P1: GDX
Journal of Chemical Ecology [joec]
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
C 2003)
Journal of Chemical Ecology, Vol. 29, No. 4, April 2003 (
QUALITATIVE AND QUANTITATIVE VARIATION IN
MONOTERPENE CO-OCCURRENCE AND COMPOSITION IN
THE ESSENTIAL OIL OF Thymus vulgaris CHEMOTYPES
JOHN D. THOMPSON,1,∗ JEAN-CLAUDE CHALCHAT,2 ANDRÉ MICHET,2
YAN B. LINHART,3 and BODIL EHLERS1
1 Centre d’Ecologie Fonctionnelle et Evolutive, CNRS
1919 Route de Mende, 34293 Montpellier Cedex 5, France
2 Laboratoire de Chimie des Huiles Essentielles
Université Blaise Pascal de Clermont
Campus des Cézeaux, 24 Avenue des Landais
63177 Aubière cedex, France
3 Department
of Environmental Population and Organismic Biology
N122 Ramaley, Campus Box 33
Boulder, Colorado 80309-0334, USA
(Received March 01, 2002; accepted December 5, 2002)
Abstract—Thymus vulgaris has a chemical polymorphism with six different
chemotypes that show marked spatial segregation in nature. Although some
populations have a single chemotype in majority, many have two or three chemotypes. In this study we analyze the quantitative variation among T. vulgaris populations in the percentage of oil composed of the dominant monoterpene(s) for
each chemotype. In general, phenolic chemotypes (thymol and carvacrol), which
occur at the end of the biosynthetic chain, have a significantly lower proportion of
their oil composed of their dominant monoterpene than nonphenolic chemotypes
(geraniol, α-terpineol, and linalool). This is due to the presence of high amounts
of precursors (γ -terpinene and paracymene) in the oil of phenolic chemotypes.
The essential oil of the nonphenolic thuyanol chemotype has four characteristic
monoterpenes that together make up a lower proportion of the oil than the single
dominant monoterpene of the other nonphenolic chemotypes. For all chemotypes, the percentage composition of the dominant monoterpene decreased significantly at sites where the chemotype is not the majority type. This decrease is
correlated with a significant increase in either the proportion of the two precursors for the thymol chemotype or the monoterpenes characteristic of the other
chemotypes at the site. The latter result suggests that a plant with dominant genes
∗
To whom correspondence should be addressed. E-mail: [email protected]
859
C 2003 Plenum Publishing Corporation
0098-0331/03/0400-0859/0 P1: GDX
Journal of Chemical Ecology [joec]
pp799-joec-461506
March 17, 2003
860
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
is responsible for the production of different monoterpenes can produce several
molecules.
Key Words—Lamiaceae, Mediterranean, adaptation, essential oil composition,
monoterpene production, polymorphism.
INTRODUCTION
The Mediterranean flora is well known for its abundance in aromatic plants, with
an estimated 49% of genera containing aromatic species in this climate zone (Ross
and Sombrero, 1991). Members of the Lamiaceae from this region have attracted
a great deal of attention due to the diversity of monoterpenes produced by different species (reviews in Richardson, 1992; Stahl-Biskup, 2002; Thompson, 2002).
This diversity occurs at three levels: among genera, among species within genera,
and as polymorphic variation within species. Indeed, different genetically based
types, or chemotypes, have been reported in Mentha spicata (Kokkini and Vokou,
1989), Origanum vulgare (Vokou et al., 1993), Rosmarinus officinalis (Granger
et al., 1973), and Thymus vulgaris (Granger and Passet, 1973). The presence of
intraspecific chemotype variation is, in fact, probably quite common in the genus
Thymus (Stahl-Biskup, 2002).
Secondary compounds, including terpenes have multiple ecological functions. The most notable of these lie in the defense they provide against herbivores
and parasites (Simeon de Bouchberg et al., 1976; Bryant et al., 1991; Shonle and
Bergelson, 2000). A number of other functions also may exist, such as the mediation of plant competition via allelopathic effects on other plant species (e.g.,
McPherson and Muller, 1969), pollinator attraction (Bergstrom, 1978; Beker et al.,
1989), leaf decomposition (Grime et al., 1996), soil microbe activity (Vokou and
Margaris, 1984), and climatic adaptation (Seufert et al., 1995). However, studies of
patterns of variation in secondary compound production in relation to ecological
variation remain rare.
Many species in the genus Thymus show evidence of polymorphic variation
in monoterpene production (Stahl-Biskup, 2002). A species in which chemotype
variation is particularly striking is the Mediterranean wild thyme, T. vulgaris. In
southern France, this species has six genetically distinct chemotypes that can be
distinguished on the basis of the dominant monoterpene produced in glandular
trichomes on the surface of the leaves (Passet, 1971; Vernet et al., 1986). Each
of the six chemotypes, geraniol (G), α-terpineol (A), thuyanol-4 (U), linalool (L),
carvacrol (C), and thymol (T), is named after its dominant monoterpene. The six
monoterpenes are all produced from geranyl pyrophosphate via a series of changes
in configuration and hydroxylation and have fairly similar molecular structures
(Figure 1). A major distinction is the phenolic nature of carvacrol and thymol, and
the nonphenolic nature of the four other monoterpenes. Presence of the dominant
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
Thymus
March 17, 2003
20:32
Style file version June 28th, 2002
861
FIG. 1. The predicted biosynthetic chain of monoterpene production in Thymus vulgaris
(modified from Passet, 1971).
monoterpene is controlled by an epistatic series of five biosynthetic loci (Vernet
et al., 1986).
Granger and Passet (1971) and Passet (1971) reported that the thuyanol
chemotype, unlike the five other chemotypes that have a single predominant
monoterpene in their essential oil, is characterized by the occurrence of four
monoterpenes (thuyanol-4, terpinen-4-ol, myrcenol-8, and linalool). They also
suggested that in the oil of the thymol and carvacrol chemotypes, the dominant
monoterpene co-occurs with relatively high levels (up to 30% of the oil) of two
precursors—γ -terpinene and para-cymene. Granger and Passet (1971) provided
an estimate of the percentage of the oil of each chemotype that is composed of
the dominant monoterpene. For geraniol, linalool, and α-terpineol chemotypes,
the dominant monoterpene occurs at levels up to 90–95% of the oil. The thuyanol
P1: GDX
Journal of Chemical Ecology [joec]
pp799-joec-461506
March 17, 2003
20:32
862
Style file version June 28th, 2002
THOMPSON ET AL.
chemotype contains up to 60–65% of a combination of thuyanol-4 and terpinen-4ol (with some linalool and myrcenol-8). The carvacrol chemotype contains up to
85% carvacrol, whereas the thymol chemotype contains no more than 65% thymol.
There has been no statistical comparison of monoterpene variation among chemotypes or sites for each chemotype, despite the fact that the different chemotypes
show marked spatial segregation in relation to a complex climatic–edaphic gradient. The two phenolic chemotypes are confined to low-elevation areas close to the
Mediterranean sea (i.e., relatively mild winters and shallower, stonier soils), and
the four nonphenolics are more abundant in areas with colder winters and deeper
soils (Granger and Passet, 1973; Vernet et al., 1977a,b; Gouyon et al., 1986).
This paper has three objectives. First, we compare the production of the dominant monoterpene among chemotypes of T. vulgaris. Second, for each chemotype,
we contrast the proportion of the dominant monoterpene in two different types of
sites—those where it is the most frequent chemotype and those where it is either
part of a mixed-chemotype population or where it is rare. Third, we quantify correlated variation among monoterpenes in plants of each chemotype. The results are
discussed in relation to the potential effects of ecological and genetic backgrounds
on monoterpene production.
METHODS AND MATERIALS
Plant Material. In May 1997 and 1998, leaf material was sampled from
plants growing in natural populations in two distinct areas in order to cover the
range of environmental variation observed in sites where Thymus vulgaris occurs
in southern France (see Table 1 for sampling details). The first area (sites 1–13)
is the St. Martin-de-Londres valley in the Hérault department of the Languedoc
region east of the Rhône valley (Figure 2). All six chemotypes have been reported
from this area, and previous work has demonstrated the occurrence of localized
spatial variation in their relative abundance (see review by Thompson et al., 1998).
Second, we sampled plants from lowland and upland sites (14 and 15) in the
Drôme department in the Rhône-Alpes region where previous sampling (Granger
and Passet, 1973; J. Lamy, personal communication) suggested the absence of
phenolic chemotypes. All plants were sampled at the end of the flowering period
to minimize phenological differences among samples.
Chemical Analyses. Analytical gas chromatography was carried out on a Delsi
121C apparatus fitted with a flame ionization detector and a CP Wax 51 fused silica
capillary column (25 × 0.3 mm; 0.15-µm film thickness). The temperature was
programmed from 50◦ C at 5 min, rising 3◦ C/min to 220◦ C. N2 was used as a carrier
gas. For GC-MS analysis, a CP Wax 51 fused silica WCOT capillary column (60 ×
0.3 mm; 0.25-µm film thickness) and 52 CB stationary phase was employed. The
initial temperature was programmed at 50◦ C for 5 min, rising 3◦ C/min to 240◦ C,
with He as a carrier gas, and the column coupled to an HP mass spectrometer.
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
Thymus
863
TABLE 1. NUMBER OF Thymus vulgaris PLANTS SAMPLED AND ANALYZED (IN
PARENTHESES) AT 15 SITES
Majority chemotypes
Population
Sample size
1. Cazevieille
2. Tourriére
3. Pic St Loup - Nord
4. Hubac-haut
5. Colline geraniol
6. Aeroport
7. Fraicinede
30 (28)
30 (28)
25 (25)
20 (20)
20 (17)
25 (23)
35 (31)
Carvacrol
Carvacrol
Carvacrol
Carvacrol + Linalool
Geraniol
Linalool/U
Linalool/Thuyanol-4/
α-terpineol
8. La Borie
9. Cabane de la Plaine
6 (6)
30 (28)
α-terpineol
Linalool
10. Gardiol
11. Hortus-januq
15 (15)
15 (15)
Thymol/Linalool
Thymol
12. Ferriéres les Verreries
13. Pompignan-haut
14. Lowland Drôme
35 (33)
20 (16)
20 (15)
Thymol
Thymol
Linalool
15. Upland Drôme
35 (32)
Linalool
a
Previous
worka
This study
Carvacrol (27), thymol (1)
Carvacrol (25), thymol (3)
Carvacrol ( 23), thymol (2)
Carvacrol (18), thymol (2)
Geraniol (16), linalool (1)
Thuyanol-4 (13), linalool (10)
Thuyanol-4 (16), linalool (8),
(α-terpineol (5), carvacrol (1),
thymol (1)
α-Terpineol (6)
Linalool (22), thymol (4),
geraniol (2)
Thymol (15)
Thymol (13), α-terpineol (2)
carvacrol (1)
Thymol (31), Carvacrol (2)
Thymol ( 16)
Linalool (7), geraniol (5),
carvacrol (3)
Linalool (27), geraniol (3),
α-terpineol (2)
From raw data used by Gouyon et al. (1986) (populations 1–13) and J. Lamy (personal communication)
(populations 14 and 15).
Samples were injected at 240◦ C and identified according to their retention time
indices and mass spectra in comparison with those of known compounds or with
published spectra.
Data Presentation and Analysis. The essential oil of T. vulgaris can contain
more than 30 different monoterpenes. In this study, plants with more than 10% of
their oil containing unidentified compounds were not included in comparisons of
monoterpene production (hence the difference between number of sampled plants
and number for which extraction was successful in Table 1). The goal here is not to
show all the diversity in monoterpene presence, since many compounds only occur as trace elements. In fact, 11 monoterpenes (geraniol, α-terpineol, thuyanol-4,
terpinen-4-ol, linalool, myrcenol-8, 1,8 cineole, γ -terpinene, paracymene, carvacrol, and thymol) regularly occur as more than 10% of the oil and form the basis
of the variation in composition among sites. β-Caryophylene is frequently present
as 5–10% of the oil and is included in our presentation of variation among and
within chemotypes.
P1: GDX
Journal of Chemical Ecology [joec]
864
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
FIG. 2. Location of the study sites. (a) Closed circles represent phenolic populations and
open circles represent nonphenolic populations. (b) Phenolic chemotypes are rare or absent
in natural populations.
To analyze the composition of the major monoterpenes in the essential oil
of Thymus vulgaris, we made a number of simplifications. First, the percentage
composition of all the major nonphenolic monoterpenes is reported as the sum of
each monoterpene and its acetate. Second, myrcenol-8 is reported as the sum of four
different forms (ac-Z -2, ac-E-2, Z -2, and E-2) of methyl-2 methylene-6 octadiene2,7-ol (see Granger et al., 1972). Third, the composition of the thuyanol-4 molecule
is the sum of the cis and trans forms. Fourth, plants that had equal proportions of
carvacrol and thymol were classed as carvacrol since they must have the dominant
gene coding for this monoterpene.
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
March 17, 2003
Thymus
20:32
Style file version June 28th, 2002
865
The number of plants of each chemotype sampled at different sites varied
due to the differences in their spatial distribution in southern France. For all six
chemotypes, we regrouped plants into two classes (home and away) that reflect
their abundance at a given site: “home” included all plants of a given chemotype
in sites where it represented the majority type, and “away” included all plants of
a given chemotype in sites where it was a minority type. These two classes were
established based on previous surveys and data in the present survey.
From the populations we sampled, we obtained a total of 26 geraniol plants (16
at home sites and 10 at away sites), 15 α-terpineol plants (6 and 9), 75 linalool plants
(49 and 26), 100 carvacrol plants (93 and 7), 88 thymol plants (75 and 13), and
29 thuyanol plants in two sites where no one chemotype predominated. Statistical
analysis of variation between home and away sites for the thuyanol chemotype was
not possible. In order to include the thuyanol chemotype in a statistical comparison
of the percentage of the oil composed of the dominant monoterpene, we excluded
plants sampled in away sites for the five other chemotypes and performed one-way
ANOVA of variation in the dominant monoterpene across chemotypes.
All statistical analyses were carried out by using SAS version 8 (SAS, 1999).
Analysis of variance (ANOVA) of mean values across chemotypes and sites were
performed with PROC GLM on arcsine square-root transformed data. Where significant differences were observed among chemotypes, mean values were compared with the Scheffé pairwise comparisons test. Correlation analyses for particular compounds within chemotypes were carried out using PROC CORR in
SAS (1999). In all tests, significance levels were adjusted to account for multiple
comparisons following the method proposed by Rice (1989).
RESULTS
Variation among Chemotypes. When the thuyanol chemotype was considered
to be a mix of terpinen-4-ol, thuyanol-4, myrcenol-8, and linalool, we found significant (F5,262 = 45.9, P < 0.001) variation among chemotypes. The geraniol,
α-terpineol, and linalool chemotypes had higher (Scheffé means test at P < 0.05)
mean values of their dominant monoterpene than did the thuyanol, carvacrol,
thymol chemotypes (Figure 3a). Within each of these two groups, there were
no significant differences in mean values. If the precursors for the two phenolic
monoterpenes are included as part of the dominant monotepene, i.e., the mean
value for the carvacrol (or thymol) chemotype is the sum of carvacrol (or thymol), paracymene, and γ -terpinene, there is significant variation among chemotypes (F5,262 = 24.3; P < 0.001) with the same two statistically different (Scheffé
means test at P < 0.05) groups of chemotypes (Figure 3b).
Variation within Chemotypes. Two-way ANOVA was carried out on the proportion of the oil made up by the dominant monoterpene in each of the different
P1: GDX
Journal of Chemical Ecology [joec]
866
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
FIG. 3. Mean percentage (±SE) of the dominant monoterpene in the essential oil of the different Thymus vulgaris chemotypes in southern France. The thuyanol chemotype is considered to have four characteristic monoterpenes (thuyanol-4, terpinen-4-ol, myrcenol-8, and
linalool), whose proportions are summed to give single value per plant. Phenolic chemotypes
are considered as either (a) the single monoterpene or (b) the monoterpene plus precursors
(λ-terpinene and paracymene). Chemotypes are: G, geraniol; A, α-terpineol; U, thuyanol;
L, linalool, C, carvacrol; and T, thymol.
chemotypes at home sites, where the chemotype was the dominant form, and away
sites, where the chemotype was either rare or in mixed-chemotype populations. We
found variation between sites (F1,294 = 87.21; P < 0.001) and differences among
chemotypes (F4,294 = 46.29; P < 0.001), but no interaction between chemotype
and site (F4,294 = 1.61; P = 0.17). For all five chemotypes, the dominant monoterpene occupied a higher fraction of the oil at home sites, where that chemotype was
the most abundant form, compared to away sites, where the chemotype was rare
or in mixed populations (Figure 4).
The decrease in the dominant monoterpene at the away sites is accompanied
by increases in one or more of the other main monoterpenes (Table 2) and by an
increase in the proportion of the range of minor monoterpenes not analyzed by
statistical comparisons. As a consequence, the 11 predominant monoterpenes tend
to occupy a smaller fraction of the total oil for most plants sampled in away sites. As
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
Thymus
March 17, 2003
20:32
Style file version June 28th, 2002
867
FIG. 4. Mean percentage (±SE) of the dominant monoterpene in the essential oil of five
Thymus vulgaris chemotypes in southern France at home sites where that chemotype is
the majority type (closed bars) and away sites where that chemotype is rare or in a mixedchemotype population (open bars). Chemotypes are: G, geraniol; A, α-terpineol; L, linalool,
C, carvacrol; and T, thymol.
we now show, this decrease is correlated with an increase in the monoterpene(s)
present in co-occurring chemotype(s). To illustrate this pattern we present the
results on a chemotype-by-chemotype basis.
Geraniol. In plants of the geraniol chemotype at away sites, there is less
geraniol in their oil and more linalool (Table 2). For the geraniol chemotype, we
obtained 10 plants from sites where this chemotype was uncommon or rare. Six
plants, sampled in sites 9 and 15 (Table 1), which are dominated by linalool plants,
had a relatively high proportion (>20%) of linalool in their oil. The four other
plants (population 14) co-occur with both the linalool and carvacrol chemotypes
(Table 1), and all four plants contained relatively high amounts (5–10%) of phenolic
compounds.
α-Terpineol. For the α-terpineol chemotype, we obtained nine plants from
away sites to compare with the six plants in the home population (Table 2). Five
plants had relatively high amounts of a combination of thuyanol-4, terpinen-4-ol,
cineole, and linalool and came from site 7 where the thuyanol chemotype occurs with linalool. Two plants had relatively high amounts of linalool (with no
thuyanol-4 or terpinenol-4-ol) and came from site 15, where linalool is the dominant chemotype. Finally, one plant had a relatively high composition of thymol
and came from site 11 where the thymol chemotype is the majority chemotype.
Thuyanol. The thuyanol chemotype occurred in two populations (populations 6 and 7) in the St. Martin-de-Londres area that are close together (roughly
2 km apart) and do not have a single majority chemotype (Tables 1 and 3). If
this chemotype is defined as having two dominant monoterpenes, thuyanol-4 and
terpinen-4-ol, there was a difference in the percentage of the essential oil made up
of these two dominant compounds between sites 6 and 7 (F1,27 = 5.04, P = 0.04).
Individually, these two compounds showed no significant variation between the
Geraniol
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -Terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
β-Caryophylene
α-Terpineol
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -Terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
Chemotype and monoterpene
73.1
0.1b
0
0
4.2
0
0.1
0.3
1.0
0
0
3.9
0
74.2
0.3
0.3
1.6
0.1
0.1
0.8
10.4
0
0.22
6
Mean
16
N
0
47.1
0
0
0.5
0
0
0
0
0
0
58.2
0
0
0
0.4
0
0
0
0
0
0
0.8
Min.
0
90.4
0.9
0.6
3.2
0.25
0.5
3.0
27.4
0
0.6
84.1
0.7
0
0
15.9
0
0.5
1.5
7.9
0
0
9.6
Max.
9
10
N
0.1
58.1
2.9
1.2
6.2
0.7
0.2
0.3
2.7
0.3
2.8
49.9
0.4
0
0
18.7
0.7
0.3
1.2
0.9
0
0
2.8
Mean
0.1
4.2
1.5
0.7
2.1
0.4
0.1
0.1
1.5
0.3
1.9
5.1
0.3
0
0
3.9
0.5
0.2
0.7
0.3
0
0
0.35
SE
0
40.9
0
0
0.9
0
0
0
0.1
0
0
23.5
0
0
0
3.8
0
0
0
0
0
0
0.3
Min.
0.9
76.7
13.9
6.2
19.2
3.3
0.8
0.9
14.0
2.4
13.2
72.7
2.5
0
0
40.8
5.3
1.5
6.9
2.6
0
0
4.2
Max.
Composition at away sites (%)
0.65
4.74(.049)
2.73
0.30
3.26
2.34
1.40
0.66
3.29
2.4
—
26.85∗
0.88 ns
—
—
21.64∗
3.44 ns
1.21 ns
0.94 ns
—
—
—
—
Fa
pp799-joec-461506
0
7.1
0.1
0.1
0.4
0.1
0.1
0.5
4.4
0
0.11
1.7
0
0
0
1.2
0
0.1
0.1
0.5
0
0
0.6
SE
Composition at home sites (%)
TABLE 2. MONOTERPENE COMPOSITION IN Thymus vulgaris CHEMOTYPES IN HOME AND AWAY SITES
P1: GDX
Journal of Chemical Ecology [joec]
March 17, 2003
20:32
868
Style file version June 28th, 2002
THOMPSON ET AL.
β-Caryophylene
Linalool
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
β-Caryophylene
Carvacrol
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -Terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
β-Caryophylene
1.6
0.5
0.1
0
81.8
0.1
0.2
0.8
1.9
0
0
3.4
0.1
1.0
0.8
0.7
3.7
8.7
5.4
57.2
3.2
0
1.0
2.8
49
93
1.48
0
0
0
0
1.1
0
0
28.9
0
0
0
0
0
0
0
0
45.4
0
0
0
0
0
0
0.9
1.02
0.9
3.9
9.4
7.1
27.1
26.8
15.4
84.1
18.2
0
3.1
6.4
30.0
5.0
1.6
0
93.8
1.0
1.9
6.8
12.3
0
0
9.5
2.0
7
26
2.9
2.6
2.1
0.4
8.0
5.0
11.7
30.9
14.1
0
0.4
3.8
1.3
2.5
4.2
2.6
63.4
0.7
0.6
1.0
2.4
1.8
1.7
3.4
2.3
1.8
1.0
1.7
0.4
3.7
1.5
4.2
2.4
4.2
0
0.4
1.2
0.8
0.6
0.3
0.5
3.1
0.2
0.2
0.3
0.9
0.4
0.6
0.6
0.5
0
0.7
0
0
3.0
0.8
0.9
21.5
3.3
0
0
0
0
0
0
0
32.2
0
0
0
0
0
0
0.5
1.3
10.3
8.2
12.2
3.0
29.9
13.0
34.6
41.1
27.2
0
3.1
10.3
16.0
14.2
20.6
8.5
83.0
2.9
3.2
6.0
23.6
7.1
10.7
12.3
6.5
38.52∗
7.76∗
0.58
7.2 (.009)
28.20∗
1.57
6.93(.01)
35.35∗
27.08∗
—
—
—
2.44
21.04∗
35.88∗
87.97∗
42.60∗
26.60∗
10.72∗
0.49
0.17
—
—
—
—
MONOTERPENES IN
pp799-joec-461506
0
01
0.1
0.1
0.3
0.7
0.4
1.3
0.4
0
0.1
0.2
0.6
0.2
0.1
0
1.3
0.1
0.1
0.2
0.4
0
0
0.3
0.14
P1: GDX
Journal of Chemical Ecology [joec]
March 17, 2003
20:32
Style file version June 28th, 2002
Thymus
869
75
N
0.3
1.5
1.1
0.7
3.9
7.2
9.9
4.1
52.5
0.1
0.8
2.7
Mean
0.1
0.2
0.1
0.1
0.1
0.6
0.8
0.5
1.4
0.1
0.1
0.2
SE
0
0
0
0.
1.1
0
0
0.8
22.4
0
0
0
Min.
8.9
6.1
3.3
5.0
8.0
21
29
26.8
72.9
1.7
3.8
6.8
Max.
13
N
0
0.7
0.6
0.7
5.1
14.1
9.7
5.0
40.1
0
0.6
4.2
Mean
0
0.2
0.1
0.4
1.1
1.3
1.4
1.0
2.8
0
0.3
0.7
SE
0
0
0
0
1.9
8.4
2.0
1.6
21.4
0
0
0
Min.
0
1.9
1.6
5.0
13.4
23.5
17.6
13.5
57.4
0
3.1
7.8
Max.
Composition at away sites (%)
3.07
2.54
8.12 (.006)
0.13
2.64
15.36∗
0.13
1.17
12.10∗
—
—
—
Fa
March 17, 2003
a
pp799-joec-461506
F values test differences between these sites. ∗ Significant after correction of the P < 0.05 threshold following the Bonferonni sequential
method (Rice, 1989). Values in parentheses are probability values before correction by the above method—Low proportions of the molecule
prevented statistical comparisons.
b 0.1 represents all trace values less than or equal to 0.1
Thymol
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
β-caryophylene
Chemotype and monoterpene
Composition at home sites (%)
TABLE 2. CONTINUED
P1: GDX
Journal of Chemical Ecology [joec]
20:32
Style file version June 28th, 2002
870
THOMPSON ET AL.
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
Thymus
871
TABLE 3. MONOTERPENE COMPOSITION IN THUYANOL-4 CHEMOTYPE OF Thymus
vulgaris IN TWO POPULATIONS
Composition (%)
at site 6 (N = 13)
Composition (%)
at site 7 (N = 16)
F1,27 a
Monoterpene
Mean
SE
Min.
Max.
Mean
SE
Min.
Max.
Geraniol
α-Terpineol
Thuyanol-4
Terpinen-4-ol
Linalool
γ -Terpinene
Paracymene
Carvacrol
Thymol
Myrcenol-8
Cineole-8
β-Caryophylene
0
3.8
28.1
15.1
14.0
1.9
0.8
2.8
1.3
13.1
0.4
3.0
0
0.4
3.5
2.7
2.4
0.5
0.3
1.6
0.7
1.2
0.1
0.6
0
2.0
12.8
2.2
3.0
0
0
0
0
5.7
0.1
0.8
0
6.5
52.2
29.1
32.5
5.0
5.0
21.4
9.3
18.7
1.0
7.5
0
7.4
19.0
14.7
8.8
1.5
0.3
0.5
0.5
3.7
3.4
7.9
0
0.8
2.7
2.3
1.4
0.3
0.1
0.3
0.2
1.3
1.4
1.5
0
3.2
1.6
3.1
2.4
0.1b
0
0
0
0
0
2.4
0
—
13.9
18.04∗
39.3
4.64 ns
29.6 < 0.01 ns
20.5
3.68 ns
4.2
0.16 ns
0.8
0.12 ns
4.4
3.04 ns
3.0
1.33 ns
12.9
26.40∗
21.2
4.66 ns
20.3
9.88∗
a ∗ Significant
b
after correction of the P < 0.05 threshold following the Bonferonni sequential method
(Rice, 1989)—Low proportions of the molecule in the essential oil prevented statistical comparisons.
0.1 represents all trace values less than or equal to 0.1.
two sites (Table 3). If the thuyanol chemotype is considered to have four main
monoterpenes (thuyanol-4, terpinen-4-ol, myrcen-8-ol, and linalool), we found a
significant (F1,27 = 30.3, P < 0.001) difference in percentage composition due
to 10% less thuyanol-4, 10% less myrcen-8-ol, and 5% less linalool at site 7,
where a greater proportion of the oil contained α-terpineol, myrcenol-8, and βcaryophylene (Table 3).
For the 54 plants of all chemotypes present at sites 6 and 7, we analyzed
correlated variation among the four different monoterpenes that characterize the
thuyanol-4 chemotype (i.e., thuyanol-4, terpinen-4-ol, myrcenol-8, and linalool).
For the six potential correlations, we found negative correlations between linalool
and both thuyanol-4 (r = −0.49, P = 0.002) and terpinen-4-ol (r = −0.45, P =
0.007) and a positive correlation between thuyanol-4 and myrcenol-8 (r = 0.62,
P < 0.001).
Linalool. When plants of the linalool chemotype occur at away sites (N = 26)
they produce less linalool and more α-terpineol, thuyanol-4, terpinen-4-ol, and
one or both of the two phenolic precursors (paracymene and γ -terpinene), but not
the dominant phenolic monoterpenes (Table 3). Sixteen plants at away sites had
thuyanol-4 and terpinen-4-ol as the most predominant secondary constituent of
their oil. These plants came from sites 6 and 7 where the thuyanol chemotype
is relatively abundant. One plant with high levels of α-terpineol and one plant
with thymol as an important secondary constituent of the oil came from site 7,
P1: GDX
Journal of Chemical Ecology [joec]
872
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
where the α-terpineol and thymol chemotypes occur. Three plants had geraniol as
a secondary constituent of the oil and occurred in sites 5 and 14, where the geraniol
chemotype is present. Finally, five plants had a mix of phenolic monoterpenes and
their precursors as secondary components of the oil and came from site 14 where
the carvacrol chemotype is present. We found no difference in the percentage of
the oil composed of the linalool monoterpene in comparisons of the two away sites
(6 and 7) or the two home sites (9 and 15). Thus, the only variation in percentage
composition of the oil of the linalool chemotype was between home and away sites.
Carvacrol. When carvacrol plants are found at sites where this chemotype is
rare, they produce less carvacrol and more of either geraniol, α-terpineol, linalool,
or thymol (Table 3). Seven carvacrol plants were obtained from sites where this
chemotype was rare. Although the two plants (2 and 3) with the highest amount
of thymol came from site 12, a site dominated by the thymol chemotype, the five
other plants showed no correlation between local chemotype and monoterpene
composition.
We found a significant (F3,89 = 17.49, P < 0.001) difference in the percentage of carvacrol for plants of this chemotype among the four sites (1–4) where this
chemotype occurred as the majority type. This difference was due to their being
two groups of populations (Figure 5a): populations 1 and 2 had higher proportions
of carvacrol than populations 3 and 4 (Scheffé means test at P < 0.05).
Thymol. Thirteen thymol plants were detected at “away” sites where they
showed a reduction in the percentage of the dominant monoterpene in their oil
(Table 3). However, they did not increase in one or more of the other dominant
monoterpenes and only the precursor, γ -terpinene, showed an increase in proportion at away sites that compensates for the decrease in thymol in the oil (Table 3).
Nevetheless, one plant at site 7, where thuyanol-4 is the most abundant chemotype, was the thymol plant with the highest concentration of thuyanol-4 in its oil.
Likewise, two plants from site 9, where linalool is the dominant chemotype, had
relatively high amounts of linalool.
We found a significant (F3,71 = 3.33, P < 0.05) difference in the percentage
of thymol in plants of the thymol chemotype among the four sites (10–13) where
this chemotype occurs as the majority type. This difference was due to a lower
proportion of thymol (and a higher proportion of precursors) at site 13 compared
to the three other sites (Schemske means test at P < 0.05) (Figure 5b).
Correlations among Phenolic Monoterpenes and Precursors. The production of the phenolic monoterpenes (thymol and carvacrol) and their precursors
(para-cymene and γ -terpinene) had a significant positive correlation between the
two precursors paracymene and γ -terpinene and a negative correlation between
the production of the monotepene characteristic of the chemotype and the two
precursors for both the thymol and carvacrol chemotypes (Table 4). The carvacrol
chemotype, but not the thymol chemotype, showed a negative correlation between
the production of thymol and carvacrol.
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
March 17, 2003
Thymus
20:32
Style file version June 28th, 2002
873
FIG. 5. Mean percentage (±SE) of the dominant monoterpene in the essential oil of the
two phenolic chemotypes carvacrol (a) and thymol (b) of Thymus vulgaris chemotypes in
four populations in southern France where the chemotype is the majority type.
DISCUSSION
Variation among Sites. The most striking result of this study of secondary
compound variation in Thymus vulgaris is the significant difference in the composition of the essential oil between sites where a chemotype is the most abundant chemotype (i.e. home sites) and those where it is rare or occurs as part of
mixed-chemotype populations (i.e., away sites). We observed this trend in five
chemotypes. Two potential causes can be invoked to explain this variation.
First, environmental differences among sites may cause differences in
monoterpene production (e.g., Lincoln and Lagenheim, 1976; Mihaliak et al.,
1989; Muzika, 1993). Hence, we cannot rule out environment as a potential cause
P1: GDX
Journal of Chemical Ecology [joec]
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
874
THOMPSON ET AL.
TABLE 4. PEARSON CORRELATION COEFFICIENTS FOR VARIATION IN MONOTERPENES
PRODUCED BY CARVACROL AND THYMOL CHEMOTYPES OF Thymus vulgaris
Monoterpenea
Chemotype
Monoterpene
Carvacrol (N = 100)
γ -terpinene
Paracymene
Carvacrol
Thymol
γ -terpinene
Paracymene
Carvacrol
Thymol
Thymol (N = 88)
λ-terpinene
Paracymene
Carvacrol
Thymol
0.360∗
−0.497∗
−0.472∗
−0.001 NS
0.053 NS
−0.449∗
0.530∗
−0.096 NS
−0.300 NS
−0.808∗
−0.585∗
−0.101 NS
a ∗P
< 0.0001, NS: not-significant at corrected threshold for multiple comparisons following Rice
(1989).
for the decrease in the production of the dominant monoterpene in T. vulgaris
chemotypes at sites where they are a minority. At such sites, the low abundance
of plants of the rare chemotype may be due to the local environment, which may
also cause plants of a given chemotype that can survive in such sites to produce
relatively low proportions of their dominant monoterpene.
An important point here is the significant variation in percentage of the dominant monoterpene among different home sites for the carvacrol and thymol chemotypes. This variation is correlated with marked ecological differences among sites.
For the four carvacrol populations, the percentage of carvacrol declines in populations 3 and 4, which are geographically the closest to the nonphenolic populations
in the St. Martin-de-Londres valley (Figure 2). These two populations occur on the
north-facing slope of the valley, whereas populations 1 and 2 occur on the southfacing slope outside the valley in a landscape where carvacrol is the dominant
chemotype across large expanses of the landscape (Vernet et al., 1977a,b; Gouyon
et al., 1986). The ridge that separates the two groups is known to separate different
ecological zones—inside the valley, where nonphenolic populations predominate
(see discussion by Gouyon et al., 1986; Thompson, 2002), winter minimum temperatures are much lower and soils are deeper and less stony than outside the valley.
The decline in the proportion of thymol at site 13 is also associated with ecological
variation, this site being drier, hotter, and on stonier soils than the three other sites
where thymol was found to be the majority chemotype.
The second potential cause of variation in dominant monoterpene composition is that genetically based differences among plants of a given chemotype
may occur among sites. Many species show genetically based differences in secondary metabolite formation (Hanover, 1966; Murray and Lincoln, 1970; Lincoln
and Lagenheim, 1976; Mihaliak et al., 1989; Mithen et al., 1995; Shonle and
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
Thymus
March 17, 2003
20:32
Style file version June 28th, 2002
875
Bergelson, 2000), including Thymus vulgaris (Vernet et al., 1986). However, in
T. vulgaris, such differences have been presented only in terms of the occurrence of
different chemotypes. No attention has been paid to the co-occurrence of monoterpenes within chemotypes (other than the thuyanol chemotype—see below), and
intermediate types have been dismissed as being rare and/or of little significance
(Granger and Passet, 1973). If the variation between home and away sites is due to
genetic differences, then such differences would provide evidence that genetically
based variation in monoterpene production may have a quantitative component
within each of the thyme chemotypes.
A result that supports the idea of genetically based variation in dominant
monoterpene composition among home and away sites in T. vulgaris concerns
the identity of the monoterpenes that occur as a high proportion of the oil at away
sites. In almost all cases, the decline in proportion of the characteristic dominant
monoterpene at away sites is correlated with a significant increase in a monoterpene
present in the co-occurring chemotypes. If a chemotype is absent from a population,
its monoterpene never occurs as a secondary constituent of the oil.
This result was particularly apparent in the four nonphenolic chemotypes
and in the carvacrol chemotype, and it shows a relationship with the epistatic
chain of monoterpene production. In T. vulgaris, the presence of the dominant
monoterpene is controlled by an epistatic series of five loci (Vernet et al., 1986).
A plant with a dominant G allele will have the geraniol chemotype, regardless
of whether it has dominant or recessive alleles at the other loci. A plant that
is homozygous recessive at the G locus (i.e., gg) with a dominant A allele will
have the α-terpineol chemotype. A plant that is homozygous recessive gg/aa with
a dominant U allele will have the thuyanol chemotype. The sequence continues in the order geraniol, α-terpineol, thuyanol, linalool, carvacrol. If all five
loci are homozygous recessive, thymol is the dominant monoterpene. Our data
illustrate that the potential relationship between monoterpene variation and the
chain of five loci may not be more complex than previously thought, since it is
clear that plants may produce significant proportions of more than one dominant
monoterpene.
Four points are important here: (1) When geraniol plants were sampled in
populations dominated by the linalool chemotype, the decrease in geraniol in their
oil was accompanied by an increase in linalool. (2) When α-terpineol plants were
sampled in a population dominated by the thuyanol and linalool chemotype, the
decrease in α-terpineol in their oil was accompanied by an increase in linalool
or thuyanol-4. (3) When linalool plants were sampled in a population with the
thuyanol chemotype, the decrease in linalool in their oil was accompanied by an
increase in thuyanol-4. (4) When carvacrol plants were sampled in a population
dominated by the thymol chemotype, the decrease in carvacrol in their oil was
accompanied by an increase in thymol, compared to an increase in linalool in
a population dominated by this chemotype. The thymol chemotype showed, in
P1: GDX
Journal of Chemical Ecology [joec]
876
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
almost all cases, a decline in thymol associated with an increase in the composition of its precursors, and not the presence of one of the other characteristic
monoterpenes. The latter result is not surprising since thymol is at the end of the
chain.
In most cases, a decline in the percentage composition of the dominant
monoterpene was associated with a significant increase in the presence of a
monoterpene produced by a dominant allele at a locus that would normally not
be expressed in the chemotype (e.g., an L allele in a geraniol plant or a U allele
in an α-terpineol plant). Three explanations can be provided to account for this
pattern. First, the presence of a “secondary” dominant gene may not be completely
switched off by the dominant gene of the chemotype prior in the chain, allowing
for limited production of a secondary monoterpene. Second, modifier genes at
loci other than that coding for the dominant allele of a particular chemotype may
allow for the production of secondary monoterpenes. Third, rather than being due
to epistasis, the genetic control of monoterpene production may be a consequence
of gene dosage effects along a chain of loci. For example, if the G locus produces
more geraniol than the A locus produces α-terpineol (and so on down the chain)
then a geraniol plant with a dominant A allele may produce small amounts of
α-terpineol.
Three lines of evidence support the dosage idea: (1) the biosynthesis of
linalool is more complex than that of geraniol due to transposition within the
molecule (Passet, 1971); (2) the gene chain is almost identical to the biosynthetic
chain, and phenolics have a significant proportion of their oil in the form of precursors, and (3) heterozygotes at the C locus are thought to produce intermediate
amounts of carvacrol. Under this hypothesis, one would predict an increase in the
production of linalool across a range of genotypes of the geraniol chemotype: from
GG/ll (no linalool), to GG/Ll, Gg/Ll, and Gg/LL. The amount of linalool will be
dependent on the genotype at the two loci and on the frequency of the dominant
alleles in the population. A similar line of reasoning can be proposed for the presence of secondary monoterpenes in the oil of the other chemotypes. This scenario
is further complicated by the fact that there may be two genes involved in the synthesis of geraniol (Vernet et al., 1986). The different loci may interact via dosage
effects that vary across the loci and allow for variable amounts of monoterpenes
in the oil.
Incomplete specificity of enzymes catalyzing different parts of the biosynthetic chain may also influence secondary monoterpene production in thyme.
Carvacrol and thymol chemotypes at away sites were occasionally observed to
produce small proportions (10–12% of the oil) of one or other of the four nonphenolic monoterpenes that are normally produced earlier in the chain. The explanation for this is that the enzyme catalyzing the formation of the precursor to
the two phenolic monoterpenes, γ -terpinene (Granger et al., 1965), is also known
to allow the production of such small proportions of the four monoterpenes that
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
Thymus
March 17, 2003
20:32
Style file version June 28th, 2002
877
characterize the four nonphenolic chemotypes of T. vulgaris (Alonso and Croteau,
1992).
The pattern of variation among home and away sites and the presence of
plants with a blend of monoterpenes rather than a single dominant monoterpene
is of particular interest in the context of the ecological functions of monoterpenes
in thyme and the causes of spatial variation in chemotype frequency. The chemotypes show marked habitat segregation (Gouyon et al., 1996; Thompson, 2002),
herbivore-specific variation in palatability (Linhart and Thompson, 1999), differences in parasite load (J. Amiot, unpublished data), and have different effects on
the germination of associated species (Tarayre et al., 1995). A particular chemotype in an away site may be less suited to the local environment, and the secondary
production of other monoterpenes could favor the persistence of plants at a given
site. In addition, at away sites, plants of each chemotype have a greater fraction of
their oil as a range of minor monoterpenes. The possibility that having more than
one monoterpene is advantageous in some situations is worth investigation.
Variation among Chemotypes. Our results provide a statistical confirmation of
the data presented by Granger and Passet (1971), who found that the mean values of
the proportion of dominant monoterpenes in the α-terpineol, geraniol, and linalool
chemotypes exceed those of the thuyanol, carvacrol, and thymol chemotypes. This
variation among chemotypes has two main components.
First, the thuyanol chemotype shows marked variation in the relative composition of its characteristic monoterpenes (thuyanol-4, terpinen-4-ol, myrenol-8,
and linalool) and has a lower proportion of its oil composed of these dominant
monoterpenes than the other nonphenolic chemotypes. This suggests that a single
enzyme is probably responsible for the production of the thuyanol-4 and terpinen4-ol monoterpenes (see also Passet, 1971). Since the proportion of linalool is negatively correlated with the production of the above two molecules, it is possible
that their production results from a different substrate and depends on a different
enzyme. The molecular structures of these two molecules and probable biosynthetic pathway leading to their formation (Passet, 1971) support this idea. There are
many known examples where a single enzyme catalyzes the secondary metabolism
of multiple products and of enzymes of secondary metabolism that can recognize
more than one substrate (see review by Pichersky and Gang, 2000). The thuyanol
chemotype of T. vulgaris would appear to be an example of this phenomenon.
Whatever the cause, the variability in the composition of the thuyanol chemotype
makes it a difficult chemotype to define (see also Passet, 1971; Granger and Passet,
1971). It is also interesting to note that as well as being the sole chemotype characterized by a mixture of different monoterpenes, the thuyanol chemotype is also the
only one that never seems to occur in monochemotype populations (Thompson,
2002).
Second, the two phenolic chemotypes also have lower proportions of their
oil composed of their dominant monoterpenes than the nonphenolic chemotypes
P1: GDX
Journal of Chemical Ecology [joec]
pp799-joec-461506
March 17, 2003
878
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
that have a single monoterpene as the characteristic component of their oil. This
is due to the fact that the precursors, paracymene and γ -terpinene, occur as components of the oil in the phenolic chemotypes, particularly in thymol plants. The
longer chain of reactions leading to the production of thymol and carvacrol would
seem to reduce the proportion of the oil composed of these monoterpenes. The
correlated variation among thymol, carvacrol, and the precursors para-cymene and
γ -terpinene in each of the carvacrol and thymol chemotypes is interesting in this
context (Table 4). First, in both chemotypes, there was a positive correlation between the production of the precursors γ -terpinene and paracymene, supporting
the claim that their production is closely linked (Granger et al., 1965; Passet, 1971)
and that both compounds are precursors to thymol and carvacrol. Second, there was
a negative correlation between the production of thymol (and carvacrol) and the
two precursors for plants of both chemotypes, suggesting incomplete transformation of the precursors. The consistency of the results suggests that both paracymene
and γ -terpinene are precursors and that paracymene is not an end product. Finally,
in the carvacrol chemotype, but not the thymol chemotype, there was a negative
correlation between the production of thymol and carvacrol. Since carvacrol plants
can be heterozygous (Cc), there can be much variation in the relative composition
of the two phenolic molecules in the carvacrol chemotype, with heterozygotes
producing less carvacrol than plants that are homozygous CC. The variation in
correlation structure among the two chemotypes is a logical result of the genetic
control of phenolic monoterpene production.
Acknowledgments—We thank Christophe Petit, Michèle Tarayre, Perrine Gauthier, Christian
Collin, and Marie Maistre for help in sampling plants and Thomas Lenormand for suggesting alternative
genetic explanations. The ”Programme Recherche Développement (Plantes Aromatiques, Médicinales
et à Parfum) – Chambre d’Agriculture de la Drôme” and the B.R.G. (contrat no. 42 dans l’appel national
à propositions 1999–2000) provided financial support.
REFERENCES
ALONSO, W. R. and CROTEAU, R. 1992. Comparison of two monoterpene cyclases isolated from higher
plants; γ -terpinene synthase from Thymus vulgaris and limonene synthase from Mentha x piperita,
pp. 239–251, in R. J. Petroski and S. P. McCormick (eds.). Secondary-Metabolite Biosynthesis
and Metabolism. Plenum Press, New York.
BEKER, R., DAFNI, A., EISIKOWITCH, D., and RAVID, U. 1989. Volatiles of two chemotypes of Majorana
syriaca L. (Labiatae) as olfactory cues for the honeybee. Oecologia 79:446–451.
BERGSTROM, G. 1978. Role of volatile chemicals in Ophrys–pollinator interactions, pp. 207–231, in
J. B. Harborne (ed.). Biochemical Aspects of Plant and Animal Coevolution. Academic Press,
London, United Kingdom.
BRYANT, J. P., PROVENZA, F. D., PASTOR, J., REICHARDT, P. B., CLAUSEN, T. P., and TOIT, J. T.
(du). 1991. Interactions between woody plants and browsing mammals mediated by secondary
metabolites. Annu. Rev. Ecol. Syst. 22:431–446.
GOUYON, P. H., VERNET, P., GUILLERM, J. L., and VALDEYRON, G.. 1986. Polymorphisms and environment: the adaptive value of the oil polymorphisms in Thymus vulgaris L. Heredity 57:59–66.
P1: GDX
Journal of Chemical Ecology [joec]
MONOTERPENES IN
pp799-joec-461506
Thymus
March 17, 2003
20:32
Style file version June 28th, 2002
879
GRANGER, R. and PASSET, J. 1971. Types chimiques de l’espèceThymus vulgaris L. C.R. Acad. Sci.
Paris 273:2350–2353.
GRANGER, R. and PASSET, J. 1973. Thymus vulgaris L. spontané de France: races chimiques et chemotaxonomie. Phytochemistry 12:1683–1691.
GRANGER, R., PASSET, J., and VERDIER, R. 1965. Présence et rôle du gamma-terpinène dans Thymus
vulgaris L. Bull. Trav. Soc. Pharm. Lyon 11:113–119.
GRANGER, R., PASSET, J., and GIRARD, J. P. 1972. Methyl-2 methylene-6 octadiene-2,7 ol isole de
Thymus vulgaris. Phytochemistry 11:2301–2305.
GRANGER, R., PASSET, J., and TEULADE-ARBOUSSET, G. 1973. Plantes medicinales à essence et chimiotaxonomie. Riv. Ital. 55:353–356.
GRIME, J. P., CORNELISSEN, J. H. H. C., THOMPSON, K., and HODGSON, J. G. 1996. Evidence of a
causal connection between anti-herbivore defence and the decomposition rate of leaves. Oikos
77:489–494.
HANOVER, J. W. 1966. Genetics of terpenes. I. Gene control of monoterpene levels in Pinus monticola
Dougl. Heredity 21:73–84.
KOKKINI, S. and VOKOU, D. 1989. Mentha spicata chemotypesgrowing wild in Greece. Econ. Bot.
43:192–202.
LINCOLN, D. E. and LANGENHEIM, J. H. 1976. Geographic patterns of monoterpenoid composition in
Satureja douglasii. Biochem. Syst. Ecol. 4:237–248.
LINHART, Y. B. and THOMPSON, J. D. 1999. Thyme is of the essence: biochemical variability and
multi-species deterence. Evol. Ecol. Res. 1:151–171.
MCPHERSON, J. K. and MULLER, C. H. 1969. Allelopathic effects of Adenostoma fasiculatum,
“chamise,” in the California chaparral. Ecol. Monogr. 39:177–198.
MIHALIAK, C. A., COUVET, D., and LINCOLN, D. E. 1989. Genetic and environmental contributions to
variation in leaf mono- and sesquiterpenes of Heterotheca subaxillaris. Biochem. Syst. and Eco.
17:529–533.
MITHEN, R., RAYBOULD, A. F., and GIAMOUSTARIS, A. 1995. Divergent selection for secondary matabolites between wild populations of Brassica oleracea and its implications for plant-herbivore interactions. Heredity 75:472–484.
MURRAY, M. J. and LINCOLN, D. E. 1970. The genetic basis of acyclic oil constituents in Mentha
citrata Ehrh. Genetics 65:457–471.
MUZIKA, R.-M. 1993. Terpenes and phenolics in response to nitrogen fertilization: a test of the carbon/nutrient hypothesis. Chemoecology 4:3–7.
PASSET, J. 1971. Thymus vulgaris L. : Chémotaxonomie et biogénèse monoterpénique. Thèse de
Doctorat. Faculté de Pharmacie, Montpellier, France.
PICHERSKY, E. and GANG, D. R. 2000. Genetics and biochemistry of secondary metabolites in plants:
an evolutionary perspective. Trends Plant Sci. 5:439–445.
RICE, W. R. 1989. Analysing tables of statistical tests. Evolution 43:223–225.
RICHARDSON, P. M. 1992. The chemistry of the Labiatae: an Introduction and overview, pp. 291–297
in R. M. Harley and T. Reynolds (eds.). Advances in Labiate Science. Royal Botanic Gardens,
Kew, United Kingdom.
ROSS, J. D. and SOMBRERO, C. 1991. Environmental control of essential oil production in Mediterranean
plants, pp. 83–94, in J. B. Harborne and F. A. Tomas-Barberan, (eds.). Ecological Chemistry and
Biochemistry of Plant Terpenoids. Clarendon Press, Oxford.
SAS. 1999. SAS/STAT Users guide. SAS, Cary, North Carolina.
SEUFERT, G., KOTZIAS, D., SPARTÀ, C., and VERSINO, B. 1995. Volatile organics in mediterranean
shrubs and their potential role in a changing environment, pp. 343–370 in J. M. Moreno and W. C.
Oechel, (eds.). Global Change and Mediterranean Type Ecosytems. Springer, New York.
SHONLE, I. and BERGELSON, J. 2000. Evolutionary ecology of the tropane alkaloids of Datura stramonium L. (Solanaceae). Evolution 54:778–788.
P1: GDX
Journal of Chemical Ecology [joec]
880
pp799-joec-461506
March 17, 2003
20:32
Style file version June 28th, 2002
THOMPSON ET AL.
SIMEON DE BOUCHBERG, M., ALLEGRINI, J., BESSIERE, C., ATTISSO, M., PASSET, J., and GRANGER,
R. 1976. Proprietès microbiologiques des huiles essentielles de chimiotypes de Thymus vulgaris
Linaeus. In Estratto dalla Rivista Italiana Essenze, Profumi, Piante officinali, Aromi, Saponi,
Cosmetici, Aerosol.
STAHL-BISKUP, E. 2002. Essential oil chemistry of the genus Thymus —a global view, In E. StahlBiskup, and F. Saez (eds.). Thyme: The genus Thymus. Taylor & Francis, London, pp. 75–124.
TARAYRE, M., THOMPSON, J. D., ESCARRÉ, J., and LINHART, Y. B. 1995. Intra-specific variation in the
inhibitory effects of Thymus vulgaris (Labiatae) monoterpenes on seed germination. Oecologia
101:110–118.
THOMPSON, J. D. 2002. Population structure and the spatial dynamics of genetic polymorphism in
thyme. In E. Stahl-Biskup, and F. Saez (eds.). Thyme: The genus Thymus. Taylor & Francis,
London, pp. 44–74.
THOMPSON, J. D., MANICACCI, D., and TARAYRE, M. 1998. Thirty five years of thyme: a tale of two
polymorphisms. Bioscience 48:805–815.
VERNET, P., GUILLERM, J. L., and GOUYON, P. H. 1977a. Le polymorphisme chimique de Thymus vulgaris L. (Labiée) I. Repartition des formes chimiques en relation avec certains facteurs
écologiques. Oecol. Plant. 12:159–179.
VERNET, P., GUILLERM, J. L., and GOUYON, P. H. 1977b. Le polymorphisme chimique de Thymus
vulgaris L. (Labiée) II. Carte à l’echelle 1/25000 des formes chimiques dans la région de SaintMartin-de-Londres (Herault-France). Oecol. Plant. 12:181–194.
VERNET, P., GOUYON, P. H., and VALDEYRON, G. 1986. Genetic control of the oil content in Thymus
vulgaris L.: a case of polymorphism in a biosynthetic chain. Genetica 69:227–231.
VOKOU, D. and MARGARIS, N. S. 1984. Effects of volatile oils from aromatic shrubs on soil microorganisms. Soil Biol. Biochem. 16:509–513.
VOKOU, D., KOKKINI, S., and BESSIERE, J. M. 1993. Geographic variation of Greek Oregano (Origanum
vulgare ssp. hirtum) essential oils. Biochem. Syst. Ecol. 21:287–295.