Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue10.Dec2000:
Biochemical Systematics and Ecology 28 (2000) 923}931
Inter-population di!erences in the essential oils
of Pinus caribaea needles
Luis Felipe Barnola!, Aragua Ceden8 o",*
!Apartado 47058, Caracas 1041-A, Caracas, Venezuela
"Facultad de Ciencias, Instituto de Zoologn& a Tropical, Universidad Central de Venezuela, Apartado 47058,
Caracas 1041-A, Venezuela
Received 10 March 1999; accepted 18 October 1999
Abstract
Di!erences in volatile terpene content of Pinus caribaea needles and soil qualities between
two pine plantations (Uverito and Sartenejas) in Venezuela were analyzed. Soils in the Uverito
pine plantation were sandy, low in nutritional quality, and de"cient in available nitrogen and
phosphorus content. Pines grown on these soils presented in their needles higher concentrations
of a-pinene, camphene, sabinene, myrcene#a-phellandrene mixture, b-phellandrene, b-caryophyllene, a-humulene, and a higher total monoterpene content than pines of the Sartenejas
plantation, where soils were nutritionally richer and higher in nitrogen and phosphorus
content. The hypothesis of the carbon/nutrient balance could explain these results. Alternatively, continuous stress on the pines of the Uverito plantation due to herbivory by the
leafcutter ant Atta laevigata may induce, in part, the di!erences observed between these pines
and those of the Sartenejas plantation. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pinus caribaea; Monoterpene; Sesquiterpene; Inter-population di!erences; Carbon/nutrient
balance hypothesis
1. Introduction
Intraspeci"c variations in volatile terpene content in leaves of certain conifers
related to the geographical distribution of the species have been reported (Rudlo! and
Rehfeldt, 1980; Hall and Langenheim, 1987; SchoK nwitz et al., 1990; Ra"i et al., 1992),
* Corresponding author. Tel.: #58-2-6051307; fax: #58-2-6051204.
E-mail address: [email protected] (A. Ceden8 o).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 3 6 - 3
924
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
in particular of the genus Pinus (Zavarin et al., 1976; Snajberk et al., 1978; Cool and
Zavarin, 1992; Nerg et al., 1994). Numerous taxonomic studies are based on the
variability of mono- and sesquiterpene content in various conifers (Zavarin et al.,
1976; Snajberk et al., 1978; Rudlo! and Rehfeldt, 1980; Ra"i et al., 1992).
Variability in volatile terpene content in leaves of certain conifers may play an
important role in their interactions with herbivorous insects (Edmunds and Alstad,
1978; Gambliel and Cates, 1995; Zou and Cates, 1995). Geographical variability in
terpene content of the oleoresin of Pinus ponderosa has been related to di!erential
selection pressures by specialist herbivores (Sturgeon, 1979; Sturgeon and Mitton,
1986).
Environmental factors such as water and nutrient (nitrogen, phosphorus) availability may in#uence the essential oil production in leaves of several plant species
(Mihaliak and Lincoln, 1985, 1989; Muzika et al., 1989; Ross and Sombrero, 1991;
Kainulainen et al., 1992; Yani et al., 1993). Variations in soil quality and fertilization
induce di!erences in leaf secondary metabolite chemistry of Inga oerstediana which
may in#uence the herbivory patterns of the leafcutter ant Atta cephalotes (NicholsOrians, 1991). Changes in leaf primary and secondary metabolite chemistry have been
related to defoliation in P. ponderosa (Wagner and Evans, 1985).
Variability in terpene content in Pinus caribaea resin has been studied (Green et al.,
1974, 1975), including taxonomic aspects of the several varieties (Coppen et al., 1993).
However, little is known with regard to terpene content in needles of this species
(Ekundayo, 1978) and its intraspeci"c variability (Barnola et al., 1994, 1997). The
selective herbivory of the leafcutter ant Atta laevigata in a plantation of Caribbean
pine was found to be related to terpene variation in pine needles (Barnola et al., 1994).
Pinus caribaea was introduced to Venezuela around 1969. One plantation has been
established in Uverito, Monagas State, and large areas in the mountains of Sartenejas,
Miranda State, have been planted with this pine species. Both the localities di!er in
climatic conditions and abiotic factors such as soil characteristics. High population
densities of Atta laevigata, which cause signi"cant economic damage, were observed in
the Uverito plantation. This ant species is also found in small numbers in the
Sartenejas woods, where LeoH n (1986) observed low herbivory intensity on Pinus
caribaea. As part of a long-term study on the relationship between Pinus caribaea and
the herbivory by Atta laevigata (Ceden8 o, 1989; Barnola et al., 1994, 1997), we investigated possible inter-relations between abiotic factors, essential oil chemistry and ant
herbivory in these two locations.
2. Materials and methods
2.1. Locations
The Uverito plantation is located in a savanna in Monagas State (8335@ N, 623 50@
W) at a mean altitude of 60 m, with an annual mean temperature of 26.33C and annual
mean precipitation of 1100 mm. Most precipitation falls between May and December
followed by a pronounced dry season from January to April. The Sartenejas woods in
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
925
Miranda State (103 25@ N, 663 53@ W) are found on hilly terrain at a mean altitude of
1300 m, with an annual mean temperature of 19.43C and annual mean precipitation of
983 mm. Greatest precipitation is between April and November followed by a dry
season from December to March.
2.2. Plant material and sampling procedure
Samples of mature needles were taken from seven Caribbean pines (Pinus caribaea
Morelet var. hondurensis Barrett and Golfari) at the Uverito plantation and 13
Caribbean pines from the Sartenejas woods. All the trees were over 15 m hight. In
order to reduce variability in terpene content due to di!erences related to canopy level
and diurnal and seasonal changes (Barnola et al., 1997), pine needle samples were
taken from the four cardinal directions from the lower canopy region ((5 m) from
each pine in both localities in the morning during the dry season (March}April, 1994).
Collections from branches within a tree were combined and immediately immersed in
liquid nitrogen until laboratory processing.
2.3. Terpene analysis
Every pine needle sample was powdered in a homogenizer (Omnimixer) containing
liquid nitrogen. Two-hundred milligrams fresh weight of frozen needle powder were
extracted in the dark with 5 ml HPLC-grade n-pentane at #43C for 96 h. Ten ll
n-tetradecane solution in n-heptane (140 ll 25 ml~1) were added to each extract as an
internal standard. Each extract was "ltered and concentrated to approximately 600 ll
under a mild stream of nitrogen and immediately analyzed by GLC. Seven microliters
of concentrated extract were injected into the GLC column [5% AT}1000 on
Chromosorb WHP 80}100 mesh, 5 m]4 mm i.d. glass column, 45 ml min~1 He
carrier gas, column temperature: 503C (16 min) to 2003C at 23C min~1, injector at
1503C, FID at 2003C]. Peak areas were measured with an electronic integrator and
quanti"ed with respect of the internal standard (n-tetradecane). Relative response
factors to n-tetradecane were determined using several mono- and sesquiterpene
authentic samples. Compound identi"cations were achieved by comparing retention
times with those of several mono- and sesquiterpene standards and by determining
their KovaH ts' indices (Davies, 1990). Identities were con"rmed by comparing relative
retention times with those obtained in a previous study (Barnola et al., 1994).
2.4. Soil analysis
Seven soil samples were taken (10}40 cm depth) from the Sartenejas woods and 17
soil samples from the Uverito plantation. For these soil samples, texture (silt, clay, and
sand percentages), percent organic matter, pH (in water, 1 : 1 w/v), interchangeable
acidity, Al3`, H` (meq 100 g~1 soil), and total nitrogen, phosphorus (as PO3~),
4
potassium, and calcium concentrations (ppm) were determined (Anderson and Ingram, 1992).
926
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
2.5. Statistics
A Mann}Whitney U-test was done between Sartenejas and Uverito plantation on
data corresponding to terpene concentrations and soil characteristics. The most
signi"cant variables were graphically represented in Figs. 1 and 2. STATISTICA 5.0
program was used for the statistical analyses.
3. Results
Individual terpene concentrations and total monoterpene content (in lg g~1 of
fresh needle) in pine needles from Uverito and Sartenejas are shown in Table 1. Major
components were b-phellandrene (8), germacrene D (16), b-caryophyllene (13),
limonene (7), a-pinene (1). According to results of the Mann}Whitney U-test in Table
1, the concentrations of a-pinene (1), camphene (2), sabinene (4), myrcene#a-phellandrene mixture (6), b-phellandrene (8), b-caryophyllene (13), and a-humulene (14) were
signi"cantly higher, and that of germacrene D (16) signi"cantly lower in the pine
needles of Uverito compared to those in Sartenejas pine samples. Fig. 1 shows
camphene and sabinene concentrations in pine needle samples from Sartenejas and
Uverito plantation. These variables were the most signi"cantly di!erent between the
two locations (Table 1).
The averages of variables in soil samples from Sartenejas and Uverito plantation
are shown in Table 2. According to the Mann}Whitney U-test in Table 2, all
variables, except percent organic matter, pH, interchangeable Al3` concentration,
Fig. 1. Graphic representation of camphene and sabinene concentrations (lg g~1) in pine needle samples
from Sartenejas and Uverito plantation.
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
927
Fig. 2. Graphic representation of nitrogen and phosphorus concentrations (ppm) in soil samples from
Sartenejas and Uverito plantation.
and sand percentage, were signi"cantly higher in the soil samples from Sartenejas
compared to those for the soil samples from Uverito plantation; and sand was
signi"cantly higher in the soil samples from Uverito plantation compared to that from
Sartenejas. Fig. 2 is a graphic representation of nitrogen and phosphorus concentrations in soil samples from Sartenejas and Uverito plantation.
4. Discussion
Results shown in Table 1 regarding the qualitative composition of essential oils in
Caribbean pine needles are similar to those found in previous studies (Barnola et al.,
1994, 1997). Also, the values of the relative silt#clay content, as well as pH values,
found for Uverito soils (Table 2) agree with the values reported by other researchers
(Fassbender et al., 1979). In general, the nutritive elements found in Uverito soils
re#ect a low fertility and limited nutritional quality, a conclusion also shared by
Fassbender et al. (1979).
These results indicate that there are important di!erences between the Uverito and
Sartenejas localities in soil characteristics and needle composition of volatile terpenes.
Mean concentrations of most terpenes are higher in pine needles of Uverito than those
in pine needles of Sartenejas (Table 1). Therefore, the low herbivory intensity by Atta
laevigata on Pinus caribaea observed by LeoH n (1986) in Sartenejas cannot be explained
on the basis of the essential oils. On the other hand, it is possible that these di!erences
in terpene concentrations are partly due to environmental factors. The soils of Uverito
928
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
Table 1
Identity and mean concentration (lg g~1 needle fresh wt) of mono- and sesquiterpenes in Caribbean pine
needles from pine plantations at Uverito and Sartenejas
Mean concentration (lg g~1)
Compound
Uverito (n"7)
Sartenejas (n"13)
Mean
S.E.
Mean
S.E.
p-level
Mann}Whitney
U-test
1 a-Pinene
2 Camphene
3 b-Pinene
4 Sabinene
5 *-3-Carene
6 Myrcene#a-Phellandrene!
7 Limonene
8 b-Phellandrene
9 c-Terpinene
10 a-Ocimene
11 Unidenti"ed
12 Bornyl acetate
13 b-Caryophyllene
14 a-Humulene
15 c-Muurolene
16 Germacrene D
17 b-Cadinene#c-Cadinene !
175
6
45
9
3
124
66
1030
9
7
4
24
366
64
17
343
33
27
1
12
1
1
14
43
132
4
7
1
6
78
14
3
33
7
86
3
59
4
5
90
204
600
4
12
3
29
192
31
15
659
25
9
1
26
1
1
10
80
74
1
8
1
8
39
7
5
141
4
0.010
0.003
0.104
0.007
0.265
0.043
0.278
0.016
0.475
0.435
0.842
0.842
0.029
0.017
0.153
0.029
0.362
Total
Monoterpenes
1503
142
1099
125
0.036
!Peaks not resolved satisfactorily.
are, in general, limited in nutritional quality compared to those of Sartenejas (Table 2).
These results are consistent with those obtained by others who reported higher total
volatile terpenoid concentrations in leaves of individuals of several plant species
growing in low nitrogen and phosphorus availability (Mihaliak and Lincoln, 1985;
Mihaliak and Lincoln, 1989; Muzika et al., 1989; Ross and Sombrero, 1991).
Nitrogen and phosphorus availability for the pines of Uverito is drastically lower
(as shown in Table 2 and Fig. 2) compared to that of the pines of Sartenejas, whereas
the concentrations of several terpenes are signi"cantly higher in pine needles of
Uverito compared to those in pine needles of Sartenejas (Table 1). These facts seem to
corroborate the carbon/nutrient balance hypothesis (Bryant et al., 1983), according to
which, in restricted growth conditions limited by nutrient availability, part of the
carbon "xed by the plant is involved in the biosynthesis of secondary metabolites,
such as essential oils (terpenes) (Bryant et al., 1983; Ross and Sombrero, 1991). Indeed,
the nutritional quality of Uverito soils is inferior compared to that of Sartenejas soils
(Table 2), and the total content of the most volatile fraction of the essential oils (total
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
929
Table 2
Mean values for soil characteristics from Sartenejas and Uverito plantation
Sartenejas
(n"7)
Mean
Sand (%)
Clay (%)
Silt (%)
Organic matter (%)
pH (in water, 1 : 1 w/v)
Interchangeable Acidity
(meq 100 g~1 soil)
Interchangeable H`
(meq 100 g~1 soil)
Interchangeable Al3`
(meq 100 g~1 soil)
Total nitrogen (ppm)
P (as PO3~) (ppm)
4
K (ppm)
Ca (ppm)
Uverito plantation
(n"17)
S.E.
Mean
S.E.
p-level
Mann}Whitney
U-test
47.3
17.4
35.3
1.6
4.6
2.8
1.6
1.7
2.9
0.6
0.1
0.3
90.4
1.6
8.0
0.7
4.5
1.1
0.6
0.2
0.5
0.1
0.1
0.2
0.00013
0.00004
0.00013
0.13549
0.20372
0.00053
2.1
0.4
0.7
0.2
0.02039
0.71
0.42
0.42
0.04
0.25255
1330
2.0
28
121
177
0.2
5
28
112
0.6
6
36
25
0.1
1
8
0.00012
0.00016
0.00016
0.01011
monoterpenes) is signi"cantly higher in pine needles of Uverito compared to that in
pine needles of Sartenejas (Table 1).
Some terpenes, like b-caryophyllene and myrcene#a-phellandrene mixture, found
in higher concentrations in the pine needles of Uverito compared to those found in
Sartenejas pine samples, may play an important role in the interactions with the
leafcutter ant Atta laevigata in the Uverito pine plantation (Barnola et al., 1994).
Results obtained in this former study seem to indicate that the herbivory by Atta
laevigata may induce changes in terpene content in the needles of defoliated Caribbean pines of the Uverito plantation, and these changes are characterized by an
increase in the concentration of certain terpenes in the pine needles. On this basis, we
speculate that the higher concentrations of several terpenes found in this study in pine
needles of Uverito (compared to those in pine needle of Sartenejas) re#ect a stressed
condition in these pines, which may be due to the low nutritional quality of the
Uverito soils, and, probably as importantly, to the constant pressure exerted by Atta
laevigata herbivory on these trees, since the population density of this ant is considerably higher in the Uverito plantation compared to that observed in Sartenejas
(personal observations). However, these hypotheses need further testing.
Acknowledgements
We are grateful to Jorge Zegarra, NapoleoH n LeoH n, AdriaH n LeoH n, Cristian Grases,
Eligio Oropeza, Masahisa Hasegawa, and Manuel Guillermo Arratia for "eld
930
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
assistance, and to Eligio Oropeza and Masahisa Hasegawa for laboratory assistance.
Ismael HernaH ndez advised on soil data interpretation and Luis Bulla on statistical
analyses. Alejandro AmormH n and VmH ctor Hugo Aguilar kindly supplied some materials.
We appreciate critical comments on the manuscript by Luisa Elena Cardozo, CeH sar
Ovalles, Oscar Barnola, Zoila GonzaH lez, Jorge Zegarra, and two anonymous reviewers. CVG-PROFORCA furnished food and lodging in the "eld. Funds were
provided by Consejo de Desarrollo CientmH "co y HumanmH stico de la Universidad
Central de Venezuela (03-313261-94) and CONICIT (S1-2257).
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Inter-population di!erences in the essential oils
of Pinus caribaea needles
Luis Felipe Barnola!, Aragua Ceden8 o",*
!Apartado 47058, Caracas 1041-A, Caracas, Venezuela
"Facultad de Ciencias, Instituto de Zoologn& a Tropical, Universidad Central de Venezuela, Apartado 47058,
Caracas 1041-A, Venezuela
Received 10 March 1999; accepted 18 October 1999
Abstract
Di!erences in volatile terpene content of Pinus caribaea needles and soil qualities between
two pine plantations (Uverito and Sartenejas) in Venezuela were analyzed. Soils in the Uverito
pine plantation were sandy, low in nutritional quality, and de"cient in available nitrogen and
phosphorus content. Pines grown on these soils presented in their needles higher concentrations
of a-pinene, camphene, sabinene, myrcene#a-phellandrene mixture, b-phellandrene, b-caryophyllene, a-humulene, and a higher total monoterpene content than pines of the Sartenejas
plantation, where soils were nutritionally richer and higher in nitrogen and phosphorus
content. The hypothesis of the carbon/nutrient balance could explain these results. Alternatively, continuous stress on the pines of the Uverito plantation due to herbivory by the
leafcutter ant Atta laevigata may induce, in part, the di!erences observed between these pines
and those of the Sartenejas plantation. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pinus caribaea; Monoterpene; Sesquiterpene; Inter-population di!erences; Carbon/nutrient
balance hypothesis
1. Introduction
Intraspeci"c variations in volatile terpene content in leaves of certain conifers
related to the geographical distribution of the species have been reported (Rudlo! and
Rehfeldt, 1980; Hall and Langenheim, 1987; SchoK nwitz et al., 1990; Ra"i et al., 1992),
* Corresponding author. Tel.: #58-2-6051307; fax: #58-2-6051204.
E-mail address: [email protected] (A. Ceden8 o).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 3 6 - 3
924
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
in particular of the genus Pinus (Zavarin et al., 1976; Snajberk et al., 1978; Cool and
Zavarin, 1992; Nerg et al., 1994). Numerous taxonomic studies are based on the
variability of mono- and sesquiterpene content in various conifers (Zavarin et al.,
1976; Snajberk et al., 1978; Rudlo! and Rehfeldt, 1980; Ra"i et al., 1992).
Variability in volatile terpene content in leaves of certain conifers may play an
important role in their interactions with herbivorous insects (Edmunds and Alstad,
1978; Gambliel and Cates, 1995; Zou and Cates, 1995). Geographical variability in
terpene content of the oleoresin of Pinus ponderosa has been related to di!erential
selection pressures by specialist herbivores (Sturgeon, 1979; Sturgeon and Mitton,
1986).
Environmental factors such as water and nutrient (nitrogen, phosphorus) availability may in#uence the essential oil production in leaves of several plant species
(Mihaliak and Lincoln, 1985, 1989; Muzika et al., 1989; Ross and Sombrero, 1991;
Kainulainen et al., 1992; Yani et al., 1993). Variations in soil quality and fertilization
induce di!erences in leaf secondary metabolite chemistry of Inga oerstediana which
may in#uence the herbivory patterns of the leafcutter ant Atta cephalotes (NicholsOrians, 1991). Changes in leaf primary and secondary metabolite chemistry have been
related to defoliation in P. ponderosa (Wagner and Evans, 1985).
Variability in terpene content in Pinus caribaea resin has been studied (Green et al.,
1974, 1975), including taxonomic aspects of the several varieties (Coppen et al., 1993).
However, little is known with regard to terpene content in needles of this species
(Ekundayo, 1978) and its intraspeci"c variability (Barnola et al., 1994, 1997). The
selective herbivory of the leafcutter ant Atta laevigata in a plantation of Caribbean
pine was found to be related to terpene variation in pine needles (Barnola et al., 1994).
Pinus caribaea was introduced to Venezuela around 1969. One plantation has been
established in Uverito, Monagas State, and large areas in the mountains of Sartenejas,
Miranda State, have been planted with this pine species. Both the localities di!er in
climatic conditions and abiotic factors such as soil characteristics. High population
densities of Atta laevigata, which cause signi"cant economic damage, were observed in
the Uverito plantation. This ant species is also found in small numbers in the
Sartenejas woods, where LeoH n (1986) observed low herbivory intensity on Pinus
caribaea. As part of a long-term study on the relationship between Pinus caribaea and
the herbivory by Atta laevigata (Ceden8 o, 1989; Barnola et al., 1994, 1997), we investigated possible inter-relations between abiotic factors, essential oil chemistry and ant
herbivory in these two locations.
2. Materials and methods
2.1. Locations
The Uverito plantation is located in a savanna in Monagas State (8335@ N, 623 50@
W) at a mean altitude of 60 m, with an annual mean temperature of 26.33C and annual
mean precipitation of 1100 mm. Most precipitation falls between May and December
followed by a pronounced dry season from January to April. The Sartenejas woods in
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
925
Miranda State (103 25@ N, 663 53@ W) are found on hilly terrain at a mean altitude of
1300 m, with an annual mean temperature of 19.43C and annual mean precipitation of
983 mm. Greatest precipitation is between April and November followed by a dry
season from December to March.
2.2. Plant material and sampling procedure
Samples of mature needles were taken from seven Caribbean pines (Pinus caribaea
Morelet var. hondurensis Barrett and Golfari) at the Uverito plantation and 13
Caribbean pines from the Sartenejas woods. All the trees were over 15 m hight. In
order to reduce variability in terpene content due to di!erences related to canopy level
and diurnal and seasonal changes (Barnola et al., 1997), pine needle samples were
taken from the four cardinal directions from the lower canopy region ((5 m) from
each pine in both localities in the morning during the dry season (March}April, 1994).
Collections from branches within a tree were combined and immediately immersed in
liquid nitrogen until laboratory processing.
2.3. Terpene analysis
Every pine needle sample was powdered in a homogenizer (Omnimixer) containing
liquid nitrogen. Two-hundred milligrams fresh weight of frozen needle powder were
extracted in the dark with 5 ml HPLC-grade n-pentane at #43C for 96 h. Ten ll
n-tetradecane solution in n-heptane (140 ll 25 ml~1) were added to each extract as an
internal standard. Each extract was "ltered and concentrated to approximately 600 ll
under a mild stream of nitrogen and immediately analyzed by GLC. Seven microliters
of concentrated extract were injected into the GLC column [5% AT}1000 on
Chromosorb WHP 80}100 mesh, 5 m]4 mm i.d. glass column, 45 ml min~1 He
carrier gas, column temperature: 503C (16 min) to 2003C at 23C min~1, injector at
1503C, FID at 2003C]. Peak areas were measured with an electronic integrator and
quanti"ed with respect of the internal standard (n-tetradecane). Relative response
factors to n-tetradecane were determined using several mono- and sesquiterpene
authentic samples. Compound identi"cations were achieved by comparing retention
times with those of several mono- and sesquiterpene standards and by determining
their KovaH ts' indices (Davies, 1990). Identities were con"rmed by comparing relative
retention times with those obtained in a previous study (Barnola et al., 1994).
2.4. Soil analysis
Seven soil samples were taken (10}40 cm depth) from the Sartenejas woods and 17
soil samples from the Uverito plantation. For these soil samples, texture (silt, clay, and
sand percentages), percent organic matter, pH (in water, 1 : 1 w/v), interchangeable
acidity, Al3`, H` (meq 100 g~1 soil), and total nitrogen, phosphorus (as PO3~),
4
potassium, and calcium concentrations (ppm) were determined (Anderson and Ingram, 1992).
926
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
2.5. Statistics
A Mann}Whitney U-test was done between Sartenejas and Uverito plantation on
data corresponding to terpene concentrations and soil characteristics. The most
signi"cant variables were graphically represented in Figs. 1 and 2. STATISTICA 5.0
program was used for the statistical analyses.
3. Results
Individual terpene concentrations and total monoterpene content (in lg g~1 of
fresh needle) in pine needles from Uverito and Sartenejas are shown in Table 1. Major
components were b-phellandrene (8), germacrene D (16), b-caryophyllene (13),
limonene (7), a-pinene (1). According to results of the Mann}Whitney U-test in Table
1, the concentrations of a-pinene (1), camphene (2), sabinene (4), myrcene#a-phellandrene mixture (6), b-phellandrene (8), b-caryophyllene (13), and a-humulene (14) were
signi"cantly higher, and that of germacrene D (16) signi"cantly lower in the pine
needles of Uverito compared to those in Sartenejas pine samples. Fig. 1 shows
camphene and sabinene concentrations in pine needle samples from Sartenejas and
Uverito plantation. These variables were the most signi"cantly di!erent between the
two locations (Table 1).
The averages of variables in soil samples from Sartenejas and Uverito plantation
are shown in Table 2. According to the Mann}Whitney U-test in Table 2, all
variables, except percent organic matter, pH, interchangeable Al3` concentration,
Fig. 1. Graphic representation of camphene and sabinene concentrations (lg g~1) in pine needle samples
from Sartenejas and Uverito plantation.
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
927
Fig. 2. Graphic representation of nitrogen and phosphorus concentrations (ppm) in soil samples from
Sartenejas and Uverito plantation.
and sand percentage, were signi"cantly higher in the soil samples from Sartenejas
compared to those for the soil samples from Uverito plantation; and sand was
signi"cantly higher in the soil samples from Uverito plantation compared to that from
Sartenejas. Fig. 2 is a graphic representation of nitrogen and phosphorus concentrations in soil samples from Sartenejas and Uverito plantation.
4. Discussion
Results shown in Table 1 regarding the qualitative composition of essential oils in
Caribbean pine needles are similar to those found in previous studies (Barnola et al.,
1994, 1997). Also, the values of the relative silt#clay content, as well as pH values,
found for Uverito soils (Table 2) agree with the values reported by other researchers
(Fassbender et al., 1979). In general, the nutritive elements found in Uverito soils
re#ect a low fertility and limited nutritional quality, a conclusion also shared by
Fassbender et al. (1979).
These results indicate that there are important di!erences between the Uverito and
Sartenejas localities in soil characteristics and needle composition of volatile terpenes.
Mean concentrations of most terpenes are higher in pine needles of Uverito than those
in pine needles of Sartenejas (Table 1). Therefore, the low herbivory intensity by Atta
laevigata on Pinus caribaea observed by LeoH n (1986) in Sartenejas cannot be explained
on the basis of the essential oils. On the other hand, it is possible that these di!erences
in terpene concentrations are partly due to environmental factors. The soils of Uverito
928
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
Table 1
Identity and mean concentration (lg g~1 needle fresh wt) of mono- and sesquiterpenes in Caribbean pine
needles from pine plantations at Uverito and Sartenejas
Mean concentration (lg g~1)
Compound
Uverito (n"7)
Sartenejas (n"13)
Mean
S.E.
Mean
S.E.
p-level
Mann}Whitney
U-test
1 a-Pinene
2 Camphene
3 b-Pinene
4 Sabinene
5 *-3-Carene
6 Myrcene#a-Phellandrene!
7 Limonene
8 b-Phellandrene
9 c-Terpinene
10 a-Ocimene
11 Unidenti"ed
12 Bornyl acetate
13 b-Caryophyllene
14 a-Humulene
15 c-Muurolene
16 Germacrene D
17 b-Cadinene#c-Cadinene !
175
6
45
9
3
124
66
1030
9
7
4
24
366
64
17
343
33
27
1
12
1
1
14
43
132
4
7
1
6
78
14
3
33
7
86
3
59
4
5
90
204
600
4
12
3
29
192
31
15
659
25
9
1
26
1
1
10
80
74
1
8
1
8
39
7
5
141
4
0.010
0.003
0.104
0.007
0.265
0.043
0.278
0.016
0.475
0.435
0.842
0.842
0.029
0.017
0.153
0.029
0.362
Total
Monoterpenes
1503
142
1099
125
0.036
!Peaks not resolved satisfactorily.
are, in general, limited in nutritional quality compared to those of Sartenejas (Table 2).
These results are consistent with those obtained by others who reported higher total
volatile terpenoid concentrations in leaves of individuals of several plant species
growing in low nitrogen and phosphorus availability (Mihaliak and Lincoln, 1985;
Mihaliak and Lincoln, 1989; Muzika et al., 1989; Ross and Sombrero, 1991).
Nitrogen and phosphorus availability for the pines of Uverito is drastically lower
(as shown in Table 2 and Fig. 2) compared to that of the pines of Sartenejas, whereas
the concentrations of several terpenes are signi"cantly higher in pine needles of
Uverito compared to those in pine needles of Sartenejas (Table 1). These facts seem to
corroborate the carbon/nutrient balance hypothesis (Bryant et al., 1983), according to
which, in restricted growth conditions limited by nutrient availability, part of the
carbon "xed by the plant is involved in the biosynthesis of secondary metabolites,
such as essential oils (terpenes) (Bryant et al., 1983; Ross and Sombrero, 1991). Indeed,
the nutritional quality of Uverito soils is inferior compared to that of Sartenejas soils
(Table 2), and the total content of the most volatile fraction of the essential oils (total
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
929
Table 2
Mean values for soil characteristics from Sartenejas and Uverito plantation
Sartenejas
(n"7)
Mean
Sand (%)
Clay (%)
Silt (%)
Organic matter (%)
pH (in water, 1 : 1 w/v)
Interchangeable Acidity
(meq 100 g~1 soil)
Interchangeable H`
(meq 100 g~1 soil)
Interchangeable Al3`
(meq 100 g~1 soil)
Total nitrogen (ppm)
P (as PO3~) (ppm)
4
K (ppm)
Ca (ppm)
Uverito plantation
(n"17)
S.E.
Mean
S.E.
p-level
Mann}Whitney
U-test
47.3
17.4
35.3
1.6
4.6
2.8
1.6
1.7
2.9
0.6
0.1
0.3
90.4
1.6
8.0
0.7
4.5
1.1
0.6
0.2
0.5
0.1
0.1
0.2
0.00013
0.00004
0.00013
0.13549
0.20372
0.00053
2.1
0.4
0.7
0.2
0.02039
0.71
0.42
0.42
0.04
0.25255
1330
2.0
28
121
177
0.2
5
28
112
0.6
6
36
25
0.1
1
8
0.00012
0.00016
0.00016
0.01011
monoterpenes) is signi"cantly higher in pine needles of Uverito compared to that in
pine needles of Sartenejas (Table 1).
Some terpenes, like b-caryophyllene and myrcene#a-phellandrene mixture, found
in higher concentrations in the pine needles of Uverito compared to those found in
Sartenejas pine samples, may play an important role in the interactions with the
leafcutter ant Atta laevigata in the Uverito pine plantation (Barnola et al., 1994).
Results obtained in this former study seem to indicate that the herbivory by Atta
laevigata may induce changes in terpene content in the needles of defoliated Caribbean pines of the Uverito plantation, and these changes are characterized by an
increase in the concentration of certain terpenes in the pine needles. On this basis, we
speculate that the higher concentrations of several terpenes found in this study in pine
needles of Uverito (compared to those in pine needle of Sartenejas) re#ect a stressed
condition in these pines, which may be due to the low nutritional quality of the
Uverito soils, and, probably as importantly, to the constant pressure exerted by Atta
laevigata herbivory on these trees, since the population density of this ant is considerably higher in the Uverito plantation compared to that observed in Sartenejas
(personal observations). However, these hypotheses need further testing.
Acknowledgements
We are grateful to Jorge Zegarra, NapoleoH n LeoH n, AdriaH n LeoH n, Cristian Grases,
Eligio Oropeza, Masahisa Hasegawa, and Manuel Guillermo Arratia for "eld
930
L. Felipe Barnola, A. CedenJ o / Biochemical Systematics and Ecology 28 (2000) 923}931
assistance, and to Eligio Oropeza and Masahisa Hasegawa for laboratory assistance.
Ismael HernaH ndez advised on soil data interpretation and Luis Bulla on statistical
analyses. Alejandro AmormH n and VmH ctor Hugo Aguilar kindly supplied some materials.
We appreciate critical comments on the manuscript by Luisa Elena Cardozo, CeH sar
Ovalles, Oscar Barnola, Zoila GonzaH lez, Jorge Zegarra, and two anonymous reviewers. CVG-PROFORCA furnished food and lodging in the "eld. Funds were
provided by Consejo de Desarrollo CientmH "co y HumanmH stico de la Universidad
Central de Venezuela (03-313261-94) and CONICIT (S1-2257).
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