily distinguished morphologically i t is doubtful i f this fact has any value in i d e n t i f i c a t i o n . It may have useful phylogenetic implications. It would be necessary to know more about the metabolic role of trehalose in these and other plants, and i t s d i s t r i b u t i o n in other plants, before these could be determined. Therefore, the metabolic role of trehalose was examined in this work. In Table I I I , possible interconversion of trehalose and sucrose is indicated in the Isoetes species which were fed radioactive CO2 for two and f i v e hours. In Isoetes occidentalis. t o t a l a c t i v i t y in the sugar f r a c t i o n was about the same after two and f i v e hours. However, after f i v e hours the per cent a c t i v i t y in sucrose had gone down by about 20% and up in trehalose by about 10%. (There was also an increase in r a d i o a c t i v i t y of about 10% in compounds near the origin.) In Isoetes bolanderi. there was a great i n -crease in t o t a l r a d i o a c t i v i t y of the sugar fraction (about s i x times); t h i s was accompanied by some increase in the r e l a t i v e r a d i o a c t i v i t y of trehalose (2%), a larger increase in the a c t i v i t y of compounds near the o r i g i n , and a decrease in the r e l a t i v e a c t i v i t y of sucrose. In Selaginella densa, there was l i t t l e difference in the d i s t r i b u -14 tion of r a d i o a c t i v i t y in sugars in those samples which were fed C 0^ for three-quarters of an hour and then allowed to photosynthesize in an open system for three-quarters of an hour, those which were fed for three-66 quarters of an hour and k i l l e d , and those which were fed for-two hours. More direct evidence of the a b i l i t y to metabolize trehalose was found in Lycopodium, Selaginella. Isoetes and Tmesipteris by feeding them radioactive trehalose. The purity of th i s trehalose was established by infra-red spectroscopy (see Figure 15). The results are shown in Table V;, Apparently a l l of the plants fed trehalose were capable of hydro-lyzing i t to glucose which could then be incorporated into sucrose or could be much more slowly incorporated into oligosaccharides which would chromatograph in the area designated "origin' 1. Some conversion of glucose to fructose occurred, probably by isomerase a c t i v i t y . Nearly twice as much radioactive trehalose was taken up by Selaginella and Isoetes as by the other plants. Trehalose may have a role in photosynthate transloca-tion in these genera solthatthey are better able to take i t up. One sample of the fern Botryehi urn vi rginianum contained a radioactive compound which co-chromatographed with trehalose. Since trehalose may therefore be present in this species, and since Tmesipteris is able to metabolize t r e h a l -ose, i t i s possible that some trehalose metabolism is common to many plants but that for some reason i t is more active in certain lycopods. It is possible that this represents a case in which selective evolutionary pres-sures altered the chemistry of a group of plants. Unfortunately the route of synthesis of trehalose in these organisms is not known. Cabib and Le l o i r (15) have shown that, in yeast, trehalose phosphate is synthesized from uridine diphosphate glucose and glucose-6-phosphate. Radioisotope incorporation studies seem to be useful in surveys of the chemical constituents of plants since they provide ready quantitative data, and since they could be useful in the metabolic studies necessary i f 67 phylogenetic implications are to be drawn from such surveys. 3. Survey of Lycopods for the Presence of Alkaloids The preliminary survey of Selaginella for the presence of alkaloids using paper chromatography indicated that small amounts of alkaloids might be present in th i s genus. Thin layer chromatography confirmed t h i s , though as can be seen from Figures 16 and 18 the amounts of alkaloids present in Selaginel1 a were very much less than the amounts present in Lycopodiurn.  Isoetes bolanderi apparently has even less, i f any. There was a f a i n t spot near the solvent front in the Tmespteris extract, and other f a i n t spots. In case these were a result of unevenness in spraying, the plate was charred as described. The spot near the front in the Tmespteris extract did repres-ent an organic compound which must have had the s o l u b i l i t y properties of an al k a l o i d . The f a i n t spots in the S. kraussiana extract, and the spots at the o r i g i n of a l l the extracts, were also organic compounds. Lycopodiurn obviously has a much higher a l k a l o i d content than any of the other genera tested. Whether or not the compounds which appeared in the Tmespteris. Selaginella and Isoetes extracts actually are alkaloids is not known. Isolation of these compounds would require extracting several kilograms of each plant. If they are alkal o i d s , i t is interesting that Tmespteris contains more than Selaginella and Isoetes. Zimmerman (64) places the Ps i l o t a l e s with the lycopods, and places the ancestors of the Psil o t a l e s nearer those of Lycopodiurn than of Selaginella or Isoetes. The d i s t r i b u -tion of supposed alkaloids f i t s t h is c l a s s i f i c a t i o n . However, before speculating about the phylogenetic implications of the presence of alkal o i d s , more should be known of their d i s t r i b u t i o n and metabolic role. 68 The presence of alkaloids with si m i l a r Rf values to those of Lycopodiurn was c l e a r l y demonstrated in the Phylloglossum drummondii ex-t r a c t . Insufficient material was available to characterize these alka-loids further. 69 SUMMARY 1. Vapour phase chromatography of methylated sugars is an unsatis-factory method for i d e n t i f i c a t i o n and estimation of sugars in large numbers of samples of plant material smaller than 20 grams, fresh weight. 2. Radioisotope incorporation studies provide a means of quantita-t i v e l y assessing the metabolic a c t i v i t y of compounds in large numbers of small samples of plant material. 3. The d i s t r i b u t i o n of r a d i o a c t i v i t y in the sugars of Lvcopodiurn. Selaginella. and Isoetes species fed C 0 2 is cha r a c t e r i s t i c for each genus. Lvcopod?urn species incorporate large amounts of r a d i o a c t i v i t y into sucrose, small amounts into glucose, fructose, and other sugars, and none into trehalose. Selaginella species incorporate large amounts of radio-a c t i v i t y into trehalose, and smal1 amounts into sucrose, glucose, fructose, and other sugars. Isoetes species incorporate most ra d i o a c t i v i t y into sucrose, some into trehalose, glucose and fructose, and small amounts into other sugars. 4. The d i s t r i b u t i o n of r a d i o a c t i v i t y in the amino acids of the species of Isoetes. Selaginella. and Lycopodiurn fed c'^02 shows no cor-rela t i o n with taxonomy. 5. Cut shoots of Lycopodium. Selaginella. Isoetes and Tmesipteris species are able to absorb trehalose from d i l u t e solution and convert i t to glucose, fructose, sucrose and other sugars. 6. 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Anal. Chem. 28: 1037. 63. Yamishito, T. and F. Sato. 1929. A chemical component in Japanese Selaginellaceae. J. Pharmaceut. Soc. Japan kS: 696-703. 6k. Zimmerman, W. 1959. Phylogenie der Pflanzen, 2nd ed. Gustav F i s -cher, Stuttgart, 777 p. 76 APPENDIX 1 Preparation of Dioxane Quenched Standards Standards were prepared c o n t a i n i n g 10 p i hexadecane-C of known r a d i o -a c t i v i t y , 15 cc dioxane s o l v e n t , and 2 cc of a c e t i c a c i d , c o n c e n t r a t i o n 100%, 66.6%, 33.3%, and 0%. The e f f i c i e n c y of each standard was p l o t t e d against the r a t i o cpm channel B/cpm channel A. The r e s u l t s are shown in Figure 19. 77 

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