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Floral odours in the Theophrastaceae
) Pergamon
BiochemicalSystematicaand Ecology,Vol. 22, No. 3, pp. 259-268, 1994
Copyright© 1994ElsevierScienceLtd Printed in GreatBritain.All rights reserved 0305-1978/94 $6.00+ 0.00
0305-1978(93)EOOO8-P
Floral Odours in the Theophrastaceae JETTE T. KNUDSEN*t$ and BERTIL STAHL§ *Department of Chemical Ecolgoy, Gbteborg University, Reutersgaten 2C, S-413 20, G6teborg, Sweden; ~:National Environmental Research Institute, Kale, Gren~vej 12, DK-8410 Rende, Denmark; §Botanical Institute, Stockholm University, S-106 91 Stockholm, Sweden
Key Word Index--Clavija; Deherainia; Jacquinia; Theophrasta; Theophrastaceae; floral odour; colour pigment degradation; sapromyophily; melittophily; head-space adsorption; GC-MS. Abstract--Floral scent was collected by head-space adsorption from seven species of the Theophrastaceae,
viz. Theophrasta americana, Deherainia smaragdina, Jacquinia keyensis, J. macrocarpa, J. sprucei, Clavija euerganea and C. repanda. The chemical composition of the floral scent was determined with coupled gas chromatography-mass spectrometry (GC-MS). The floral scent composition accord with the generic delimitation within the Theophrastaceae; Theophrasta is characterized by a fatty acid derived alcohol and an acid, Deherainia by fatty acid derived esters and an acid, and pyrazines, Jacquinia by benzenoids and phenyl propenoids with additionally trimethylcyclohexanes-derivatives of carotenoids in the orange-flowered species (J. macrocarpa, J. sprucely, and Clavija mainly by sesquiterpene hydrocarbons. We suggest that the trimethylcyclohexenes in the floral scent of the orange-flowered Jacquinia species are formed during anthesis through degradation of carotenoids in the corolla pigments. Floral morphology and scent chemistry suggest that Theophras~aand Deherainia are sapromyophilous, while Jacquinia and Clavija are melittophilous. Melittophily is suggested to be the ancient condition in the family, while the derived condition, sapromyophily, may have evolved independently in Deherainia and Theophrasta.
Introduction
The neotropical family Theophrastaceae, which generally is regarded as a natural entity of the Primulales (Cronquist, 1988), includes six genera and about 90 species of shrubs and small trees. Despite its relatively small size the family exhibits a large variation in many floral features, including shape, size and colour of the corolla as well as the apparent scent. Flowers of Clavija, the largest and chiefly South American genus, are usually unisexual or functionally unisexual and emit a faint, though quite distinct, fruity scent; the corolla is subrotate and pale to dark orange (St&hi, 1991). In Jacquinia, a genus occurring mainly in arid areas of the Antilles, Mesoamerica, and northern South America, the flowers have a strong, almost perfumed scent; the corolla is campanulate or urceolate and, depending on the species, whitish, orange, or brick-red (St~hl, 1989; 1992; and unpubl.). Deherainia, with two species distributed in humid forests from southern Mexico to Honduras, is often cultivated as a curiosity because of its large, green flowers; the corolla is broadly campanulate and emit a very unpleasant, sour smell (Chandler, 1911; St~hl, 1989). In Theophrasta, with two species in the island of Hispaniola, the corolla is campanulate and white to yellowish white; in Theophrasta, and probably also Neomezia, the flowers have a rather foetid smell (Mez, 1903; St~hl, 1987). Votschia, with a single, rare species in Panama, has broadly campanulate flowers, the scent of which as yet are unknown (St~hl, 1993). During the last decade a large number of works dealing with the chemical composition of floral odours have been published and to date more than 700 compounds have been identified in head-space samples from different groups of plants (Knudsen et al., 1993). Although the Orchidaceae are better known as regards tAuthor to whom correspondence should be addressed.
(Received 22 June 1993) 259
260
J.T. KNUDSEN AND B. STAHL
floral odours than any other group of plants (Kaiser, 1993), most families are still poorly investigated, if at all. This paper includes the first reports on the chemical composition of the floral odour in the Theophrastaceae. The purpose of the study was to identify important and/or dominating chemical compounds and to compare the chemical data with other floral features. We also wanted to find out if, or in what degree, the chemical composition of floral odours from different taxa would reflect taxonomic/phylogenetic relationships. For example, a close resemblance is expected between the "sweet smelling" and orange-flowered species of Jacquinia, but is this resemblance also extended to orange-flowered and "sweet smelling" species of Clavija, and to other "sweet smelling" but white-flowered species of Jacquinia? Furthermore, is the unpleasant smell emitted by flowers of Deherainia and Theophrasta caused by similar or different chemical compounds? Materials and Methods Floral scent of seven species belonging to four genera of the Theophrastaceae was investigated. The investigated taxa are listed in Table 1 along with information on important floral features and general distribution; additional collecting data are given in Table 2. Scent samples of one species, Jacquinia spruce~ was collected in the field in western Ecuador in January 1990; scent samples of the remaining species were obtained from plants cultivated in greenhouses at the G6teborg Botanical Garden and the Botanical Institute, University of G6teborg. Dried vouchers of all taxa are deposited in the Botanical Museum, G6teborg (GB). Inflorescences were enclosed in polyacetate bags and the scent containing air was passed through and trapped on adsorbents in cartridges by a battery operated membrane pump. A 200 mg of a 1:1 by weight mixture of Tenax-TA (mesh size 20-35) and Carbotrap (mesh size 20--40) was used as adsorbent. Blank samples of empty bags or from green plant parts were collected in parallel. The adsorbed scent was extracted with 2 ml of pentane; methyl stearate was added as internal standard. The samples were analysed on a Varian 3400 (GC) with on-column injector connected to a Finnigan Ion Trap Detector (MS). A 25 m fused silica GC-column with an internal diameter of 0.25 mm, coated with CP-Wax 52 CB at a film density of 0.2 pm, was used as a stationary phase. The on-column injector was programmed for 1 min at 40=C, then steady at 230°C for 40 min. The GC was programmed for 5 rain at 40°C, increased with 8~C min -1 to 230°C, and then steady for 15 rain at 230°C. Helium was used as carrier gas. Identifications were made by comparing obtained mass spectra with computer library spectra, with spectra in Stenhagen et al. (1974), Adams (1989), and R6del and Petrzika (1991), and with GC-ratention times and spectra of authentic reference compounds. The similarity in floral scent composition between two samples was calculated using S~rensens index of similarity: Is= (2Mw/ Ma+ Mb) × 100, where M w is the sum of the smaller values of compounds present in the samples, and M a and M b are the sum of all compounds present in both a and b.
Results Table 2 shows the relative and total amounts of volatiles collected from the studied species. The compounds are ordered in chemical classes largely reflecting their biosynthetic origin. Fatty acid derivatives, mainly alcohols and esters, characterize the floral scent of Theophrasta americana and Deherainia smaragdina, constituting 69.5% TABLE 1. TAXA EXAMINED; IMPORTANTFLORAL FEATURESAND DISTRIBUTION Taxa*
Corolla colour
Corolla shape
Flora scent
Distribution
Theophrasta americana L. Deherainia smaragdina (Planch.)
whitish green
campanulate broadly campanulate
foetid foetid, sour
Hispaniola S Mexico, N and C Am
white dark orange
campanulate campanulate
sweet, aromatic sweet, aromatic
Florida-Jamaica Mexico, N and C Am
dark orange
campanulate
sweet, aromatic
orange orange
subrotate subrotate
sweet sweet
W Ecuador, NW Peru S Ecuador, N Peru S Ecuador
Decne. ssp smaragdina Jacquinia keyensis Mez J. macrocarpa Cav. ssp. macrocarpa J. spruceiMez Clavija euerganea Macbr. C. repanda St~hl
*For illustrations and full descriptions, see Standley and Williams, 1966 (D. smaragdina; J. macrocarpa, "J. aurantiaca"), St~hl, 1986 (C. repanda), 1987 (T. americana), 1990 (C. euerganea; J. spruce1), and 1992 (J. keyensis).
261
FLORAL ODOURS IN THE THEOPHRASTACEAE
TABLE 2. RELATIVE AND TOTAL AMOUNTS OF CHEMICAL COMPOUNDS COLLECTED FROM FLOWERS OF THE THEOPHRASTACEAE. Theophrasta americana (T. ame.), Deherainia smaragdina ssp. smaragdina (D. sma.), Jacquinia keyensis (J. key.), J. macrocarpa (J. mac.), J. sprucei(J, spr.), C/avijaeuerganea (C. eue.) and C. repanda (C. rep.). RT: compound identified by retention time and mass spectra of authentic reference samples, tr: trace amounts
No. individuals (No. samples) collected Total amount of volatile collected/ flower/12 h (ng)
T. ame.
D. sma.
J. key.
3 (3) 0.91
1 (1) 600
1 (2) 1500
J. mac. 1 (1) 20,0000*
J. spr.
C. eue.
C. rep.
3 (3) 6100
2 (2) 800t 4OO$
1 (1) 500
Fatty acid derivatives RT RT RT RT RT
Butyl acetate 3-Methylbutyl acetate Pentyl acetate 3-Methyl-l-butanol 3-Hydmxy-2-butanone RT cis-3-Hexenylacetate RT Hexanol
tr
83.2 3.0 2.8
67.4 0.7
RT cis-3-Hexenol FIT 1-Octanol RT 3-Methylbutanoic acid
2.1
3.3
3.8
1.0
0.3 0.9 4.1 4.4 0.4
2.2 0.2
Benzenoids RT RT RT RT RT RT RT RT FIT RT RT RT RT RT
Benzaldehyde Ethyl benzoate Benzyl acetate 1,4-Dimethoxybenzene 1,3-Dimethoxybenzene Methyl 2-hydroxybenzoate 3,5-Dimethoxytoluene Benzyl alcohol 2-Phenylethanol 4-Methoxybenzaldehyde p-Cresol Methyl 4-methoxybenzoate 4-Methoxybenzyl acetate 4-Methoxybenzyl alcohol
23.4
6.8
2.0 0.6
3-Phenylpropyl acetate Methyl cis-cinnamate Ethyl cis-cinnamate 3-Phenylpropanol RT trans-Cinnamicaldehyde RT Methyl trans-cinnamate RT Ethyl trans-cinnamate RT Cinnamic acetate RT Eugenol RT trans-Cinnamic alcohol 1648 4-Meth oxyphenylpropanol 1714 4-Methoxycinnamic aldehyde
6.8
37.4 7.4 0.1 0.9
0.3
0.03
0.7
0.8 0.8
3.0 0.9
0.04
1.0 0.2
0.4 0.3 26.9 0.2 23.0 0.1 0.1 0.8 12.9
Phenyl propanoids 11 cis-Cinnamic aldehyde FIT ¶ ¶ RT
9.2 0.4
3.3 2.2
O.3 4.0
0.1 0.3 0.2 0.1 0.1 0.4
0.3 0.3 1.8 0.2
1.8 0.2
Isoprenoids Monoterpenes RT J3-Pinene RT Sabinene RT Myrcene RT Limonene RT 3'-Terpinene RT O-ans-J~-Ocimene
1.7
0.3
8.1
0.7 0.5 0.5 0.8 0.6 0.1
#-Cymene RT trans-4-Thujanol RT Linalool
RT cis-4-Thujanol RT Lavandulyl acetate RT Lavandulol RT c(-Terpineol
3.7 8.0 1.1
0.4
1.0
6.8
1.8 0.8 0.7
3.2
3.2
262
J, T. KNUDSEN AND B. ST,~,HL
TABLE 2--CONTINUED T. ame.
D. sma.
Irre£1ular terpenes Rr 4,8-Dimethyl-l,3(Z),7-nonatriene RT 4,8-Dimethyl-l,3(E),7-nonatriene RT 6-Methyl-5-hepten-2-one RT Geranylacetone
J. key.
J. mac.
J. spr.
C. eue.
C. rep.
0.3 0.2 5.3 1.7
0.4
Derivatives of carotenoids 0.5
949 135,39,41,107,79,123,91,67 RT 2,6,6-trimethyl-l-cyclohexen-1carboxaldehyde (I]-Cyclocitral) 1038 41,43,39,56,85,125,69,153,(168) RT 2,6,6-Trimethyl-2-cyclohexen-l,4dione (4-Oxoisophorone) 1071 39,41,91,107,79,135,55,67,122,150 1091 1,3,4-Trimethyl-3-cyclohexene-1carboxaldehyde RT 2,2,6-Trimethylcyclohexane-l,4dione 1455 43,125,153,55,79,91,109,137,183 1479 43,41,39,69,55,111,153,107
23 7.4 23.1
1.6 17.2 0.5
Sesquiterpenes RT c~-Cubebene RT Caryophyllene R'F Humulene RT I]-Selinene RT (Z),(Z)-Farnesol RT (E),(E)-Farnesol
0.2
0.6
5.7 1.6 1.0
4.3
0.02 53.7 6.8 0.2
53.2 7.9 0.5
1.9 0.4
Types of unknown sesquiterpenes§ 1.2 1.5 0.6 1.1 1.1 8.2 1.8 8.0 2.2 0.6 1.3 4.6 0.7
929 I~-Cubebene type 954 Aromadendrene type
958 cis-c~-Bergamotene 959 Caryophyllene type 969 Bergamotene type 976 trans-c-Bergamotene 1099 ~]-Bisabolene type 1126 I]-Elemene type 1135 [8-Sesquiphellandrene 1258 Caryophyllene oxide type 1298 Caryophyllene oxide type 1305 Caryophyllene oxide type 1585 Santalol type 1621 Santalol type
0.6
0.7 0.5
4.5 1.0 20.7 2.0 0.2 0.5 1.5 0.2 0.2
Nitrogen containing compounds 2.4 1.7
RT Trirnethylpyrazine RT Tetramethylpyrazine Unknowns§ 236 Monoterpene hydrocarbon 269 43,41,71,39,55 557 43,41,45,39,31,57,67,89 784 135,79,91,39,150,107 850 43,57,41,71,72,101,85 966 41,39,70,69 1001 42,41,39,87,56 1093 39,79,108,91,43,106,51,65,124,150 1281 43,41,39,67,55,107 1580 43,41,45,39,108,93,67,55,79 1652 77,105,51,182,59
1.0 1.4 1.0 0.7 0.9 1.6 1.9 0.5 O.6 1.4
0.1 0.3
0.5
*Amount of volatiles collected per inflorescence; tamount of volatiles collected in a male flower, and :[:in a female flower; ITprobably the cis stereoisomer, it has the same mass spectrum as the trans stereoisomer; §only those compounds which occurred in a relative amount > 1% in any sample are listed.
FLORAL ODOURS IN THE THEOPHRASTACEAE
263
and 92.3% of the floral scent in respective species. Benzenoids, especially benzaldehyde, benzyl alcohol, and methoxybenzenes, together with phenyl propanoids (mainly derivatives of cinnamic acid) characterize the floral scent of Jacquinia keyensis (97.6%) and constitute a large part of the floral scents of J. sprucei (57.8%) and J. macrocarpa (27.5%). In the other genera benzenoids are very sparse (Deherainia, Theophrasta) or present in trace amounts only (Clavija). Isoprenoids are present as monoterpenes in all species, whereas sesquiterpenes are found almost exclusively in Clavija and then in very large amounts, i.e. 95.9% in C. euerganea and 94.0% in C. repanda. Irregular terpenes, especially trimethylcyclohexenes derivatives of carotenoids, are characteristic components of the floral scent of the orange-flowered species of Jacquinia (J. sprucei and J. macrocarpa). The floral scent of Theophrasta americana is characterized by 3-methyl-l-butanol (67.4%), monoterpenes, benzaldehyde, and one of the three individuals investigated contained the very unpleasant smelling acid 3-methylbutanoic acid. In Deherainia smaragdina the floral scent contains mainly butyl acetate (83.2%), but the very unpleasant, sour odour is due to the presence of 3-methylbutanoic acid, and probably also tri- and tetramethylpyrazine. The white-flowered Jacquinia keyensis has a scent characterized by benzenoids and phenyl propanoids, especially a series of compounds with a methoxy-group in the 4-position on the aromatic ring, and some of the corresponding compounds without this substitution (Fig. 1). The floral scent of the orange-flowered J. spruceiand J. macrocarpa contains many of the same benzenoids and phenyl propanoids as that of J. keyensis, but in lower amounts, except for 3,5-dimethoxytoluene, which is the main compound in J. sprucei. In addition, the floral scent of J. macrocarpa and J. sprucei contains a series of derivatives of carotenoids, i.e. 2,6,6-trimethyl-l-cyclohexen-l-carboxaldehyde, 2,6,6-trimethyl-2cyclohexen-l,4-dione, 2,2,6-trimethylcyclohexane-l,4-dione (Fig. 2), and some unidentified compounds of the same type; that of J. macrocarpa also contains two sesquiterpenes, viz, (Z),(Z)- and (E),(E)-farnesol. The floral scent of Clavija euerganea and C. repanda contains almost exclusively sesquiterpenes with caryophyllene as the main compound, constituting 53.7% and 53.2% of the floral scent in respective species. We have only been able to verify the identification of a few of the sesquiterpenes with authentic reference compounds and, consequently, most of them are reported here only as types according to their main mass fragments. The similarity in floral scent composition of male and female individuals of C. euerganea was 71.6%. All the compounds present in C. repanda except one, were also found in C. euerganea, but the number of compounds were fewer in the former. The similarity in floral scent composition of the male individuals of C. euerganea and C. repanda was 78.3%, and between female of C. euerganea and male of C. repanda 73.3% Discussion Although only seven species in four genera were investigated, the sample still covers most of the variation in floral morphology and corolla colour present in the family. Clavija is very homogenous as regards floral morphology and colour (St~hl, 1991) and in Jacquinia representatives of both existing colour morphs, white and orange, were studied. Living plants of the monotypic genera Neomezia and Votschiahave not been available for this study, nor have any information on the floral scent in these genera been reported in literature or on herbarium labels. However, Neomezia appears to have a floral biology very similar to that of its nearest relative, Theophrasta (St~hl, 1987). The genus Votschia, a recent segregate from Jacquinia (St~hl, 1993), is so far known only from two collections. It has a corolla similar in size and shape to that of Deherainia, but being yellow its flowers are not likely to produce a foetid scent as do flowers of that genus.
264
J.T. KNUDSEN AND B. STAHL
Jacquinia
Jacquinia keyensis
Me
Me
o a ~ 0 II ,.H C"
H2C h i ~
OMe 0
II,.H
Me d ()
C'
I0~
C II o
I Me
1
O Me CH2OH
XC /
II
O! Me O%c/O~
CH2OH
Me
0
H2C /
Me
O Me !
OH
O ! Me
~
CH2OH MeO~
Deherainia $maragdina
m
.N~
CH2OH
Theophrasta americana
CH2 OH
P
0 H
Me
Me Clavija
M
N
Me
Me
_N.
Me
Me
N
M
FIG. 1. CHARACTERISTIC AND DOMINANT COMPOUNDS ISOLATED IN THE FLORAL SCENT OF SPECIES OF THEOPHRASTACEAE. In Jacquinia: (a) 3,5-dimethoxytoluene; (b) benzaldehyde; (c) 4-methoxybenzaldehyde; (d) 1,4-dimethoxybenzene; (e) 4-methoxybenzyl alcohol; (f) benzyl alcohol; and (g) 3-phenylpropanol; in J. keyens/s: (h) 4~methoxybenzyl acetate; (i) benzyl acetate; (j) p-cresol; (k) methyl 4-methoxybenzoate; and (I) 1,4-met~oxyphenylpropanol; in Deherainia smaragdina: (m) butyl acetate; (n) 2,3,5-trimethylpyrazine; and (o) 2,3,5,6-tetramethylpyrazine; in Theophrastaamericana: (p} 3-methyl-l-butanol; and in species of C/aw~: (q) caryophyllene; and (r) humulene.
Floral scent chemistry
Most of the compounds in the floral scent of the Theophrastaceae are common in head-space samples of a large number of angiosperms (Knudsen et al., 1993). However, 3-methylbutanoic acid was only recently reported in the floral scent of two species of Masdevallia (Orchidaceae; Kaiser, 1993), methyl 4-methoxybenzoate in Moneses unMora (Pyrolaceae; Knudsen and Tollsten, 1991), and 4-methoxybenzyl
FLORAL ODOURS IN THE THEOPHRASTACEAE
265
(or-Carotene) Cleaving enzyme(s)
H
~
R
R=CHOor (R= COOH)
trans-Crocetindialdehyde ( trans-Crocetinhalbaldehyde )
-tCHO
2,6,6-Trimethyl-1-cyclohexen1-carboxaldehyde (IB-cyclocitral)
o•O o•O
2,6,6-Trimethyl-2-cyclohexen1,4-dione (4-Oxoisophorone)
2,2,6-Trimethyl-1,4-cyclohexanedione
FIG. 2. FLORAL SCENT COMPOUNDS OF ORANGE-FLOWERED SPECIES OF JACQUINIA POSTULATED TO BE DERIVED FROM CAROTENOIDS. ( Trans-crocetindi- and halbaldehyde identified in dried flowers of J. nervosa by Eugster et ak, 1969).
alcohol in a species of Cucurbita (Cucurbitaceae; Andersen and Metcalf, 1987). Triand tetramethylpyrazine, found in D. smaragdina, are here reported as new constituents in floral scent. Of the three identified derivatives of carotenoids, 4-oxoisophorone has been reported in Freesia (Iridaceae; Lamparsky, 1985), in seven species of orchids (Kaiser, 1993), and in Angelica (Apiaceae; Tollsten eta/., 1994), 2,6,6trimethyl-l-cyclohexen-l-carboxaldehyde (~-cyclocitral) in Freesia (Lamparsky, 1985), and 2,2,6-trimethyl-l,4-cyclohexanedione in Encylcia (Orchidaceae; Kaiser, 1993). Extracts of dried stigmas of Crocus sativus (Iridaceae) have been found to contain the above mentioned compounds, but with 2,6,6-trimethyl-l-cyclohexene-1carboxaldehyde glycocidally bound in the 4-position (R6del and Petrzika, 1991; Himeno and Sano, 1987). The floral scent composition of the studied species accord with the .generic delimitations in the Theophrastaceae. That of Theophrasta is distinguished by an aliphatic alcohol; Deherainia by aliphatic esters, and two pyrazines. However, Theophrasta and Deherainia have 3-methylbutanoic acid in common; Jacquinia by aromatics and, in the orange-flowered species, additional derivatives of carotenoids; and, finally, Clavija by various sesquiterpenes. Within Jacquinia, each species has a characteristic floral scent composition. Jacquinia spruceidiffers from J. rnacrocarpa in having methyl and ethyl cinnamate and much higher amounts of 3,5-dimethoxytoluene; these two orange-flowered species are distinguished from the whiteflowered J. keyensis by containing carotenoid derived scent compounds. The chemical composition of the floral scents of Clavija euerganea (from both male and female plants) and C. repanda is very similar and cannot be used to distinguish between these two species.
266
J.T. KNUDSEN AND B. ST~,HL
Floral scent and pigments The corolla colour of Jacquinia macrocarpa and J. spruceichanges from orange to almost brick-red as the flowers age, suggesting a chemical change in the pigments. Orange colours usually indicate presence of large amounts of ~-carotene (Harborne, 1988). However, Eugster et aL (1969) did not find this pigment in the dried flowers of J. nervosa Presl ("J. angustifolia"), a close relative of J. sprucei (St~hl, unpubl.). Instead they found trans-crocetindialdehyde and trans-crocetinhalbaldehyde (Fig. 2). Most floral scent compounds with a trimethylcyclohexene structure, as those reported here in J. macrocarpa and J. spruce~ are suggested to be derived from 0¢- or ~-carotene through enzymatical cleavage and oxidation of the polyene chain (e.g. Croteau and Karp 1991; Kaiser, 1993). Similar compounds or compounds with the same general structure have been identified as volatile components in flowers of saffron (Crocus sativus; RSdel and Petrzika, 1991), but, in that particular case, zeaxanthin, a dihydroxylated I]-carotene, was presumed to be the parent compound (Himeno and Sano, 1987; Zarghami and Heinz, 1971). The absence of 0¢- or I]-carotene in the dried flowers of J. nervosa, the presence of degradation products of similar structure as those from the degradation of zeaxanthin (crocin), and the colour change in flowers of many species of Jacquinia strongly suggest that the emitted trimethylcyclohexenes are formed at anthesis through degradation of the flowers own colour pigments (Fig. 2). Additional evidence supporting this hypothesis is provided by J. keyensis. The floral scent of this white-flowered species is very similar to those of the orange-flowered J. sprucei and J. macrocarpa, except for the lack of trimethylcyclohexenes. The floral scent of Clavija consists almost exclusively of sesquiterpenes, suggesting that the orange colour of its flowers originates from other pigments than those found in Jacquinia or that the cleaving enzymes producing the trimethylcyclohexenes from carotenes are not present or non-functional. Pollination biology There are no reliable observations of pollinators to the Theophrastaceae and conclusions about their pollination biology have to be drawn mainly from indirect evidence. Since all members have dry, nectarless flowers (St~hl, unpubl.), pollination will have to be effected by pollen gathering insects or through deceit. Self pollination may occur in the hemaphrodite genera but then hardly within the same flower since these are protandrous and have extrorsely dehiscent anthers. Most or perhaps all of the hermaphrodite genera also seem to be self-sterile (Chandler, 1911; St&hi, unpubl.). The Mesoamerican species Jacquinia nervosa, whose flowers are very similar to those of the orange-flowered species of Jacquinia studied here, is believed to be hummingbird pollinated (Janzen, 1983; "J. pungens"), but the lack of nectar make this seem very unlikely. Large green flowers as those of Deherainia are unusual among the angiosperms. They are met with in some bat-pollinated Bignoniaceae (e.g. Crescent~a; Gentry, 1974) and Lecythidaceae (Lecythis poiteaui, L. barnebyi; Mori and Prance, 1990), as well as in the Orchidaceae (e.g. Lycaste; Kaiser, 1983). Since Deherainia flowers are lacking nectar and are more or less hidden by the foliage, they are hardly pollinated by bats. It seems more likely, as already suggested by Pohl (1931), that Deherainia smaragdina is pollinated by flies, but this has still to be observed in the field. The floral scent of Deherainia resembles that of some sapromyophilous species of Masdevallia (Orchidaceae; Kaiser, 1993) in containing the characteristic smelling 3-methylbutanoic acid, an otherwise rare compound in floral scents. Although not evident in the greenhouse material used for this study, flowers of Theophrasta americana observed in the field were reported to have a fungal scent (St~hl, 1987). Together with other pieces of evidence (more or less hidden flowers, pale brown corollas, uneven surfaces of the corolla walls, and presence of Diptera
FLORALODOURSIN THETHEOPHRASTACEAE
267
larvae) the species was suggested to be pollinated by Diptera that normally visit m u s h r o o m s to breed a n d / o r lay their eggs. The chemical composition of a floral scent reminiscent of m u s h r o o m s is only k n o w n from Dracula chestertonii (Orchidaceae; Kaiser, 1993), The scent of this species was f o u n d to contain the m u s h r o o m smelling 1-octen-3-01 as the main c o m p o u n d . However, w e did not find this nor any of the other of its volatiles in the scent of Th. americana. Using the classification of Faegri and van der Pijl (1979), the more or less sweetscented flowers of Jacquinia and Clavija m a y be described as melittophilous, whereas the flowers of Theophrasta and Deherainia, w i t h their less agreeable scents, as sapromyophilous. In this case, however, large differences in ecology, floral features, and not least, floral scent chemistry indicate that this classification is a rather crude simplification. Being prevalent in the rest of the Primulales, melittophily represents probably the ancient condition in the Theophrastaceae, although it is n o w quite differently expressed in Clavija and Jacquinia. The sapromyophilous condition in Deherainia, Theophrasta, and probably Neomezia should consequently be regarded as a derived state in the Theophrastaceae. This hypothesis accords also w i t h supposed phylogenetic relationships w i t h i n the family based on morphological evidence (St,~hl, 1991). However, Deherainia and Theophrasta are only distantly related and must have acquired the s a p r o m y o p h i l o u s condition independently, given that the morphological study is correct. However, despite considerable differences in floral scent chemistry between Deherainia and Theophrasta, the present analysis does not lend full s u p p o r t to the i n d e p e n d e n t origins of s a p r o m y o p h i l y in these t w o genera since the scent of Deherainia contains the acetate of 3-methyl-l-butanol, the main c o m p o u n d in Theophrasta, and both have the acid.
Acknowledgements--Wethank B. Bro and J. P. Fell for help in the field, R. Kaiser for generously providing reference substances, and the staff at the G6teborg Botanical Garden for nursing the plants. The study was financially supported by the Regnell Foundation (KVA).
References Adams, R. P. (1989) Identification of Essential Oils by Ion TrapMass Spectroscopy.Academic Press, San Diego. Andersen, J. F. and Metcalf, R. L. (1987) Factors influencing the distribution of Diabrotica and Acalymma spp. from blossoms of cultivated Cucurbitassp. J. Chem.Ecol. 13, 681-699. Chandler, B. (1911)DeherainiasmaragdinaDcne. Notes Roy.Bot. Gard.Edinburgh5, 4,9-56. Cronquist, A. (1988) The Evolution and Classification of FloweringPlants.The New York Botanical Garden, New York. Croteau, R. and Karp, F. (1981)Origin of natural odorants. In Perfumes:Art, Scienceand Technology(MUller, P. M. and Lamparsky, D., eds), pp. 101-126. Elsevier Applied Science, Oxford and New York. Eugster, C. H., HSrlimann, H. and Leuenberger, H. J. (1969)90. Crocetindialdehyd und Crocetinhalbaldehydals BlStenfarbstoffe von Jacquiniaangus#'folia.Helvetica Chem.Acta 52, 806-807. Faegri, K. and van der Pijl, L. (1979) The Principles of Pollina~'onEcology. Pergamon Press, Oxford. Gentry, A. H. (1974) Flowering phenology and diversity in tropical Bignoniaceae.Biotropica6, 64-68. Harborne, J. B. (1988) Introduction to EcologicalBiochemistry, 3rd edn. Academic Press, London. Himeno, H. and Sano, K. (1987) Synthesis of crocin, picrocrocin and safranal by saffron stigma-like structures proliferated in vitro. Agric. Biol. Chem.51, 2395-2400. Janzen, D. H. (1983) Jacquiniapungens, In Costa Rican Natural History (Janzen, D. H., ed.), pp. 265-268. The University of Chicago Press, Chicago. Kaiser, R. (1993) The Scent of Orchids. Olfactory and ChemicalInvestigations. Elsevier,Amsterdam. Knudsen, J. T. and Tollsten, L. (1991) Floral scent and intrafloral scent differentiation in Monesesand Pyrola (Pyrolaceae). PI. Syst. Evol. 177, 81-91. Knudsen, J. T., Tollsten, L. and Bergstr~m, L. G. (1993)Floral scents--a checklist of volatile compounds isolated by head-space techniques. Phytochemistry33, 253-280. Lamparsky, D. (1985) Headspace technique as a versatile complementary tool to increase knowledge about con~ituents of domestic or exotic flowers and fruits. In EssentialOils and Aromatic Plants (Svendsen,A. B. and Scheffer, J. J. C., eds), pp. 79-92. Martinus Nijhoff/Dr. Junk Publisher,Dordrecht. Mez, C. (1903) Theophrastaceae. In Das Pflanzenreich (Engler, A., ed.), 15, (IV, 236a), pp. 1-48. Engelman, Leipzig.
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J.T. KNUDSENAND B. STAHL
Mori, S. A. and Prance, G. T. (1990) Lecythidaceae--Part I1. FI. Neotropica Monogr. 21 (11)pp. 1-376. The New York Botanical Garden, New York. Pohl, F. (1931) 0ber sich 5ffnende Kristallr~iume in den Antheren von Deherainia smaragdina. Jahrb. Wiss. Bot. 75, 481-493. RSdel, W. and Petrzika, M. (1991) Analysis of the volatile components of Saffron J. High Resolution Chromatogr. 14, 771-774. St&hi, B. (1986) Two new species of Clavija (Theophrastaceae) from NW South America Nordo J. Bot. 6, 769772. St~hl, B. (1987) The genus Theophrasta (Theophrastaceae). Foliar structures, floral biology and taxonomy. Nord. J. Bot. 7, 529-538. St~hl, B. (1989) A synopsis of Central American Theophrastaceae. Nord. J. Bot. 9, 15-30. St~hl, B. (1990) Theophrastaceae. In FI. Ecuador(Harling, G. and Andersson, L., eds), 39, pp. 1-21. Department of Systematic Botany, University of G6teborg, G6teborg. St~hl, B. (1991) A revision of Clavija (Theophrastaceae).Opera Bot. 107, 1-77. St~hl, B. (1982) On the identity of Jacquinia armillaris (Theophrastaceae) and related species. Brittonia 44, 5460. St~hl, B. (1983) Votschia, a new genus of Theophrastaceae from southeastern Panama. Brittonia 45, 204-207. Standley, P. C. and Williams, L. O. (1966) Flora of Guatemala. Fieldiana Bot. 24(8), 179-206. Stenhagen, E., Abrahamsson, S. and McLafferty, F. W. (1974) Regisb'y of Mass SpectralData. John Wiley and Sons, New York. Tollsten, K., Knudsen, J. T. and BergstrSm, L. G. (1994) Floral scent in generalistic Angelica--an adaptive character (Apiaceae) Biochem. Syst. Ecol. 22, 161-169. Zarghami, N. S. and Heinz, D. E. (1971) Monoterpene aldehydes and isophorone-related compounds of saffron. Phytochemistry 10, 2755-2761.
BiochemicalSystematicaand Ecology,Vol. 22, No. 3, pp. 259-268, 1994
Copyright© 1994ElsevierScienceLtd Printed in GreatBritain.All rights reserved 0305-1978/94 $6.00+ 0.00
0305-1978(93)EOOO8-P
Floral Odours in the Theophrastaceae JETTE T. KNUDSEN*t$ and BERTIL STAHL§ *Department of Chemical Ecolgoy, Gbteborg University, Reutersgaten 2C, S-413 20, G6teborg, Sweden; ~:National Environmental Research Institute, Kale, Gren~vej 12, DK-8410 Rende, Denmark; §Botanical Institute, Stockholm University, S-106 91 Stockholm, Sweden
Key Word Index--Clavija; Deherainia; Jacquinia; Theophrasta; Theophrastaceae; floral odour; colour pigment degradation; sapromyophily; melittophily; head-space adsorption; GC-MS. Abstract--Floral scent was collected by head-space adsorption from seven species of the Theophrastaceae,
viz. Theophrasta americana, Deherainia smaragdina, Jacquinia keyensis, J. macrocarpa, J. sprucei, Clavija euerganea and C. repanda. The chemical composition of the floral scent was determined with coupled gas chromatography-mass spectrometry (GC-MS). The floral scent composition accord with the generic delimitation within the Theophrastaceae; Theophrasta is characterized by a fatty acid derived alcohol and an acid, Deherainia by fatty acid derived esters and an acid, and pyrazines, Jacquinia by benzenoids and phenyl propenoids with additionally trimethylcyclohexanes-derivatives of carotenoids in the orange-flowered species (J. macrocarpa, J. sprucely, and Clavija mainly by sesquiterpene hydrocarbons. We suggest that the trimethylcyclohexenes in the floral scent of the orange-flowered Jacquinia species are formed during anthesis through degradation of carotenoids in the corolla pigments. Floral morphology and scent chemistry suggest that Theophras~aand Deherainia are sapromyophilous, while Jacquinia and Clavija are melittophilous. Melittophily is suggested to be the ancient condition in the family, while the derived condition, sapromyophily, may have evolved independently in Deherainia and Theophrasta.
Introduction
The neotropical family Theophrastaceae, which generally is regarded as a natural entity of the Primulales (Cronquist, 1988), includes six genera and about 90 species of shrubs and small trees. Despite its relatively small size the family exhibits a large variation in many floral features, including shape, size and colour of the corolla as well as the apparent scent. Flowers of Clavija, the largest and chiefly South American genus, are usually unisexual or functionally unisexual and emit a faint, though quite distinct, fruity scent; the corolla is subrotate and pale to dark orange (St&hi, 1991). In Jacquinia, a genus occurring mainly in arid areas of the Antilles, Mesoamerica, and northern South America, the flowers have a strong, almost perfumed scent; the corolla is campanulate or urceolate and, depending on the species, whitish, orange, or brick-red (St~hl, 1989; 1992; and unpubl.). Deherainia, with two species distributed in humid forests from southern Mexico to Honduras, is often cultivated as a curiosity because of its large, green flowers; the corolla is broadly campanulate and emit a very unpleasant, sour smell (Chandler, 1911; St~hl, 1989). In Theophrasta, with two species in the island of Hispaniola, the corolla is campanulate and white to yellowish white; in Theophrasta, and probably also Neomezia, the flowers have a rather foetid smell (Mez, 1903; St~hl, 1987). Votschia, with a single, rare species in Panama, has broadly campanulate flowers, the scent of which as yet are unknown (St~hl, 1993). During the last decade a large number of works dealing with the chemical composition of floral odours have been published and to date more than 700 compounds have been identified in head-space samples from different groups of plants (Knudsen et al., 1993). Although the Orchidaceae are better known as regards tAuthor to whom correspondence should be addressed.
(Received 22 June 1993) 259
260
J.T. KNUDSEN AND B. STAHL
floral odours than any other group of plants (Kaiser, 1993), most families are still poorly investigated, if at all. This paper includes the first reports on the chemical composition of the floral odour in the Theophrastaceae. The purpose of the study was to identify important and/or dominating chemical compounds and to compare the chemical data with other floral features. We also wanted to find out if, or in what degree, the chemical composition of floral odours from different taxa would reflect taxonomic/phylogenetic relationships. For example, a close resemblance is expected between the "sweet smelling" and orange-flowered species of Jacquinia, but is this resemblance also extended to orange-flowered and "sweet smelling" species of Clavija, and to other "sweet smelling" but white-flowered species of Jacquinia? Furthermore, is the unpleasant smell emitted by flowers of Deherainia and Theophrasta caused by similar or different chemical compounds? Materials and Methods Floral scent of seven species belonging to four genera of the Theophrastaceae was investigated. The investigated taxa are listed in Table 1 along with information on important floral features and general distribution; additional collecting data are given in Table 2. Scent samples of one species, Jacquinia spruce~ was collected in the field in western Ecuador in January 1990; scent samples of the remaining species were obtained from plants cultivated in greenhouses at the G6teborg Botanical Garden and the Botanical Institute, University of G6teborg. Dried vouchers of all taxa are deposited in the Botanical Museum, G6teborg (GB). Inflorescences were enclosed in polyacetate bags and the scent containing air was passed through and trapped on adsorbents in cartridges by a battery operated membrane pump. A 200 mg of a 1:1 by weight mixture of Tenax-TA (mesh size 20-35) and Carbotrap (mesh size 20--40) was used as adsorbent. Blank samples of empty bags or from green plant parts were collected in parallel. The adsorbed scent was extracted with 2 ml of pentane; methyl stearate was added as internal standard. The samples were analysed on a Varian 3400 (GC) with on-column injector connected to a Finnigan Ion Trap Detector (MS). A 25 m fused silica GC-column with an internal diameter of 0.25 mm, coated with CP-Wax 52 CB at a film density of 0.2 pm, was used as a stationary phase. The on-column injector was programmed for 1 min at 40=C, then steady at 230°C for 40 min. The GC was programmed for 5 rain at 40°C, increased with 8~C min -1 to 230°C, and then steady for 15 rain at 230°C. Helium was used as carrier gas. Identifications were made by comparing obtained mass spectra with computer library spectra, with spectra in Stenhagen et al. (1974), Adams (1989), and R6del and Petrzika (1991), and with GC-ratention times and spectra of authentic reference compounds. The similarity in floral scent composition between two samples was calculated using S~rensens index of similarity: Is= (2Mw/ Ma+ Mb) × 100, where M w is the sum of the smaller values of compounds present in the samples, and M a and M b are the sum of all compounds present in both a and b.
Results Table 2 shows the relative and total amounts of volatiles collected from the studied species. The compounds are ordered in chemical classes largely reflecting their biosynthetic origin. Fatty acid derivatives, mainly alcohols and esters, characterize the floral scent of Theophrasta americana and Deherainia smaragdina, constituting 69.5% TABLE 1. TAXA EXAMINED; IMPORTANTFLORAL FEATURESAND DISTRIBUTION Taxa*
Corolla colour
Corolla shape
Flora scent
Distribution
Theophrasta americana L. Deherainia smaragdina (Planch.)
whitish green
campanulate broadly campanulate
foetid foetid, sour
Hispaniola S Mexico, N and C Am
white dark orange
campanulate campanulate
sweet, aromatic sweet, aromatic
Florida-Jamaica Mexico, N and C Am
dark orange
campanulate
sweet, aromatic
orange orange
subrotate subrotate
sweet sweet
W Ecuador, NW Peru S Ecuador, N Peru S Ecuador
Decne. ssp smaragdina Jacquinia keyensis Mez J. macrocarpa Cav. ssp. macrocarpa J. spruceiMez Clavija euerganea Macbr. C. repanda St~hl
*For illustrations and full descriptions, see Standley and Williams, 1966 (D. smaragdina; J. macrocarpa, "J. aurantiaca"), St~hl, 1986 (C. repanda), 1987 (T. americana), 1990 (C. euerganea; J. spruce1), and 1992 (J. keyensis).
261
FLORAL ODOURS IN THE THEOPHRASTACEAE
TABLE 2. RELATIVE AND TOTAL AMOUNTS OF CHEMICAL COMPOUNDS COLLECTED FROM FLOWERS OF THE THEOPHRASTACEAE. Theophrasta americana (T. ame.), Deherainia smaragdina ssp. smaragdina (D. sma.), Jacquinia keyensis (J. key.), J. macrocarpa (J. mac.), J. sprucei(J, spr.), C/avijaeuerganea (C. eue.) and C. repanda (C. rep.). RT: compound identified by retention time and mass spectra of authentic reference samples, tr: trace amounts
No. individuals (No. samples) collected Total amount of volatile collected/ flower/12 h (ng)
T. ame.
D. sma.
J. key.
3 (3) 0.91
1 (1) 600
1 (2) 1500
J. mac. 1 (1) 20,0000*
J. spr.
C. eue.
C. rep.
3 (3) 6100
2 (2) 800t 4OO$
1 (1) 500
Fatty acid derivatives RT RT RT RT RT
Butyl acetate 3-Methylbutyl acetate Pentyl acetate 3-Methyl-l-butanol 3-Hydmxy-2-butanone RT cis-3-Hexenylacetate RT Hexanol
tr
83.2 3.0 2.8
67.4 0.7
RT cis-3-Hexenol FIT 1-Octanol RT 3-Methylbutanoic acid
2.1
3.3
3.8
1.0
0.3 0.9 4.1 4.4 0.4
2.2 0.2
Benzenoids RT RT RT RT RT RT RT RT FIT RT RT RT RT RT
Benzaldehyde Ethyl benzoate Benzyl acetate 1,4-Dimethoxybenzene 1,3-Dimethoxybenzene Methyl 2-hydroxybenzoate 3,5-Dimethoxytoluene Benzyl alcohol 2-Phenylethanol 4-Methoxybenzaldehyde p-Cresol Methyl 4-methoxybenzoate 4-Methoxybenzyl acetate 4-Methoxybenzyl alcohol
23.4
6.8
2.0 0.6
3-Phenylpropyl acetate Methyl cis-cinnamate Ethyl cis-cinnamate 3-Phenylpropanol RT trans-Cinnamicaldehyde RT Methyl trans-cinnamate RT Ethyl trans-cinnamate RT Cinnamic acetate RT Eugenol RT trans-Cinnamic alcohol 1648 4-Meth oxyphenylpropanol 1714 4-Methoxycinnamic aldehyde
6.8
37.4 7.4 0.1 0.9
0.3
0.03
0.7
0.8 0.8
3.0 0.9
0.04
1.0 0.2
0.4 0.3 26.9 0.2 23.0 0.1 0.1 0.8 12.9
Phenyl propanoids 11 cis-Cinnamic aldehyde FIT ¶ ¶ RT
9.2 0.4
3.3 2.2
O.3 4.0
0.1 0.3 0.2 0.1 0.1 0.4
0.3 0.3 1.8 0.2
1.8 0.2
Isoprenoids Monoterpenes RT J3-Pinene RT Sabinene RT Myrcene RT Limonene RT 3'-Terpinene RT O-ans-J~-Ocimene
1.7
0.3
8.1
0.7 0.5 0.5 0.8 0.6 0.1
#-Cymene RT trans-4-Thujanol RT Linalool
RT cis-4-Thujanol RT Lavandulyl acetate RT Lavandulol RT c(-Terpineol
3.7 8.0 1.1
0.4
1.0
6.8
1.8 0.8 0.7
3.2
3.2
262
J, T. KNUDSEN AND B. ST,~,HL
TABLE 2--CONTINUED T. ame.
D. sma.
Irre£1ular terpenes Rr 4,8-Dimethyl-l,3(Z),7-nonatriene RT 4,8-Dimethyl-l,3(E),7-nonatriene RT 6-Methyl-5-hepten-2-one RT Geranylacetone
J. key.
J. mac.
J. spr.
C. eue.
C. rep.
0.3 0.2 5.3 1.7
0.4
Derivatives of carotenoids 0.5
949 135,39,41,107,79,123,91,67 RT 2,6,6-trimethyl-l-cyclohexen-1carboxaldehyde (I]-Cyclocitral) 1038 41,43,39,56,85,125,69,153,(168) RT 2,6,6-Trimethyl-2-cyclohexen-l,4dione (4-Oxoisophorone) 1071 39,41,91,107,79,135,55,67,122,150 1091 1,3,4-Trimethyl-3-cyclohexene-1carboxaldehyde RT 2,2,6-Trimethylcyclohexane-l,4dione 1455 43,125,153,55,79,91,109,137,183 1479 43,41,39,69,55,111,153,107
23 7.4 23.1
1.6 17.2 0.5
Sesquiterpenes RT c~-Cubebene RT Caryophyllene R'F Humulene RT I]-Selinene RT (Z),(Z)-Farnesol RT (E),(E)-Farnesol
0.2
0.6
5.7 1.6 1.0
4.3
0.02 53.7 6.8 0.2
53.2 7.9 0.5
1.9 0.4
Types of unknown sesquiterpenes§ 1.2 1.5 0.6 1.1 1.1 8.2 1.8 8.0 2.2 0.6 1.3 4.6 0.7
929 I~-Cubebene type 954 Aromadendrene type
958 cis-c~-Bergamotene 959 Caryophyllene type 969 Bergamotene type 976 trans-c-Bergamotene 1099 ~]-Bisabolene type 1126 I]-Elemene type 1135 [8-Sesquiphellandrene 1258 Caryophyllene oxide type 1298 Caryophyllene oxide type 1305 Caryophyllene oxide type 1585 Santalol type 1621 Santalol type
0.6
0.7 0.5
4.5 1.0 20.7 2.0 0.2 0.5 1.5 0.2 0.2
Nitrogen containing compounds 2.4 1.7
RT Trirnethylpyrazine RT Tetramethylpyrazine Unknowns§ 236 Monoterpene hydrocarbon 269 43,41,71,39,55 557 43,41,45,39,31,57,67,89 784 135,79,91,39,150,107 850 43,57,41,71,72,101,85 966 41,39,70,69 1001 42,41,39,87,56 1093 39,79,108,91,43,106,51,65,124,150 1281 43,41,39,67,55,107 1580 43,41,45,39,108,93,67,55,79 1652 77,105,51,182,59
1.0 1.4 1.0 0.7 0.9 1.6 1.9 0.5 O.6 1.4
0.1 0.3
0.5
*Amount of volatiles collected per inflorescence; tamount of volatiles collected in a male flower, and :[:in a female flower; ITprobably the cis stereoisomer, it has the same mass spectrum as the trans stereoisomer; §only those compounds which occurred in a relative amount > 1% in any sample are listed.
FLORAL ODOURS IN THE THEOPHRASTACEAE
263
and 92.3% of the floral scent in respective species. Benzenoids, especially benzaldehyde, benzyl alcohol, and methoxybenzenes, together with phenyl propanoids (mainly derivatives of cinnamic acid) characterize the floral scent of Jacquinia keyensis (97.6%) and constitute a large part of the floral scents of J. sprucei (57.8%) and J. macrocarpa (27.5%). In the other genera benzenoids are very sparse (Deherainia, Theophrasta) or present in trace amounts only (Clavija). Isoprenoids are present as monoterpenes in all species, whereas sesquiterpenes are found almost exclusively in Clavija and then in very large amounts, i.e. 95.9% in C. euerganea and 94.0% in C. repanda. Irregular terpenes, especially trimethylcyclohexenes derivatives of carotenoids, are characteristic components of the floral scent of the orange-flowered species of Jacquinia (J. sprucei and J. macrocarpa). The floral scent of Theophrasta americana is characterized by 3-methyl-l-butanol (67.4%), monoterpenes, benzaldehyde, and one of the three individuals investigated contained the very unpleasant smelling acid 3-methylbutanoic acid. In Deherainia smaragdina the floral scent contains mainly butyl acetate (83.2%), but the very unpleasant, sour odour is due to the presence of 3-methylbutanoic acid, and probably also tri- and tetramethylpyrazine. The white-flowered Jacquinia keyensis has a scent characterized by benzenoids and phenyl propanoids, especially a series of compounds with a methoxy-group in the 4-position on the aromatic ring, and some of the corresponding compounds without this substitution (Fig. 1). The floral scent of the orange-flowered J. spruceiand J. macrocarpa contains many of the same benzenoids and phenyl propanoids as that of J. keyensis, but in lower amounts, except for 3,5-dimethoxytoluene, which is the main compound in J. sprucei. In addition, the floral scent of J. macrocarpa and J. sprucei contains a series of derivatives of carotenoids, i.e. 2,6,6-trimethyl-l-cyclohexen-l-carboxaldehyde, 2,6,6-trimethyl-2cyclohexen-l,4-dione, 2,2,6-trimethylcyclohexane-l,4-dione (Fig. 2), and some unidentified compounds of the same type; that of J. macrocarpa also contains two sesquiterpenes, viz, (Z),(Z)- and (E),(E)-farnesol. The floral scent of Clavija euerganea and C. repanda contains almost exclusively sesquiterpenes with caryophyllene as the main compound, constituting 53.7% and 53.2% of the floral scent in respective species. We have only been able to verify the identification of a few of the sesquiterpenes with authentic reference compounds and, consequently, most of them are reported here only as types according to their main mass fragments. The similarity in floral scent composition of male and female individuals of C. euerganea was 71.6%. All the compounds present in C. repanda except one, were also found in C. euerganea, but the number of compounds were fewer in the former. The similarity in floral scent composition of the male individuals of C. euerganea and C. repanda was 78.3%, and between female of C. euerganea and male of C. repanda 73.3% Discussion Although only seven species in four genera were investigated, the sample still covers most of the variation in floral morphology and corolla colour present in the family. Clavija is very homogenous as regards floral morphology and colour (St~hl, 1991) and in Jacquinia representatives of both existing colour morphs, white and orange, were studied. Living plants of the monotypic genera Neomezia and Votschiahave not been available for this study, nor have any information on the floral scent in these genera been reported in literature or on herbarium labels. However, Neomezia appears to have a floral biology very similar to that of its nearest relative, Theophrasta (St~hl, 1987). The genus Votschia, a recent segregate from Jacquinia (St~hl, 1993), is so far known only from two collections. It has a corolla similar in size and shape to that of Deherainia, but being yellow its flowers are not likely to produce a foetid scent as do flowers of that genus.
264
J.T. KNUDSEN AND B. STAHL
Jacquinia
Jacquinia keyensis
Me
Me
o a ~ 0 II ,.H C"
H2C h i ~
OMe 0
II,.H
Me d ()
C'
I0~
C II o
I Me
1
O Me CH2OH
XC /
II
O! Me O%c/O~
CH2OH
Me
0
H2C /
Me
O Me !
OH
O ! Me
~
CH2OH MeO~
Deherainia $maragdina
m
.N~
CH2OH
Theophrasta americana
CH2 OH
P
0 H
Me
Me Clavija
M
N
Me
Me
_N.
Me
Me
N
M
FIG. 1. CHARACTERISTIC AND DOMINANT COMPOUNDS ISOLATED IN THE FLORAL SCENT OF SPECIES OF THEOPHRASTACEAE. In Jacquinia: (a) 3,5-dimethoxytoluene; (b) benzaldehyde; (c) 4-methoxybenzaldehyde; (d) 1,4-dimethoxybenzene; (e) 4-methoxybenzyl alcohol; (f) benzyl alcohol; and (g) 3-phenylpropanol; in J. keyens/s: (h) 4~methoxybenzyl acetate; (i) benzyl acetate; (j) p-cresol; (k) methyl 4-methoxybenzoate; and (I) 1,4-met~oxyphenylpropanol; in Deherainia smaragdina: (m) butyl acetate; (n) 2,3,5-trimethylpyrazine; and (o) 2,3,5,6-tetramethylpyrazine; in Theophrastaamericana: (p} 3-methyl-l-butanol; and in species of C/aw~: (q) caryophyllene; and (r) humulene.
Floral scent chemistry
Most of the compounds in the floral scent of the Theophrastaceae are common in head-space samples of a large number of angiosperms (Knudsen et al., 1993). However, 3-methylbutanoic acid was only recently reported in the floral scent of two species of Masdevallia (Orchidaceae; Kaiser, 1993), methyl 4-methoxybenzoate in Moneses unMora (Pyrolaceae; Knudsen and Tollsten, 1991), and 4-methoxybenzyl
FLORAL ODOURS IN THE THEOPHRASTACEAE
265
(or-Carotene) Cleaving enzyme(s)
H
~
R
R=CHOor (R= COOH)
trans-Crocetindialdehyde ( trans-Crocetinhalbaldehyde )
-tCHO
2,6,6-Trimethyl-1-cyclohexen1-carboxaldehyde (IB-cyclocitral)
o•O o•O
2,6,6-Trimethyl-2-cyclohexen1,4-dione (4-Oxoisophorone)
2,2,6-Trimethyl-1,4-cyclohexanedione
FIG. 2. FLORAL SCENT COMPOUNDS OF ORANGE-FLOWERED SPECIES OF JACQUINIA POSTULATED TO BE DERIVED FROM CAROTENOIDS. ( Trans-crocetindi- and halbaldehyde identified in dried flowers of J. nervosa by Eugster et ak, 1969).
alcohol in a species of Cucurbita (Cucurbitaceae; Andersen and Metcalf, 1987). Triand tetramethylpyrazine, found in D. smaragdina, are here reported as new constituents in floral scent. Of the three identified derivatives of carotenoids, 4-oxoisophorone has been reported in Freesia (Iridaceae; Lamparsky, 1985), in seven species of orchids (Kaiser, 1993), and in Angelica (Apiaceae; Tollsten eta/., 1994), 2,6,6trimethyl-l-cyclohexen-l-carboxaldehyde (~-cyclocitral) in Freesia (Lamparsky, 1985), and 2,2,6-trimethyl-l,4-cyclohexanedione in Encylcia (Orchidaceae; Kaiser, 1993). Extracts of dried stigmas of Crocus sativus (Iridaceae) have been found to contain the above mentioned compounds, but with 2,6,6-trimethyl-l-cyclohexene-1carboxaldehyde glycocidally bound in the 4-position (R6del and Petrzika, 1991; Himeno and Sano, 1987). The floral scent composition of the studied species accord with the .generic delimitations in the Theophrastaceae. That of Theophrasta is distinguished by an aliphatic alcohol; Deherainia by aliphatic esters, and two pyrazines. However, Theophrasta and Deherainia have 3-methylbutanoic acid in common; Jacquinia by aromatics and, in the orange-flowered species, additional derivatives of carotenoids; and, finally, Clavija by various sesquiterpenes. Within Jacquinia, each species has a characteristic floral scent composition. Jacquinia spruceidiffers from J. rnacrocarpa in having methyl and ethyl cinnamate and much higher amounts of 3,5-dimethoxytoluene; these two orange-flowered species are distinguished from the whiteflowered J. keyensis by containing carotenoid derived scent compounds. The chemical composition of the floral scents of Clavija euerganea (from both male and female plants) and C. repanda is very similar and cannot be used to distinguish between these two species.
266
J.T. KNUDSEN AND B. ST~,HL
Floral scent and pigments The corolla colour of Jacquinia macrocarpa and J. spruceichanges from orange to almost brick-red as the flowers age, suggesting a chemical change in the pigments. Orange colours usually indicate presence of large amounts of ~-carotene (Harborne, 1988). However, Eugster et aL (1969) did not find this pigment in the dried flowers of J. nervosa Presl ("J. angustifolia"), a close relative of J. sprucei (St~hl, unpubl.). Instead they found trans-crocetindialdehyde and trans-crocetinhalbaldehyde (Fig. 2). Most floral scent compounds with a trimethylcyclohexene structure, as those reported here in J. macrocarpa and J. spruce~ are suggested to be derived from 0¢- or ~-carotene through enzymatical cleavage and oxidation of the polyene chain (e.g. Croteau and Karp 1991; Kaiser, 1993). Similar compounds or compounds with the same general structure have been identified as volatile components in flowers of saffron (Crocus sativus; RSdel and Petrzika, 1991), but, in that particular case, zeaxanthin, a dihydroxylated I]-carotene, was presumed to be the parent compound (Himeno and Sano, 1987; Zarghami and Heinz, 1971). The absence of 0¢- or I]-carotene in the dried flowers of J. nervosa, the presence of degradation products of similar structure as those from the degradation of zeaxanthin (crocin), and the colour change in flowers of many species of Jacquinia strongly suggest that the emitted trimethylcyclohexenes are formed at anthesis through degradation of the flowers own colour pigments (Fig. 2). Additional evidence supporting this hypothesis is provided by J. keyensis. The floral scent of this white-flowered species is very similar to those of the orange-flowered J. sprucei and J. macrocarpa, except for the lack of trimethylcyclohexenes. The floral scent of Clavija consists almost exclusively of sesquiterpenes, suggesting that the orange colour of its flowers originates from other pigments than those found in Jacquinia or that the cleaving enzymes producing the trimethylcyclohexenes from carotenes are not present or non-functional. Pollination biology There are no reliable observations of pollinators to the Theophrastaceae and conclusions about their pollination biology have to be drawn mainly from indirect evidence. Since all members have dry, nectarless flowers (St~hl, unpubl.), pollination will have to be effected by pollen gathering insects or through deceit. Self pollination may occur in the hemaphrodite genera but then hardly within the same flower since these are protandrous and have extrorsely dehiscent anthers. Most or perhaps all of the hermaphrodite genera also seem to be self-sterile (Chandler, 1911; St&hi, unpubl.). The Mesoamerican species Jacquinia nervosa, whose flowers are very similar to those of the orange-flowered species of Jacquinia studied here, is believed to be hummingbird pollinated (Janzen, 1983; "J. pungens"), but the lack of nectar make this seem very unlikely. Large green flowers as those of Deherainia are unusual among the angiosperms. They are met with in some bat-pollinated Bignoniaceae (e.g. Crescent~a; Gentry, 1974) and Lecythidaceae (Lecythis poiteaui, L. barnebyi; Mori and Prance, 1990), as well as in the Orchidaceae (e.g. Lycaste; Kaiser, 1983). Since Deherainia flowers are lacking nectar and are more or less hidden by the foliage, they are hardly pollinated by bats. It seems more likely, as already suggested by Pohl (1931), that Deherainia smaragdina is pollinated by flies, but this has still to be observed in the field. The floral scent of Deherainia resembles that of some sapromyophilous species of Masdevallia (Orchidaceae; Kaiser, 1993) in containing the characteristic smelling 3-methylbutanoic acid, an otherwise rare compound in floral scents. Although not evident in the greenhouse material used for this study, flowers of Theophrasta americana observed in the field were reported to have a fungal scent (St~hl, 1987). Together with other pieces of evidence (more or less hidden flowers, pale brown corollas, uneven surfaces of the corolla walls, and presence of Diptera
FLORALODOURSIN THETHEOPHRASTACEAE
267
larvae) the species was suggested to be pollinated by Diptera that normally visit m u s h r o o m s to breed a n d / o r lay their eggs. The chemical composition of a floral scent reminiscent of m u s h r o o m s is only k n o w n from Dracula chestertonii (Orchidaceae; Kaiser, 1993), The scent of this species was f o u n d to contain the m u s h r o o m smelling 1-octen-3-01 as the main c o m p o u n d . However, w e did not find this nor any of the other of its volatiles in the scent of Th. americana. Using the classification of Faegri and van der Pijl (1979), the more or less sweetscented flowers of Jacquinia and Clavija m a y be described as melittophilous, whereas the flowers of Theophrasta and Deherainia, w i t h their less agreeable scents, as sapromyophilous. In this case, however, large differences in ecology, floral features, and not least, floral scent chemistry indicate that this classification is a rather crude simplification. Being prevalent in the rest of the Primulales, melittophily represents probably the ancient condition in the Theophrastaceae, although it is n o w quite differently expressed in Clavija and Jacquinia. The sapromyophilous condition in Deherainia, Theophrasta, and probably Neomezia should consequently be regarded as a derived state in the Theophrastaceae. This hypothesis accords also w i t h supposed phylogenetic relationships w i t h i n the family based on morphological evidence (St,~hl, 1991). However, Deherainia and Theophrasta are only distantly related and must have acquired the s a p r o m y o p h i l o u s condition independently, given that the morphological study is correct. However, despite considerable differences in floral scent chemistry between Deherainia and Theophrasta, the present analysis does not lend full s u p p o r t to the i n d e p e n d e n t origins of s a p r o m y o p h i l y in these t w o genera since the scent of Deherainia contains the acetate of 3-methyl-l-butanol, the main c o m p o u n d in Theophrasta, and both have the acid.
Acknowledgements--Wethank B. Bro and J. P. Fell for help in the field, R. Kaiser for generously providing reference substances, and the staff at the G6teborg Botanical Garden for nursing the plants. The study was financially supported by the Regnell Foundation (KVA).
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Mori, S. A. and Prance, G. T. (1990) Lecythidaceae--Part I1. FI. Neotropica Monogr. 21 (11)pp. 1-376. The New York Botanical Garden, New York. Pohl, F. (1931) 0ber sich 5ffnende Kristallr~iume in den Antheren von Deherainia smaragdina. Jahrb. Wiss. Bot. 75, 481-493. RSdel, W. and Petrzika, M. (1991) Analysis of the volatile components of Saffron J. High Resolution Chromatogr. 14, 771-774. St&hi, B. (1986) Two new species of Clavija (Theophrastaceae) from NW South America Nordo J. Bot. 6, 769772. St~hl, B. (1987) The genus Theophrasta (Theophrastaceae). Foliar structures, floral biology and taxonomy. Nord. J. Bot. 7, 529-538. St~hl, B. (1989) A synopsis of Central American Theophrastaceae. Nord. J. Bot. 9, 15-30. St~hl, B. (1990) Theophrastaceae. In FI. Ecuador(Harling, G. and Andersson, L., eds), 39, pp. 1-21. Department of Systematic Botany, University of G6teborg, G6teborg. St~hl, B. (1991) A revision of Clavija (Theophrastaceae).Opera Bot. 107, 1-77. St~hl, B. (1982) On the identity of Jacquinia armillaris (Theophrastaceae) and related species. Brittonia 44, 5460. St~hl, B. (1983) Votschia, a new genus of Theophrastaceae from southeastern Panama. Brittonia 45, 204-207. Standley, P. C. and Williams, L. O. (1966) Flora of Guatemala. Fieldiana Bot. 24(8), 179-206. Stenhagen, E., Abrahamsson, S. and McLafferty, F. W. (1974) Regisb'y of Mass SpectralData. John Wiley and Sons, New York. Tollsten, K., Knudsen, J. T. and BergstrSm, L. G. (1994) Floral scent in generalistic Angelica--an adaptive character (Apiaceae) Biochem. Syst. Ecol. 22, 161-169. Zarghami, N. S. and Heinz, D. E. (1971) Monoterpene aldehydes and isophorone-related compounds of saffron. Phytochemistry 10, 2755-2761.