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Variability of foliage terpenes of Fitzroya cupressoides
BiochemicalSystematicsand Ecology,VoL 19, No. 5, pp. 421-432, 1991.
Printed in GreatBritain,
0305-1978/91$3.00+ 0,00 © 1991 PergamonPressplc.
Variability of Foliage Terpenes of Fitzroya cupressoides LAURENCE G. COOL, ARIEL B. POWER and EUGENE ZAVARIN University of California, Forest Products Laboratory, Richmond, CA 94804, U.S.A.
Key Word Index--Fitzroya cupressoides; Cupressaceae; alerce; monoterpenes; sesquiterpenes; foliage; chemotype; chemotaxonomy. Abstract--Foliage of 57 Fitzroya cupressoides trees from six locations in Chile was analysed for monoterpenes and sesquiterpenes. A new monoterpene, ipsdienyl acetate (2-methyl-4-acetoxy-6-methylene-octa2,7-diene), was found. No geographic variability in monoterpene composition was evident. Three distinct sesquiterpene chemotypes were found in the areas sampled. The basic chemotype produced sizeable amounts of germacrene D, bicyclogermacrene, caryophyllene and several sesquiterpene alcohols including trans-nerolidol. The second chemotype elaborated in addition ¢-Iongipinene and an unknown sesquiterpene alcohol, as well as some I~-Iongipinene, which has not been previously reported in higher plants. The third chemotype produced ~- and l]-acorenol and a number of cedrene-related hydrocarbons. Minor geographic variability in the sesquiterpene composition of the basic chemotype was indicated.
Introduction
Fitzroya cupressoides (Mol.) Johnst. (Span. "alerce', Cupressaceae), is endemic to the rain forests of southern Chile and adjacent Argentina. The genus is monotypic and there are only two other native Cupressaceae in South America: Pilgerodendron uviferum (D. Don) Florin and Austrocedrus chilensis (D. Don) Pic.-Ser. et Bizz. Both species are also found in Chile and their ranges partially overlap that of F.cupressoides. The taxonomic relationship between these conifers is controversial. The distribution of alerce is now confined to the Chilean Coastal range and Chilo6 island between 39°50'S and 42°30'S and the Andean range between 40°00'S and 43°30'S; the formerly extensive stands in the more accessible central lowlands have been almost completely logged. Because of its heavy exploitation, alerce is now in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [1] and on the U.S. Department of the Interior list of threatened species (U.S. Endangered Species Act of 1973). Despite its economic importance, threatened status, and sheer magnificence (reminiscent of California's giant redwoods), F. cupressoides has received relatively little scientific study, with only a few references to chemistry. Erdtman and Tsuno analysed the heartwood extractives of a wood sample of unknown provenance [2]; several other groups have analysed the foliage biflavones of planted specimens (references in [3]). To date, no studies of the morphological or chemical variability of F. cupressoides have been reported, nor has the foliage essential oil apparently been analysed. The present study was undertaken to investigate the variability of its foliage terpene composition. Materials and Methods Foliage from a total of 57 trees was collected from six locations in the range of Fitzroya cupressoides and stored in aluminium foil and plastic bags while being transported to this laboratory. Each tree sampled was at least 100 m from the next. Samples for GC analysis were prepared by removing woody twigs and actively growing tips from the green branchlets. Approximately 2 g of foliage was chopped with a razor blade, covered with n-pentane in a small stoppered flask, and held for several days at --10°C. The clear light green solution was drawn off and concentrated under nitrogen flow at room temperature to about 0.1 ml.
(Received31 January 1991) 421
422
L.G. COOL ETAL.
GC analysis Glass SCOT columns: (a) 54 m by 0.5 mm i.d. OV-17 and (b) 43 m by 0.5 mm i.d. Carbowax 20M. Carrier gas: He, 25 cm sec 1. Temperature (both columns): 70°C isothermal for 5 min, 6°C rain -~ program to 220°C. Split injection (20:1 ), injector at 200°C. Detector: FID, 250°C. Some samples were re-analysed on WCOT columns: (a) 30 m by 0.25 mm i.d. SE-54 and (b) 30 m by 0.25 mm i.d. Carbowax 20M. Carrier: He, 30 cm sec -1. Temperature (both columns): 50°C isothermal for 3 min, 6°C min 1 program to 250°C. Split injection (70:1), injector at 180°C. Detector: FID, 250°C. Kovats' retention indices (KI) given below were calculated with reference to n-alkane peaks from isothermal DB-5 and Carbowax 20M (C20M) chromatograms at 165°C unless otherwise indicated. GC-MS analysis WCOT column, 30 m by 0.2 mm i.d. DB-5. Carrier: He, 30 cm sec 1. Temperature: 60°C isothermal for 3 min, 6°C min 1 to 250°C. Split injection (100:1), injector at 280°C. MS detection: El at 70 eV. Ipsdienylacetate. GC retention indices (135°): KI = 1243 (DB-5), 1594 (C20M). GC-MS m/z (rel. int,): 134(18), 127(5), 119(31), 93(9), 92(12), 91(30), 86(5), 85(100), 79(21), 78(5), 77(13), 67(10), 65(9), 55(9), 53(13), 51 (7), 45(6), 43(78), 41 (37), no parent ion. Bicyclogermacrene. Since bicyclogermacrene had originally been reported as a major component in Citrus junos peel oil [4] a sample of the fruit peel of this species was extracted and analysed by GC-MS. Both the retention time and mass spectrum of the F. cupressoides unknown matched those of a major component of C junos, thus substantiating the identification. Bicycloelemene. A minor sesquiterpene component noted on GC-MS analysis was tentatively identified as bicycloelemene. This peak was only observed when the injector temperature was high (280°C versus 200°C used in routine GC analysis) and was assumed to be an artifact produced by Cope rearrangement of bicyclogermacrene at the high injector temperature used in GC-MS [4]. This phenomenon was recently noted elsewhere [5]; the reported mass spectrum and retention times for bicycloelemene agree with those found by us. ~-Longipinene. GC retention indices (135°): KI= 1418 (DB-5), 1599 (C20M). GC-MS m/z (rel. int.): [M] * = 204(27), 189(27), 161(57), 147(26), 134(23), 133(55), 121(32), 120(37), 119(45), 109(34), 107(52), 106(24), 105(66), 95(25), 93(75), 92(42), 91 (92), 82(18), 81 (35), 80(21 ), 79(66), 77(54), 69(34), 67(43), 65(23), 57(21 ), 55(55), 53(31 ), 43(22), 41 (100). Thujopsene, ~J-cedrene, cuparene. An extract of Juniperus virginiana wood [6] was analysed by GC-MS to further substantiate identifications of these components, whose identities had been deduced from published retention and mass spectral data [7-9]. In all cases the data for major components of the reference species matched those for the three unknowns of Type 2. F. cupressoides. Germacrene A, GC-MS analysis revealed the presence of a minor component whose distinctive mass spectrum was very similar to that of ~-elemene. The proposed identity of this unknown is based on this similarity and its late retention time typical of the germacrenes. UnknownO-1, sesquiterpene hydrocarbon. This compound was not well separated from ~,-muurolene on GC or GC-MS analysis. o¢-, ~J-Acorenol. GC retention indices: KI = 1657, 1660 (DB-5), 2135, 2161 (C20M). GC-MS m/z (rel. int.): [M] + = 222(2), 204(25), 161 (23), 121 (44), 119(100), 107(24), 105(36), 93(51 ), 91 (31), 81 (28), 79(37), 77(23), 69(20), 67(24), 59(74), 55(28), 43(58), 41 (48). Mass spectra of both isomers were identical. 4cL-Hydroxygermacra-1 (10), 5-diene. GC retention indices: KI = 1603 (DB-5), 2056 (C20M). GC-MS m/z (rel. int.): [M] += 222(1), 207(9), 204(8), 161(19), 123(17), 121(12), 119(12), 109(12), 105(19), 95(15), 93(16), 91(14), 81 (100), 80(17), 79(19), 71 (15), 69(15), 67(19), 55(21 ), 53(12), 43(77), 41 (43). Unknown 0-2, sesquiterpene alcohol. GC retention indices: KI = 1545 (DB-5), 1953 (C20M). GC-MS m/z (rel. int.): [M] + =222(3), 208(11), 207(66), 204(28), 162(13), 161(68), 137(12), 135(12), 133(13), 123(24), 121(25), 119(24), 109(18), 107(22), 105(44), 95(23), 93(37), 91(38), 81(31), 79(34), 77(25), 71(11), 69(17), 67(20), 55(32), 53(15), 43(100), 41 (60). Unknown 0-3, oxygenated sesquiterpene, GC retention indices: KI = 1607 (DB-5), 2122 (C20M). GC-MS m/z (rel. int.): [ M ] * = 220(3), 205(31), 202(8), 187(14), 177(8), 162(16), 159(22), 149(9), 147(16), 146(12), 145(11), 133(12), 131 (14), 121 (13), 119(32), 117(12), 107(22), 105(30), 93(30), 91 (43), 81 (26), 79(32), 77(24), 71 (14), 69(24), 67(24), 65(12), 55(24), 53(19), 43(100), 41 (61). Unknown O-4a, b, sesquiterpene alcohols. GC retention indices: KI = 1618 (DB5); 2071, 2074 (C20M). Peaks unresolved in GC-MS. Unknown 0-5, sesqu/terpene alcohol. GC retention indices: KI = 1702 (DB-5), 2237 (C20M). GC-MS m/z (rel. int.): [M] + = 222(3), 204(10), 137(65), 121(17), 119(51), 110(10), 109(26), 107(11), 105(11), 95(25), 93(30), 91(15), 84(54), 83(27), 82(16), 81 (15), 79(15), 77(15), 69(47), 67(25), 56(16), 55(51 ), 53(16), 43(25), 41 (100). Unknown 1-1, sesquiterpene alcohol GC retention indices: KI = 1653 (DB-5), 2169 (C20M). GC-MS m/z (rel. int.): 204(55), 179(100), 161 (95), 121 (26), 119(74), 110(24), 109(35), 107(23), 105(62), 91 (45), 93(43), 95(43), 81 (47), 79(37), 77(29), 69(38), 67(28), 55(50), 43(82), 41 (86), no parent ion. Unknown 1-2, sesquiterpene alcohol GC retention indices: KI = 1673 (DB-5), 2110 (C20M). GC-MS m/z (rel. int.): [M] ~ = 222(15), 204(30), 121(36), 119(100), 111 (20), 110(36), 109(40), 107(26), 105(41), 95(41), 93(46), 91 (41), 82(35), 81 (25), 79(28), 69(39), 67(31), 56(25), 55(67), 53(24), 43(52), 41 (98). Unknown 2-1, sesquiterpene hydrocarbon. GC retention index: K1=1480 (DB-5). GC-MS m/z (rel. int.):
TERPENES OF FITZROYACUPRESSOIDES
423
[M] + = 204(20), 162(16), 161(100), 159(2), 147(7), 133(13), 119(29), 117(12), 115(8), 105(44), 93(18), 91(46), 81(22), 79(20), 77(17), 67(12), 65(11), 55(16), 43(12), 41 (36).
Results and Discussion This study was based on the analysis of 57 trees from six populations in Chile: three from the coastal range, two from the Andes, and one from the central lowland area. The locations of the populations sampled are shown in Table 1 and Fig. 1. The monoterpene composition of each population is presented in Table 2. The monoterpenes consisted of high levels of 0{-pinene (>82% in all trees), with myrcene, limonene and I~-pinene accounting for most of the balance. Oxygenated monoterpenes were minor constituents (each < 1%). In addition to the ubiquitous terpinyl and bornyl acetates, verbenone was detected in very small amounts in all GC-MS analyses but was not quantified, due to poor GC separation from larger components. Ipsdienyl acetate (2-methyl-4-acetoxy-6-methylene-octa-2,7-diene, 1, Fig. 2), which has not been previously reported, was identified by GC-MS comparison with an authentic sample prepared by acetylation of commercial ipsdienol. The monoterpene composition of F. cupressoides was uniform from population to population: discriminant function analysis on the individual tree monoterpene data grouped by population gave no significant separation among populations. The sesquiterpene composition proved to be more interesting. First, the sesquiterpenes were relatively abundant, their total amounting to an average of 87% of the monoterpenes. Of this total, about one-third was composed of sesquiterpene alcohols. The diversity of sesquiterpenes was also great, with over 40 compounds detected. Finally, every population sampled had some trees which were qualitatively different from the others; three distinct chemotypes were identified. The compositions of these chemotypes are summarized in Table 3. Also included is the analysis of a single tree (NAR4) which contained the sesquiterpenes of all chemotypes. The basic chemotype, denoted Type 0, contained high levels of germacrene D, with substantial amounts of bicylcogermacrene, caryophyllene, ~-copaene, 0c-muurolene, 0~-humulene, ~)-cadinene, trans-nerolidol, and several other sesquiterpene alcohols. Several Type 0 sesquiterpenes had frequency distributions which deviated significantly from normal (Shapiro-Wilk test, P<0.01): caryophyllene, ~.-humulene, 0cmuurolene, unknown 0-3 and unknown 0-5 (Fig. 3). The caryophyllene distribution suggests monogenic control of the level of this terpene, as has been found in Pinus pinaster [10]. One of the major sesquiterpene alcohols was identified as 40~-hydroxygermacral(10),5-diene (2, Fig. 2). The concentration of this alcohol correlated positively with germacrene D and bicyclogermacrene (R = 0.59 and 0.65 respectively, significant at P<0.0001, for all trees). It had a mass spectrum which agreed with that for the enantiomer of this compound (Dr William Fenical, personal communication) and with published data (only eight peaks reported, [11]). The Kovats index also matched that calculated from the chromatogram in [11]. TABLE 1. DESCRIPTION OF POPULATIONS SAMPLED
Population
Location
Latitude
Longitude
Elevation (m)
Number of trees
1 2 3 4 5 6
La Uni6n Pargua Chilo~ Island Puerto Montt Lake Chaiquenes Rio Negro
40°08'S 41°45'S 42"38'S 41°20'S 41°30'S 41°55'S
73°20'W 73°40'W 74°05'W 72°53'W 72°35'W 72°25'W
800 25 400 105 800 630
10 10 10 10 8 9
424
L. G. COOL E T A L , 74°
40 °
73 °
72 °
+
+
40 °
,,
01
CHILE
41 °
41 °
tt
,
•
6
+ ~
42 °
'
") ISLAND
42 o
G U L F OF I
,
0
20
40
60
I
I
I
I
km
i I r
2
74 °
73 °
72 °
FIG. 1. LOCATION OF POPULATIONS SAMPLED.
In all trees there was a group of minor constituents derived from a J]-acorenyl precursor: 13-funebrene (3, Fig 2), J]-acorenol (7, Fig. 4) and J]-acoradiene (8, Fig. 4). j]-Funebrene, by which we mean the 1,7-diepimer (or its enantiomer, the 2,5-diepimer) of iS-cedrene, was identified from literature retention time and mass spectral data [12, 13]. In further support of this identification is the co-occurrence of J]-acorenol and j]-acoradiene: these are likely congeners of the funebrenes [14, 15]. Surprisingly, even though I]-acorenol was present in much larger amounts in Type 2 trees (see below), I]-funebrene and 13-acoradiene levels showed no increase. It seems reasonable to assume that different enzymes are responsible for the biosynthesis of the lS-acorenyl sesquiterpenes in Type 0 and Type 2 trees. The second chemotype (Type 1), in addition to the Type 0 compounds, elaborated substantial amounts of 0¢-Iongipinene and an unknown sesquiterpene alcohol, as well as lesser amounts of several other Iongipinene-related hydrocarbons. On the basis of retention time (J] isomers generally elute somewhat later than their c(counterparts) and the excellent correlation with 0¢-Iongipinene (Table 4), it was deduced that one of the Type 1 hydrocarbons was 6-1ongipinene (4, Fig. 2), which although hitherto unreported in vascular plants has been found to occur naturally in the liverwort Scapania undulata as the (-) enantiomer [16]. To verify this identification, an authentic sample of Iongipinanol, which readily dehydrates on GC analysis to c~- and J]-Iongipinene [16], was analysed by GC and GC-MS. The first hydrocarbon to elute was 0¢-Iongipinene;
0.3 89.3 0.3 1.3 0.5 4.2 3.0 0,1 0,2 0.3 0.3 0,2
0.04 1.91 0.05 0.55 0.08 1.41 0.82 0.12 0.07 0.14 0.26 0.06
S.D, 0,3 89,7 0.3 1.0 0.6 3,8 2.6 0,1 0.2 0,3 0.5 0.2 tr.
2 Mean 0.02 2.65 0.02 0.16 0.04 1,93 0.87 0.09 0,06 0.11 0.29 0.12
S.D. 0,3 88.3 0,2 0.8 0.4 5,7 2,7 0,1 0,1 0.1 0,3 0.2 tr,
3 Mean 0.10 2.95 0,09 0.43 0.16 3.0t 0,91 0.09 0,17 0,08 0.26 0.18
S.D.
Population
0.3 88.2 0,3 1.2 0.5 5.0 2.7 0.1 0.2 0.2 0.2 0.2
4 Mean 0.05 1,81 0.04 0.51 0,08 1.61 1.18 0,17 0.08 0,11 0,19 0,09
S.D. 0.3 91.1 0.3 0.9 0.5 3,7 2.3 0,1 0,2 0.2 0,2 0.1 tr.
5 Mean 0.05 2.03 0.01 0.24 0.05 1.41 0.52 0.07 0.07 0.04 0.22 0.08
S.D.
0.03 89.8 0.3 1.0 0.5 4.5 2.5 0.1 0.2 0.2 0,2 0.2
6 Mean
0.05 2.19 0.03 0.31 0.04 1.33 0.88 0.12 0.10 0.06 0.21 0.15
S.D.
B B B B B B B B B A B B A
Identityt
*As per cent of total monoterpenes, tldentification codes: A = GC-MS comparison with authentic substance, B = GC-MS comparison with component of thoroughly characterized plant extract run under identicaJ conditions, tr. = trace amounts,
Tricyclene ~-Pinene Camphene ~-Pinene Sabinene Myrcene Limonene ~-Phellandrene Terpinolene Ipsdienyl acetate =-Terpinyl acetate Bornyl acetate Verbenone
Compound
1 Mean
TABLE 2. FOLIAGE MONOTERPENE COMPOSITION* OF flTZROYA CUPRESSOIDESPOPULATIONS
z
426
L.G. COOL ETAL.
Ac0 ]
2
H 3
4
FIG. 2. TERPENE STRUCTURES FROM FITZROYA CUPRESSOIDES.
the retention times and mass spectrum of the second hydrocarbon (I]-Iongipinene) were identical to those of the £ cupressoides component. Since the higher plants are reported to invariably produce Iongifolene-relatives antipodal to those found in the liverworts, it is expected that both the 0{- and j]-Iongipinene found here are the (+) enantiomers. As can be seen in Table 4, 0c-ylangene also appears among the Type 1 sesquiterpenes and correlated positively with 0~-Iongipinene. This biosynthetic relationship, which has not been noted before, will be explored in more detail in a subsequent publication. The third chemotype, Type 2, was distinguished by the appearance of 0{- and ~Bcedrene and several related hydrocarbons, and by large amounts of two isomeric sesquiterpene alcohols. These were identified as 0~- and I]-acorenol (5, 7 in Fig. 4) by GC-MS comparison with an extract of Juniperus rigida wood, from which the acorenols were first isolated [17, 18]. Analysis of the J. rigida extract also provided substantiating evidence for the identities of 0c- and ~B-acoradiene (6, 8 in Fig. 4), both of which had also been originally isolated from this source. In the case of the component identified as ~'-acoradiene (9, Fig. 4), also known in the (--) form as 0~-alaskene* [19], the very distinctive mass spectrum (base m/z = 121, major m/z = 136) pointed either to this compound or to 6-acoradiene (10, Fig. 4). The retention indices in [20] enabled us to identify our unknown as the former isomer. As can be seen in Table 3, the three chemotypes of F. cupressoides are quite well defined: when the distinguishing sesquiterpenes for Type 1 or Type 2 appear at all, they appear in substantial amounts. Also, the percentages of the various Type 1 and Type 2 sesquiterpenes are positively correlated inter se (Tables 4 and 5). These facts are strongly suggestive of single-gene control of the biosynthesis of the Iongipinaneand acorane-related compounds in F. cupressoides, as has been demonstrated in the *A note on the nomenclature of the acorane hydrocarbons: in 1970 the structures of the alaskenes [19J and the acoradienes [17, 181 were both reported, c(-Alaskene and "F-acoradiene (9, Fig. 4) proved to be synonymous, while ~B-alaskene was the enantiomer of 5-acoradiene (10, Fig. 4). In order to reduce the number of trivial names and to avoid confusion, we would suggest that all of these compounds be given the more general name acoradienes. Thus c(- and ~-alaskene would be replaced by (--) "~-acoradiene and (--)6-acoradiene, respectively.
427
TERPENES OF FITZROYACUPRESSOIDES TABLE 3. FOLIAGE SESQUITERPENE COMPOSITION* OF FITZROYACUPRESSOIDESCHEMOTYPES Type 0
Type 1
Compound
(40 trees) Mean Range
(9 trees) Mean
Range
(7 trees) Mean
Range
Tree NAR4
c¢-Cubebene
0.1
0.0-0.4
0.1
0.1-0.2
0.1
0.0-0.2
0.1
B
~-Copaene
2.0
0.3-4.3
1.7
0.8-2.4
1.9
1.3-2.9
1.0
A
~]-Cubebene
0.3
0.0-0.7
0.3
0.2-0.4
0.3
0.2-0.4
0.2
C
J]-Elemene
0.3
0.0-0.5
0.3
0.1-0.5
0.2
0.2-0.5
0.1
A
~-Funebrene
0.1
0.0-0.4
0.1
0.0-0.3
0.2
0.2-0.4
0.0
C
Caryophyllene
9.8
1.7-27.0
7.3
1.6--23.8
1.8-21.0
5.7
A
~-Farnesene
1.0
0.0-2.3
0.8
0.2-1.3
0.7
0.4-1,3
1.0
B
~-Humulene
2.3
1.0-6.4
1.8
0.9-3.4
2.2
1.2-5.2
1.1
B
~-Acoradiene
0.5
0.0-1.3
0.5
0,2-0.6
0.3
0.0-1.0
0.7
B
0,9 35.8
0.1-2.0 18.0-55.3
0.7 28.8
0.5-1.0 21.7-40.0
0.3 22.1
0.0-0.7 10.8-37.6
0.9 35.3
3"-Muurolene+unknown 0-1 Germacrene D
Type 2
12.7
Identityt
B, E B
~-Selinene
tr.
--
tr.
--
tr.
--
--
A
~.-Muurolene
2.3
0.1-6.3
2.2
1.4-3.0
2.3
1.3-4.2
1.2
A
Bicyclogermacrene
9.9
3.6-18.2
7.7
3.9-12.0
6.2
3.6-12.5
9.5
B
(Germacrene A)
tr.
--
tr.
--
tr.
--
--
E
~'-Cadinene
0.9
0.0-2.1
0.6
0.2-1.0
0.7
0.4-1.1
0.9
B
5-Cadinene
2.9
1.3-5.2
2.4
1.6-3.5
2.5
2.0-3.3
1.9
B
trans-Nerolidol
4.8
0.1-11.3
3.3
0.8-7.2
2.6
1.2-5.2
1.1
A
4~,-Hydroxygermacra-
5.8
3.2-11.0
5.3
2.7-7.2
3.4
2.1-4.7
5.2
C
Unknown 0 - 2
12.0
0.5-26.8
9.3
5.0-15.2
10.7
7.9-13.8
6.4
E
Unknown 0-3
2.5
0.6-6.4
1.9
0.8-3.0
1.4
0.2-2.2
1.3
E
Unknown 0-4a,b
2.5
0.1-5.6
2.0
1.0-3.2
1.8
0.2-3.0
1.2
E
Unknown 0 - 5
2.5
0.0-9.8
1.7
0.1-3.7
0.8
0.1-2.0
3.0
E
c¢-Ylangene
0.0
--
0.1
0.0-0.2
0.0
--
0.1
B
0~-Longipinene
0.0
--
8.9
3.4-13,6
0.0
--
5.8
A
~-Longipinene
0.0
--
0.6
0.2-0.9
0.0
--
0.4
A
Longifolene
0.0
--
0.6
0.0-1.3
0.0
--
0,5
A
~-Himachalene
0.0
--
2.8
0.2-4.8
0.0
--
1.4
C
Unknown 1-1
0.0
--
0.6
0.1-1.5
0.0
--
0.3
E
Unknown 1-2
0.0
--
7.8
2.7-13,3
0.0
--
5.8
E
(Bergamotene isomer)
0.0
--
0.0
--
0.2
0.1-0.2
0.1
D
0~-Cedrene
0.0
--
0.0
--
0.6
0.2-1.1
0.1
A
1 (10),5-diene
~-Cedrene
0.0
--
0.0
--
1.5
0.6-3.1
0.3
B
Thujopsene
0.0
--
0.0
--
0.1
0.0-0.2
0.0
B
c¢-Acoradiene
0.0
--
0.0
--
1,8
1,1-3,0
0.3
B
3'-Acoradiene
0.0
--
0.0
--
0.2
0.4-1.2
0.4
C
U n k n o w n 2-1
0.0
--
0.0
--
tr.
--
tr.
E
~-Bisabolene
tr.
--
tr.
--
0.4
0.2-0.7
0.1
A
Cuparene
0.0
--
0.0
--
tr.
--
tr.
8
Cedrol
tr.
--
tr.
--
0.3
0.1-0.5
0.1
A
12.9
9.2-17.1
4.1
B
8.5
5.6-11.2
2.8
B
¢-Acorenol
0.0
--
0.0
--
~-Acorenol
0.8
0.0-1,9
0.7
0.4-1.4
* A s per cent of total sesquiterpenes. tldentification codes: A = GC-MS comparison with authentic substance run under identical conditions, B = GC-MS comparison with c o m p o n e n t of thoroughly characterized plant extract run under identical conditions, C = GC-MS comparison with published retention time and M S data, D = (tentative identification) M S comparison with published data, E = see note in Materials and Methods.
case of the Iongifolene/0c-longipinene in Pinus pinaster [10]. Though the data are few, the frequency distributions of the Type 1 and Type 2 compounds (see Fig. 5 for 0¢-Iongipinene and 0¢-acorenol) are not inconsistent with the trimodal distribution that would be expected from the single-gene model. The biosynthetic implications of this model--and the identities of unknown compounds--will be explored in a subsequent publication.
L. G. COOL ETAL.
428 2o
2o
o~1o
~z0
0
o
2 4 a-HUNULENE (%)
10 20 CARYOPHYLLENE (%)
6
2o
15,
E-,
~o
I 2 a-MUUROLENE
8
4
2 4 UNKNOWN o - s (~.)
(Z)
0 0
6
5 UNKNOWN 0 - 5 (Z)
io
FIG. 3. FREQUENCY DISTRIBUTIONS OF SESQUITERPENE LEVELS IN FITZROYA CUPRESSOIDES.
5
R = OH,
6
R = CH 2
CH~
7
R = OH.
8
R = CH 2
g
CH 3
Io
FIG. 4. ACORANE SESQUITERPENES.
The sesquiterpene composition of Type 0 trees (Table 6) showed only modest geographic variability among populations, with a significant (P<0.03 level, MannWhitney test, 21 coastal vs. 11. Andean trees) trend being a west to east decrease of caryophyllene. Although principal component analysis of the individual-tree sesquiterpene data revealed no obvious clustering of populations, discriminant function analysis did show some separation of populations 4 and 6 from each other and from the rest (Fig. 6).
429
TERPENES OF FITZROYACUPRESSOIDES TABLE 4. CORRELATION BETWEEN PERCENTAGES OF LONGIPINANE-RELATED SESQUITERPENES
Kendall Rank Correlations, 10 trees (R/signif.) 0~-ylangene ~-Iongipinene Iongifolene c~-Longipinene
0.689 0.006
0¢-Ylangene
0.911 0.000 0.778 0.002
0.778 0.002 0.733 0.003 0.778 0.002
~-Longipinene Longifolene
~-himachalene
Unknown 1-1
Unknown 1-2
0.867 0.001 0.644 0.010 0.867 0.001 0.822 0.001
0.600 0.016 0.733 0.003 0.600 0.016 0.467 0.060 0.467 0.060
0,956 0,000 0,733 0.003 0,867 0.001 0,822 0,001 0,822 0,001 0,556 0,025
I]-Himachalene Unknown 1-1
TABLE 5. CORRELATION BETWEEN PERCENTAGES OF ACORANE-RELATED SESQUITERPENES
Kendall Rank Correlations, 8 trees (R/signif.) ~-cedrene ~.-acoradiene "(-acoradiene c~-Cedrene
~-Cedrene 0~-Acoradiene "~-Acoradiene ~-Bisabolene 0~-Acorenol
~-Acorenol
0.909 0.002
0.982 0.001 0.857 0.003
0.982 0.001 0.929 0.001 0.929 0.001
I~-bisabolene o¢-acorenol
I~-acorenol
cedrol
0.618 0.030 0.571 0.048 0.571 0.048 0.643 0.026
0.982 0.001 0.857 0.003 1.000 0,001 0.929 0.001 0.571 0.048 1.000 0.001
0.963 0.001 0.837 0.003 0.982 0.001 0.909 0.002 0.618 0.028 0.982 0.001 0.982 0.001
0.982 0.001 0.857 0.003 1.000 0.001 0.929 0.001 0.571 0.048
There were too few data for each of the other two chemotypes (nine trees of Type 1, seven of Type 2, and the tree representing both) for a similar statistical analysis of their geographic variability. However, by assuming the single-gene model for each chemotype, allele frequencies (Lp--, Lp+ for low and high Iongipinanes; Ac--, Ac+ for low and high acoranes) could be calculated for each population and some tentative conclusions made about the geographic distribution of these alleles. To calculate allele frequencies, the following assumptions were made: codominance of the Lp--/Lp+ and Ac--/Ac+ alleles at their respective loci, independence of the two loci, validity of the Hardy-Weinberg law within each population, and identical phenotypic ranges in all populations for each genotype. The phenotypic ranges were chosen to optimize overall agreement with the Hardy-Weinberg law and are indicated in the histograms for 0¢-Iongipinene (Lp--Lp-- 0-1%, Lp--Lp+ 1-12%, Lp+Lp+ above 12%) and 0¢-acorenol (Ac--Ac- 0-1%, Ac--Ac-t- 1-16.5%, Ac+Ac+ above 16.5%) (Fig. 5). Finally, the presumed frequencies for the Lp and Ac alleles for each population were calculated, as shown in Tables 7 and 8.
430
L. G. COOL ETAL.
sol , Lp-Lpo
Lp-Lp+
Lp+Lp+
,10.~
0
5
10
~-LONGIPINENE ( ~ )
Ac-Ac+
5
Ac+Ac+
10
~-ACORENOL
15
(%)
FIG. 5. FREQUENCYDISTRIBUTIONSOF a-LONGIPINENEAND c(-ACORENOL LEVELSiN FITZROYACUPRESSOIDES,SHOWING PHENOTYPIC RANGES CORRESPONDINGTO EACH GENOTYPE.
In the case of the Lp--Lp+ and Lp-t-Lp+ genotypes, i.e. the nine Type 1 trees and the double heterozygote NAR4, the Lp+ allele frequency of population 2 seemed unusually high (25%) compared to the other populations (average 7o4%). However, this difference was not significant at the P<0.05 level (modified Chi-square procedure of Ref. [21]). The Ac--Ac-I- and A c + A c + trees (seven Type 2 and NAR4), like Type 1, were scattered throughout the range of the six populations sampled. Although the low frequency for the Ac-I- allele made meaningful statistical comparisons impossible, the data do not suggest any geographic trends in the distribution of this gene. In summary, the monoterpene data indicate that in the range sampled F. cupressoides is quite uniform, while the sesquiterpene data show some geographic variability. It will be possible to better assess this variability when further sampling (more populations and more trees per population) over the entire range of the species is undertaken.
431
TERPENES OF FITZROYA CUPRESSQIDES TABLE 6. GEOGRAPHICVARIATIONOF TYPE 0 SESQUITERPENECOMPOSITION*
Compound 0~-Cubebene ~.-Copaene ~-Cubebene ~-Elemene [3-Funebrene Caryophyllene ~-Farnesene c(-Humulene ~-Acoradiene T-Muurolene+unknown 0-1 Germacrene D 0¢-Muurolene Bicylcogermacrene T-Cadinene 5-Cadinene trans-Nerolidol ~-Acorenol 4¢-Hydroxygermacra1(10), 5-diene Unknown 0-2 Unknown 0-3 Unknown 0-4a,b Unknown 0-5
1 (7 trees) Mean S . D .
2 (6 trees) Mean S . D .
Population 3 4 (8 trees) (8 trees) Mean S . D . Mean S . D .
5 (5 trees) Mean S . D .
6 (6 trees) Mean S.D.
0.1 1.9 0.4 0.3 0.2 10.5 0.9 2.8 0.6 0.9 35.1 2.0 9.7 1.0 2.6 3.7 0.9 6.3
0.04 0.90 0.15 0.08 0.09 7.61 0.37 1.41 0.28 0.23 5.31 0.91 1.70 0.36 0.75 2.37 0.23 1.45
0.2 2.6 0.4 0.3 0.t 16.4 0.8 2.2 0.6 1.0 28.6 2.7 6.7 0.9 3.1 5.9 0.9 4.9
0.10 0.83 0.17 0.06 0.05 9.26 0.38 0.31 0.23 0.39 4.02 1.70 2.34 0.49 0.99 2.52 0.35 1.17
0.1 2.1 0.3 0.2 0.1 13.7 0.5 2.2 0.4 0.7 35.9 2.1 9.7 0.6 2.8 5.9 0.6 5.2
0.07 0.98 0.11 0.16 0.13 9.56 0.27 0.84 0.19 0.27 10.30 1.02 3.75 0.29 0.63 2.34 0.30 1.51
0.2 2.2 0.4 0.3 0.0 4.3 0.2 2.7 0.2 0.7 41.6 2.6 10.8 0.4 3.3 2.6 0.7 6.2
0.07 1.14 0.16 0.09 0.08 1.83 0.31 1.83 0.18 0.38 9.24 1.69 4.31 0.45 1.04 1.92 0.28 1.50
0.1 1.6 0.2 0.2 0.1 6.6 0.7 1.6 0.5 1.0 40.8 1.6 13.2 0.8 2.7 4.1 0.8 7.3
0.03 0.32 0.04 0.16 0.13 4.06 0.30 2.00 0.24 0.31 2.33 0.36 3.08 0.29 0.53 3.14 0.31 2.31
0.2 2.0 0.3 0.2 0.1 7.0 1.4 2.0 0.8 1.1 31.7 3.0 10.0 1.2 3.0 6.8 1.3 5.4
0.10 1.16 0.18 0.11 0.15 8.91 0.51 0.58 0.40 0.38 8.21 1.80 4.37 0.49 1.24 1.68 0.43 1.30
11.3 2.6 2.5 3.2
5.30 0.93 1.08 0.65
13.1 3.2 2.7 2.9
6.98 1.70 1.46 1.60
10.8 2.5 1.8 1.1
5.34 1.33 1.04 0.70
13.9 2.4 3.1 0.8
7.15 1.50 1.66 0.86
10.1 1.6 2.2 2.4
1.84 0.57 0.76 0.85
12.2 2.4 2.6 5.4
6.66 1.15 1.61 2.43
*As per cent of total sesquiterpenes.
5 ~ Z o
4
rj Z
2.
6
/
36
",,
/
6
1-
'~'~
~ o. z - 1N
"
~
/4
6
4..I, 4
i i i
II I
/
I "6
~,,
1
5
1 1
5155
~-2.
3
19 5
r~
N-3-
23
2~ 3
33
22
3
2
--4"
-8
-8
-'4
-'2
6
2
8
DISCRIMINANT FUNCTION 1 FIG. 6. PLOT OF SESQUITERPENEDISCRIMINANT FUNCTIONS 1 AND 2 FOR FITZROYA CUPRESSOIDESTYPE O. TABLE 7. LONGIPINANEALLELE FREQUENCIES
Population
Assumed genotype frequency Lp-Lp-Lp-Lp+
1 2 3 4 5 6
0.90 0.60 1.00 0.90 0.75 0.78
0.10 0.30 0.00 0.00 0.25 0.22
Lp+Lp+
Assumed allele frequency LpLp+
Calculated Hardy-Weinberg 9enotype frequency Lp-LpLp--Lp+ Lp+Lp÷
0.00 0.10 0.00 0.10 0~00 0.00
0.95 0.75 1.00 0.90 0.87 0.89
0.90 0.56 1.00 0.81 0.76 0.79
0.05 0.25 0.00 0.10 0.13 0,11
0.10 0.38 0.00 0.18 0.22 0.20
0.00 0.06 0.00 0.01 0.02 0.01
432
L. G. COOL ETAL.
TABLE 8. ACORANE ALLELE FREQUENCIES
Population
Assumed genotype frequency Ac--Ac- Ac-Ac+
1 2 3 4 5 6
0.80 1.00 0.80 0.90 0.74 0.89
0.20 0.00 0.20 0.10 0.13 0.11
Ac+Ac+
Assumed allele frequency Ac-Ac+
Calculated Hardy-Weinberg genotype frequency Ac Ac- Ac-Ac+ Ac+Ac+
0.00 0.00 0.00 0.00 0.13 0.00
0.90 1.00 0.90 0.95 0.81 0.94
0.81 1.00 0.81 0.90 0.65 0.89
0.10 0.00 0.10 0.05 0.19 0.06
0.18 0.00 0.18 0.10 0.31 0.11
0.01 0.00 0.01 0.00 0.04 0.00
Acknowledgements--The authors are indebted to the Committee for the Defense of the Flora and Fauna of Chile--with special thanks to Sr Hern&n Verscheure--for their enthusiastic assistance with the collection of samples. We wish to thank Dr Rainer Scora for providing samples of Citrusjunos, Dr Niels H. Andersen for a sample of Iongipinanol, Mr Ken Hobson for a sample of ipsdienol, Dr William Fenical for spectral data of 4c(hydroxygermacra-l(10),5-diene, and Dr Robert Flath for listings of sesquiterpene retention times and mass spectral data. We are also grateful to Dr Constance Millar for reviewing the manuscript and providing valuable suggestions, and to Mr Charles Berolzheimer and the California Cedar Products Company for a grant supporting this work.
References 1. Convention on International Trade in Endangered Species of Wild Fauna and Flora. (1984) U.S. Department of the Interior, Fish and Wildlife Service, Publication 50 CFR 23.23. 2. Erdtman, H. and Tsuno, K. (1969) Acta Chem. Scand. 23, 2021. 3. Naqvi, S. W. I., Parveen, N., Parveen, M. and Khan, N. U. (1987) Curr. Sci. 56, 480. 4. Nishimura, K., Shinoda, N. and Hirose, Y. (1969) Tetrahedron Lett., 3097. 5. Le Quere, J.-L and Latrasse, A. (1990) J. Agric, Food Chem. 38, 3. 6. Wenninger, J. A., Yates, R. L. and Dolinsky, M. (1967) J.A.O.A.C. 50, 1304. 7. Heller, S. R. and Milne, G. W. A. (1978) EPA/NIHMass Spectra/Database, National Standard Data Reference Series #63, vol. 2. U.S. Department of Commerce, National Bureau of Standards. Note: the spectrum for l]-cedrene is incorrect, as are those for c(-cedrene and cedrol (correct spectra of ~-cedrene and cedrol in [8], ~-cedrene in [9]). 8. Stenhagen, E., Abrahamsson, S. and McLafferty, F. W. (1974) Registry of Mass Spectral Data, vol. 2. John Wiley, New York. Note: the spectrum for IB-cedrene is incorrect; correct spectrum in [9]. 9. Ramaswami, S. K., Briscese, P., Gargiullo, R. J. and yon Geldern, 1". (1986) in Flavors and Fragrances." A World Perspective (Lawrence, B. M., Mookherjee, B. D. and Willis, B. J., eds), p. 951. Proceedings of the 10th International Congress of Essential Oils, Fragrances and Flavors, Washington DC, U.S.A. 10. Marpeau, A., Baradat, Ph. and Bernard-Dagan, C. (1975) Ann. Scl~ Forest. 32, 185. 11. Schmaus, G., Schultze, W. and Kubeczka, K.-H. (1989) Planta Med. 55, 482. 12. Norin, T., Sundin, S., Karlsson, B., Kierkegaard, P., Pilotti, A.-M. and Wiehager, A.-C. (1973) Tetrahedron Lett., 17. 13. Piovetti, L. and Dia ra, A. (1977) Phytochemistry 16, 103. 14. Kirtany, J. K. and Paknikar, S. K. (1973) Indian J. Chem. 11, 508. 15. Piovetti, L. and Diara, A. (1980) Tetrahedron Lett., 1453. 16. Andersen, N. H., Bissonette, P., Liu, C.-B., Shunk, B., Ohta, Y., Tseng, C.-L. W., Moore, A. and Huneck, S. (1977) Phytochemistry 16, 1731. 17. Tomita, B. and Hirose, Y. (1970) Tetrahedron Lett., 143. 18. Tomita, B., Isono, T. and Hirose, Y. (1970) Tetrahedron Lett., 1371. 19. Andersen, N. H. and Syrdal, D. D. (1970) Tetrahedron Lett., 2277. 20. Andersen, N. H., Syrdal, D. D., Lawrence, B. M., Terhune, S. J. and Hogg, J. W. (1973) Phytochemistry 12, 827. 21. Dixon, W. J. and Massey, F. J. Jr (1957) Introduction to StatisticalAnalysis, p. 232. McGraw-Hill, New York.
Note added in proof." Unknown 2 has been identified by GC-MS as cubebol [thanks to Dr R. P. Adams for a sample of ayou oil; ref. P. Weyerstahl et aL (1989), Flavour and Fragrance J. 4, 93. Unknown 3 is spathulenol [Adams, R. P. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press, San Diego].
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0305-1978/91$3.00+ 0,00 © 1991 PergamonPressplc.
Variability of Foliage Terpenes of Fitzroya cupressoides LAURENCE G. COOL, ARIEL B. POWER and EUGENE ZAVARIN University of California, Forest Products Laboratory, Richmond, CA 94804, U.S.A.
Key Word Index--Fitzroya cupressoides; Cupressaceae; alerce; monoterpenes; sesquiterpenes; foliage; chemotype; chemotaxonomy. Abstract--Foliage of 57 Fitzroya cupressoides trees from six locations in Chile was analysed for monoterpenes and sesquiterpenes. A new monoterpene, ipsdienyl acetate (2-methyl-4-acetoxy-6-methylene-octa2,7-diene), was found. No geographic variability in monoterpene composition was evident. Three distinct sesquiterpene chemotypes were found in the areas sampled. The basic chemotype produced sizeable amounts of germacrene D, bicyclogermacrene, caryophyllene and several sesquiterpene alcohols including trans-nerolidol. The second chemotype elaborated in addition ¢-Iongipinene and an unknown sesquiterpene alcohol, as well as some I~-Iongipinene, which has not been previously reported in higher plants. The third chemotype produced ~- and l]-acorenol and a number of cedrene-related hydrocarbons. Minor geographic variability in the sesquiterpene composition of the basic chemotype was indicated.
Introduction
Fitzroya cupressoides (Mol.) Johnst. (Span. "alerce', Cupressaceae), is endemic to the rain forests of southern Chile and adjacent Argentina. The genus is monotypic and there are only two other native Cupressaceae in South America: Pilgerodendron uviferum (D. Don) Florin and Austrocedrus chilensis (D. Don) Pic.-Ser. et Bizz. Both species are also found in Chile and their ranges partially overlap that of F.cupressoides. The taxonomic relationship between these conifers is controversial. The distribution of alerce is now confined to the Chilean Coastal range and Chilo6 island between 39°50'S and 42°30'S and the Andean range between 40°00'S and 43°30'S; the formerly extensive stands in the more accessible central lowlands have been almost completely logged. Because of its heavy exploitation, alerce is now in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [1] and on the U.S. Department of the Interior list of threatened species (U.S. Endangered Species Act of 1973). Despite its economic importance, threatened status, and sheer magnificence (reminiscent of California's giant redwoods), F. cupressoides has received relatively little scientific study, with only a few references to chemistry. Erdtman and Tsuno analysed the heartwood extractives of a wood sample of unknown provenance [2]; several other groups have analysed the foliage biflavones of planted specimens (references in [3]). To date, no studies of the morphological or chemical variability of F. cupressoides have been reported, nor has the foliage essential oil apparently been analysed. The present study was undertaken to investigate the variability of its foliage terpene composition. Materials and Methods Foliage from a total of 57 trees was collected from six locations in the range of Fitzroya cupressoides and stored in aluminium foil and plastic bags while being transported to this laboratory. Each tree sampled was at least 100 m from the next. Samples for GC analysis were prepared by removing woody twigs and actively growing tips from the green branchlets. Approximately 2 g of foliage was chopped with a razor blade, covered with n-pentane in a small stoppered flask, and held for several days at --10°C. The clear light green solution was drawn off and concentrated under nitrogen flow at room temperature to about 0.1 ml.
(Received31 January 1991) 421
422
L.G. COOL ETAL.
GC analysis Glass SCOT columns: (a) 54 m by 0.5 mm i.d. OV-17 and (b) 43 m by 0.5 mm i.d. Carbowax 20M. Carrier gas: He, 25 cm sec 1. Temperature (both columns): 70°C isothermal for 5 min, 6°C rain -~ program to 220°C. Split injection (20:1 ), injector at 200°C. Detector: FID, 250°C. Some samples were re-analysed on WCOT columns: (a) 30 m by 0.25 mm i.d. SE-54 and (b) 30 m by 0.25 mm i.d. Carbowax 20M. Carrier: He, 30 cm sec -1. Temperature (both columns): 50°C isothermal for 3 min, 6°C min 1 program to 250°C. Split injection (70:1), injector at 180°C. Detector: FID, 250°C. Kovats' retention indices (KI) given below were calculated with reference to n-alkane peaks from isothermal DB-5 and Carbowax 20M (C20M) chromatograms at 165°C unless otherwise indicated. GC-MS analysis WCOT column, 30 m by 0.2 mm i.d. DB-5. Carrier: He, 30 cm sec 1. Temperature: 60°C isothermal for 3 min, 6°C min 1 to 250°C. Split injection (100:1), injector at 280°C. MS detection: El at 70 eV. Ipsdienylacetate. GC retention indices (135°): KI = 1243 (DB-5), 1594 (C20M). GC-MS m/z (rel. int,): 134(18), 127(5), 119(31), 93(9), 92(12), 91(30), 86(5), 85(100), 79(21), 78(5), 77(13), 67(10), 65(9), 55(9), 53(13), 51 (7), 45(6), 43(78), 41 (37), no parent ion. Bicyclogermacrene. Since bicyclogermacrene had originally been reported as a major component in Citrus junos peel oil [4] a sample of the fruit peel of this species was extracted and analysed by GC-MS. Both the retention time and mass spectrum of the F. cupressoides unknown matched those of a major component of C junos, thus substantiating the identification. Bicycloelemene. A minor sesquiterpene component noted on GC-MS analysis was tentatively identified as bicycloelemene. This peak was only observed when the injector temperature was high (280°C versus 200°C used in routine GC analysis) and was assumed to be an artifact produced by Cope rearrangement of bicyclogermacrene at the high injector temperature used in GC-MS [4]. This phenomenon was recently noted elsewhere [5]; the reported mass spectrum and retention times for bicycloelemene agree with those found by us. ~-Longipinene. GC retention indices (135°): KI= 1418 (DB-5), 1599 (C20M). GC-MS m/z (rel. int.): [M] * = 204(27), 189(27), 161(57), 147(26), 134(23), 133(55), 121(32), 120(37), 119(45), 109(34), 107(52), 106(24), 105(66), 95(25), 93(75), 92(42), 91 (92), 82(18), 81 (35), 80(21 ), 79(66), 77(54), 69(34), 67(43), 65(23), 57(21 ), 55(55), 53(31 ), 43(22), 41 (100). Thujopsene, ~J-cedrene, cuparene. An extract of Juniperus virginiana wood [6] was analysed by GC-MS to further substantiate identifications of these components, whose identities had been deduced from published retention and mass spectral data [7-9]. In all cases the data for major components of the reference species matched those for the three unknowns of Type 2. F. cupressoides. Germacrene A, GC-MS analysis revealed the presence of a minor component whose distinctive mass spectrum was very similar to that of ~-elemene. The proposed identity of this unknown is based on this similarity and its late retention time typical of the germacrenes. UnknownO-1, sesquiterpene hydrocarbon. This compound was not well separated from ~,-muurolene on GC or GC-MS analysis. o¢-, ~J-Acorenol. GC retention indices: KI = 1657, 1660 (DB-5), 2135, 2161 (C20M). GC-MS m/z (rel. int.): [M] + = 222(2), 204(25), 161 (23), 121 (44), 119(100), 107(24), 105(36), 93(51 ), 91 (31), 81 (28), 79(37), 77(23), 69(20), 67(24), 59(74), 55(28), 43(58), 41 (48). Mass spectra of both isomers were identical. 4cL-Hydroxygermacra-1 (10), 5-diene. GC retention indices: KI = 1603 (DB-5), 2056 (C20M). GC-MS m/z (rel. int.): [M] += 222(1), 207(9), 204(8), 161(19), 123(17), 121(12), 119(12), 109(12), 105(19), 95(15), 93(16), 91(14), 81 (100), 80(17), 79(19), 71 (15), 69(15), 67(19), 55(21 ), 53(12), 43(77), 41 (43). Unknown 0-2, sesquiterpene alcohol. GC retention indices: KI = 1545 (DB-5), 1953 (C20M). GC-MS m/z (rel. int.): [M] + =222(3), 208(11), 207(66), 204(28), 162(13), 161(68), 137(12), 135(12), 133(13), 123(24), 121(25), 119(24), 109(18), 107(22), 105(44), 95(23), 93(37), 91(38), 81(31), 79(34), 77(25), 71(11), 69(17), 67(20), 55(32), 53(15), 43(100), 41 (60). Unknown 0-3, oxygenated sesquiterpene, GC retention indices: KI = 1607 (DB-5), 2122 (C20M). GC-MS m/z (rel. int.): [ M ] * = 220(3), 205(31), 202(8), 187(14), 177(8), 162(16), 159(22), 149(9), 147(16), 146(12), 145(11), 133(12), 131 (14), 121 (13), 119(32), 117(12), 107(22), 105(30), 93(30), 91 (43), 81 (26), 79(32), 77(24), 71 (14), 69(24), 67(24), 65(12), 55(24), 53(19), 43(100), 41 (61). Unknown O-4a, b, sesquiterpene alcohols. GC retention indices: KI = 1618 (DB5); 2071, 2074 (C20M). Peaks unresolved in GC-MS. Unknown 0-5, sesqu/terpene alcohol. GC retention indices: KI = 1702 (DB-5), 2237 (C20M). GC-MS m/z (rel. int.): [M] + = 222(3), 204(10), 137(65), 121(17), 119(51), 110(10), 109(26), 107(11), 105(11), 95(25), 93(30), 91(15), 84(54), 83(27), 82(16), 81 (15), 79(15), 77(15), 69(47), 67(25), 56(16), 55(51 ), 53(16), 43(25), 41 (100). Unknown 1-1, sesquiterpene alcohol GC retention indices: KI = 1653 (DB-5), 2169 (C20M). GC-MS m/z (rel. int.): 204(55), 179(100), 161 (95), 121 (26), 119(74), 110(24), 109(35), 107(23), 105(62), 91 (45), 93(43), 95(43), 81 (47), 79(37), 77(29), 69(38), 67(28), 55(50), 43(82), 41 (86), no parent ion. Unknown 1-2, sesquiterpene alcohol GC retention indices: KI = 1673 (DB-5), 2110 (C20M). GC-MS m/z (rel. int.): [M] ~ = 222(15), 204(30), 121(36), 119(100), 111 (20), 110(36), 109(40), 107(26), 105(41), 95(41), 93(46), 91 (41), 82(35), 81 (25), 79(28), 69(39), 67(31), 56(25), 55(67), 53(24), 43(52), 41 (98). Unknown 2-1, sesquiterpene hydrocarbon. GC retention index: K1=1480 (DB-5). GC-MS m/z (rel. int.):
TERPENES OF FITZROYACUPRESSOIDES
423
[M] + = 204(20), 162(16), 161(100), 159(2), 147(7), 133(13), 119(29), 117(12), 115(8), 105(44), 93(18), 91(46), 81(22), 79(20), 77(17), 67(12), 65(11), 55(16), 43(12), 41 (36).
Results and Discussion This study was based on the analysis of 57 trees from six populations in Chile: three from the coastal range, two from the Andes, and one from the central lowland area. The locations of the populations sampled are shown in Table 1 and Fig. 1. The monoterpene composition of each population is presented in Table 2. The monoterpenes consisted of high levels of 0{-pinene (>82% in all trees), with myrcene, limonene and I~-pinene accounting for most of the balance. Oxygenated monoterpenes were minor constituents (each < 1%). In addition to the ubiquitous terpinyl and bornyl acetates, verbenone was detected in very small amounts in all GC-MS analyses but was not quantified, due to poor GC separation from larger components. Ipsdienyl acetate (2-methyl-4-acetoxy-6-methylene-octa-2,7-diene, 1, Fig. 2), which has not been previously reported, was identified by GC-MS comparison with an authentic sample prepared by acetylation of commercial ipsdienol. The monoterpene composition of F. cupressoides was uniform from population to population: discriminant function analysis on the individual tree monoterpene data grouped by population gave no significant separation among populations. The sesquiterpene composition proved to be more interesting. First, the sesquiterpenes were relatively abundant, their total amounting to an average of 87% of the monoterpenes. Of this total, about one-third was composed of sesquiterpene alcohols. The diversity of sesquiterpenes was also great, with over 40 compounds detected. Finally, every population sampled had some trees which were qualitatively different from the others; three distinct chemotypes were identified. The compositions of these chemotypes are summarized in Table 3. Also included is the analysis of a single tree (NAR4) which contained the sesquiterpenes of all chemotypes. The basic chemotype, denoted Type 0, contained high levels of germacrene D, with substantial amounts of bicylcogermacrene, caryophyllene, ~-copaene, 0c-muurolene, 0~-humulene, ~)-cadinene, trans-nerolidol, and several other sesquiterpene alcohols. Several Type 0 sesquiterpenes had frequency distributions which deviated significantly from normal (Shapiro-Wilk test, P<0.01): caryophyllene, ~.-humulene, 0cmuurolene, unknown 0-3 and unknown 0-5 (Fig. 3). The caryophyllene distribution suggests monogenic control of the level of this terpene, as has been found in Pinus pinaster [10]. One of the major sesquiterpene alcohols was identified as 40~-hydroxygermacral(10),5-diene (2, Fig. 2). The concentration of this alcohol correlated positively with germacrene D and bicyclogermacrene (R = 0.59 and 0.65 respectively, significant at P<0.0001, for all trees). It had a mass spectrum which agreed with that for the enantiomer of this compound (Dr William Fenical, personal communication) and with published data (only eight peaks reported, [11]). The Kovats index also matched that calculated from the chromatogram in [11]. TABLE 1. DESCRIPTION OF POPULATIONS SAMPLED
Population
Location
Latitude
Longitude
Elevation (m)
Number of trees
1 2 3 4 5 6
La Uni6n Pargua Chilo~ Island Puerto Montt Lake Chaiquenes Rio Negro
40°08'S 41°45'S 42"38'S 41°20'S 41°30'S 41°55'S
73°20'W 73°40'W 74°05'W 72°53'W 72°35'W 72°25'W
800 25 400 105 800 630
10 10 10 10 8 9
424
L. G. COOL E T A L , 74°
40 °
73 °
72 °
+
+
40 °
,,
01
CHILE
41 °
41 °
tt
,
•
6
+ ~
42 °
'
") ISLAND
42 o
G U L F OF I
,
0
20
40
60
I
I
I
I
km
i I r
2
74 °
73 °
72 °
FIG. 1. LOCATION OF POPULATIONS SAMPLED.
In all trees there was a group of minor constituents derived from a J]-acorenyl precursor: 13-funebrene (3, Fig 2), J]-acorenol (7, Fig. 4) and J]-acoradiene (8, Fig. 4). j]-Funebrene, by which we mean the 1,7-diepimer (or its enantiomer, the 2,5-diepimer) of iS-cedrene, was identified from literature retention time and mass spectral data [12, 13]. In further support of this identification is the co-occurrence of J]-acorenol and j]-acoradiene: these are likely congeners of the funebrenes [14, 15]. Surprisingly, even though I]-acorenol was present in much larger amounts in Type 2 trees (see below), I]-funebrene and 13-acoradiene levels showed no increase. It seems reasonable to assume that different enzymes are responsible for the biosynthesis of the lS-acorenyl sesquiterpenes in Type 0 and Type 2 trees. The second chemotype (Type 1), in addition to the Type 0 compounds, elaborated substantial amounts of 0¢-Iongipinene and an unknown sesquiterpene alcohol, as well as lesser amounts of several other Iongipinene-related hydrocarbons. On the basis of retention time (J] isomers generally elute somewhat later than their c(counterparts) and the excellent correlation with 0¢-Iongipinene (Table 4), it was deduced that one of the Type 1 hydrocarbons was 6-1ongipinene (4, Fig. 2), which although hitherto unreported in vascular plants has been found to occur naturally in the liverwort Scapania undulata as the (-) enantiomer [16]. To verify this identification, an authentic sample of Iongipinanol, which readily dehydrates on GC analysis to c~- and J]-Iongipinene [16], was analysed by GC and GC-MS. The first hydrocarbon to elute was 0¢-Iongipinene;
0.3 89.3 0.3 1.3 0.5 4.2 3.0 0,1 0,2 0.3 0.3 0,2
0.04 1.91 0.05 0.55 0.08 1.41 0.82 0.12 0.07 0.14 0.26 0.06
S.D, 0,3 89,7 0.3 1.0 0.6 3,8 2.6 0,1 0.2 0,3 0.5 0.2 tr.
2 Mean 0.02 2.65 0.02 0.16 0.04 1,93 0.87 0.09 0,06 0.11 0.29 0.12
S.D. 0,3 88.3 0,2 0.8 0.4 5,7 2,7 0,1 0,1 0.1 0,3 0.2 tr,
3 Mean 0.10 2.95 0,09 0.43 0.16 3.0t 0,91 0.09 0,17 0,08 0.26 0.18
S.D.
Population
0.3 88.2 0,3 1.2 0.5 5.0 2.7 0.1 0.2 0.2 0.2 0.2
4 Mean 0.05 1,81 0.04 0.51 0,08 1.61 1.18 0,17 0.08 0,11 0,19 0,09
S.D. 0.3 91.1 0.3 0.9 0.5 3,7 2.3 0,1 0,2 0.2 0,2 0.1 tr.
5 Mean 0.05 2.03 0.01 0.24 0.05 1.41 0.52 0.07 0.07 0.04 0.22 0.08
S.D.
0.03 89.8 0.3 1.0 0.5 4.5 2.5 0.1 0.2 0.2 0,2 0.2
6 Mean
0.05 2.19 0.03 0.31 0.04 1.33 0.88 0.12 0.10 0.06 0.21 0.15
S.D.
B B B B B B B B B A B B A
Identityt
*As per cent of total monoterpenes, tldentification codes: A = GC-MS comparison with authentic substance, B = GC-MS comparison with component of thoroughly characterized plant extract run under identicaJ conditions, tr. = trace amounts,
Tricyclene ~-Pinene Camphene ~-Pinene Sabinene Myrcene Limonene ~-Phellandrene Terpinolene Ipsdienyl acetate =-Terpinyl acetate Bornyl acetate Verbenone
Compound
1 Mean
TABLE 2. FOLIAGE MONOTERPENE COMPOSITION* OF flTZROYA CUPRESSOIDESPOPULATIONS
z
426
L.G. COOL ETAL.
Ac0 ]
2
H 3
4
FIG. 2. TERPENE STRUCTURES FROM FITZROYA CUPRESSOIDES.
the retention times and mass spectrum of the second hydrocarbon (I]-Iongipinene) were identical to those of the £ cupressoides component. Since the higher plants are reported to invariably produce Iongifolene-relatives antipodal to those found in the liverworts, it is expected that both the 0{- and j]-Iongipinene found here are the (+) enantiomers. As can be seen in Table 4, 0c-ylangene also appears among the Type 1 sesquiterpenes and correlated positively with 0~-Iongipinene. This biosynthetic relationship, which has not been noted before, will be explored in more detail in a subsequent publication. The third chemotype, Type 2, was distinguished by the appearance of 0{- and ~Bcedrene and several related hydrocarbons, and by large amounts of two isomeric sesquiterpene alcohols. These were identified as 0~- and I]-acorenol (5, 7 in Fig. 4) by GC-MS comparison with an extract of Juniperus rigida wood, from which the acorenols were first isolated [17, 18]. Analysis of the J. rigida extract also provided substantiating evidence for the identities of 0c- and ~B-acoradiene (6, 8 in Fig. 4), both of which had also been originally isolated from this source. In the case of the component identified as ~'-acoradiene (9, Fig. 4), also known in the (--) form as 0~-alaskene* [19], the very distinctive mass spectrum (base m/z = 121, major m/z = 136) pointed either to this compound or to 6-acoradiene (10, Fig. 4). The retention indices in [20] enabled us to identify our unknown as the former isomer. As can be seen in Table 3, the three chemotypes of F. cupressoides are quite well defined: when the distinguishing sesquiterpenes for Type 1 or Type 2 appear at all, they appear in substantial amounts. Also, the percentages of the various Type 1 and Type 2 sesquiterpenes are positively correlated inter se (Tables 4 and 5). These facts are strongly suggestive of single-gene control of the biosynthesis of the Iongipinaneand acorane-related compounds in F. cupressoides, as has been demonstrated in the *A note on the nomenclature of the acorane hydrocarbons: in 1970 the structures of the alaskenes [19J and the acoradienes [17, 181 were both reported, c(-Alaskene and "F-acoradiene (9, Fig. 4) proved to be synonymous, while ~B-alaskene was the enantiomer of 5-acoradiene (10, Fig. 4). In order to reduce the number of trivial names and to avoid confusion, we would suggest that all of these compounds be given the more general name acoradienes. Thus c(- and ~-alaskene would be replaced by (--) "~-acoradiene and (--)6-acoradiene, respectively.
427
TERPENES OF FITZROYACUPRESSOIDES TABLE 3. FOLIAGE SESQUITERPENE COMPOSITION* OF FITZROYACUPRESSOIDESCHEMOTYPES Type 0
Type 1
Compound
(40 trees) Mean Range
(9 trees) Mean
Range
(7 trees) Mean
Range
Tree NAR4
c¢-Cubebene
0.1
0.0-0.4
0.1
0.1-0.2
0.1
0.0-0.2
0.1
B
~-Copaene
2.0
0.3-4.3
1.7
0.8-2.4
1.9
1.3-2.9
1.0
A
~]-Cubebene
0.3
0.0-0.7
0.3
0.2-0.4
0.3
0.2-0.4
0.2
C
J]-Elemene
0.3
0.0-0.5
0.3
0.1-0.5
0.2
0.2-0.5
0.1
A
~-Funebrene
0.1
0.0-0.4
0.1
0.0-0.3
0.2
0.2-0.4
0.0
C
Caryophyllene
9.8
1.7-27.0
7.3
1.6--23.8
1.8-21.0
5.7
A
~-Farnesene
1.0
0.0-2.3
0.8
0.2-1.3
0.7
0.4-1,3
1.0
B
~-Humulene
2.3
1.0-6.4
1.8
0.9-3.4
2.2
1.2-5.2
1.1
B
~-Acoradiene
0.5
0.0-1.3
0.5
0,2-0.6
0.3
0.0-1.0
0.7
B
0,9 35.8
0.1-2.0 18.0-55.3
0.7 28.8
0.5-1.0 21.7-40.0
0.3 22.1
0.0-0.7 10.8-37.6
0.9 35.3
3"-Muurolene+unknown 0-1 Germacrene D
Type 2
12.7
Identityt
B, E B
~-Selinene
tr.
--
tr.
--
tr.
--
--
A
~.-Muurolene
2.3
0.1-6.3
2.2
1.4-3.0
2.3
1.3-4.2
1.2
A
Bicyclogermacrene
9.9
3.6-18.2
7.7
3.9-12.0
6.2
3.6-12.5
9.5
B
(Germacrene A)
tr.
--
tr.
--
tr.
--
--
E
~'-Cadinene
0.9
0.0-2.1
0.6
0.2-1.0
0.7
0.4-1.1
0.9
B
5-Cadinene
2.9
1.3-5.2
2.4
1.6-3.5
2.5
2.0-3.3
1.9
B
trans-Nerolidol
4.8
0.1-11.3
3.3
0.8-7.2
2.6
1.2-5.2
1.1
A
4~,-Hydroxygermacra-
5.8
3.2-11.0
5.3
2.7-7.2
3.4
2.1-4.7
5.2
C
Unknown 0 - 2
12.0
0.5-26.8
9.3
5.0-15.2
10.7
7.9-13.8
6.4
E
Unknown 0-3
2.5
0.6-6.4
1.9
0.8-3.0
1.4
0.2-2.2
1.3
E
Unknown 0-4a,b
2.5
0.1-5.6
2.0
1.0-3.2
1.8
0.2-3.0
1.2
E
Unknown 0 - 5
2.5
0.0-9.8
1.7
0.1-3.7
0.8
0.1-2.0
3.0
E
c¢-Ylangene
0.0
--
0.1
0.0-0.2
0.0
--
0.1
B
0~-Longipinene
0.0
--
8.9
3.4-13,6
0.0
--
5.8
A
~-Longipinene
0.0
--
0.6
0.2-0.9
0.0
--
0.4
A
Longifolene
0.0
--
0.6
0.0-1.3
0.0
--
0,5
A
~-Himachalene
0.0
--
2.8
0.2-4.8
0.0
--
1.4
C
Unknown 1-1
0.0
--
0.6
0.1-1.5
0.0
--
0.3
E
Unknown 1-2
0.0
--
7.8
2.7-13,3
0.0
--
5.8
E
(Bergamotene isomer)
0.0
--
0.0
--
0.2
0.1-0.2
0.1
D
0~-Cedrene
0.0
--
0.0
--
0.6
0.2-1.1
0.1
A
1 (10),5-diene
~-Cedrene
0.0
--
0.0
--
1.5
0.6-3.1
0.3
B
Thujopsene
0.0
--
0.0
--
0.1
0.0-0.2
0.0
B
c¢-Acoradiene
0.0
--
0.0
--
1,8
1,1-3,0
0.3
B
3'-Acoradiene
0.0
--
0.0
--
0.2
0.4-1.2
0.4
C
U n k n o w n 2-1
0.0
--
0.0
--
tr.
--
tr.
E
~-Bisabolene
tr.
--
tr.
--
0.4
0.2-0.7
0.1
A
Cuparene
0.0
--
0.0
--
tr.
--
tr.
8
Cedrol
tr.
--
tr.
--
0.3
0.1-0.5
0.1
A
12.9
9.2-17.1
4.1
B
8.5
5.6-11.2
2.8
B
¢-Acorenol
0.0
--
0.0
--
~-Acorenol
0.8
0.0-1,9
0.7
0.4-1.4
* A s per cent of total sesquiterpenes. tldentification codes: A = GC-MS comparison with authentic substance run under identical conditions, B = GC-MS comparison with c o m p o n e n t of thoroughly characterized plant extract run under identical conditions, C = GC-MS comparison with published retention time and M S data, D = (tentative identification) M S comparison with published data, E = see note in Materials and Methods.
case of the Iongifolene/0c-longipinene in Pinus pinaster [10]. Though the data are few, the frequency distributions of the Type 1 and Type 2 compounds (see Fig. 5 for 0¢-Iongipinene and 0¢-acorenol) are not inconsistent with the trimodal distribution that would be expected from the single-gene model. The biosynthetic implications of this model--and the identities of unknown compounds--will be explored in a subsequent publication.
L. G. COOL ETAL.
428 2o
2o
o~1o
~z0
0
o
2 4 a-HUNULENE (%)
10 20 CARYOPHYLLENE (%)
6
2o
15,
E-,
~o
I 2 a-MUUROLENE
8
4
2 4 UNKNOWN o - s (~.)
(Z)
0 0
6
5 UNKNOWN 0 - 5 (Z)
io
FIG. 3. FREQUENCY DISTRIBUTIONS OF SESQUITERPENE LEVELS IN FITZROYA CUPRESSOIDES.
5
R = OH,
6
R = CH 2
CH~
7
R = OH.
8
R = CH 2
g
CH 3
Io
FIG. 4. ACORANE SESQUITERPENES.
The sesquiterpene composition of Type 0 trees (Table 6) showed only modest geographic variability among populations, with a significant (P<0.03 level, MannWhitney test, 21 coastal vs. 11. Andean trees) trend being a west to east decrease of caryophyllene. Although principal component analysis of the individual-tree sesquiterpene data revealed no obvious clustering of populations, discriminant function analysis did show some separation of populations 4 and 6 from each other and from the rest (Fig. 6).
429
TERPENES OF FITZROYACUPRESSOIDES TABLE 4. CORRELATION BETWEEN PERCENTAGES OF LONGIPINANE-RELATED SESQUITERPENES
Kendall Rank Correlations, 10 trees (R/signif.) 0~-ylangene ~-Iongipinene Iongifolene c~-Longipinene
0.689 0.006
0¢-Ylangene
0.911 0.000 0.778 0.002
0.778 0.002 0.733 0.003 0.778 0.002
~-Longipinene Longifolene
~-himachalene
Unknown 1-1
Unknown 1-2
0.867 0.001 0.644 0.010 0.867 0.001 0.822 0.001
0.600 0.016 0.733 0.003 0.600 0.016 0.467 0.060 0.467 0.060
0,956 0,000 0,733 0.003 0,867 0.001 0,822 0,001 0,822 0,001 0,556 0,025
I]-Himachalene Unknown 1-1
TABLE 5. CORRELATION BETWEEN PERCENTAGES OF ACORANE-RELATED SESQUITERPENES
Kendall Rank Correlations, 8 trees (R/signif.) ~-cedrene ~.-acoradiene "(-acoradiene c~-Cedrene
~-Cedrene 0~-Acoradiene "~-Acoradiene ~-Bisabolene 0~-Acorenol
~-Acorenol
0.909 0.002
0.982 0.001 0.857 0.003
0.982 0.001 0.929 0.001 0.929 0.001
I~-bisabolene o¢-acorenol
I~-acorenol
cedrol
0.618 0.030 0.571 0.048 0.571 0.048 0.643 0.026
0.982 0.001 0.857 0.003 1.000 0,001 0.929 0.001 0.571 0.048 1.000 0.001
0.963 0.001 0.837 0.003 0.982 0.001 0.909 0.002 0.618 0.028 0.982 0.001 0.982 0.001
0.982 0.001 0.857 0.003 1.000 0.001 0.929 0.001 0.571 0.048
There were too few data for each of the other two chemotypes (nine trees of Type 1, seven of Type 2, and the tree representing both) for a similar statistical analysis of their geographic variability. However, by assuming the single-gene model for each chemotype, allele frequencies (Lp--, Lp+ for low and high Iongipinanes; Ac--, Ac+ for low and high acoranes) could be calculated for each population and some tentative conclusions made about the geographic distribution of these alleles. To calculate allele frequencies, the following assumptions were made: codominance of the Lp--/Lp+ and Ac--/Ac+ alleles at their respective loci, independence of the two loci, validity of the Hardy-Weinberg law within each population, and identical phenotypic ranges in all populations for each genotype. The phenotypic ranges were chosen to optimize overall agreement with the Hardy-Weinberg law and are indicated in the histograms for 0¢-Iongipinene (Lp--Lp-- 0-1%, Lp--Lp+ 1-12%, Lp+Lp+ above 12%) and 0¢-acorenol (Ac--Ac- 0-1%, Ac--Ac-t- 1-16.5%, Ac+Ac+ above 16.5%) (Fig. 5). Finally, the presumed frequencies for the Lp and Ac alleles for each population were calculated, as shown in Tables 7 and 8.
430
L. G. COOL ETAL.
sol , Lp-Lpo
Lp-Lp+
Lp+Lp+
,10.~
0
5
10
~-LONGIPINENE ( ~ )
Ac-Ac+
5
Ac+Ac+
10
~-ACORENOL
15
(%)
FIG. 5. FREQUENCYDISTRIBUTIONSOF a-LONGIPINENEAND c(-ACORENOL LEVELSiN FITZROYACUPRESSOIDES,SHOWING PHENOTYPIC RANGES CORRESPONDINGTO EACH GENOTYPE.
In the case of the Lp--Lp+ and Lp-t-Lp+ genotypes, i.e. the nine Type 1 trees and the double heterozygote NAR4, the Lp+ allele frequency of population 2 seemed unusually high (25%) compared to the other populations (average 7o4%). However, this difference was not significant at the P<0.05 level (modified Chi-square procedure of Ref. [21]). The Ac--Ac-I- and A c + A c + trees (seven Type 2 and NAR4), like Type 1, were scattered throughout the range of the six populations sampled. Although the low frequency for the Ac-I- allele made meaningful statistical comparisons impossible, the data do not suggest any geographic trends in the distribution of this gene. In summary, the monoterpene data indicate that in the range sampled F. cupressoides is quite uniform, while the sesquiterpene data show some geographic variability. It will be possible to better assess this variability when further sampling (more populations and more trees per population) over the entire range of the species is undertaken.
431
TERPENES OF FITZROYA CUPRESSQIDES TABLE 6. GEOGRAPHICVARIATIONOF TYPE 0 SESQUITERPENECOMPOSITION*
Compound 0~-Cubebene ~.-Copaene ~-Cubebene ~-Elemene [3-Funebrene Caryophyllene ~-Farnesene c(-Humulene ~-Acoradiene T-Muurolene+unknown 0-1 Germacrene D 0¢-Muurolene Bicylcogermacrene T-Cadinene 5-Cadinene trans-Nerolidol ~-Acorenol 4¢-Hydroxygermacra1(10), 5-diene Unknown 0-2 Unknown 0-3 Unknown 0-4a,b Unknown 0-5
1 (7 trees) Mean S . D .
2 (6 trees) Mean S . D .
Population 3 4 (8 trees) (8 trees) Mean S . D . Mean S . D .
5 (5 trees) Mean S . D .
6 (6 trees) Mean S.D.
0.1 1.9 0.4 0.3 0.2 10.5 0.9 2.8 0.6 0.9 35.1 2.0 9.7 1.0 2.6 3.7 0.9 6.3
0.04 0.90 0.15 0.08 0.09 7.61 0.37 1.41 0.28 0.23 5.31 0.91 1.70 0.36 0.75 2.37 0.23 1.45
0.2 2.6 0.4 0.3 0.t 16.4 0.8 2.2 0.6 1.0 28.6 2.7 6.7 0.9 3.1 5.9 0.9 4.9
0.10 0.83 0.17 0.06 0.05 9.26 0.38 0.31 0.23 0.39 4.02 1.70 2.34 0.49 0.99 2.52 0.35 1.17
0.1 2.1 0.3 0.2 0.1 13.7 0.5 2.2 0.4 0.7 35.9 2.1 9.7 0.6 2.8 5.9 0.6 5.2
0.07 0.98 0.11 0.16 0.13 9.56 0.27 0.84 0.19 0.27 10.30 1.02 3.75 0.29 0.63 2.34 0.30 1.51
0.2 2.2 0.4 0.3 0.0 4.3 0.2 2.7 0.2 0.7 41.6 2.6 10.8 0.4 3.3 2.6 0.7 6.2
0.07 1.14 0.16 0.09 0.08 1.83 0.31 1.83 0.18 0.38 9.24 1.69 4.31 0.45 1.04 1.92 0.28 1.50
0.1 1.6 0.2 0.2 0.1 6.6 0.7 1.6 0.5 1.0 40.8 1.6 13.2 0.8 2.7 4.1 0.8 7.3
0.03 0.32 0.04 0.16 0.13 4.06 0.30 2.00 0.24 0.31 2.33 0.36 3.08 0.29 0.53 3.14 0.31 2.31
0.2 2.0 0.3 0.2 0.1 7.0 1.4 2.0 0.8 1.1 31.7 3.0 10.0 1.2 3.0 6.8 1.3 5.4
0.10 1.16 0.18 0.11 0.15 8.91 0.51 0.58 0.40 0.38 8.21 1.80 4.37 0.49 1.24 1.68 0.43 1.30
11.3 2.6 2.5 3.2
5.30 0.93 1.08 0.65
13.1 3.2 2.7 2.9
6.98 1.70 1.46 1.60
10.8 2.5 1.8 1.1
5.34 1.33 1.04 0.70
13.9 2.4 3.1 0.8
7.15 1.50 1.66 0.86
10.1 1.6 2.2 2.4
1.84 0.57 0.76 0.85
12.2 2.4 2.6 5.4
6.66 1.15 1.61 2.43
*As per cent of total sesquiterpenes.
5 ~ Z o
4
rj Z
2.
6
/
36
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6
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1 1
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3
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33
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DISCRIMINANT FUNCTION 1 FIG. 6. PLOT OF SESQUITERPENEDISCRIMINANT FUNCTIONS 1 AND 2 FOR FITZROYA CUPRESSOIDESTYPE O. TABLE 7. LONGIPINANEALLELE FREQUENCIES
Population
Assumed genotype frequency Lp-Lp-Lp-Lp+
1 2 3 4 5 6
0.90 0.60 1.00 0.90 0.75 0.78
0.10 0.30 0.00 0.00 0.25 0.22
Lp+Lp+
Assumed allele frequency LpLp+
Calculated Hardy-Weinberg 9enotype frequency Lp-LpLp--Lp+ Lp+Lp÷
0.00 0.10 0.00 0.10 0~00 0.00
0.95 0.75 1.00 0.90 0.87 0.89
0.90 0.56 1.00 0.81 0.76 0.79
0.05 0.25 0.00 0.10 0.13 0,11
0.10 0.38 0.00 0.18 0.22 0.20
0.00 0.06 0.00 0.01 0.02 0.01
432
L. G. COOL ETAL.
TABLE 8. ACORANE ALLELE FREQUENCIES
Population
Assumed genotype frequency Ac--Ac- Ac-Ac+
1 2 3 4 5 6
0.80 1.00 0.80 0.90 0.74 0.89
0.20 0.00 0.20 0.10 0.13 0.11
Ac+Ac+
Assumed allele frequency Ac-Ac+
Calculated Hardy-Weinberg genotype frequency Ac Ac- Ac-Ac+ Ac+Ac+
0.00 0.00 0.00 0.00 0.13 0.00
0.90 1.00 0.90 0.95 0.81 0.94
0.81 1.00 0.81 0.90 0.65 0.89
0.10 0.00 0.10 0.05 0.19 0.06
0.18 0.00 0.18 0.10 0.31 0.11
0.01 0.00 0.01 0.00 0.04 0.00
Acknowledgements--The authors are indebted to the Committee for the Defense of the Flora and Fauna of Chile--with special thanks to Sr Hern&n Verscheure--for their enthusiastic assistance with the collection of samples. We wish to thank Dr Rainer Scora for providing samples of Citrusjunos, Dr Niels H. Andersen for a sample of Iongipinanol, Mr Ken Hobson for a sample of ipsdienol, Dr William Fenical for spectral data of 4c(hydroxygermacra-l(10),5-diene, and Dr Robert Flath for listings of sesquiterpene retention times and mass spectral data. We are also grateful to Dr Constance Millar for reviewing the manuscript and providing valuable suggestions, and to Mr Charles Berolzheimer and the California Cedar Products Company for a grant supporting this work.
References 1. Convention on International Trade in Endangered Species of Wild Fauna and Flora. (1984) U.S. Department of the Interior, Fish and Wildlife Service, Publication 50 CFR 23.23. 2. Erdtman, H. and Tsuno, K. (1969) Acta Chem. Scand. 23, 2021. 3. Naqvi, S. W. I., Parveen, N., Parveen, M. and Khan, N. U. (1987) Curr. Sci. 56, 480. 4. Nishimura, K., Shinoda, N. and Hirose, Y. (1969) Tetrahedron Lett., 3097. 5. Le Quere, J.-L and Latrasse, A. (1990) J. Agric, Food Chem. 38, 3. 6. Wenninger, J. A., Yates, R. L. and Dolinsky, M. (1967) J.A.O.A.C. 50, 1304. 7. Heller, S. R. and Milne, G. W. A. (1978) EPA/NIHMass Spectra/Database, National Standard Data Reference Series #63, vol. 2. U.S. Department of Commerce, National Bureau of Standards. Note: the spectrum for l]-cedrene is incorrect, as are those for c(-cedrene and cedrol (correct spectra of ~-cedrene and cedrol in [8], ~-cedrene in [9]). 8. Stenhagen, E., Abrahamsson, S. and McLafferty, F. W. (1974) Registry of Mass Spectral Data, vol. 2. John Wiley, New York. Note: the spectrum for IB-cedrene is incorrect; correct spectrum in [9]. 9. Ramaswami, S. K., Briscese, P., Gargiullo, R. J. and yon Geldern, 1". (1986) in Flavors and Fragrances." A World Perspective (Lawrence, B. M., Mookherjee, B. D. and Willis, B. J., eds), p. 951. Proceedings of the 10th International Congress of Essential Oils, Fragrances and Flavors, Washington DC, U.S.A. 10. Marpeau, A., Baradat, Ph. and Bernard-Dagan, C. (1975) Ann. Scl~ Forest. 32, 185. 11. Schmaus, G., Schultze, W. and Kubeczka, K.-H. (1989) Planta Med. 55, 482. 12. Norin, T., Sundin, S., Karlsson, B., Kierkegaard, P., Pilotti, A.-M. and Wiehager, A.-C. (1973) Tetrahedron Lett., 17. 13. Piovetti, L. and Dia ra, A. (1977) Phytochemistry 16, 103. 14. Kirtany, J. K. and Paknikar, S. K. (1973) Indian J. Chem. 11, 508. 15. Piovetti, L. and Diara, A. (1980) Tetrahedron Lett., 1453. 16. Andersen, N. H., Bissonette, P., Liu, C.-B., Shunk, B., Ohta, Y., Tseng, C.-L. W., Moore, A. and Huneck, S. (1977) Phytochemistry 16, 1731. 17. Tomita, B. and Hirose, Y. (1970) Tetrahedron Lett., 143. 18. Tomita, B., Isono, T. and Hirose, Y. (1970) Tetrahedron Lett., 1371. 19. Andersen, N. H. and Syrdal, D. D. (1970) Tetrahedron Lett., 2277. 20. Andersen, N. H., Syrdal, D. D., Lawrence, B. M., Terhune, S. J. and Hogg, J. W. (1973) Phytochemistry 12, 827. 21. Dixon, W. J. and Massey, F. J. Jr (1957) Introduction to StatisticalAnalysis, p. 232. McGraw-Hill, New York.
Note added in proof." Unknown 2 has been identified by GC-MS as cubebol [thanks to Dr R. P. Adams for a sample of ayou oil; ref. P. Weyerstahl et aL (1989), Flavour and Fragrance J. 4, 93. Unknown 3 is spathulenol [Adams, R. P. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press, San Diego].