Comparison of Mediterranean Pistacia lentiscus genotypes by random amplified polymorphic DNA, chemical, and morphological analyses

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C 2003) Journal of Chemical Ecology, Vol. 29, No. 8, August 2003 (°

COMPARISON OF MEDITERRANEAN Pistacia lentiscus GENOTYPES BY RANDOM AMPLIFIED POLYMORPHIC DNA, CHEMICAL, AND MORPHOLOGICAL ANALYSES

OZ BARAZANI,1 NATIV DUDAI,2 and AVI GOLAN-GOLDHIRSH1,∗ 1 Albert

Katz Department of Dryland Biotechnologies Desert Plant Biotechnology Laboratory Jacob Blaustein Institute for Desert Research Ben-Gurion University of the Negev Sede Boker Campus 84990, Israel 2 Aromatic, Medicinal and Spice Crops Unit Newe Ya’ar Research Center Agricultural Research Organization Ramat Yishay 30095, Israel

(Received July 22, 2002; accepted April 11, 2003)

Abstract—Characterization of the genetic variability of Mediterranean Pistacia lentiscus genotypes by RAPD, composition of essential oils, and morphology is presented. High polymorphism in morphological parameters was found among accessions, with no significant differences in relation to geographical origin, or to gender. GC-MS analysis of leaves extracted by t-butyl methyl ether, showed 12 monoterpenes, seven sesquiterpenes, and one linear nonterpenic compound. Cluster analysis divided the accessions into two main groups according to the relative content of the major compounds, with no relation to their geographical origin. In contrast, a dendrogram based on RAPD analysis gave two main clusters according to their geographical origins. Low correlation was found between genetic and essential oil content matrices. High morphological and chemical variability on one hand, and genotypic polymorphism on the other, provide ecological advantages that might explain the distribution of Pistacia lentiscus over a wide range of habitats. The plants under study were grown together in the same climatic and environmental conditions, thus pointing to the plausible genetic basis of the observed phenotypic differences. Key Words—Essential oil, genetic diversity, germplasm, morphology, Pistacia lentiscus, RAPD.



To whom correspondence should be addressed. E-mail: [email protected]

1939 C 2003 Plenum Publishing Corporation 0098-0331/03/0800-1939/0 °

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BARAZANI, DUDAI, AND GOLAN-GOLDHIRSH INTRODUCTION

Pistacia lentiscus L. (Anacardiaceae), an evergreen dioecious shrub, is widely distributed along the Mediterranean basin shores (Zohary, 1952). It is a member of a heterogeneous family of eleven species. Zohary (1952), in his monographic study of the genus, included P. lentiscus in the section EU Lentiscus and disputed the varietal subdivision of this species, based mainly on size, shape, and number of leaflets. P. lentiscus (mastic tree) is well known in Mediterranean countries for its resin, mastic gum, used since antiquity for incense, as a chewing gum for pleasant breath, for spicing liqueurs and jam, and in the cosmetic industry (Browicz, 1987). The therapeutic effect of mastic gum was demonstrated in healing of duodenal ulcers (Al-Habbal et al., 1984; Al-Said et al., 1986). Antimicrobial activity of the essential oil and resin was also reported (Magiatis et al., 1999). On the Greek island Chios, P. lentiscus (referred as P. lentiscus var. chia by Browicz, 1987) is cultivated and vegetatively propagated by the locals who collect the resin from the bark (Browicz, 1987). Chemical analysis of the resin and/or essential oils of P. lentiscus was reported from different locations around the Mediterranean basin including: Spain (Boelens and Jimenez, 1991), Greece (Papageorgiou et al., 1991), Egypt (De Pooter et al., 1991), Israel (Fleisher and Fleisher, 1992), and Corsica (Castola et al., 2000). Comparison of these reports revealed variation in the chemical composition of the leaf essential oils. α-Pinene and myrcene were major components in the Spanish resin (Boelens and Jimenz, 1991). In Corsica, leaf oil was composed mainly of tepinene-4-ol and α-pinene (Castola et al., 2000). In Greece, α-pinene constituted 58.9–70% of the essential oil of mastic gum (Papageorgiou et al., 1991). Analysis of the essential oil from Mt. Carmel, Israel was characterized by a high relative content of α-terpineol (Fleisher and Fleisher, 1992). Cultivated P. lentiscus accessions grown in Egypt, had car-3-ene as the major component, constituting 65% of the essential oil (De Pooter et al., 1991). P. lentiscus is a leading component of the low altitude Mediterranean maquis. Its distribution around the Mediterranean basin extends to North and Eastern Africa and Madeira Island (Zohary, 1996). It was suggested that ecotypic differentiation enabled Israeli P. lentiscus to grow in diverse habitats (Shaviv, 1978). At the J. Blaustein Institute for Desert Research (BIDR), located in the Negev desert, Sede Boker, Israel, a live germplasm collection of Pistacia spp. was established (Golan-Goldhirsh and Kostiukovsky, 1998). Accessions of P. lentiscus collected from different locations around the Mediterranean basin (Israel, Cyprus, Spain, and Tunisia) are maintained together under similar conditions. The germplasm, therefore, offers the possibility to assess the genetic basis for the phenotypic variation. We report the comparison of chemical and morphological variation among accessions, and the use of random amplified polymorphic DNA (RAPD) analysis,

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to evaluate the genetic differences among genotypes of Mediterranean P. lentiscus growing in the germplasm collection. METHODS AND MATERIALS

Plant Material. Leaves of Pistacia lentiscus were collected from the live germplasm collection at Blaustein Institute for Desert Research (BIDR) (GolanGoldhirsh and Kostiukovsky, 1998). The accessions originated in Israel (Adoraim), Cyprus (Emba, Limasol area), Spain (Lleida Province) and from three locations in Tunisia (Aindrham, Takrouna, and Ain Sebaa) (Table 1). A total of 16 accessions were studied, both male and female trees. Plants are grown under field conditions in a 3 × 4 m area in row and between rows, respectively (Golan-Goldhirsh and Kostiukovsky, 1998). The collection is located at the Negev desert highland (elevation 525 m); with average precipitation of ca. 90 mm. The average day and night temperatures in summer are 33◦ C and 15◦ C, respectively, with winter average temperatures of 15◦ C and 5◦ C, respectively, typical of a subtropical desert. During summer, plants are irrigated approx. once a week by a controlled drip irrigation system. In winter, run-off is supplemented with irrigation once every 2–3 wk. RAPD Analysis. A total of 120 primers were pretested for the study of the genetic relations among Pistacia species and 39 revealed polymorphism (data not shown). Of the 39 primers, nine showed polymorphism among individuals of P. lentiscus. These were selected for further RAPD analysis with all 16 accessions TABLE 1. Pistacia lentiscus GROWING IN THE GERMPLASM COLLECTION FIELD IN SEDE BOKER AND THEIR ORIGIN No.

Country

Site of origin

Gender

Abbreviation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Israel Israel Cyprus Cyprus Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Spain Spain Spain Spain Spain Spain

Adoraim Adoraim Limassol area Limassol area Aindrham Takrouna Takrouna Ain Sebaa Takrouna Ain Sebaa Lleida Province Lleida Province Lleida Province Lleida Province Lleida Province Lleida Province

Male Female Male Female Male Male Female Female Female Female Male Male Female Female Female Female

Is-m Is-f Cy-m Cy-f Tn-m1 Tn-m2 Tn-f1 Tn-f2 Tn-f3 Tn-f4 Sp-m1 Sp-m2 Sp-f1 Sp-f2 Sp-f3 Sp-f4

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(Table 1). A DNA sample from P. atlantica was used in the RAPD analysis as an out-group species. DNA extraction followed the protocol of Doyle and Doyle (1987) with modification by Hormaza et al. (1994). Optimization of reaction conditions for RAPD analysis was based on previous work (Khandka et al., 1997) and consisted of a 25-µl reaction mixture containing 10 mM Tris-HCl (pH 8.0), 20 ng of genomic DNA, 0.1 mM of each dNTP, 50 mM KCl, 2 mM MgCl2 , and 1 U of Taq DNA polymerase (Bioline). Amplification was carried out in a BIO-RAD iCycler under the following conditions: 94◦ C for 4 min, 1 cycle; 94◦ C for 1 min, 35◦ C for 1 min, 72◦ C for 2 min, 45 cycles; 72◦ C for 7 min, 1 cycle. Amplified products were analyzed by electrophoresis in a 1.2% agarose gel after staining with ethidium bromide (0.5 µg/ml) and viewing under UV light. All reactions were repeated twice, and only reproducible bands were scored for statistical analysis. Chemical Analysis. Following Lewinsohn et al. (1993), the essential oil composition of Pistacia lentiscus leaves was determined by GC-MS analysis of a t-butyl methyl ether (MTBE) extract of the leaves. Leaflets from each sample were cut and mixed. One gram of each sample was shaken in MTBE for 2 hr in a ratio of 1:5 (w/v), containing 10 µg/ml isobutylbenzene (Aldrich, USA) as an internal standard and kept in the solvent overnight. The extract was cleaned by passing it through a small column (Pasteur pipette) containing anhydrous Na2 SO4 and silicic acid (Silicagel 60, 230–400 mesh, Merck, Germany), to dry the sample and to remove high molecular weight polar substances that interfere with the GC-analyses. An Hewlett Packard G 1800 B GCD GC-MS system, with an electron ionization detector, was used for the essential oil analysis. Diluted MTBE extracts were injected on to the Rtx-5SIL MS column equilibrated with He gas at a flow rate of 1.0 ml/min. Temperature was programmed to start at 70◦ C for 2 min, followed by a gradient of 4◦ C/min from 70 to 200◦ C. The injector temperature was set to 250◦ C and the detector set to 280◦ C. The scanning range was recorded from 45 to 450 m/z, with an ionization energy of 70 eV. Identification of the main components was by coinjection of authentic standards and comparison of EI-MS spectra of authentic standards with computerized libraries. Quantification of the compounds was based on total ion chromatographic peak size, as related to the internal standard. Morphological Measurements. Morphological traits, quantitative and qualitative, were measured and evaluated. For each accession, five measurements of leaf length, number of leaflets per leaf, leaflet length, and width were recorded. Qualitative traits included: leaflet shape, color, and texture and were assessed according to an evaluation scale, 1 (light green) to 5 (dark green) for color, 1 (flexible) to 5 (leathery) for leaf texture, and lanceolate (1) to ovate (5) for leaflet shape. Data Analysis. RAPD data as well as the relative content of essential oil compounds were subjected to similarity matrix and cluster analyses, using the Numerical Taxonomy and Multivariate Analysis System program package for PC

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(NTSYS-pc, version 2). The relative content of essential oil constituents was normalized by transformed values (arcsin), and the similarity index was constructed using similarity for interval data with Euclidean distances. For RAPD analysis, amplified bands on a gel were scored as 1 (present) or 0 (absent), and a similarity matrix was constructed using simple matching coefficient function. Both dendrograms were constructed by applying the unweighted pair group method with arithmetic averages (UPGMA) to the similarity matrix (Nei, 1987). A Mantel test, with 1000 permutations was conducted by NTSYS-pc to compare the chemical and genetic similarity matrices. RESULTS

RAPD Analysis. A total of 77 bands were scored, ranging between 300 bp and 2 kb, an average of ca. 9 bands for each primer (Table 2). Seventy out of the 77 scored bands were polymorphic (Table 2). On the basis of these polymorphisms, similarity values were calculated, showing genetic relations among the accessions (Table 3). High similarity values were obtained among the Spanish accessions (0.76–0.89), Tunisian (0.81–0.91), Israeli (0.94), and Cyprian accessions (0.86). High similarity values were found between Tunisian and Spanish accessions, (0.68–0.86), indicating close relations between P. lentiscus from these two countries. The Israeli accessions showed higher similarity values to Tunisian accessions (0.69–0.80) than to the Spanish accessions (0.58–0.73). The Cyprian accessions showed lower similarity values to all other accessions (0.56–0.68). The similarity values between all 16 accessions and P. atlantica, as an out-group, was relatively low (0.35–0.50) (Table 3). A dendrogram, divided the 16 accessions into two distinct genetic groups (Figure 1). One group included the Tunisian, Spanish, and Israeli accessions and TABLE 2. PRIMERS USED FOR RAPD ANALYSIS AND THE NUMBER OF SCORED BANDS Primer

50 -30 Sequence

Total bands

Polymorphic bands

OPA -08 OPB-01 OPC-02 OPD-03 OPD-05 OPD-07 OPD-13 OPD-20 OPE-06

GTGACGTAGG GTTTCGCTCC GTGAGGCGTC GTCGCCGTCA TGAGCGGACA TTGGCACGGG GGGGTGACGA ACCCGGTCAC AAGACCCCTC

12 3 16 12 10 8 8 4 4

10 2 14 12 9 8 8 3 4

77

70

Total

1.00 0.73 0.69 0.78 0.75 0.76 0.79 0.80 0.78 0.63 0.71 0.66 0.65 0.73 0.70 0.44 1.00 0.86 0.65 0.60 0.64 0.64 0.68 0.63 0.58 0.66 0.59 0.58 0.63 0.65 0.38 1.00 0.66 0.56 0.63 0.65 0.64 0.64 0.59 0.65 0.59 0.56 0.61 0.64 0.38 1.00 0.85 0.84 0.81 0.83 0.83 0.70 0.76 0.69 0.73 0.78 0.73 0.50 1.00 0.84 0.84 0.90 0.88 0.68 0.74 0.69 0.73 0.75 0.68 0.44 1.00 0.83 0.86 0.81 0.74 0.83 0.75 0.71 0.84 0.76 0.45 1.00 0.86 0.91 0.74 0.83 0.75 0.74 0.81 0.74 0.44 1.00 0.88 0.70 0.81 0.71 0.70 0.80 0.73 0.48 1.00 0.78 0.86 0.79 0.80 0.85 0.75 0.44 1.00 0.86 0.84 0.88 0.83 0.83 0.39

1.00 0.83 0.86 0.89 0.84 0.41

1.00 0.76 0.84 0.75 0.35

1.00 0.83 0.80 0.39

1.00 0.83 0.38

1.00 0.40

1.00

Cy-m Cy-f Tn-m1 Tn-m2 Tn-f1 Tn-f2 Tn-f3 Tn-f4 Sp-m1 Sp-m2 Sp-f1 Sp-f2 Sp-f3 Sp-f4 P. atlantica

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For abbreviations see Table 1.

1.00 0.94 0.68 0.66 0.74 0.69 0.71 0.74 0.75 0.73 0.58 0.66 0.60 0.59 0.69 0.65 0.41

Is-m Is-f Cy-m Cy-f Tn-m1 Tn-m2 Tn-f1 Tn-f2 Tn-f3 Tn-f4 Sp-m1 Sp-m2 Sp-f1 Sp-f2 Sp-f3 Sp-f4 P. atlantica

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Accessiona

TABLE 3. MATRIX OF SIMILARITY VALUES OBTAINED FROM RAPD ANALYSIS OF Pistacia lentiscus ACCESSIONS

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FIG. 1. Dendrogram of genetic RAPD analysis relations among Pistacia lentiscus accessions, obtained by applying unweighted pair group method with arithmetic averages (UPGMA). See Table 1 for abbreviations.

the second genetic group, the Cyprian. The Tunisian accessions were clustered together in the same phylogenic subgroup of the Spanish accessions (Figure 1). The Israeli accessions were genetically closer to the later two than to accessions from Cyprus. P. atlantica was in a separate outgroup, distinctly different from the rest (Figure 1). Chemical Composition of Essential Oils. GC-MS analysis of MTBE extract from leaves of Pistacia lentiscus showed 20 main constituents, including 12 different monoterpene hydrocarbons, seven sesquiterpene hydrocarbons, and one linear nonterpenic compound (Table 4). Among them, α-pinene, sabinene, limonene, caryophyllene, and germacrene D were the major compounds (Table 4). Cluster analysis (Figure 2) divided the 16 accessions into two main groups according to the relative content of one of the five main compounds. The first group was characterized by a high relative content of limonene (28.7–45.5%), and was further divided into two subgroups: (I) the limonene/α-pinene subgroup, included the Israeli and Cyprian male accessions and exhibited a high relative content of α-pinene (13.8–24.1%), and (II) the limonene/caryophyllene subgroup, included two Spanish (Sp-m2, Sp-f1) and Israeli female accessions and exhibited a high content of the sesquiterpene caryophyllene (13.2–22.4%). The second main group was divided into three subgroups: (I) the Cyprian female and two Tunisian

For abbreviations see Table 1. Retention indices.

545

1181

1084

0.0 49.0 1.2 12.2 8.6 1.0 0.8 6.1 6.4 0.0 0.0 0.2 0.0 0.0 3.7 0.7 0.0 10.0 0.0 0.0 100

Cy-f

1305

0.5 18.7 0.0 32.8 14.1 0.8 0.7 5.4 4.9 0.0 0.6 1.0 0.8 0.0 3.7 1.4 1.3 11.8 0.0 1.4 100 706

0.0 42.7 0.9 12.6 4.6 0.6 1.3 6.7 5.6 1.7 5.2 0.0 0.5 0.0 7.1 0.0 0.0 10.4 0.0 0.0 100 829

0.0 35.1 0.0 19.9 3.0 1.1 2.8 15.4 9.9 1.8 5.3 0.2 0.0 0.9 1.1 0.0 0.0 3.7 0.0 0.0 100 671

0.5 19.0 0.4 24.1 6.3 0.8 4.7 9.3 8.6 0.0 1.9 0.5 0.0 0.0 2.8 0.5 1.0 18.7 0.0 0.8 100 1347

0.0 22.0 0.0 42.3 5.1 0.8 2.3 8.1 8.2 0.0 0.0 0.0 0.0 0.6 0.8 0.4 0.5 8.3 0.0 0.7 100 683

0.0 26.0 0.7 45.8 2.0 1.2 1.3 7.3 6.4 0.0 1.8 0.0 0.8 0.0 3.1 0.0 0.0 3.4 0.0 0.0 100 793

11.1 22.7 2.8 24.3 4.4 1.1 4.0 3.3 3.0 0.0 0.0 0.2 0.0 0.0 10.7 1.8 0.0 10.2 0.0 0.3 100 904

0.0 5.1 0.0 11.0 0.0 1.3 0.0 28.7 0.9 0.0 0.0 0.0 0.0 0.8 22.4 3.2 1.1 23.7 0.5 1.3 100 762

0.0 6.5 0.0 10.2 0.9 1.5 1.1 29.7 1.2 0.0 1.4 0.0 0.5 0.8 20.7 2.9 1.0 20.3 0.0 1.1 100 749

0.0 22.3 0.9 9.6 7.3 0.3 0.0 1.6 1.5 0.0 0.7 0.0 0.6 0.0 6.0 3.0 2.2 41.1 0.8 2.1 100

1040

12.3 19.4 2.7 28.6 3.5 1.6 0.3 3.7 3.1 0.0 0.0 0.0 0.2 0.7 9.0 1.8 1.7 9.7 0.6 1.3 100

1514

0.0 13.2 1.3 21.7 2.1 0.4 0.0 0.7 1.0 0.0 0.0 0.0 0.8 0.9 6.8 2.7 1.0 45.9 0.4 1.1 100

Tn-m1 Tn-m2 Tn-f1 Tn-f2 Tn-f3 Tn-f4 Sp-m1 Sp-m2 Sp-f1 Sp-f2 Sp-f3 Sp-f4

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974

0.3 24.1 0.0 1.2 7.4 1.6 0.0 40.6 1.2 0.0 0.0 0.0 0.3 2.9 0.0 1.2 0.4 18.8 0.0 0.0 100

Cy-m

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Is-m

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923 0.0 0.0 933 13.8 16.2 950 0.5 1.0 972 0.7 0.7 979 1.9 3.9 988 1.9 1.1 1007 1.1 0.4 1029 45.5 29.5 1031 3.3 4.2 1034 0.0 0.2 1044 0.0 0.6 1057 0.0 0.0 1293 1.1 0.7 1388 1.1 0.0 1418 5.4 13.2 1454 1.3 2.4 1476 0.0 0.0 1480 22.3 26.0 1498 0.0 0.0 1519 0.0 0.0 100 100

RIb

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α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene Limonene β-Phellandrene β-Ocimene Z β-Ocimene E γ -Terpinene 2-Undecanone β-Elemene Caryophyllene α-Humulene γ -Muurolene Germacrene D α-Muurolene δ-Cadinene Total

Compound

Essential oil content (%)a

TABLE 4. ESSENTIAL OIL CONTENT OF Pistacia lentiscus LEAVES

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FIG. 2. Dendrogram of relations of essential oil content among Pistacia lentiscus accessions, obtained by applying unweighted pair group method with arithmetic averages (UPGMA). See Table 1 for abbreviations.

(Tn-f1, Tn-m2) accessions were clustered together in α-pinene group (35.1– 49.0%); (II) Characterized by relatively high concentration of sabinene (24.1– 45.8%), four Tunisian (Tn-m1, Tn-f2, Tn-f3, Tn-f4) and two Spanish (Sp-m1, Sp-f3) accessions were clustered together; and (III) Germacrene D group (41.1– 45.9%), clustering two Spanish female accessions (Sp-f4, Sp-f2) (Figure 2). Four of the six Spanish accessions were clustered in separate subgroups within each of the limonene dominant group and the sabinene subgroup (Figure 2). None of the six Tunisian accessions was clustered in the limonene group, nor did the Israeli or the Cyprian accessions clustered in the sabinene subgroup (Figure 2). In addition, α-muurolene was found in a low relative concentration (0.4–0.8%) just in four of the Spanish accessions; cis-β-ocimene was present in three accessions, one Israeli (0.2%) and two Tunisian (Tn-m2, Tn-f1) accessions (1.7–1.8%) (Table 4). High relative content of α-thujene (11.1–12.3%) was present in two Spanish accessions, Sp-m1 and Sp-f3 (Table 4). Of the 1000 permutations, the Mantel test, conducted to analyze the relation between the genetic and chemical content matrices indicated low correlation (R = −0.25) with no significant relation between the matrices (P = 0.02). Morphological Characteristics. The number of leaflet pairs ranged from 2 to 3 in Cyprian accessions, 3 to 4 in the Israeli, and 4 to 6 in both Spanish and Tunisian

4.0 (0.0) 3.4 (0.5) 2.0 (0.0) 3.2 (0.8) 5.8 (0.4) 5.0 (0.0) 5.6 (0.5) 4.6 (0.5) 4.0 (0.0) 4.4 (0.5) 5.6 (0.5) 5.6 (0.5) 4.0 (0.0) 4.2 (0.4) 4.0 (0.0) 4.8 (0.4)

2.0 (0.1) 2.0 (0.0) 2.6 (0.3) 2.3 (0.7) 2.2 (0.2) 2.1 (0.1) 2.0 (0.3) 2.3 (0.1) 2.7 (0.2) 2.7 (0.2) 1.9 (0.3) 3.3 (0.2) 2.2 (0.0) 2.3 (0.3) 2.8 (0.1) 2.5 (0.2)

0.8 (0.0) 0.9 (0.1) 1.0 (0.0) 1.0 (0.0) 0.8 (0.1) 0.8 (0.0) 0.6 (0.1) 0.8 (0.1) 0.8 (0.1) 1.1 (0.1) 0.6 (0.1) 1.2 (0.1) 0.9 (0.0) 0.8 (0.1) 0.9 (0.0) 0.8 (0.1)

3.7 2.8 3.4 3.6 2.3 4.8 3.6 2.9 3.1 3.8 3.5 3.3 3.3 3.4 5.6 4.5

79.0 93.0 125.0 61.0 80.0 96.0 75.0 68.0 77.0 83.0 93.0 156.0 135.0 76.0 82.0 95.0

1.7 1.9 1.0 2.5 0.9 1.3 1.7 1.0 1.4 1.3 1.4 1.9 2.3 1.1 1.8 0.5

3 4 5 4 2 3 3 2 2 2 2 2 2 3 2 2

4 4 2 3 1 3 2 4 3 3 4 2 3 5 2 3

3 2 4 3 2 2 1 2 1 3 1 1 2 3 2 1

Length (cm) Width (cm) Trunk diameter (cm) Plant height (cm) Canopy area (m2 ) Texture Color Shape

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Note: Number in brackets indicates standard deviation of five measurements of each accession. For abbreviations see Table 1. Leaflet texture, color, and shape were evaluated by a scale of 1–5: 1 (light green) and 5 (dark green) for color, 1 (flexible) and 5 (leathery) for leaf texture, and from lanceolate (1) and ovate (5) for leaflet shape.

3.2 (0.3) 3.5 (0.3) 3.0 (0.0) 3.0 (0.7) 6.9 (0.6) 4.9 (0.4) 4.2 (0.3) 4.4 (0.4) 4.5 (0.3) 5.7 (0.5) 4.2 (0.4) 7.8 (1.1) 3.6 (0.4) 4.1 (0.2) 5.7 (0.2) 5.3 (0.6)

No.

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Is-m Is-f Cy-m Cy-f Tn-m1 Tn-m2 Tn-f1 Tn-f2 Tn-f3 Tn-f4 Sp-m1 Sp-m2 Sp-f1 Sp-f2 Sp-f3 Sp-f4

Leaf length (cm)

Qualitative traitsb

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Leaflets

TABLE 5. MORPHOLOGICAL CHARACTERISTICS OF MEDITERRANEAN ACCESSIONS OF Pistacia lentiscus GROWING AT BIDR

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accessions (Table 5). In five accessions (Is-m, Cy-m, Tn-m2, Tn-f3, Sp-f1, and Sp-f3), the number of leaflet pairs was equal in all the counted leaves, in others, variability was found (Table 5). A terminal leaflet was observed in a few leaves of some of the accessions. In some accessions, e.g., Sp-m1, Tn-f2, and others, an uneven number of leaflets was also found. A “wing-like” rachis of ca. 1–2 mm was measured in all accessions. Leaf length ranged from 3.0 cm (Cy-m, Cy-f) to 7.8 cm (Sp-m2), and leaflet length and width between 1.9 and 0.6 cm (Sp-m1) to 3.3 and 1.2 cm (Sp-m2), respectively (Table 5). Accession Tn-m1 had the thin trunk diameter (2.3 cm), while Sp-f3 was widest (5.6 cm). Plant height ranged from 61 cm (Cy-f) to 156 cm (Sp-m2), and canopy area from 0.5 m2 (Sp-f4) to 2.5 m2 (Cy-f). ANOVA analysis conducted to compare all accessions, when sorted by gender, on the basis of geographic origin, or chemical composition, did not show significant differences in any of the quantitative morphological traits. Leaflet shape ranged from almost lanceolate (e.g., Sp-m1) to ovate shape (Cy-m) (Table 5). Leaves from Cyprus and Israel accessions were characterized as having the most leathery leaflets, while others had a more flexible texture. Leaf color ranged from light green in the Tunisian accession Tn-m1, to dark green in the Spanish accession Sp-f2 (Table 5).

DISCUSSION

A phylogenetic tree of P. lentiscus analyzed by RAPD divided the accessions according to their geographic origin (Figure 1). Interestingly, the Israeli accessions were closer genetically to Tunisian and Spanish accessions than to the Cyprian accessions. The latter were clustered into a separate group, genetically distinct from the rest, suggesting the genetic isolation of P. lentiscus growing in Cyprus and the development of distinctive genotypes. Phytochemical and morphological characters showed high phenotypic variability among accessions of P. lentiscus, and did not reflect differences by geographic location (Tables 4 and 5). On the basis of the relative content of the 20 constituents of the leaf essential oil, cluster analysis divided the different accessions according to what appears to be chemotypic groups rather than to their geographic origin or gender (Figure 2), thus indicating close resemblance in the chemical content of P. lentiscus around the Mediterranean. The Mantel test, used to compare essential oil content to the genetic matrices, indicated a low, nonsignificant relation between the matrices, suggesting that there was no chemotypic differentiation. However, it is possible that the dominant nature of RAPD markers and the limitation of the technique to detect polymorphism in cases of heterozygosity (Parker et al., 1998) may account for the low correlation between the matrices. Alternatively, genetic RAPD analysis was correlated with the volatile oil constituents

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of accessions, as in the cases of Ocimum gratissimum (Vieira et al., 2001), and Cymbopogon sp. (Sangwan et al., 2001). Therefore, it is possible that using more accessions, and/or additional differential primers, could produce better correlation between the two matrices. In comparison to other reports on P. lentiscus (e.g., Boelens and Jimenez, 1991; Fleisher and Fleisher, 1992; Castola et al., 2000), relatively high content of sabinene and germacrene D was found in leaf extracts, while oxygenated compounds were absent. It is possible that extraction with methyl t-butyl ether may be the cause for the observed differences. However, the effect of environmental conditions on phenotypic plasticity is likely. The effect of stress on the content of essential oil has been reported in several studies. Llusia and Penuelas (1998) reported that under drought conditions total terpene concentration in P. lentiscus, grown under irrigated conditions, was increased by 21.3% compared to control. Drought and nutrient deficiency (phosphorus and/or nitrogen) enhanced allocation of fixed carbon into the biosynthesis of essential oils in Rosmarinus officinalis and Lavandula latifolia (Ross and Sombrero, 1991). UV-B radiation enhanced the level of most of the major volatiles in Ocimum basilicum (Johnson et al., 1999). Therefore, it is possible that under the extreme desert conditions at our germplasm collection in the Negev desert, the concentration of specific compounds (e.g., sabinene and germacrene D) increased. However, to verify this assumption, the effect of environmental stress on the content of P. lentiscus essential oil should be further studied under controlled conditions. More definite indication to the effect of the environmental desert conditions on P. lentiscus was supplemented by morphological characteristics. Asymmetry is an indicator of environmental stress in both plants and animals (Graham et al., 1993). Therefore, the uneven number of paired lateral leaflets and appearance of terminal leaflet, suggests that P. lentiscus is not well adapted to growth under desert conditions. Morphological variability was expressed in most of the analyzed parameters, e.g., leaflet characteristics, plant height, trunk diameter, and canopy area (Table 5). When comparisons were done after sorting the accessions by geographic origin or gender, statistical analysis did not show significant differences in any of the parameters, reflecting the high variability within accessions from each of the geographic regions. Similarly, morphological differences were not observed between genders of P. lentiscus in Portugal (Barradas and Correia, 1999). The large morphological and chemical variability found among accessions of P. lentiscus on one hand, and the genotypic differentiation on the other hand, may contribute to the wide distribution of P. lentiscus around the Mediterranean basin. P. lentiscus was found to be one of the most drought tolerant plants among other evergreen species in the Mediterranean maquis (Gratani, 1995). Different ecophysiological characteristics such as drought resistance, fast regeneration after fires, and resistance to herbivores contribute to the potential of P. lentiscus for forestry in our region. This emphasizes the importance in preserving the genetic

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variability of this species. The data reported in this work are based on accessions available in the live germplasm collection at BIDR and are not yet enough to extrapolate from these results to the genetic variability in nature. Further sampling is needed to determine the genetic variability and establishment of conservation management practices. The fact that the plants were grown together under similar conditions reduced the effect of environmental variation on the measured differences. Thus, the Pistacia spp. live germplasm collection at BIDR enabled study of the genetic basis of observed and measured phenotypic polymorphisms. While genetic analysis separated the accessions according to their geographic origin, significant phenotypic differences, i.e., content of essential oil and morphological parameters, were not observed. This points to the importance of DNA molecular marker techniques when ecotypic or chemotypic differentiation is discussed. Acknowledgments—Our thanks to colleagues around the Mediterranean basin, who assisted in seed collection. The technical assistance of Mrs Olga Larkov in GC-MS analyses is greatly appreciated. This research work was supported partially by AID-CDR grant TA-MOU-98-CA17-028 and the Bona Terra Foundation.

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