Chemical variability of peel and leaf essential oils of 15 species of mandarins

Biochemical Systematics and Ecology 29 (2001) 77}104 Chemical variability of peel and leaf essential oils of 15 species of mandarins Marie-Laure Lota, Dominique de Rocca Serra, FeH lix Tomi, Joseph Casanova* Universite& de Corse - Equipe Chimie et Biomasse, URA CNRS 2053, Route des Sanguinaires, 20000 Ajaccio, France Received 1 December 1999; received in revised form 24 February 2000; accepted 28 February 2000 Abstract Peel and leaf oils of 58 mandarin cultivars, belonging to 15 di!erent species were obtained from fruits and leaves collected on mandarin-trees submitted to the same pedoclimatic and cultural conditions. Their chemical composition was investigated by capillary GC, GC/MS and C NMR and the results were submitted to a cluster analysis and a discriminant analysis. Three major chemotypes, limonene, limonene/γ-terpinene and linalyl acetate/limonene, were distinguished for peel oils while three other chemotypes, sabinene/linalool, γ-terpinene/linalool and methyl N-methylanthranilate, were observed for leaf oils. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Rutaceae; Citrus; Mandarin; Peel oil; Leaf oil; Essential oil composition; GC; GC/MS; C NMR; Statistical analysis 1. Introduction Mandarins (Rutaceae family, Citrus genus) predominate with oranges the fresh fruit market. According to Tanaka (1961), they are classi"ed into more than 30 species, comprising from one to several tens of varieties. Cultivars of mandarin present a great diversity of morphological and horticultural characters. * Corresponding author. Tel.: 33-4-95-52-41-21; fax: 33-4-95-52-41-42. E-mail address: casanova@vignola.univ-corse.fr (J. Casanova). 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 2 9-6

78 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 As for most of the Citrus, mandarin peel oil and leaf or `petit graina oil can be obtained, respectively, by cold pressing and hydrodistillation of the fresh material. Studies concerning the chemical composition of peel and leaf oils of mandarins have been reviewed (Shaw, 1979; Lawrence, 1995a, b, 1996; Mondello et al., 1997). Nevertheless, in most cases, the species and/or the varieties remained unspeci"ed and the results are not useful from the taxonomic point of view. The aim of our work was to study the inter- and intraspeci"c chemical variability of the large mandarin group. We divided our work into two parts: the "rst concerned 41 cultivars from C. reticulata Blanco, (Lota et al., 2000); the second dealt with 15 other species of mandarin, including C. clementina Hort. ex Tan. We report our results on the chemical variability of these 15 species represented by 58 cultivars of mandarin. We will compare the chemical composition of peel oils on the one hand and that of leaf oils on the other. We will discuss chemical variability including our previous results concerning cultivars from C. reticulata Blanco (Lota et al., 2000). 2. Materials and methods 2.1. Plant materials Clonal propagated trees, grafted on Troyer citrange rootstock, were 12 years old and grown in the same pedoclimatic and cultural conditions in the germplasm collection orchard of the `Station de Recherches Agronomiquesa of INRA-CIRAD, located at San Ghjulianu (Corsica, France). The Citrus varieties collection of INRA-CIRAD in Corsica is one of the FAO recognized Citrus collection in the world. In this arboretum, each tree has a computerized identi"cation number. Geographic and climatic characteristics were: average per year: rainfall 840 mm and temperature 15.23C, soil derived from alluvial deposits and classi"ed as fersiallitic, ph range 5.0}5.6. Trees were in good vigor, disease-free and without visible insect infestation. 2.2. Sampling, peel and leaf essential oil For each cultivar of mandarin, about 500 g of leaves from the last autumn leaf #ush and at least 30 ripe fruits were collected from many parts round the same tree, early in the morning and only by dry weather during the period November 1996 to April 1997. The peel of fresh fruits was cold-pressed and then the essential oil was separated from the crude-extract by centrifugation (10 min at 15000 rpm). Fresh leaves were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. Yield ranged between 0.05 and 0.60%.

2.3. GC, GC/MS and C NMR analyses Identi"cation of components and data processing were carried out as previously reported for peel and leaf oils from C. reticulata (Lota et al., 2000). All peel and leaf oils were investigated by GC. Five peel oils and seven leaf oils were analysed by GC/MS while 23 peel oils and 25 leaf oils were analysed by C NMR, following a methodology "rst reported by FormaH cek and Kubeczka (1982), developed in our laboratory (Tomi et al., 1995) and well-suited for chemical polymorphism studies (Salgueiro et al., 1997; Corticchiato et al., 1998; Castola et al., 2000). Samples submitted to GC/MS and/or C NMR analysis were selected on the basis of their chromatographic pro"le. Note that 56 peel oils instead of 58 were analysed because the mandarin-trees of `ougona and `shekwashaa cultivars do not produce fruits in the climatic conditions of San Ghjulianu. 2.4. Data analyses M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 79 The data (components *1% for peel and leaf oils, respectively) were processed by cluster analysis using hierarchical clustering (Ward`s technique and Euclidean distance measure) and were submitted to discriminant analysis. These processing were performed with the xlstat-pro software. 3. Results The 58 following cultivars, belonging to 15 species, were investigated. For the convenience of comparison of the present results with the previous ones for 41 varieties from C. reticulata (Lota et al., 2000), we numbered the samples of this study from 42 to 99. Citrus clementina Hort. ex Tan. species : MA3 (no. 42), Nules (no. 43), MA2 (no. 44), Hernandina (no. 45), Tardia Villareal (no. 46), Reina (no. 47), Ca$n (no. 48), MacBean (no. 49), Oroval (no. 50), Monreal (no. 51), Bruno (no. 52), Tomatera (no. 53), Commune (no. 54), Marisol (no. 55), Ragheb (no. 56), Guillermina (no. 57), C. deliciosa Ten. species : Late Emperor (no. 58), Empress (no. 59), Emperor (no. 62), Peau rugueuse (no. 63), Peau lisse (no. 76), Commune (no. 79), de Chios (no. 92), Avana Apireno (no. 93), Willow leaf (no. 90), Tardivo di Ciaculli (no. 94), C. nobilis Lour. species : Geleking (no. 74), Yellowking (no. 75), King of Siam (no. 80), Du Japon (no. 85), King (no. 86), Rode king (no. 87), Kunembo (no. 89), C. tangerina Hort. ex Tan. species : Vohangisahy (no. 60), Beauty of Glen Retreat (no. 61), Brickaville (no. 64), Dancy (no. 66), Redskin (no. 68), Swatow (no. 78), C. unshiu Mac. Mark. species : Wase (no. 65), Clausellina (no. 70), URSS (no. 71), Owari (no. 72), C. suhuiensis Hort. ex Tan. species : Sihue Gan (no. 73), Szibat (no. 91), Szinkom (no. 96); C. temple Hort. ex Y. Tan. species : Temple Sue Linda (no. 81), Temple Temple (no. 83), Temple (no. 84),

80 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 C. paratangerina Hort. ex Tan. species : Ladu (no. 69), Ladu Ordinary (no. 77), C. amblycarpa Hassk. Ochse. species : Nasnaran (no. 88), C. depressa Hay. species : Shekwasha (no. 98), C. erythrosa Hort. ex Tan. species : Fuzhu (no. 67), C. reshni Hort. ex Tan. species : Cleopatra (no. 82), C. suavissima Hort. ex Tan. species : Ougon (no. 99), C. sunki Hort. ex Tan. : Sunki (no. 95), C. yatsushiro Hort. ex Tan. : Yatsushiro (no. 97). In order to simplify the discussion, we will "rst describe our results for C. clementina Hort. ex Tan. All other taxa will be reported in Section 3.2. 3.1. Citrus clementina Hort. ex Tan. 3.1.1. Peel oils The chemical composition of the 16 investigated samples are presented in Table 1. The 30 identi"ed components accounted for 97.3}99.5% of the total amount of oil. Peel oils consisted almost exclusively of hydrocarbons with limonene as the major component (89.1}95.5%) with sabinene (0.3}4.0%) and myrcene (1.4}2.0%). α-pinene, β-phellandrene, β-pinene, (E)-β-ocimene, 3-carene and γ-terpinene were identi"ed in almost all samples at low amounts (tr-0.6%). The oxygenated fraction was made up of linalool (0.6}2.3%), octanal, decanal, citronellal, α-terpineol, α-sinensal and β-sinensal ()0.7% for each one). The homogeneous composition of our 16 samples is similar to that reported in the literature (percentage of limonene: 92}97%) for `communea and `nulesa cultivars from Uruguay (Verzera et al., 1998), for `orovala, `monreala and `communea cultivars from Italy (Calabria) (Verzera et al., 1997) and for unspeci"ed cultivars from Italy (Calabria, Sicily) (Mondello et al., 1995), Algeria (Baaliouamer et al., 1992) and unspeci"ed cultivars from unspeci"ed origin (Calvarano et al., 1974; Huet, 1991; Gazea et al., 1998; Ruberto et al., 1993, 1994, 1997). 3.1.2. Leaf oils The 45 identi"ed components accounted for 96.1 to 99.8% of the total amount of oil (Table 2). All samples exhibited a high sabinene/linalool composition (33.1}49.8%/16.6}24.7%). The other main components of the ole"nic fraction (20.9}28.2%) were limonene, 3-carene, (E)-β-ocimene, myrcene, β-pinene, γ-terpinene, α-pinene, terpinolene, α-terpinene and β-phellandrene (0.8}6.9% each). Terpinen-4-ol, α-terpineol, trans-sabinene hydrate, citronellal, citronellol, geranyl acetate, α-sinensal and β-sinensal were also identi"ed in almost all samples (0.1}4.8%). The oxygenated fraction represented less than 40% of the whole oil. The composition of a few leaf oils from clementin are reported in the literature. Three samples (unspeci"ed cultivar) from Italy were characterized by a β- pinene/linalool composition (approximately 45%/15%) (Di Giacomo et al., 1982). Spanish oils from 12 cultivars `rufatinaa, `clemennullesa, `clemenvillaa, `esbala, `"naa, `guillermaa, `hernandinaa, `monreala, `orovala and `tomateraa exhibited a major component of unknown structure (19.2}48.8%) associated with linalool

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 81 Table 1 Chemical composition of clementin peel oils Constituents BP-20 BP-1 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 α-pinene 1021 930 0.3 0.4 0.3 0.3 0.4 0.3 0.3 0.4 0.3 0.5 0.5 0.5 0.5 0.3 0.6 * β-pinene 1109 971 tr tr tr tr tr tr tr t r tr 0.1 0.1 0.1 0.1 0.1 0.2 0.1 Sabinene 1119 964 0.3 0.7 0.4 0.6 1.0 0.9 0.9 0.6 0.9 1.3 1.8 1.8 1.2 1.8 4.0 2.8 3-Carene 1145 1005 * tr 0.1 tr tr tr tr 0.1 0.1 0.1 0.1 0.1 * tr 0.1 0.1 Myrcene 1157 978 1.6 1.7 1.6 1.4 1.6 1.6 1.7 1.6 1.6 1.8 1.8 1.8 1.7 1.6 2.0 1.6 Limonene 1199 1021 95.5 95.0 95.0 94.6 94.1 94.1 94.1 93.9 93.5 92.9 92.7 92.2 91.8 91.2 90.1 89.1 β-phellandrene 1208 1021 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 γ-terpinene 1241 1048 tr tr tr * tr tr tr 0.1 tr 0.1 tr 0.1 tr tr tr tr (E)-β-Ocimene 1245 1035 tr tr tr tr tr tr 0.2 tr tr 0.1 0.1 0.1 tr tr 0.1 0.1 Octanal 1285 978 0.1 0.1 tr 0.1 0.3 0.2 0.2 * 0.2 0.2 0.3 0.3 0.3 0.3 0.2 0.1 trans-sabinene hydrate 1458 1053 * tr * tr tr tr tr tr tr tr tr tr * 0.1 tr 0.2 Citronellal 1473 1130 tr tr * tr tr 0.1 tr tr 0.1 tr tr tr tr 0.1 tr 0.1 α-copaene 1484 1379 tr tr tr tr tr tr tr 0.1 tr tr tr tr tr tr tr 0.1 Decanal 1492 1183 0.2 0.2 0.1 0.1 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.5 Linalool 1539 1082 0.8 0.6 0.8 0.8 1.2 0.9 0.8 1.0 1.5 0.8 1.0 1.1 0.8 2.3 0.7 1.9 trans-α-bergamotene 1580 1432 * * * tr * tr tr 0.1 tr * * * tr tr tr * (E)-β-Farnesene 1654 1448 * * * tr * tr tr * * * * * tr tr tr 0.1 α-terpineol 1688 1172 0.1 tr tr 0.1 0.1 0.1 tr 0.1 0.1 tr 0.1 0.1 * 0.1 0.1 0.2 Germacrene-D 1704 1480 tr tr tr tr tr tr tr tr tr tr tr tr * tr tr 0.1 Geranial 1742 1242 * * tr tr tr * * 0.1 * 0.1 * * * * 0.1 * δ-cadinene 1746 1516 tr tr tr * tr * * 0.1 tr tr tr * * tr * 0.1 β-sinensal 2225 1673 tr 0.1 tr 0.1 tr 0.1 0.2 * 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.3 α-sinensal 2323 1726 0.1 0.3 0.2 0.1 0.2 0.3 0.4 0.4 0.4 0.3 0.4-0.3 0.5 0.4 0.7 Total 99.3 99.4 98.8 98.4 99.4 99.2 99.3 99.1 99.3 98.9 99.5 98.8 97.3 99.0 99.3 98.5 Other compounds ( tr): p-cymene, α-phellandrene, terpinolene, nonanal, cis-limonene-1,2-oxide, trans-limonene-1,2-oxide, citronellol. Cultivars : MA3 (42), Nules (43), MA2 (44), Hernandina (45), Tardia Villareal (46), Reina (47), Ca$n (48), Mac Bean (49), Oroval (50), Monreal (51), Bruno (52), Tomatera (53), Commune (54), Marisol (55), Ragheb (56), Guillerma (57). Order of elution and percentages of components are given on BP-20 column. All the components were identi"ed by GC-RI on polar and apolar columns. All the compounds of samples no. 49 and 54 were also identi"ed by GC/MS. The major components (bold letters) of samples no. 51, 53, 54, 56 and 57 were identi"ed by C NMR.

82 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 2 Chemical composition of clementin leaf oils Constituents BP-20 BP-1 56 43 47 53 46 52 55 45 44 48 51 49 54 42 50 57 α-thujeneh 1021 922 0.3 0.3 0.3 0.3 0.4 0.4 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.4 0.3 0.4 α-pineneh 1021 930 1.3 1.6 1.4 1.4 1.1 1.5 0.7 1.6 1.5 1.5 0.8 1.7 1.5 1.6 1.5 1.2 Camphene 1066 944 tr tr tr 0.1 tr tr tr tr tr tr tr tr tr tr tr tr β-pinene 1109 971 1.7 1.8 1.7 1.8 1.9 1.9 1.8 2.2 1.9 2.0 2.1 2.1 2.1 2.0 2.2 2.3 Sabinene 1119 964 33.1 35.1 34.3 35.2 38.1 37.9 36.9 40.1 39.9 41.8 43.5 42.5 42.0 41.9 47.0 49.8 3-Carene 1145 1005 4.5 6.5 6.5 6.2 4.8 4.4 5.1 3.8 3.8 2.6 3.3 3.8 4.1 3.8 2.4 3.0 Myrcene 1157 978 2.8 3.3 3.2 3.2 3.0 3.1 3.0 3.1 3.0 3.0 3.0 3.2 3.3 3.2 3.0 3.2 α-phellandrene 1162 997 0.5 0.7 0.6 0.6 0.5 0.5 0.5 0.4 0.4 0.3 0.4 0.4 0.4 0.4 0.3 0.4 α-terpinene 1179 1009 1.3 1.3 0.9 1.2 1.0 1.4 1.0 1.2 1.0 1.2 1.2 1.2 1.2 1.0 0.9 1.1 Limonene 1199 1021 6.9 2.5 2.7 3.1 2.8 2.9 4.3 4.8 3.8 3.3 3.6 2.9 3.5 4.0 4.2 3.0 β-phellandrene 1208 1021 0.8 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.9 (Z)-β-Ocimene 1228 1024 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.2 0.1 0.1 γ-terpinene 1241 1048 2.1 1.9 1.5 1.9 1.6 2.2 1.5 1.9 1.5 2.0 2.0 1.9 1.9 1.6 1.5 1.7 (E)-β-Ocimene 1245 1035 3.8 5.5 5.6 4.8 4.8 5.4 5.4 3.8 3.9 4.2 3.6 4.4 4.5 4.6 3.8 2.5 p-cymene 1267 1012 tr tr 0.3 tr 0.2 tr 0.1 tr tr tr tr tr 0.1 tr tr 0.1 Terpinolene 1278 1078 1.2 1.5 1.4 1.5 1.2 1.2 1.2 1.1 1.0 0.9 1.0 1.1 1.1 1.0 0.8 1.0 6-Methylhept-5- en-2-one 1332 963 tr 0.1 tr 0.1 tr * tr * * - * * * * * tr trans-sabinene hydrate 1458 1053 0.7 0.6 1.0 0.9 1.4 0.9 1.0 0.8 1.2 0.9 0.9 1.0 1.2 1.0 1.1 1.3 Citronellal 1473 1130 3.4 1.6 3.7 3.0 1.4 1.2 2.2 1.5 1.5 0.9 1.8 1.6 0.9 1.1 0.3 1.7 Decanal 1492 1183 tr tr * tr * tr tr tr tr tr * * tr 0.1 0.1 tr Linalool 1539 1082 23.5 19.4 21.7 22.9 24.7 22.7 21.8 22.6 23.9 24.2 21.5 20.5 21.0 21.6 22.0 16.6 cis-p-menth-2-en-1-ol 1555 1107 0.3 0.3 0.2 0.3 0.3 0.3 0.2 0.3 0.2 0.3 0.3 0.3 0.3 0.2 0.2 0.3

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 83 (E)-Caryophyllene 1588 1420 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 tr 0.1 0.1 0.2 0.1 tr Terpinen-4-ol 1595 1162 4.6 4.6 3.6 3.7 4.3 4.8 3.6 4.0 3.5 4.3 4.6 4.2 4.1 3.6 3.3 4.5 trans-p-menth- 1618 1123 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 2-en-1-ol (E)-β-Farnesene 1654 1448 tr tr tr * tr * tr * 0.1 * tr tr * 0.1 tr * Neral 1674 1213 0.2 0.6 0.4 0.4 0.1 0.2 0.3 tr 0.1 tr 0.1 tr 0.1 tr tr 0.1 α-terpineol 1688 1172 1.1 1.4 1.2 1.2 1.3 1.0 1.0 0.8 1.1 0.7 0.7 0.7 0.8 0.8 0.7 1.1 Neryl acetate 1725 1340 0.1 * * 0.1 * 0.3 0.4 * * 0.1 * tr * * * 0.2 Bicyclogermacrene 1727 1494 0.1 0.1 * * * * * * * * * 0.1 tr tr * * Geranial 1742 1242 0.3 0.8 0.6 0.6 0.2 0.1 * * 0.2 0.1 0.1 * 0.1 * tr 0.1 Geranyl acetate 1748 1358 0.7 0.4 0.5 0.4 0.2 0.2 1.0 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.2 Citronellol 1756 1207 0.7 0.3 0.7 0.5 0.3 0.3 0.6 0.4 0.4 0.3 0.4 0.3 0.3 0.3 0.1 0.5 Nerol 1790 1207 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 tr 0.1 Geraniol 1837 1232 0.1 0.5 0.1 0.1 tr 0.1 0.2 tr tr tr tr tr tr tr tr tr (E)-Nerolidol 2031 1547 0.1 tr 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 tr 0.1 0.1 0.1 0.1 tr β-sinensal 2225 1673 2.0 1.2 2.2 1.8 1.6 1.9 2.0 2.1 2.1 2.5 1.8 2.2 2.2 2.6 2.2 0.8 α-sinensal 2323 1726 0.4 0.6 1.1 0.9 0.8 1.0 1.0 0.7 0.8 0.8 0.6 0.7 1.0 1.0 0.9 0.3 Total 99.2 96.1 98.9 99.8 99.5 99.3 98.5 98.9 98.6 99.7 99.1 98.8 99.8 99.5 99.6 98.6 Other compounds (tr): octanal, nonanal, citronellyl acetate, α-humulene, (E,E)-α-farnesene, δ-cadinene, thymol. Cultivars : Ragheb (56), Nules (43), Reina (47), Tomatera (53), Tardia Villareal (46), Bruno (52), Marisol (55), Hernandina (45), MA2 (44), Ca$n(48), Monreal (51), Mac Bean (49), Commune (54), MA3 (42), Oroval (50), Guillerma (57). Order of elution and percentages of components are given on BP-20 column, except compounds with an asterisk (percentages given on BP-1 column). All the components were identi"ed by GC-RI on polar and apolar columns. All the compounds of samples no. 47, 54 and 56 were also identi"ed by GC/MS. The major components (bold letters) of samples no. 50, 53, 54 and 56 were identi"ed by C NMR.

84 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 (16.7}27.1%) (Ortiz Marcide et al., 1983). It is likely if not certain that the unidenti"ed major component reported by Ortiz Marcide is sabinene. The sabinene/linalool chemotype, found in our sampling, is con"rmed unambiguously for the "rst time for C. clementina leaf oils in this study. 3.2. The other species of mandarin 3.2.1. Peel oils The total of the 44 identi"ed components accounted for 95.8}99.7% of the oil (Table 3). Even though the composition was dominated by limonene (55.8}96.7%) for 39 samples over 40, the content of the major components varied considerably from sample to sample. Several other monoterpene hydrocarbons were frequently identi- "ed at appreciable contents: γ-terpinene (tr-19.9%), p-cymene (tr-12.0%), myrcene (0.7}24.0%), β-pinene (tr-14.2%), sabinene (0.1}8.7%), α-pinene (0.2}2.2%) and β- phellandrene (0.2}0.8%). Among the oxygenated compounds, linalool was present in all the samples (0.1}10.7%) whereas percentages of octanal, α-terpineol and decanal were not over 0.5%. The contents of citronellal (9.9%) and of linalyl acetate (48.7%) were important, respectively, in two samples (no. 88, `Nasnarana cultivar from C. amblycarpa species and no. 97, `Yatsushiroa cultivar from C. yatsushiro species). The dendrogram, obtained from cluster analysis and reported in Fig. 1, suggested the existence of three principal clusters (I, II and III) within the essential oil of the individuals of mandarin. Discriminant analysis (Fig. 2) con"rmed this clustering with respect to the contents of limonene, γ-terpinene and linalyl acetate (Fig. 3). Limonene chemotype : most of the peel oils (no. 58}87) were characterized by a very high amount of limonene (87.1}96.7%). This chemotype (cluster I) could be divided into two subgroups on the basis of the content of γ-terpinene, which was very low ()0.9%) in the subgroup IA (samples no. 79}87) and higher (3.5}5.8%) in the subgroup IB (samples no. 58}78) Limonene/γ-terpinene chemotype: nine samples (no. 88}96) belonged to this chemotype (cluster II). The composition was dominated by limonene (55.8}79.0%) associated with γ-terpinene (0.1}19.9%) and p-cymene (0}12.0%). Three samples (no. 88, 89 and 96), which belonged to the same group, exhibited quantitative di!erences in their composition. Indeed, the sample no. 88 was discriminated by higher contents of β-pinene (14.2% vs 0.2}1.6%), sabinene (8.7% vs 0.1}2.1%) and citronellal (9.9% vs tr-0.1%). Similarly, samples no. 89 and 96 were distinguished, respectively, by important amounts of myrcene (24.0% vs 1.2}1.7%) and p-cymene (12.0% vs 0}6.9%). Linalyl acetate/limonene chemotype: only one sample (no. 97) belonged to the cluster III and was characterized by the predominance of linalyl acetate (48.7%) over limonene (22.8%), γ-terpinene (5.6%) and β-pinene (5.4%). A few studies are reported on the chemical composition of peel mandarin oils from species other than C. reticulata and C. clementina. Essential oils of C. unshiu were reviewed by Lawrence (1989) and divided into two chemotypes: (i) limonene (78}96%) from `praecoxa cultivar from Japan (Lawrence, 1989), unspeci"ed cultivar from Japan (Yamanishi et al., 1968; Yajima et al., 1979), from Georgia, Russia and from unspeci"ed origin (Lawrence, 1989), (ii) limonene/p-cymene (42}79%/4}27%)

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 85 Table 3 Chemical composition of peel mandarin oils Constituents BP-20 BP-1 IB 58 59 60 61 62 63 64 65 66 67 68 69 70 71 α-thujeneh 1021 922 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.1 α-pineneh 1021 930 0.7 0.6 0.9 0.6 0.6 0.4 0.5 0.5 0.5 0.6 0.7 0.9 0.5 0.5 β-pinene 1109 971 0.3 0.3 0.4 0.3 0.3 0.2 0.3 0.4 0.3 0.4 0.3 0.4 0.4 0.4 Sabinene 1119 964 0.2 0.2 0.1 0.1 0.3 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Myrcene 1157 978 1.6 1.6 1.7 1.6 1.6 1.5 1.5 1.7 1.5 1.6 1.6 1.7 1.8 1.6 α-terpinene 1179 1009 tr 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 tr 0.1 0.1 Limonene 1199 1021 91.4 90.8 90.9 90.2 90.4 90.5 90.2 90.3 87.1 88.4 89.2 87.6 88.0 88.0 β-phellandrene 1208 1021 0.3 0.2 * 0.2 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.3 0.2 0.2 γ-terpinene 1241 1048 3.4 4.0 3.9 4.3 4.2 4.3 4.7 4.3 5.2 4.9 4.4 4.6 5.3 5.8 (E)-β-Ocimene 1245 1035 tr 0.1 0.1 0.2 0.1 0.2 0.2 tr 0.2 0.1 0.1 0.2 0.1 0.1 p-cymene 1267 1012 0.2 0.3 0.4 0.2 0.4 0.5 0.2 0.1 0.2 0.1 0.3 0.4 0.2 0.2 Terpinolene 1278 1078 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.3 Octanal 1285 978 0.2 tr 0.1 0.1 0.1 * 0.1 tr 0.2 0.2 0.1 0.2 * tr trans-limonene- 1,2-oxide 1451 1120 tr tr tr * tr tr * * *- * * * * * Citronellal 1473 1130 * tr * tr * * tr tr tr tr * tr * tr Linalool 1539 1082 0.5 0.6 0.4 0.7 0.6 0.4 0.6 0.2 2.2 1.9 1.7 0.8 0.3 0.2 Linalyl acetate 1549 1239 * * * * * * * * tr tr * tr * * β-elemene 1586 1388 * tr * * * * * 0.2 0.1 tr * * 0.6 0.4 (E)-β-Farnesene 1654 1448 * * * * * * * * * * * * * * α-terpineol 1688 1172 0.1 0.3 tr 0.1 tr 0.1 0.1 tr 0.1 0.1 0.1 0.1 tr tr β-bisabolene 1721 1500 * * * * * * * * 0.1 * * * * * Neryl acetate 1725 1340 * * * tr * * * * * * * * * * (E,E)-α-Farnesene 1744 1500 * * * * * * * 0.3 * * * * 0.7 0.6 Geranyl acetate 1748 1358 tr tr * * tr tr * * tr tr * tr * * Citronellol 1756 1207 * tr * * * tr * * tr * * * * * Methyl N- 2082 1385 * *- * * * * * * * * * * * * methylanthranilate α-sinensal 2323 1726 tr tr 0.1 0.2 * 0.2 0.2 * 0.2 0.1 0.1 0.2 * * Total 99.3 99.5 99.6 99.3 99.5 99.2 99.5 99.1 98.7 99.3 99.4 98.0 99.2 99.1 *continued

86 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 3*continued IB IA Constituents BP-20 BP-1 72 73 74 75 76 77 78 79 80 81 82 83 84 85 α-thujeneh 1021 922 0.2 0.2 0.1 0.1 0.1 0.2 0.1 * tr tr * * tr * α-pineneh 1021 930 0.8 0.7 0.6 0.5 0.5 0.8 0.6 0.6 0.9 0.5 0.3 0.5 0.5 0.4 β-pinene 1109 971 0.4 0.4 0.3 0.3 0.3 0.4 0.3 1.5 0.2 tr tr tr tr tr Sabinene 1119 964 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.3 0.4 1.0 1.1 0.4 0.9 0.2 Myrcene 1157 978 1.8 1.7 1.6 1.6 1.5 1.7 1.6 1.8 1.5 1.8 1.7 1.7 1.7 1.6 α-terpinene 1179 1009 0.1 0.1 0.1 0.1 0.1 tr 0.1 * tr * * * * * Limonene 1199 1021 88.8 89.1 89.2 89.2 89.6 89.6 89.6 92.6 93.1 93.3 93.6 93.9 90.7 95.4 β-phellandrene 1208 1021 0.3 0.2 0.3 0.3 0.3 0.2 0.3 0.3 0.8 0.2 0.3 0.3 0.2 0.3 γ-terpinene 1241 1048 5.5 5.3 5.1 5.1 4.7 4.8 5.2 * 0.9 * tr tr tr tr (E)-β-Ocimene 1245 1035 tr 0.2 0.5 0.6 0.2 0.2 tr 0.3 0.1 0.1 0.1 0.1 0.1 * p-cymene 1267 1012 0.2 0.2 0.2 0.2 0.4 0.1 0.1 * 0.3 * * * * * Terpinolene 1278 1078 0.3 0.2 0.2 0.2 0.2 0.2 0.2 * 0.1 tr tr tr tr * Octanal 1285 978 * 0.1 0.3 tr * 0.1 0.1 0.1 * 0.4 0.1 0.3 0.4 0.4 trans-limonene- 1,2-oxide 1451 1120 * * * * tr * tr * * * * * * * Citronellal 1473 1130 tr tr tr tr tr * tr tr * tr * tr tr 0.1 Linalool 1539 1082 0.1 0.8 0.2 0.2 0.6 0.7 0.6 0.1 0.2 1.7 1.0 1.1 4.0 0.6 Linalyl acetate 1549 1239 * * * * tr * * * 0.2 * * * tr * β-elemene 1586 1388 0.3 * * * * * tr * * * * * * * (E)-β-Farnesene 1654 1448 tr * * * * * * tr 0.1 * * * * 0.1 α-terpineol 1688 1172 * 0.1 0.1 0.1 0.1 0.2 0.1 tr 0.1 tr 0.1 0.1 0.1 tr β-bisabolene 1721 1500 * * * * * * * tr tr * * * * * Neryl acetate 1725 1340 * * tr * * 0.1 tr * * * - 0.1 0.2 * (E,E)-α-Farnesene 1744 1500 * * tr * * * * * * * * * * * Geranyl acetate 1748 1358 * tr * * * tr tr 0.2 * * * * * * Citronellol 1756 1207 * * * * tr * tr * * * * * * * Methyl N- 2082 1385 * * * * * * * * * * * * * * methylanthranilate α-sinensal 2323 1726 * 0.1 * * 0.3 0.1 tr * * tr * * 0.1 * Total 99.5 99.6 99.3 99.0 99.2 99.5 99.3 98.5 98.3 99.3 98.5 98.7 99.2 99.4

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 87 Constituents IA II III BP-20 BP-1 86 87 88 89 90 91 92 93 94 95 96 97 α-thujeneh 1021 922 * * 0.1 tr 0.5 0.9 0.6 0.6 0.5 0.1 0.4 0.2 α-pineneh 1021 930 0.4 0.5 1.3 0.2 1.3 2.2 1.9 0.7 1.8 0.4 1.7 0.7 β-pinene 1109 971 tr tr 14.2 0.2 1.3 1.6 1.3 1.3 1.3 0.5 1.3 5.4 Sabinene 1119 964 0.1 0.1 8.7 0.1 2.1 0.2 0.2 0.2 0.2 0.1 0.3 0.9 Myrcene 1157 978 1.7 1.7 1.7 24.0 1.6 1.6 1.7 1.7 1.5 1.3 1.2 0.7 α-terpinene 1179 1009 * * * 0.1 0.2 0.4 0.4 0.3 0.1 0.1 * 0.1 Limonene 1199 1021 96.7 96.7 55.8 63.7 65.3 68.9 73.1 75.3 74.5 79.0 75.0 22.8 β-phellandrene 1208 1021 0.3 0.3 0.5 0.3 0.2 0.2 0.2 0.2 0.3 0.2 0.4 0.2 γ-terpinene 1241 1048 tr tr 0.1 5.8 15.9 19.9 17.3 16.7 13.7 11.5 0.7 5.6 (E)-β-Ocimene 1245 1035 tr tr 0.2 * * * * tr tr * * 0.3 p-cymene 1267 1012 tr * * 0.2 6.9 1.2 0.5 0.5 2.8 1.3 12.0 0.1 Terpinolene 1278 1078 * * tr 0.3 0.7 0.8 0.8 0.7 0.5 0.4 tr 0.2 Octanal 1285 978 0.1 0.1 0.5 * 0.1 0.1 0.1 0.1 tr 0.1 0.2 tr trans-limonene- 1,2-oxide 1451 1120 * * * * tr tr * * 0.1 tr 0.7 * Citronellal 1473 1130 * tr 9.9 0.1 tr tr tr tr tr 0.1 tr * Linalool 1539 1082 0.1 0.1 0.4 0.1 0.6 0.2 0.2 0.2 0.2 1.4 0.8 10.7 Linalyl acetate 1549 1239 * * tr * * * * * tr * * 48.7 β-elemene 1586 1388 * * 0.3 * tr * * * tr 0.3 * * (E)-β-Farnesene 1654 1448 tr tr * 0.4 * * * * * * * 0.1 α-terpineol 1688 1172 tr tr * 0.1 0.4 tr 0.2 0.2 0.2 0.2 0.3 * β-bisabolene 1721 1500 * * * * * * * * * * 0.3 0.4 Neryl acetate 1725 1340 * * * 0.9 tr * * * * 0.1 * 0.5 (E,E)-α-Farnesene 1744 1500 tr * 1.5 * * 0.9 * * * * * * Geranyl acetate 1748 1358 0.1 0.1 * 1.6 * tr * * tr 0.2 tr 0.3 Citronellol 1756 1207 * * 0.7 0.2 * tr tr * tr tr * * Methyl N- methylanthranilate 2082 1385 * * * * 1.3 * 0.4 0.3 * * * * α-sinensal 2323 1726 * * 0.8 * 0.3 * 0.2 0.2 0.3 * 0.1 * Total 99.6 99.7 97.9 98.4 99.0 99.3 99.3 99.3 98.2 97.7 95.8 99.1 *¹able footnote continued overleaf

88 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 3*footnote continued Other compounds (tr-0.3%): camphene, α-phellandrene, (Z)-β-ocimene, nonanal, cis-limonene-1,2-oxide, trans-sabinene hydrate, octyl acetate, α-copaene, decanal, trans-α-bergamotene, (E)-caryophyllene, terpinen-4-ol, α-humulene, neral, α-terpinyl acetate, germacrene-d, bicyclogermacrene. Cultivars : Late Emperor (58, C. deliciosa), Empress (59, C. deliciosa), Vohangisahy (60, C. tangerina), Beauty of Glen Retreat (61, C. tangerina), Emperor (62, C. deliciosa), Peau rugueuse (63, C. deliciosa), Brickaville (64, C. tangerina), Wase (65, C. unshiu), Dancy (66, C. tangerina), Fuzhu (67, C. erythrosa), Redskin (68, C. tangerina), Ladu (69, C. paratangerina), Clauselina (70, C. unshiu), URSS (71, C. unshiu), Owari (72, C. unshiu), Sihue Gan (73, C. suhuiensis), Geleking (74, C. nobilis), Yellowking (75, C. nobilis), Peau lisse (76, C. deliciosa), Ladu Ordinary (77, C. paratangerina), Swatow (78, C. tangerina), Commune (79, C. deliciosa), King of Siam (80, C. nobilis), Temple Sue Linda (81, C. temple), Cleopatra (82, C. reshni), Temple x Temple (83, C. temple), Temple (84, C. temple), Du Japon (85, C. nobilis), King (86, C. nobilis), Rode king (87, C. nobilis), Nasnaran (88, C. amblycarpa), Kunembo (89, C. nobilis), Willow leaf (90, C. deliciosa), Szibat (91, C. suhuiensis), de Chios (92, C. deliciosa), Avana Apireno (93, C. deliciosa), Tardivo di Ciaculli (94, C. deliciosa), Sunki (95, C. sunki), Szinkom (96, C. suhuiensis), Yatsushiro (97, C. yatsushiro). Order of elution and percentages of components are given on BP-20 column, except compounds with an asterisk (percentages given on BP-1 column). All the components were identi"ed by GC-RI on polar and apolar columns. All the compounds of samples no. 88, 96 and 97 were also identi"ed by GC/MS. The major components (bold letters) of samples no. 65, 66, 67, 69, 75, 77, 78, 80, 82, 83, 84, 88, 90, 92, 93, 94, 96 and 97 were identi"ed by C NMR.

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 89 Fig. 1. Dendrogram obtained from the cluster analysis of 56 mandarin peel oils. Samples are clustered using Ward's technique with an Euclidean distance measure. for four samples from an unspeci"ed japanese cultivar (Lawrence, 1989). To our knowledge, only one study concerns C. deliciosa species. Two samples (unspeci"ed variety) from Italy and Spain were characterized by the limonene/γ-terpinene chemotype (72}77%/14}19%) (Boelens and Jimenez, 1989). It should be pointed out that a very large number of commercial oils from Italy of certainly known but not reported species and variety (400 samples, Dugo, 1994), as well as oils from Argentina (Retamar, 1986) or from unspeci"ed origin (Lawrence, 1996) exhibited this last chemotype. 3.2.2. Leaf oils The 58 identi"ed components accounted for 97.1}99.9% of the total amount of oil (Tables 4). We observed an important chemical variability with the occurrence of sabinene (0.1}57.3%), γ-terpinene (0.1}67.4%), linalool (tr-59.3%) and methyl N- methylanthranilate (0}78.7%). For most samples (29 over 42), the content of monoterpene hydrocarbons was important. The best represented components were sabinene, γ-terpinene, myrcene, limonene and (E)-β-ocimene. Conversely, for the 13 other samples, the oxygenated fraction was dominant with high contents of linalool, thymol or methyl N-methylanthranilate. The dendrogramm (Fig. 4) suggested the existence of three principal groups. The discriminant analysis con"rmed this repartition with respect to the contents of sabinene, γ-terpinene, linalool and methyl N-methylanthranilate (Figs. 5 and 6).

90 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Fig. 2. Discriminant analysis scatterplott of 56 mandarin peel oils. ( ) Cluster I; ( ) Cluster II, ( ) Cluster III. Fig. 3. Discriminant analysis scatterplot of the peel oil constituents.

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 91 Table 4 Chemical composition of leaf mandarin oils Constituents BP-20 BP-1 IB IA 74 75 86 87 80 83 82 63 84 62 59 78 81 58 α-thujeneh 1021 922 0.6 0.8 0.5 0.5 0.5 0.5 0.5 0.6 0.4 0.5 0.5 1.2 0.3 0.5 α-pineneh 1021 930 1.7 2.1 0.9 0.9 1.3 2.2 1.9 1.0 1.9 1.9 2.0 1.4 1.4 1.8 β-pinene 1109 971 2.6 2.9 3.4 3.3 3.5 2.7 2.7 2.8 2.5 2.3 2.4 2.3 1.9 2.2 Sabinene 1119 964 46.5 47.8 55.5 57.3 56.0 55.6 49.7 48.3 51.5 37.9 40.5 37.8 39.0 37.4 3-Carene 1145 1005 tr tr tr tr tr tr tr tr 1.5 tr tr tr 2.2 tr Myrcene 1157 978 3.5 3.6 3.5 3.8 3.6 3.4 3.6 3.2 3.2 2.8 2.9 2.8 2.7 2.7 α-terpinene 1179 1009 1.7 1.9 1.5 1.6 1.6 1.2 1.8 1.2 1.2 1.2 0.9 1.5 1.1 0.9 Limonene 1199 1021 1.8 1.9 7.7 5.6 8.6 1.7 1.6 2.9 2.0 2.3 1.8 2.4 1.4 1.9 β-phellandrene 1208 1021 0.7 0.7 0.7 0.7 0.7 0.8 0.6 0.5 0.8 0.4 0.4 0.5 0.7 0.4 (Z)-β-Ocimene 1228 1024 0.5 0.5 0.2 0.2 0.2 0.3 0.3 0.4 0.2 0.4 0.4 0.3 0.3 0.4 γ-terpinene 1241 1048 4.3 4.4 2.4 2.8 2.7 2.1 3.1 2.8 1.9 2.7 2.3 3.3 1.8 2.2 (E)-β-Ocimene 1245 1035 16.0 14.0 6.5 6.6 5.9 7.1 6.9 6.9 5.0 9.1 9.0 5.6 6.2 7.7 p-cymene 1267 1012 0.6 0.7 tr tr 0.1 0.2 * 0.4 0.1 0.4 0.4 0.5 0.1 0.4 Terpinolene 1278 1078 0.8 0.8 0.6 0.7 0.7 0.5 0.7 0.6 0.7 0.6 0.5 0.7 0.7 0.5 p-cymenene 1432 1072 0.1 0.1 tr tr * * tr 0.4 * 0.3 0.4 0.3 * 0.3 trans-sabinene hydrate 1458 1053 0.9 0.9 0.8 0.7 0.8 0.7 0.7 0.7 0.9 0.7 0.6 0.9 0.8 0.8 Citronellal 1473 1130 * * * * * * * tr 0.2 tr tr tr 0.5 tr Decanal 1492 1183 * tr * * tr * tr 0.1 tr tr tr 0.1 tr tr Linalool 1539 1082 5.4 3.4 6.7 5.3 3.1 10.6 13.0 17.0 17.2 26.2 25.5 25.6 27.1 31.0 cis-p-menth-2-en-1-ol 1555 1107 0.5 0.5 0.3 0.4 0.4 0.3 0.5 0.3 0.3 0.3 0.2 0.4 0.3 0.2 β-elemene 1586 1388 0.5 0.4 0.6 0.8 0.7 tr 0.2 0.1 * 0.1 0.1 0.1 tr 0.1 (E)-Caryophyllene 1588 1420 0.2 0.2 0.2 0.2 0.2 0.3 0.3 * 0.2 * * * * * Thymyl methyl ether 1589 1214 * * * * * * * 1.0 * 1.1 1.2 1.0 * 1.0 Terpinen-4-ol 1595 1162 7.4 7.4 5.4 6.1 6.6 4.5 7.9 3.8 4.3 4.3 3.2 6.2 4.2 3.2 *continued

92 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 4*continued Constituents BP-20 BP-1 IB IA 74 75 86 87 80 83 82 63 84 62 59 78 81 58 trans-p-menth-2-en-1-ol 1618 1123 0.3 0.3 0.2 0.2 0.2 0.2 * 0.1 0.1 0.1 0.1 0.2 0.1 0.1 Citronellyl acetate 1654 1332 * * * * * tr * * * * * * tr * α-humulene 1660 1453 0.1 0.1 0.1 0.1 0.1 tr 0.1 tr tr tr * tr tr tr Neral 1674 1213 * tr tr * * * * * tr * 0.1 * 0.3 * α-terpineol 1688 1172 0.3 0.3 0.2 0.2 0.2 0.3 0.6 0.2 0.4 0.4 0.3 0.5 0.7 0.5 α-bisabolene 1724 1496 * * * * * 0.1 * 0.4 * 0.5 0.4 0.4 * * Neryl acetate 1725 1340 * * * * * * * * * * * * 0.3 * Bicyclogermacrene 1727 1494 * 0.1 0.1 0.1 * * 0.1 * * * * * * 0.4 Geranyl acetate 1748 1358 * tr * * * * * * * * * * * * Citronellol 1756 1207 * * * * * 0.1 * * 0.2 * * * 0.2 * Nerol 1790 1207 0.1 0.1 * * * tr tr tr 0.6 tr * * 0.8 * Geraniol 1837 1232 * * * * * tr tr * tr * * tr 0.1 * Methyl N- methylanthranilate 2082 1385 * tr tr tr 0.1 * * * * * * * * * Thymol 2189 1266 0.4 0.3 0.2 0.1 0.1 * * 0.1 * 0.1 * 0.1 * 0.2 β-sinensal 2225 1673 0.5 0.5 tr tr tr 1.5 * 1.3 1.1 1.5 1.3 1.2 1.7 1.4 α-sinensal 2323 1726 0.9 0.8 tr * tr 1.6 * 0.9 0.8 0.6 0.6 0.6 0.9 0.7 Total 99.3 97.8 98.7 98.7 98.3 99.1 97.2 98.4 99.5 99.1 98.3 98.2 98.7 99.4

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 93 Table 4*continued Constituents BP-20 BP-1 IA IIB 68 85 88 89 87 70 71 65 72 67 95 91 96 98 α-thujeneh 1021 922 0.5 0.4 0.2 1.2 1.2 1.5 1.9 1.3 1.5 2.1 2.1 1.8 2.6 2.2 α-pinene* 1021 930 1.6 1.6 0.8 2.6 2.4 4.2 4.0 3.3 3.9 4.7 4.3 3.5 5.3 4.3 β-pinene 1109 971 2.4 2.2 3.0 8.1 8.0 17.1 14.5 13.7 15.4 4.2 4.2 4.5 5.3 5.2 Sabinene 1119 964 43.1 42.8 21.2 1.1 1.2 2.5 2.2 1.8 2.0 0.3 0.4 0.4 0.4 0.5 3-Carene 1145 1005 tr 3.1 tr * * * * tr * * * * * * myrcene 1157 978 2.9 3.4 1.5 15.5 19.4 1.1 1.1 1.0 1.0 1.2 1.1 1.0 1.4 1.1 α-terpinene 1179 1009 1.3 1.4 0.4 0.3 0.5 0.8 0.9 0.9 1.0 0.3 0.7 0.8 1.5 1.1 Limonene 1199 1021 2.0 2.2 2.2 15.9 7.7 4.7 5.2 4.8 4.5 4.0 4.1 4.1 4.6 4.1 β-phellandrene 1208 1021 0.4 0.7 0.3 0.2 0.2 0.3 0.2 0.3 0.4 tr tr tr tr 0.1 (Z)-β-Ocimene 1228 1024 0.3 0.4 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.7 0.3 0.1 0.1 0.1 γ-terpinene 1241 1048 2.9 2.3 0.7 31.9 32.4 39.0 38.8 37.5 35.8 11.7 28.3 34.3 67.4 53.4 (E)-β-Ocimene 1245 1035 6.0 7.8 1.2 2.4 3.3 4.2 3.3 3.4 3.4 10.3 7.5 1.2 3.4 * p-cymene 1267 1012 0.5 0.1 tr 6.6 1.5 14.3 16.0 15.0 16.6 3.2 4.9 6.4 1.3 4.1 Terpinolene 1278 1078 0.7 1.1 0.2 1.0 1.2 2.1 2.1 2.1 2.1 1.9 1.9 2.2 2.8 2.2 p-cymenene 1432 1072 0.5 * * tr * 1.7 2.0 1.6 1.7 2.1 0.6 1.5 * 0.1 trans-sabinene hydrate 1458 1053 0.7 0.7 0.3 0.1 tr * * * * tr tr 0.1 * 0.1 Citronellal 1473 1130 tr 0.3 47.8 1.2 2.8 * * tr * 0.2 * tr * * Decanal 1492 1183 tr * * 0.4 0.2 * 0.1 0.1 * tr * 0.1 * tr Linalool 1539 1082 22.1 16.7 6.6 2.0 3.3 1.6 4.4 5.8 5.3 27.6 19.5 15.0 0.3 11.4 cis-p-menth-2-en-1-ol 1555 1107 0.4 0.3 0.1 * * tr tr tr tr tr tr tr * * β-elemene 1586 1388 0.1 0.6 0.1 0.6 0.7 1.5 0.8 2.1 1.9 0.2 * * tr * (E)-Caryophyllene 1588 1420 * * 0.2 0.1 0.1 0.4 0.2 0.5 0.5 * * tr 0.3 * Thymyl methyl ether 1589 1214 0.9 0.2 * * * * * * * 10.8 17.3 * * 8.0 Terpinen-4-ol 1595 1162 6.1 5.2 1.5 0.2 0.2 0.3 0.3 0.6 0.6 0.2 0.2 0.3 0.1 0.3 *continued

94 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 4*continued Constituents BP-20 BP-1 IA IIB 68 85 88 89 87 70 71 65 72 67 95 91 96 98 trans-p-menth-2-en-1-ol 1618 1123 0.2 0.2 * * tr tr tr tr * * tr tr * tr Citronellyl acetate 1654 1332 * * 0.1 0.3 0.5 * * * * * * * * * α-humulene 1660 1453 * * tr 0.1 0.1 0.2 0.1 0.3 0.2 * * tr tr * Neral 1674 1213 0.1 * tr 0.4 1.5 * * 0.1 * * * 0.1 * * α-terpineol 1688 1172 0.3 0.6 0.3 0.1 0.2 0.1 0.1 0.3 0.5 0.3 0.4 0.5 0.1 0.6 α-bisabolene 1724 1496 * * * * * 0.1 0.1 * 0.1 * * 0.1 0.3 0.1 Neryl acetate 1725 1340 0.3 0.3 * 1.9 2.8 * * * * * * * * * Bicyclogermacrene 1727 1494 * * 0.1 * * * * * * 0.6 * * * * Geranyl acetate 1748 1358 0.1 tr 0.1 1.4 3.2 * * * * * * * * * Citronellol 1756 1207 * 0.3 7.7 0.1 0.2 * * * * * * * * * Nerol 1790 1207 tr 0.5 * 0.3 0.6 * * tr * tr tr 0.1 tr * Geraniol 1837 1232 tr 0.1 0.3 0.2 0.5 * * tr * 0.1 tr * * * Methyl N- 2082 1385 0.1 tr * tr * * * * * * * * * * methylanthranilate Thymol 2189 1266 0.1 tr * * * 0.1 tr * 0.1 9.5 0.4 20.5 * 0.1 β-sinensal 2225 1673 1.2 1.5 * * * * * * * 0.2 * * * * α-sinensal 2323 1726 0.5 * 0.2 * * tr * * * 0.8 * * 1.3 * Total 98.6 97.6 97.2 97.5 99.3 98.6 99.1 97.6 97.1 97.7 98.4 98.8 98.7 99.6

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 95 Table 4*continued Constituents BP-20 BO-1 IIB IIA III 73 77 64 76 60 61 69 66 99 93 92 90 79 94 α-thujeneh 1021 922 2.0 1.3 1.1 1.1 1.2 1.3 1.2 0.3 tr 0.7 0.6 2.8 0.9 1.0 α-pineneh 1021 930 3.7 2.8 2.7 2.5 2.4 2.7 2.4 0.7 0.2 1.5 1.3 1.6 2.0 2.3 β-pinene 1109 971 4.7 2.9 2.7 2.6 2.7 2.9 2.6 1.1 0.1 1.8 1.3 2.5 2.0 2.2 Sabinene 1119 964 0.5 0.2 0.2 0.2 0.3 0.3 0.3 0.3 1.4 0.2 0.1 0.2 0.9 0.2 3-Carene 1145 1005 * * * * * * * * * * * * tr * Myrcene 1157 978 1.1 0.8 0.7 0.7 0.8 0.8 0.8 0.4 0.4 0.5 0.5 0.7 0.5 0.6 α-terpinene 1179 1009 1.4 0.3 0.3 0.3 0.3 0.3 0.3 0.1 tr 0.2 0.2 0.2 0.3 0.3 Limonene 1199 1021 4.3 2.5 4.3 2.5 2.6 2.8 2.1 4.5 11.8 4.6 8.9 8.3 6.3 7.9 β-phellandrene 1208 1021 tr tr tr tr 0.5 tr tr 0.1 tr tr tr 0.1 tr tr (Z)-β-Ocimene 1228 1024 0.1 0.5 0.5 0.5 0.6 0.6 9.8 0.3 0.7 0.2 0.1 0.2 0.1 0.2 γ-terpinene 1241 1048 59.7 15.2 14.5 15.7 12.0 13.4 10.7 3.0 0.1 21.4 16.5 28.6 22.6 24.1 (E)-β-Ocimene 1245 1035 2.4 9.4 9.3 10.3 11.0 10.1 0.5 4.1 1.7 0.6 0.5 0.5 0.5 0.5 p-cymene 1267 1012 1.4 3.0 3.2 3.5 2.7 3.6 2.9 3.1 * 3.4 2.2 3.9 2.7 3.7 Terpinolene 1278 1078 2.4 1.4 1.1 1.1 1.2 1.2 1.1 0.7 tr 0.7 0.5 0.8 0.7 0.7 p-cymenene 1432 1072 tr 1.2 0.5 0.6 0.8 1.4 1.2 1.5 * 0.1 0.1 0.1 0.1 0.1 trans-sabinene hydrate 1458 1053 tr tr tr * tr tr tr tr tr tr * * tr * Citronellal 1473 1130 tr * * * * * * 0.1 * * * * * * Decanal 1492 1183 * * * * * 0.1 * * 0.1 tr tr tr * tr linalool 1539 1082 13.7 37.6 42.9 42.4 41.4 40.9 47.0 59.3 tr 0.6 0.3 0.2 0.9 0.4 cis-p-menth-2-en-1-ol 1555 1107 tr * * * 0.2 * tr * * * * * * tr β-elemene 1586 1388 * tr * * * * tr tr * * * 0.1 * * (E)-Caryophyllene 1588 1420 0.2 0.6 0.4 0.4 0.3 * 0.3 * 0.4 0.1 0.4 0.4 0.1 0.3 Thymyl methyl ether 1589 1214 * 0.3 0.4 0.4 0.4 0.4 0.3 8.8 * 0.1 * * 0.1 * Terpinen-4-ol 1595 1162 0.3 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.1 0.1 0.2 0.2

96 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Table 4*continued Constituents BP-20 BP-1 IA IIB 68 85 88 89 87 70 71 65 72 67 95 91 96 98 trans-p-menth-2-en-1-ol 1618 1123 tr tr tr * * 0.1 tr * * * * * * * Citronellyl acetate 1654 1332 * * * * * * * tr * * * * * * α-humulene 1660 1453 tr 0.1 * * tr tr tr * tr * * tr * tr Neral 1674 1213 * * * * * * * * * * tr 0.1 * 0.1 α-terpineol 1688 1172 0.4 0.3 0.1 0.1 0.3 0.2 0.3 0.2 * 0.2 0.1 0.1 0.2 0.2 α-bisabolene 1724 1496 * 0.2 * 0.1 0.1 * 0.1 * tr * tr tr * tr Neryl acetate 1725 1340 0.1 * * * * * * * * * * * * * Bicyclogermacrene 1727 1494 * * 0.2 * * 0.1 * * * * * * * * Geranyl acetate 1748 1358 * tr * * * * * * * * * * * * Citronellol 1756 1207 * * * * * tr * * * * * * * * Nerol 1790 1207 tr tr * * * 0.1 tr * * * tr * * tr Geraniol 1837 1232 * tr tr tr 0.2 tr tr tr * * * * * * Methyl N-methylanthranilate 2082 1385 * * * * * 0.1 * * 78.7 62.3 66.1 48.0 57.7 54.5 Thymol 2189 1266 * 16.2 12.2 12.5 15.3 13.0 14.1 8.4 0.1 0.3 0.1 0.3 0.1 0.1 β-sinensal 2225 1673 * * * * * * * * 2.7 * * * * * α-sinensal 2323 1726 0.6 1.4 1.4 1.5 1.8 0.6 0.6 0.5 * * * * * * Total 99.1 98.6 98.9 99.2 99.4 97.4 98.9 98.1 98.8 99.6 99.9 99.8 98.2 99.6 Other compounds ( tr-0.3%): camphene, α-phellandrene, octanal, 6-methylhept-5-en-2-one, nonanal, cis-limonene-1,2-oxide, trans-limonene-1,2-oxide, (E)-β-farnesene, germacrene-d, (E,E)-α-farnesene, δ-cadinene, caryophyllene oxide, (E)-nerolidol, elemol, spathulenol, τ-cadinol, α-cadinol, manoyl oxide. Cultivars : Geleking (74, C. nobilis), Yellow King (75, C. nobilis), King (86, C. nobilis), Rode king (87, C. nobilis), King of Siam (80, C. nobilis), Temple Temple (83, C. temple), Cleopatra (82, C. reshni), Peau rugueuse (63, C. deliciosa), Temple (84, C. temple), Emperor (62, C. deliciosa), Empress (59, C. deliciosa), Swatow (78, C. tangerina), Temple Sue Linda (81, C. temple), Late Emperor (58, C. deliciosa), Redskin (68, C. tangerina), Du Japon (85, C. nobilis), Nasnaran (88, C. amblycarpa), Kunembo (89, C. nobilis), Yatsushiro (97, C. yatsushiro), Clausellina (70, C. unshiu), URSS (71, C. unshiu), Wase (65, C. unshiu), Owari (72, C. unshiu), Fuzhu (67, C. erythrosa), Sunki (95, C. sunki), Szibat (91, C. suhuiensis), Szinkom (96, C. suhuiensis), Shekwasha (98, C. depressa), Sihue Gan (73, C. suhuiensis), Ladu Ordinary (77, C. paratangerina), Brickaville (64, C. tangerina), Peau lisse (76, C. deliciosa), Vohangisahy (60, C. tangerina), Beauty of Glen Retreat (61, C. tangerina), Ladu (69, C. paratangerina), Dancy (66, C. tangerina), Ougon (99, C. suavissima), Avana Apireno (93, C. deliciosa), de Chios (92, C. deliciosa), Willow leaf (90, C. deliciosa), Commune (79, C. deliciosa), Tardivo di Ciacculli (94, C. deliciosa). Order of elution and percentages of components are given on BP-20 column, except compounds with an asterisk (percentages given on BP-1 column). All the components were identi"ed by GC-RI on polar and apolar columns. All the compounds of samples no. 58, 62, 97 and 93 were also identi"ed by GC/MS. The major components (bold letters) of samples no. 75, 78, 81, 82, 83, 65, 67, 72, 88, 89, 91, 95, 96, 97, 66, 69, 76, 79, 92, 93 and 94 were identi"ed by C NMR.

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 97 Fig. 4. Dendrogram obtained from the cluster analysis of 58 mandarin leaf oils. Samples are clustered using Ward's technique with an Euclidean distance measure. Sabinene/linalool chemotype: 17 cultivars (no. 58,59,62,63,68,74,75,78,80}88) belonged to this group. Essential oils were characterized by a high content of sabinene (21.2}57.3%), associated with linalool (3.1}31.0%) and terpinen-4-ol (1.5}7.9%). This cluster could be divided into three subgroups. Subgroups IA and IB are di!erentiated on the basis of sabinene content: 21}43% for seven samples (no. 58,59,62,68,78,81,85, subgroup IA) and 46}57% for the nine others (no. 63,74,75,80,82-84,86,87, subgroup IB). For most samples of subgroup IA, linalool exhibited a higher percentage (17}31%) that those of subgroup IB (3}17%). It is noticeable that the samples no. 74 and 75 exhibited higher content of (E)-β-ocimene (14.0 and 16.0% vs 5.0}7.1%). The remaining sample (no. 88, subgroup IC) exhibited an atypical composition dominated by citronellal (47.8%) with an appreciable amount of sabinene (21.2%) and moderate contents of citronellol (7.7%) and linalool (6.6%). γ-terpinene/linalool chemotype: 19 essential oils (samples no. 60,61,64}67, 69}73,76,77}89,91,95}98) belonged to this chemotype (cluster II). This cluster could be readily divided into two subgroups on the basis of linalool/γ-terpinene ratio. The "rst subgroup (IIA), to which belonged seven samples (no. 60,61,64,66,69,76,77) exhibited linalool as major component (37.6}59.3%) with appreciable contents of γ-terpinene

98 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Fig. 5. Discriminant analysis scatterplott of 58 mandarin leaf oils. Fig. 6. Discriminant analysis scatterplot of the leaf oil constituents.

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 99 (3.0}15.7%) and thymol (8.4}16.2%). The second subgroup (IIB), to which belonged the 12 other samples (no. 65,67,70}73,89,91,95}98) exhibited an appreciable amount of γ-terpinene (28.3}67.4%) except for the oil no. 67 (11.4%). The content of linalool was not over 27.6%. We noticed that several components were sometimes important: (Z)-β-ocimene (9.8% vs 0.3}0.6%) associated with a low content of (E)-β-ocimene (0.5% vs 4.1}11.0%) in the sample no. 69 (subgroup IIA); β-pinene (13.7}17.1% vs 4.2}8.1%) and p-cymene (14.3}16.6% vs 1.3}6.6%) in the oils no. 65, 70}72 (subgroup IIB); myrcene (15.5}19.4% vs 1.0}1.4%) and limonene (7.7}15.9% vs 4.0}5.2%) associated with a slightly higher content of oxygenated acyclic monoterpenes: citronellal, neral, neryl acetate and geranyl acetate in the oils no. 89 and 97 (subgroup IIB); thymol (20.5% vs 0}9.5%) in the sample no. 67 (subgroup IIB); thymyl methyl ether (8.0}17.3% vs 0}0.4%) in the sample no. 66 of subgroup IIA and samples no. 67, 95 and 98 of subgroup IIB. Methyl N-methylanthranilate chemotype: only six oils (no. 79, 90, 92}94 and 99) were dominated by methyl N-methylanthranilate (48.0}78.7%) with appreciable amounts of γ-terpinene (0.1}28.6%) and limonene (4.6}11.8%). It is noticeable that the sample no. 99 (C. suavissima) exhibited a higher percentage of methyl N-methylanthranilate (78.7%), a very low content of γ-terpinene (0.1%) and an appreciable amount of β-sinensal (2.7%). In the literature, three di!erent compositions were described: (i) cis- and translinalool oxide (12}60%), associated with linalool (14}36%) for `miyacawaa, `okitsua (C. unshiu) and `dahongpaoa cultivars (C. tangerina) from China (Lin and Hua, 1992); (ii) γ-terpinene (11}38%) and p-cymene (14}41%) associated with β-pinene (6}14%) (Kamiyama, 1968; Lawrence, 1995b) or linalool (23%) (Kamiyama, 1967) for samples from Japan, or associated with β-elemene (11}25%) and linalool (3}15%) (Lawrence, 1995a) for the samples from unspeci"ed cultivars (C. unshiu); (iii) methyl N-methylanthranilate (42}52%) associated with γ-terpinene (24}29%) for commercial oils (Dugo et al., 1996; Mondello et al., 1996a, b, 1997). The γ-terpinene/p-cymene/β-pinene composition described for our samples no. 65, 70}72 from C. unshiu (`wasea, `clausellinaa, `URSSa, and `owaria cultivars) was close to that reported in the literature for three samples from the same species (Kamiyama, 1968). 4. Discussion To our knowledge, this is the "rst time that the chemical composition of peel and leaf oils of nearly 100 cultivars of mandarin from 16 species has been reported. Peel oils of mandarin belonged to three major chemotypes: limonene, limonene/γterpinene and linalyl acetate/limonene. Fig. 7 shows that the limonene chemotype was more widespread than the limonene/γ-terpinene chemotype (80 varieties from 11 species vs 16 varieties from 6 species). The monovarietal C. yatsushiro exhibited an atypical composition dominated by linalyl acetate and limonene.

100 M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 Fig. 7. Inter- and intraspeci"c di!erentiation on the basis of three chemotypes distinguished for peel mandarin oils. For "ve species (C. clementina, C. tangerina, C. unshiu, C. temple and C. paratangerina), all the varieties investigated exhibited the limonene chemotype. Conversely, a chemical intraspeci"c variability was observed for C. reticulata, C. deliciosa, C. nobilis and C. suhuensis. Concerning C. reticulata and C. deliciosa (41 and 10 samples, respectively), the limonene and limonene/γ-terpinene compositions appeared at a ratio and. C. nobilis is dominated by the limonene chemotype while two samples over three, belonging to C. suhuiensis, exhibited the limonene/γ-terpinene composition. Concerning the monovarietal species, the situation appears more variable. The oils from C. erythrosa and C. reshni exhibited limonene as a major component whereas the samples from C. amblycarpa and C. sunki on the one hand, the sample from C. yatsushiro on the other hand showed limonene/γ-terpinene and linalyl acetate/ limonene compositions respectively. It has to be pointed out that three samples, belonging to the limonene/γ-terpinene chemotype, were distinguished by atypical compositions: limonene/β-pinene for the sample no. 88 from the monovarietal C. amblycarpa, limonene/myrcene for the oil no. 89 from C. nobilis and limonene/p-cymene for the sample no. 96 from C. suhuiensis. In our sampling, domination of the oils by limonene was common. This has been reported before for the oils of `dancya, `malvasioa, `ortaniquea, `ellendalea and `cravoa cultivars from C. reticulata (Ashoor and Bernhard, 1967; Moshonas and

M.-L. Lota et al. / Biochemical Systematics and Ecology 29 (2001) 77}104 101 Fig. 8. Inter- and intraspeci"c di!erentiation on the basis of three chemotypes distinguished for leaf mandarin oils. Shaw, 1974; Koketsu et al., 1983; Dellacassa et al., 1992; Calvarano et al., 1989), `communea, `nulesa, `orovala, `monreala cultivars from C. clementina (Verzera et al., 1997, 1998), `praecoxa cultivar from C. unshiu (Lawrence, 1989) and for some samples from unspeci"ed varieties and/or species (Shaw, 1979; Lawrence, 1989, 1995b). The limonene/γ-terpinene chemotype has been described for a great number of commercial oils (Verzera et al., 1992; Dugo, 1994) and for three cultivars `sandersona, `comuna and `malvasioa from C. reticulata (Ashoor and Bernhard, 1967; Dellacassa et al., 1992). To our knowledge, the linalyl acetate/limonene, the limonene/β-pinene and the limonene/myrcene compositions have never been reported for the mandarin peel oils. The chemical composition of the leaf oils was characterized by a great variability. The 99 oils investigated in this and the previous studies (Lota et al., 2000), exhibited three major chemotypes: sabinene/linalool, γ-terpinene/linalool and methyl N- methylanthranilate. Fig. 8 shows that the sabinene/linalool composition was the most frequently observed (59 samples belonging to 8 species) followed by the γ-terpinene/linalool chemotype (31 varieties belonging to 11 species) and the methyl N-methylanthranilate chemotype (9 varieties which belonged to 3 di!erent species). We observed a chemical variability for the C. reticulata and C. deliciosa cultivars, which exhibited the three major compositions while two chemotypes were found in