Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol29.Issue2.Feb2001:
Biochemical Systematics and Ecology 29 (2001) 189}198
Volatile oil variability in Thymus serpylloides ssp.
gadorensis growing wild in Southeastern Spain
Francisco SaH ez*
Departamento de Biologn& a Vegetal (Bota& nica), Facultad de Biologn& a, Universidad de Murcia,
30,100-Espinardo (Murcia), Spain
Received 21 April 1998; accepted 27 October 1999
Abstract
Volatile oils from single plants of Thymus serpylloides ssp. gadorensis were collected from
Southeastern Spain and studied to check for chemical variability using gas chromatography
(GC) and gas chromatography}mass spectrometry (GC}MS). Many of the samples showed
a phenolic chemotype, while another important group had signi"cant levels of linalool.
Geraniol, myrcene, caryophyllene oxide, terpinen-4-ol and 1,8-cineole were commonly present.
Principal Component Analysis (PCA) and Cluster Analysis (CA) of this chemical variability
separated two groups of plants characterized by either phenols or linalool, and an isolated third
type with geraniol. A few samples were found to have both phenolic and non-phenolic
compounds in high quantities, thus showing a mixed chemotype. Multidimensional scaling
analysis (MDS) of the percentage concentration for each component of the essential oil showed
that thymol, linalool, 1,8-cineole, borneol and geraniol have clear divergent vectors. ( 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Thymus serpylloides ssp. gadorensis; Lamiaceae; Essential oil; Thymol; Linalool; Geraniol;
Myrcene; Caryophyllene oxide
1. Introduction
The genus Thymus L. is distributed over the Eurasian continent, the northern part of
Africa and southern Greenland, although it has been spread by man all over the
* Correspondence address. Geology Department, Faculty of Mathematics and Natural Sciences, Bergen
University, AlleH gaten, 41, 5007-Bergen, Norway.
E-mail address: francisco.saez@geol.uib.no (F. SaH ez).
0305-1978/01/$ - see front matter ( 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 0 - 5
190
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
world. However, when Section Serpyllum (with clearly di!erent ecological preferences,
at places with little or no summer drought) is not considered, the Mediterranean basin
becomes its dispersion area, with many species being adapted to both hot summer and
cold continental winter conditions (Morales, 1986, 1989, 1993). The Iberian Peninsula
is one of the most diversi"ed territories for this genus, where the morphological and
chemical variability of thyme is as large as the number of environmental conditions
displayed (Rivas-MartmH nez, 1988). Southeast Spain has traditionally been a source of
wild raw material, not only of thyme but also a large number of other aromatic plants
(Alcaraz et al., 1988), to supply the #avour, cosmetics, pharmaceutical and condiment
industries (Adzet et al., 1987; Stahl-Biskup, 1991; Lawrence, 1992). The economic
pressure on these wild resources sometimes leads to an over-exploitation that
threatens ecosystems, especially where semiarid weather conditions do not allow
a quick regeneration of shrub populations.
Thymus serpylloides Bory ssp. gadorensis (Pau) Jalas has a discontinuous distribution over southeastern Iberian Peninsula, mainly in Granada Province, but also in
AlmermH a, Murcia and Alicante, in mountain ranges higher than 1500 m, growing on
scarcely developed soils with a varied chemical composition, from calcareous to
clayish. It contains 58 chromosomes in somatic cells, and di!ers from the type
subspecies in having a denser indument and being less prostrate, although it generally
does not grow higher than 20 cm. Genetic relationships with other species of thyme
are found frequently, leading to hybrids such as T.]aitanae ("T. serpylloides ssp.
gadorensis]T. vulgaris ssp. vulgaris), T.]hieronymi ("T. serpylloides ssp. gadorensis]T. mastichina) and T.]pastoris ("T. serpylloides ssp. gadorensis]T. zygis ssp.
gracilis) (Morales, 1995).
Recent studies (SaH ez, 1995a, b, 1996, 1998, 1999) have shown a complex chemical
picture for some Thymus species in the studied area, where the proximity of high
mountains to the sea shore, the geological mosaic (metamorphic materials adjacent to
siliceous or calcareous extensions, and Quaternary depressions with abundant badlands and karstic formations), and the bioclimatological conditions (with the high
Betic rangelands performing a barrier to stop the wet wind originated at Atlantic
ocean), cause considerable ecological variability that leads to chemical variability.
Consequently, T. serpylloides ssp. gadorensis often meets the possibility of producing:
(a) the above mentioned botanical hybrids, not studied here, despite of their great
botanical and chemical interest; (b) specimens that morphologically correspond to the
type subspecies but present some chemical characteristics that deviate from the
general guidelines for it, namely the presence of 1,8-cineole. This variability in the
essential oil is sometimes explained if other species of thyme and the potential from
hybridization are taken into account.
The volatile oil of T. serpylloides ssp. gadorensis has previously been studied from one
sample from Granada Province (Crespo et al., 1988) revealing a carvacrol (34%)
chemotype that proved to be e!ective in vitro against several Gram-positive and
Gram-negative bacteria (Crespo et al., 1990). One sample from AlmermH a Province was
studied by Morales (Morales, 1986) and characterized as the carvacrol chemotype.
Arrebola et al. (1995) studied one population from Granada that showed a predominance
of carvacrol in almost all the individuals, similar to ssp. serpylloides (Arrebola et al., 1994).
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
191
2. Materials and methods
2.1. Plant material
Aerial parts of #owering Thymus serpylloides ssp. gadorensis were collected from the
Spanish southeast (Fig. 1) from May to July of 1990}1993. The number of individuals
(single plants) taken (1}5) from each locality depended on morphological variability
observed. The plant material was dried at room temperature (1}2 months) and steam
distilled for 2 h in a Clevenger-type apparatus. Volatile oils were kept in sealed glass
tubes at 43, without anhydrous Na SO , until analysis. Voucher specimens from each
2 4
locality are kept at the Herbarium of Murcia University (MUB). The determination of
the taxonomical status for each sample was performed in situ. No morphological
hybrids were collected for this study, although a certain variability was detected, both
inter- and intra-populations.
2.2. Gas chromatography
Analysis was carried out in a Hewlett Packard Series II 5890 gas chromatograph
equipped with FID using a 25 m]0.2 mm &Carbowax 20M' capillary column with the
Fig. 1. Localization of populations at the study area. Dispersal area of Thymus serpylloides ssp. gadorensis is
restricted to the highest altitudes. Numbers inside parenthesis stand for the number of samples taken at
each locality.
192
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
following temperature program: 703C isothermal for 1 min, rising by 103C min~1 up
to 903C, isothermal for 1 min, followed by an increase of 103C min~1 up to 2103C.
Injector and detector temperature was 2503C. Carrier gas was He with a #ow rate of
1 ml min~1. Peak area and concentrations were calculated with a Hewlett Packard
3396A integrator. Essential oil component identi"cation was performed by IR spectra,
Rt comparison with pure standards, and GC}MS comparison with bibliography
(Jennings and Shibamoto, 1980).
For IR identi"cation, preparative GC was performed using a Perkin}Elmer 3B
chromatograph (TCD detector) with a Carbowax 20M column (4 m]3 mm), a temperature program of 703C}2153C at a rate of 23C min~1, and He as a carrier gas at
25 ml min~1. Injector and detector temperature was 2403C. Infrared spectrophotometer Beckman IR 4280 was used to obtain spectra. GC/MS analysis was
performed on a Hewlett-Packard 5890 gas chromatograph using a 70 eV mass
selective detector HP 5971, with the same column and chromatographic parameters
as stated for GC. The percentage of identi"ed compounds in each sample ranged from
90 to 99%.
2.3. Statistical analysis
Ma!ei et al. (1993a, b) have previously made extensive use of statistical methods to
interpret di!erent aspects of the metabolism of aromatic plants, demonstrating the
usefulness of cluster analysis (CA) and principal component analysis (PCA). Another
technique, multidimensional scaling (MDS), is allegedly very robust (Minchin, 1987)
due to its iterative diminishment of a goodness-of-"t statistic that represents the
normalized discrepancies between distances in the MDS plot and the smoothed
distances predicted from dissimilarities within data being used. In the present study,
all data were statistically processed with a Systat 5 software for PC. Trace quantities
((0.01%) were considered as zero value. The analysis included: (a) cluster analysis
(CA; Euclidean distance, single linkage method) to establish the di!erent
groups/chemotypes within the individual essential oils; (b) principal component
analysis (PCA, based on a Euclidean correlation matrix) to check for partition among
the identi"ed compounds; (c) multidimensional scaling (MDS, based on a Spearman
correlation matrix) to study the relationships among the di!erent compounds.
3. Results and discussion
The statistical analysis of the data set shows most individuals adhered to two
chemotypes, and the rest formed smaller groups or stayed separate due to the presence
c
Fig. 2. Cluster analysis of the studied 34 individuals with remarks on the essential oil component(s) that
characterize the major subgroups. Letters A}F refer to samples highlighted in Fig. 3.
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
193
Compound
3(2)
6(1)
8(3)
8(4)
10(1)
10(2)
*
0.40
0.38
0.06
0.03
0.17
0.03
0.06
0.12
0.31
*
1.08
0.09
0.10
0.78
0.15
1.02
1.99
1.52
0.14
0.27
2.95
1.05
0.10
1.95
79.99
0.32
0.05
0.24
0.10
0.97
0.13
*
0.74
0.31
0.15
0.07
1.40
1.37
0.40
0.35
5.98
10.33
30.85
0.12
0.19
0.10
0.06
0.90
1.27
0.08
*
0.06
1.46
0.05
0.42
*
0.26
1.03
*
0.05
0.46
18.50
20.13
0.03
1.62
0.77
0.37
1.04
0.13
5.07
0.84
4.05
11.70
*
11.32
1.80
1.18
7.13
29.38
1.33
0.09
2.25
0.13
0.34
0.54
0.15
0.02
0.05
0.25
0.31
0.07
0.15
0.07
11.55
0.77
0.16
0.74
4.22
0.24
0.06
0.83
0.07
0.95
0.25
0.73
*
1.61
0.05
0.45
7.48
24.47
39.39
1.04
0.13
0.73
2.48
2.15
1.02
1.32
0.04
1.24
0.48
0.05
0.02
0.07
0.90
0.87
*
3.54
4.66
0.84
*
17.11
*
*
10.52
1.82
*
15.10
*
*
1.31
1.54
*
0.48
*
10.82
0.54
1.54
7.13
*
*
*
6.28
*
*
2.04
3.56
1.37
*
3.02
5.86
*
*
30.39
*
*
10.19
*
*
*
*
*
*
*
*
*
*
11.71
27.02
2.36
4.89
*
*
*
1.74
*
2.81
*
*
*
!3(2)"Buitre 2; 6(1)"Tetica 1; 8(3)"CambroH n 3; 8(4)"CambroH n 4; 10(1)"Calar Mundo 1; 10(2)"Calar Mundo 2.
Max. %
Mean$SEM
0.38
4.21
7.93
2.37
1.56
30.39
5.07
5.61
13.52
20.85
10.33
40.46
1.80
28.91
9.99
79.74
39.39
11.10
5.22
28.05
27.02
10.44
7.13
17.70
1.95
79.99
14.10
0.93
2.81
5.36
56.97
27.73
0.1$0.0
1.7$0.2
1.7$0.4
0.5$0.1
0.3$0.1
5.0$1.5
0.8$0.2
1.2$0.3
3.2$0.7
5.4$0.9
0.6$0.4
14.7$2.3
0.2$0.1
1.3$0.8
2.3$0.5
17.4$4.5
2.7$1.2
1.4$0.4
0.7$0.2
3.2$1.0
1.5$0.8
1.7$0.3
1.2$0.3
0.8$0.5
0.2$0.1
7.4$3.9
1.9$0.6
0.1$0.0
0.2$0.1
0.5$0.2
11.9$2.5
3.3$1.2
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
a-thujene
a-pinene
Camphene
b-pinene
Sabinene
Myrcene
a-terpinene
Limonene
1,8-cineole
c-terpinene
tr-ocimene
p-cymene
Terpinolene
tr-sabinene hyd.
Camphor
Linalool
Linalyl ac.
Isobornyl ac.
b-caryophyllene
Terpinen-4-ol
a-terpineol
Borneol
Geranial
Geranyl ac.
Citronellol
Geraniol
Caryophyllene ox.
Viridi#orol
Elemol
Spathulenol
Thymol
Carvacrol
% Site (sample)
194
Table 1
Essential oil composition of the most di!erent samples of Thymus serpylloides ssp. gadorensis. Data refers to percentage of total essential oil at selected sites, and
the maximum and mean percentages for each compound from 34 samples!
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
195
of unusual concentrations of particular compounds. Thus, cluster analysis develops
a total of "ve groups, marked with vertical lines at Fig. 2, and outlined as follows:
(a) The group characterized by phenols is the largest one, with the highest level of
thymol in the individual named &Aulago 1' (57%), and with &Aulago 2' presenting the
maximum for carvacrol (27%). Both phenols were found to co-occur not only at the
same site (di!erent plants), but also in the same sample, such as &Tetica 1' (Table 1) and
'MarmH a 1' (13% thymol and 17% carvacrol). Medium to high proportions of phenolic
precursors (p-cymene and c-terpinene) are also characteristic throughout the group.
(b) The linalool group is also well represented with many samples, although
chemical variability here is greater than in the phenol group. The most diverse
samples are &CambroH n 4' (24% linalool #39% linalyl ac.) and Bacares 1 (39%
linalool #29% tr-sabinene hydrate), with &CambroH n 3' and &MorroH n 1' being peripheral due to the simultaneous presence of phenols and linalool.
(c) Myrcene characterizes a group of "ve samples, which, with the exception of
&Tetica' are geographically close. Levels of myrcene range from 17 to 30%, and
caryophyllene oxide, 2 to 14%) are present.
(d) Three samples were found with geraniol levels from 75 to 80%. They were
collected from &Buitre' and &La Ragua' sites, which are relatively near to each other at
the southwestern part of the study area.
(e) The remaining three samples in Fig. 2 present considerable chemical di!erences
and have been placed apart from the rest due to an unusual combinations of essential
oil components. Thus, &Aitana 2' has a mixture of phenolic and non-phenolic compounds (18% carvacrol#10% 1,8-cineole#10% borneol), while &Bacares 3' is characterized by terpinen-4-ol (28%), and &Calar Mundo bajo 2' has myrcene (30%),
a-terpineol (27%), terpinen-4-ol (12%) and 1,8-cineole (10%).
The great variability in the volatile oil of T. serpylloides ssp. gadorensis is also
demonstrated by Principal Components Analysis (Fig. 3), where samples marked with
&C' and &D' represent the linalool group, &B' and the neighbouring plots are devoted to
phenols, and the lower part of the graph contains the myrcene group. Plots &A', &E' and
&F' represent the three unusual combinations of components. Complete essential oil
composition of all samples represented with a letter is shown at Table 1, also including
maximum and mean values for each compound. Factor 1 re#ects 34.7% of the total
variability, while Factor 2 re#ects 24.2% and Factor 3, 13.0%, jointly making 71.9% of
the total. Fig. 4 shows vectors for the di!erent compounds presenting more than 8% at
least in one studied sample, after MDS analysis. There is a high number of vectors
represented, if this analysis is compared with similar statistical studies performed for
other species of thyme growing at the same area (SaH ez, 1995a, b, 1996, 1998, 1999).
Vectors representing phenols, linalool, geraniol and myrcene clearly diverge, while other
components such as 1,8-cineole or terpinen-4-ol take up intermediate positions.
4. Conclusion
Two concluding remarks can be made from these results. The "rst deals with the
chemotypes to be recognized for T. serpylloides ssp. gadorensis. This species has been
196
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
Fig. 3. Localization of individuals for (a) factors 1 and 2, and (b) factors 1 and 3 after PCA. A"Buitre 2;
B"Tetica 1; C"CambroH n 3; D"CambroH n 4; E"Calar del Mundo bajo 1; F"Calar del Mundo bajo
2. Samples A and F overlap at a.
previously reported to be exclusively phenolic, thus, the presence of 1,8-cineole and
linalool in plants morphologically within the type subspecies (not hybrids) could be
regarded as "ngerprints of introgression processes with other species of thyme
growing nearby, that present these chemotypes themselves (namely, T. vulgaris and
T. zygis ssp. gracilis).
Secondly, a geographical distribution of chemotypes must be noted, especially for
myrcene and geraniol, with the former appearing at sites 10 and 11 (the farthest from
the sea), and the latter in the western-most samples, from &Buitre' and &La Ragua'. This
clear separation of chemotypes is well represented by the vectors for thymol, linalool,
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
197
Fig. 4. Multidimensional Scaling Analysis. Only compounds with 8% in at least one sample are represented.
geraniol, myrcene and caryophyllene oxide in Figs. 3 and 4 (with easily detectable
chemical/geographical groups). The comparison of data reported here with the
previously published data shows a much wider variability in essential oil composition
of T. serpylloides ssp. gadorensis than earlier expected. The punctual appearance of
medium levels of tr-sabinene hydrate, terpinen-4-ol, caryophyllene oxide and other
compounds, and the remarks stated above suggest that investigations with much
more plant material per site is required, to improve the knowledge about the
occurrence of these components in the essential oil of this economically interesting
species. Speci"c focusing of future works on morphological hybrids will presumably
lead to the description of new mixed chemotypes.
Acknowledgements
The author thanks Dr. M.C. GarcmH a-Vallejo and Dr. M.C. Soriano for their help
with analytical methods. Dr. P. SaH nchez kindly modi"ed maps from Rivas-MartmH nez.
Thanks are also due to RAMON SABATER, S.A., the Laboratory of Palynology at
Murcia University, and &FundacioH n SEND ECA'
References
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sa signi"cation taxonomique. Biochem. Syst. Ecol. 5, 269}272.
Alcaraz, F., SaH nchez-GoH mez, P., Correal, E., 1988. CataH logo de las plantas aromaH ticas, condimentarias
y medicinales de la RegioH n de Murcia. MonografmH as I.N.I.A. No. 67. Madrid.
Arrebola, M.L., Navarro, M.C., JimeH nez, J., Ocan8 a FA., 1994. Yield and composition of the essential oil of
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Volatile oil variability in Thymus serpylloides ssp.
gadorensis growing wild in Southeastern Spain
Francisco SaH ez*
Departamento de Biologn& a Vegetal (Bota& nica), Facultad de Biologn& a, Universidad de Murcia,
30,100-Espinardo (Murcia), Spain
Received 21 April 1998; accepted 27 October 1999
Abstract
Volatile oils from single plants of Thymus serpylloides ssp. gadorensis were collected from
Southeastern Spain and studied to check for chemical variability using gas chromatography
(GC) and gas chromatography}mass spectrometry (GC}MS). Many of the samples showed
a phenolic chemotype, while another important group had signi"cant levels of linalool.
Geraniol, myrcene, caryophyllene oxide, terpinen-4-ol and 1,8-cineole were commonly present.
Principal Component Analysis (PCA) and Cluster Analysis (CA) of this chemical variability
separated two groups of plants characterized by either phenols or linalool, and an isolated third
type with geraniol. A few samples were found to have both phenolic and non-phenolic
compounds in high quantities, thus showing a mixed chemotype. Multidimensional scaling
analysis (MDS) of the percentage concentration for each component of the essential oil showed
that thymol, linalool, 1,8-cineole, borneol and geraniol have clear divergent vectors. ( 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Thymus serpylloides ssp. gadorensis; Lamiaceae; Essential oil; Thymol; Linalool; Geraniol;
Myrcene; Caryophyllene oxide
1. Introduction
The genus Thymus L. is distributed over the Eurasian continent, the northern part of
Africa and southern Greenland, although it has been spread by man all over the
* Correspondence address. Geology Department, Faculty of Mathematics and Natural Sciences, Bergen
University, AlleH gaten, 41, 5007-Bergen, Norway.
E-mail address: francisco.saez@geol.uib.no (F. SaH ez).
0305-1978/01/$ - see front matter ( 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 0 - 5
190
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
world. However, when Section Serpyllum (with clearly di!erent ecological preferences,
at places with little or no summer drought) is not considered, the Mediterranean basin
becomes its dispersion area, with many species being adapted to both hot summer and
cold continental winter conditions (Morales, 1986, 1989, 1993). The Iberian Peninsula
is one of the most diversi"ed territories for this genus, where the morphological and
chemical variability of thyme is as large as the number of environmental conditions
displayed (Rivas-MartmH nez, 1988). Southeast Spain has traditionally been a source of
wild raw material, not only of thyme but also a large number of other aromatic plants
(Alcaraz et al., 1988), to supply the #avour, cosmetics, pharmaceutical and condiment
industries (Adzet et al., 1987; Stahl-Biskup, 1991; Lawrence, 1992). The economic
pressure on these wild resources sometimes leads to an over-exploitation that
threatens ecosystems, especially where semiarid weather conditions do not allow
a quick regeneration of shrub populations.
Thymus serpylloides Bory ssp. gadorensis (Pau) Jalas has a discontinuous distribution over southeastern Iberian Peninsula, mainly in Granada Province, but also in
AlmermH a, Murcia and Alicante, in mountain ranges higher than 1500 m, growing on
scarcely developed soils with a varied chemical composition, from calcareous to
clayish. It contains 58 chromosomes in somatic cells, and di!ers from the type
subspecies in having a denser indument and being less prostrate, although it generally
does not grow higher than 20 cm. Genetic relationships with other species of thyme
are found frequently, leading to hybrids such as T.]aitanae ("T. serpylloides ssp.
gadorensis]T. vulgaris ssp. vulgaris), T.]hieronymi ("T. serpylloides ssp. gadorensis]T. mastichina) and T.]pastoris ("T. serpylloides ssp. gadorensis]T. zygis ssp.
gracilis) (Morales, 1995).
Recent studies (SaH ez, 1995a, b, 1996, 1998, 1999) have shown a complex chemical
picture for some Thymus species in the studied area, where the proximity of high
mountains to the sea shore, the geological mosaic (metamorphic materials adjacent to
siliceous or calcareous extensions, and Quaternary depressions with abundant badlands and karstic formations), and the bioclimatological conditions (with the high
Betic rangelands performing a barrier to stop the wet wind originated at Atlantic
ocean), cause considerable ecological variability that leads to chemical variability.
Consequently, T. serpylloides ssp. gadorensis often meets the possibility of producing:
(a) the above mentioned botanical hybrids, not studied here, despite of their great
botanical and chemical interest; (b) specimens that morphologically correspond to the
type subspecies but present some chemical characteristics that deviate from the
general guidelines for it, namely the presence of 1,8-cineole. This variability in the
essential oil is sometimes explained if other species of thyme and the potential from
hybridization are taken into account.
The volatile oil of T. serpylloides ssp. gadorensis has previously been studied from one
sample from Granada Province (Crespo et al., 1988) revealing a carvacrol (34%)
chemotype that proved to be e!ective in vitro against several Gram-positive and
Gram-negative bacteria (Crespo et al., 1990). One sample from AlmermH a Province was
studied by Morales (Morales, 1986) and characterized as the carvacrol chemotype.
Arrebola et al. (1995) studied one population from Granada that showed a predominance
of carvacrol in almost all the individuals, similar to ssp. serpylloides (Arrebola et al., 1994).
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
191
2. Materials and methods
2.1. Plant material
Aerial parts of #owering Thymus serpylloides ssp. gadorensis were collected from the
Spanish southeast (Fig. 1) from May to July of 1990}1993. The number of individuals
(single plants) taken (1}5) from each locality depended on morphological variability
observed. The plant material was dried at room temperature (1}2 months) and steam
distilled for 2 h in a Clevenger-type apparatus. Volatile oils were kept in sealed glass
tubes at 43, without anhydrous Na SO , until analysis. Voucher specimens from each
2 4
locality are kept at the Herbarium of Murcia University (MUB). The determination of
the taxonomical status for each sample was performed in situ. No morphological
hybrids were collected for this study, although a certain variability was detected, both
inter- and intra-populations.
2.2. Gas chromatography
Analysis was carried out in a Hewlett Packard Series II 5890 gas chromatograph
equipped with FID using a 25 m]0.2 mm &Carbowax 20M' capillary column with the
Fig. 1. Localization of populations at the study area. Dispersal area of Thymus serpylloides ssp. gadorensis is
restricted to the highest altitudes. Numbers inside parenthesis stand for the number of samples taken at
each locality.
192
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
following temperature program: 703C isothermal for 1 min, rising by 103C min~1 up
to 903C, isothermal for 1 min, followed by an increase of 103C min~1 up to 2103C.
Injector and detector temperature was 2503C. Carrier gas was He with a #ow rate of
1 ml min~1. Peak area and concentrations were calculated with a Hewlett Packard
3396A integrator. Essential oil component identi"cation was performed by IR spectra,
Rt comparison with pure standards, and GC}MS comparison with bibliography
(Jennings and Shibamoto, 1980).
For IR identi"cation, preparative GC was performed using a Perkin}Elmer 3B
chromatograph (TCD detector) with a Carbowax 20M column (4 m]3 mm), a temperature program of 703C}2153C at a rate of 23C min~1, and He as a carrier gas at
25 ml min~1. Injector and detector temperature was 2403C. Infrared spectrophotometer Beckman IR 4280 was used to obtain spectra. GC/MS analysis was
performed on a Hewlett-Packard 5890 gas chromatograph using a 70 eV mass
selective detector HP 5971, with the same column and chromatographic parameters
as stated for GC. The percentage of identi"ed compounds in each sample ranged from
90 to 99%.
2.3. Statistical analysis
Ma!ei et al. (1993a, b) have previously made extensive use of statistical methods to
interpret di!erent aspects of the metabolism of aromatic plants, demonstrating the
usefulness of cluster analysis (CA) and principal component analysis (PCA). Another
technique, multidimensional scaling (MDS), is allegedly very robust (Minchin, 1987)
due to its iterative diminishment of a goodness-of-"t statistic that represents the
normalized discrepancies between distances in the MDS plot and the smoothed
distances predicted from dissimilarities within data being used. In the present study,
all data were statistically processed with a Systat 5 software for PC. Trace quantities
((0.01%) were considered as zero value. The analysis included: (a) cluster analysis
(CA; Euclidean distance, single linkage method) to establish the di!erent
groups/chemotypes within the individual essential oils; (b) principal component
analysis (PCA, based on a Euclidean correlation matrix) to check for partition among
the identi"ed compounds; (c) multidimensional scaling (MDS, based on a Spearman
correlation matrix) to study the relationships among the di!erent compounds.
3. Results and discussion
The statistical analysis of the data set shows most individuals adhered to two
chemotypes, and the rest formed smaller groups or stayed separate due to the presence
c
Fig. 2. Cluster analysis of the studied 34 individuals with remarks on the essential oil component(s) that
characterize the major subgroups. Letters A}F refer to samples highlighted in Fig. 3.
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
193
Compound
3(2)
6(1)
8(3)
8(4)
10(1)
10(2)
*
0.40
0.38
0.06
0.03
0.17
0.03
0.06
0.12
0.31
*
1.08
0.09
0.10
0.78
0.15
1.02
1.99
1.52
0.14
0.27
2.95
1.05
0.10
1.95
79.99
0.32
0.05
0.24
0.10
0.97
0.13
*
0.74
0.31
0.15
0.07
1.40
1.37
0.40
0.35
5.98
10.33
30.85
0.12
0.19
0.10
0.06
0.90
1.27
0.08
*
0.06
1.46
0.05
0.42
*
0.26
1.03
*
0.05
0.46
18.50
20.13
0.03
1.62
0.77
0.37
1.04
0.13
5.07
0.84
4.05
11.70
*
11.32
1.80
1.18
7.13
29.38
1.33
0.09
2.25
0.13
0.34
0.54
0.15
0.02
0.05
0.25
0.31
0.07
0.15
0.07
11.55
0.77
0.16
0.74
4.22
0.24
0.06
0.83
0.07
0.95
0.25
0.73
*
1.61
0.05
0.45
7.48
24.47
39.39
1.04
0.13
0.73
2.48
2.15
1.02
1.32
0.04
1.24
0.48
0.05
0.02
0.07
0.90
0.87
*
3.54
4.66
0.84
*
17.11
*
*
10.52
1.82
*
15.10
*
*
1.31
1.54
*
0.48
*
10.82
0.54
1.54
7.13
*
*
*
6.28
*
*
2.04
3.56
1.37
*
3.02
5.86
*
*
30.39
*
*
10.19
*
*
*
*
*
*
*
*
*
*
11.71
27.02
2.36
4.89
*
*
*
1.74
*
2.81
*
*
*
!3(2)"Buitre 2; 6(1)"Tetica 1; 8(3)"CambroH n 3; 8(4)"CambroH n 4; 10(1)"Calar Mundo 1; 10(2)"Calar Mundo 2.
Max. %
Mean$SEM
0.38
4.21
7.93
2.37
1.56
30.39
5.07
5.61
13.52
20.85
10.33
40.46
1.80
28.91
9.99
79.74
39.39
11.10
5.22
28.05
27.02
10.44
7.13
17.70
1.95
79.99
14.10
0.93
2.81
5.36
56.97
27.73
0.1$0.0
1.7$0.2
1.7$0.4
0.5$0.1
0.3$0.1
5.0$1.5
0.8$0.2
1.2$0.3
3.2$0.7
5.4$0.9
0.6$0.4
14.7$2.3
0.2$0.1
1.3$0.8
2.3$0.5
17.4$4.5
2.7$1.2
1.4$0.4
0.7$0.2
3.2$1.0
1.5$0.8
1.7$0.3
1.2$0.3
0.8$0.5
0.2$0.1
7.4$3.9
1.9$0.6
0.1$0.0
0.2$0.1
0.5$0.2
11.9$2.5
3.3$1.2
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
a-thujene
a-pinene
Camphene
b-pinene
Sabinene
Myrcene
a-terpinene
Limonene
1,8-cineole
c-terpinene
tr-ocimene
p-cymene
Terpinolene
tr-sabinene hyd.
Camphor
Linalool
Linalyl ac.
Isobornyl ac.
b-caryophyllene
Terpinen-4-ol
a-terpineol
Borneol
Geranial
Geranyl ac.
Citronellol
Geraniol
Caryophyllene ox.
Viridi#orol
Elemol
Spathulenol
Thymol
Carvacrol
% Site (sample)
194
Table 1
Essential oil composition of the most di!erent samples of Thymus serpylloides ssp. gadorensis. Data refers to percentage of total essential oil at selected sites, and
the maximum and mean percentages for each compound from 34 samples!
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
195
of unusual concentrations of particular compounds. Thus, cluster analysis develops
a total of "ve groups, marked with vertical lines at Fig. 2, and outlined as follows:
(a) The group characterized by phenols is the largest one, with the highest level of
thymol in the individual named &Aulago 1' (57%), and with &Aulago 2' presenting the
maximum for carvacrol (27%). Both phenols were found to co-occur not only at the
same site (di!erent plants), but also in the same sample, such as &Tetica 1' (Table 1) and
'MarmH a 1' (13% thymol and 17% carvacrol). Medium to high proportions of phenolic
precursors (p-cymene and c-terpinene) are also characteristic throughout the group.
(b) The linalool group is also well represented with many samples, although
chemical variability here is greater than in the phenol group. The most diverse
samples are &CambroH n 4' (24% linalool #39% linalyl ac.) and Bacares 1 (39%
linalool #29% tr-sabinene hydrate), with &CambroH n 3' and &MorroH n 1' being peripheral due to the simultaneous presence of phenols and linalool.
(c) Myrcene characterizes a group of "ve samples, which, with the exception of
&Tetica' are geographically close. Levels of myrcene range from 17 to 30%, and
caryophyllene oxide, 2 to 14%) are present.
(d) Three samples were found with geraniol levels from 75 to 80%. They were
collected from &Buitre' and &La Ragua' sites, which are relatively near to each other at
the southwestern part of the study area.
(e) The remaining three samples in Fig. 2 present considerable chemical di!erences
and have been placed apart from the rest due to an unusual combinations of essential
oil components. Thus, &Aitana 2' has a mixture of phenolic and non-phenolic compounds (18% carvacrol#10% 1,8-cineole#10% borneol), while &Bacares 3' is characterized by terpinen-4-ol (28%), and &Calar Mundo bajo 2' has myrcene (30%),
a-terpineol (27%), terpinen-4-ol (12%) and 1,8-cineole (10%).
The great variability in the volatile oil of T. serpylloides ssp. gadorensis is also
demonstrated by Principal Components Analysis (Fig. 3), where samples marked with
&C' and &D' represent the linalool group, &B' and the neighbouring plots are devoted to
phenols, and the lower part of the graph contains the myrcene group. Plots &A', &E' and
&F' represent the three unusual combinations of components. Complete essential oil
composition of all samples represented with a letter is shown at Table 1, also including
maximum and mean values for each compound. Factor 1 re#ects 34.7% of the total
variability, while Factor 2 re#ects 24.2% and Factor 3, 13.0%, jointly making 71.9% of
the total. Fig. 4 shows vectors for the di!erent compounds presenting more than 8% at
least in one studied sample, after MDS analysis. There is a high number of vectors
represented, if this analysis is compared with similar statistical studies performed for
other species of thyme growing at the same area (SaH ez, 1995a, b, 1996, 1998, 1999).
Vectors representing phenols, linalool, geraniol and myrcene clearly diverge, while other
components such as 1,8-cineole or terpinen-4-ol take up intermediate positions.
4. Conclusion
Two concluding remarks can be made from these results. The "rst deals with the
chemotypes to be recognized for T. serpylloides ssp. gadorensis. This species has been
196
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
Fig. 3. Localization of individuals for (a) factors 1 and 2, and (b) factors 1 and 3 after PCA. A"Buitre 2;
B"Tetica 1; C"CambroH n 3; D"CambroH n 4; E"Calar del Mundo bajo 1; F"Calar del Mundo bajo
2. Samples A and F overlap at a.
previously reported to be exclusively phenolic, thus, the presence of 1,8-cineole and
linalool in plants morphologically within the type subspecies (not hybrids) could be
regarded as "ngerprints of introgression processes with other species of thyme
growing nearby, that present these chemotypes themselves (namely, T. vulgaris and
T. zygis ssp. gracilis).
Secondly, a geographical distribution of chemotypes must be noted, especially for
myrcene and geraniol, with the former appearing at sites 10 and 11 (the farthest from
the sea), and the latter in the western-most samples, from &Buitre' and &La Ragua'. This
clear separation of chemotypes is well represented by the vectors for thymol, linalool,
F. Sa& ez / Biochemical Systematics and Ecology 29 (2001) 189}198
197
Fig. 4. Multidimensional Scaling Analysis. Only compounds with 8% in at least one sample are represented.
geraniol, myrcene and caryophyllene oxide in Figs. 3 and 4 (with easily detectable
chemical/geographical groups). The comparison of data reported here with the
previously published data shows a much wider variability in essential oil composition
of T. serpylloides ssp. gadorensis than earlier expected. The punctual appearance of
medium levels of tr-sabinene hydrate, terpinen-4-ol, caryophyllene oxide and other
compounds, and the remarks stated above suggest that investigations with much
more plant material per site is required, to improve the knowledge about the
occurrence of these components in the essential oil of this economically interesting
species. Speci"c focusing of future works on morphological hybrids will presumably
lead to the description of new mixed chemotypes.
Acknowledgements
The author thanks Dr. M.C. GarcmH a-Vallejo and Dr. M.C. Soriano for their help
with analytical methods. Dr. P. SaH nchez kindly modi"ed maps from Rivas-MartmH nez.
Thanks are also due to RAMON SABATER, S.A., the Laboratory of Palynology at
Murcia University, and &FundacioH n SEND ECA'
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