chemotaxonomic significance of leaf wax n-alkanes in the umbelliferae, cruciferae and leguminosae...

Pergamon

PII: S0305-1978(96)00037-3

Biochemical S),stema#'cs and Ecology, VoL 24, No. 6, pp. 531-545,1996 Copyright © 1996 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0305-1978/96 $15.00+0.00

Chemotaxonomic Significance of Leaf Wax n-Alkanes in the Umbelliferae, Cruciferae and Leguminosae (Subf. Papilionoideae)

MASSIMO MAFFEI Department of Plant Biology, University of Turin, Viale Mattioli, 25, 1 0125, Turin, Italy

Key Word Index--Umbelliferae; Cruciferae; Leguminosae; leaf wax n-alkanes; chemotaxonomy; cluster analysis.

Abstract- -Leaf wax alkanes were extracted from 41 genera and 58 species belonging to the Umbelli- ferae, from 35 genera and 61 species belonging to the Cruciferae and from 32 genera and 78 species belonging to the Leguminosae. n-Alkane composition was determined by GC and GC-MS. All families were characterized by the presence of C29 and C31; however, in the Umbelliferae, the C29 content was greater and in the Cruciferae, it was equal and in the Leguminosae, it was lower than that of Cal. The data matrix of the n-alkane percentages and total content was used to calculate for each family a cluster analysis. The results obtained confirmed the usefulness of leaf wax alkanes as chemotaxonomic characters at the familial, subfamilial and tribal levels. Copyright © 1996 Elsevier Science Ltd

In t roduc t ion Plant surfaces are covered by a waxy layer which represents the first physico- chemical barrier to the entry of pathogens and to adverse environmental factors. The cuticle, which represents this barrier, consists of cross-esterified polymerized hydroxy-fatty acids (the cutin) and a mixture of non-polar lipids which are called cuticular waxes. On the cuticle surface there is an epicuticular layer consisting of linear and branched alkanes and other aliphatic compounds (Kolattukudy, 1981; Schnable et al., 1994). Alkane composition has been used to estimate the species composition of herbage mixtures (Dove, 1992), pasture sampling for the estimation of herbal intake (Vulich et al., 1993), leaf feeding patterns (Bergman et al., 1991 ; Bodnaryk, 1992), chilling injury (Maffei etal., 1993a and refs cited therein), edible oil characterization (McGill et al., 1993) and as bioindicators of the general degree of pollution of the air both in plants (Tuomisto and Neuvonen, 1993 and refs cited therein) and lichens (Zygadlo et al., 1993). Recently, the chemotaxonomic significance of wax alkanes has been demonstrated in studies on the Sola- naceae (Zygadlo et al., 1994), Crassulaceae (Stevens et al., 1994), Labiatae and four related plant families (Maffei, 1994a), Compositae (Maffei, 1994b), and Gramineae (Maffei, 1996), while Schnable et al. (1994) and G01z (1994) have reviewed the genetics of cuticular wax biosynthesis and the epicuticular leaf waxes in the evolution of the plant kingdom, respectively.

Little information is available on the leaf wax alkanes of other important plant families, such as the Umbelliferae, Cruciferae and Leguminosae. To my knowledge no infomation is available on Umbelliferae leaf wax alkanes. The Cruciferae are a large natural family of major economic importance due to the presence of a wide array of crop plants used for human nutrition and animal feeding (Heywood, 1978). In the genus Brassica, leaf wax alkanes have been found to be an important antixenotic factor affecting the rate and pattern of feeding of flea beetles

(Received 24 January 1996; accepted 11 March 1996)

531

532 M. MAFFEI

(Bodnaryk, 1992). In this family, leaf wax alkanes of the genus Coincya are domi- nated by carbon atoms with an odd number and similar results were found for the genus Brassica (Vioque et al., 1994 and refs cited therein). A mixture of n-alkanes with odd numbers has been found also in some Leguminosae, such as alfalfa (Bergman et al., 1991 ), lucerne and subterranean clover (Dove, 1992).

The aim of the present work was to evaluate the leaf wax n-alkane content, composition and chemotaxonomic significance in some species belonging to the Umbelliferae, the Cruciferae and the Leguminosae (Subf. Papilionoideae).

Mater ials and Methods Perennial species were grown for more than 20 years in experimental plots of the Botanical Garden of the University of Turin, while annuals were sown and cultivated in the same place as perennials. Voucher specimens of all species are deposited at the Herbarium Generale (TO) of the Department of Plant Biology of the University of Turin, Italy. Exotic plants were grown from seeds obtained from other Botanical Gardens. The species studied were:

Umbell i ferae: Aegopodium podagraria L, Angelica archangelica L., A. syIvestris L, Anthriscus vulgaris Pers., Apium graveolens L., Astran~'a major L., Athamanta sicula L., Bifora radians Bieb., Bunium bulbocas- tanum L., Bupleurum fructicosum L., B. stellatum L., Cachrys trifida Miller, Carum carvi L., Chaerophyllum aroma~cum L., C. hirsutum L., Conium maculatum L., Crithmum maritimum L., Daucus carota L., Dorema ammoniacum D. Don., Eryngium amethystinum L., E. maritimum L., E. planum L., Falearia vulgaris Bernh., Ferula communis L, F. samarkandica Korovin, Ferulago campestris (Bess.) Grec., F. galbanifera Koch., Foe- niculum vulgate Mill., Heracleum alpinum Georgi, H. mantegazdanum Sommier et Levier, H. sphondylium L., Laserpi~um gallicum L., L. latifolium L., L. prutenicum L., L. slier L., Levis~cum officinale W. D. J. Koch, Meum athamanticum Jacq., Myrrhis odorata (L ) Scop., Oenanthe crocata L., O. pimpinelloides L., Opopanax chironium (L ) W. D. J. Koch, Pastinaca sativa L., Petroselinum hortense Hoffm., Peucedanum cervaria (L.) Lapeyr., P. officinale L, P. ostruthium (L.) Koch., Pimpinella saxifraga L., Scandix peeten-veneris L., Seseli tortuosum L., S. montanum L., Slum latifolium L., Smyrnium perfoliatum L., Thapsia garganica L., Torilis anthriscus Gmel, T. japonica (Houtt.) DC, Trinia glauca (L.) Dumort and Trochiscanthes nodiflorus (Viii.) W. D. J. Koch.

Cruciferae: Aethionema grandiflorum Boiss. and Hohen, Alliaria officinalis Andrz., A. pe~olata (Bieb.) C av. et G rande, Alyssoides u~riculata (L.) M edicus, Alyssum montanum L., A. saxatile L., Arabis alpina D C, A. brassica (Leers) Rauschert, A. caucasica Willd., A. ciliata Clairv., A. collina Ten., A. nova Viii., A. saxa~Tis All., A. stelleri (japonica) DC, A. turrita L., Aubrieta deltoidea D.C., Barbarea vulgaris R. Br., Biscutella levigata L., Brassica nigra (L.) Koch., Bunias orientalis L., Capsella bursa-pastoris (L.) Medicus, Cardamine amara L, C. hirsuta L., Cardaria draba (L.) Desv., Cheiranthus cheiri L., C. scoparius Brouss., Cochlearia armocacia L., Crambe cordifolia Stev., C. maritima L., C. tataria Jacq., Diplotaxis muralis (L ) DC, D. tenuifolia (L.) DC, Draba aizoides L., D. dubia Suter, D. muralis L., Erucastrum nasturtiifolium (Poiret) O. E. Schulz, Erysimum cheiranthoides L., Erysimum pumilum D.C., Fibigia clypeata (L.) Medicus, Hesperis matronalis L., Iberis pruitii Tineo, I. sempervirens L., Isatis tinctoria L., Kernera saxatilis (L.) Rchb., Lepidium latifolium L., L. virginicum L., Lunaria annua L, L redlviva L., Moricandia arvensis (L.) DC, Nasturtium silvestre R. Br., Petrocallis pyr- enaica R. Br., Ptilotrichum spinosum Boiss., Rhaphanus rhaphanistrum L., Sinapis alba L., S. arvensis L., S. nigra L., Sisymbrium alliaria Scop., S. strictissimum L., Thlaspi arvense L. and T. brachypetalum Jordan.

Leguminosae: Amorpha fructicosa L., Anthyllis hermanniae L., A.vulneraria L., Astragalus campestris L., A. cicer L., A. glycyphyllus L., Astragalus neglectus Fisch., Bap~'a australis Hort. ex Lemm., Chamaecytisus hirsutus Link, Coronilla coronata L, Coronilla emerus L., Coronilla varia L., Cytisus alpinus Lam., Cytisus grandiflorus DC, Cytisus sessilifolius L., Desmodium canadense DC, Galega officinalis L., Genista cinema (Viii) DC, Genista germanica L., Genista horrida DC, Genista sagittalis L, Genista scorpius Georgi Reise, Genista ~nctoria L., Glycyrrhiza echinata L., Glycyrrhiza lepidota Pursh, Glycyrrhiza paucifoliata Hence, GIy- cyrrhiza uralensis Fisch (ex DC), Hedysarum obscurum L., Hippocrepis comosa L., Laburnum alpinum Miller, Lespedeza bicolor Turcz., Lespedeza juncea Pers., Lathyrus hirsutus L., Lathyrus montanus Bernh., Lathyrus pratensis L, Lathyrus sylvestris L., Lathyrus tuberosus L., Lathyrus vernus (L.) Bernh., Lotus alpinus (DC) Schleicher, Lotus corniculatus L., Lotus siliquosus L., Medicago falcata L., Medicago lupulina L., Medicago sativa L, Melilotus alba Medicus, Melilotus altissima Thuill., Melilotus officinalis (L ) Pallas., Onobrychis alpina L., Onobrychis viciaefolia Scop., Ononis natrix L., Ononis repens L., Ononis spinosa L., Oxytropis halleri Bunge, Oxytropis helvetica Scheele, Phaseolus vulgaris L., Pisum satlvus L., Robinia pseudoacacia L., Sophora japonica L., Spartium junceum L., Thermopsis fabacea DC, Trifolium alpestre L, Trifolium alpinum L., Trifolium badium Scheber, Trifolium campestre Scheber, Trifolium montanum L, Trifolium pannonicum Jacq, Trifolium pratense L., Trifolium repens L., Trifolium thalii Viii., Ulex europaeus L., Vicia aurantia Boiss., Vicia cracca L., Vicia onobrychioides L., Vicia pisiformis L., Vicia sa~va L., Vicia tenuifolia Roth and Wisteria sinensis (Sims.) Sweet.

LEAF WAX ALKANES OF UMBELLIFERAE, CRUCIFERAE AND LEGUMINOSAE 533

The taxonomic subdivision of the species belonging to the three families was as described by Pimenov and Leonov (1993) for the Umbelliferae, Heywood (1978) for the Cruciferae and Polhil and Raven (1981 ) for the Leguminosae.

Fully developed non-senescing leaves were randomly collected from at least 10 plants from each species and 5 g of fresh material was immediately extracted with 5 ml pentane-hexane (5:1) for 60 s; 30 pg of n-tricosane was then added as internal standard. The extract was concentrated by a gentle stream of N2, passed through a column of anhydrous MgS04 and then analysed by GC. The extract (1 pl) was injected into an on-column injector of a Varian 3700 gas chromatograph equipped with FID using the conditions previously described (Maffei, 1994a). An average of three injections was done for each sample. Peak identification was based on both R t comparison with pure standards and GC-MS, as described earlier (Maffei, 1990). Abbreviations and diagnostic ions (m/z) of the identified alkanes were as reported previously (Maffei, 1994a, 1996).

All data were statistically processed using a Systat 5.2 software for Macintosh as described previously (Maffei et aL, 1993b; Maffei, 1994a).

Results and Discussion In general, the leaf wax alkane composition of the families studied was repre-

sented by linear alkanes ranging from Cls to C36 (Table 1 ).

Leaf wax alkanes of the Umbelliferae The Umbelliferae examined (41 genera and 58 species) belong to two sub-

families, the Saniculoideae and the Apioideae. Only one tribe of the Saniculoideae (Saniculeae) was represented by the taxa studied, while the Apioideae were represented by taxa belonging to the tribes Angeliceae, Apieae, Caucalideae, Cor- iandreae, Laserpitieae, Peucedaneae, Scandiceae, Smyrnieae and Tordylieae. The highest leaf wax alkane content was found for Bo bulbocastanurn (2.91 mg g-1 fresh wt) while high percentages of low carbon-numbered alkanes (C18,-'C22) were found for S. libanotis (25.9), M. odorata (18.3), O. chironium (2.0) and C. trifida (16.3 and 8.4) (Table 1 ). Intermediate chain-length alkanes (C23-C28) were particularly high in P. saxifraga (9.8), F. campestris (12.9), F. galbanifera (15.6), S. montanum (15.0), P. cervaria (21.4) and P. sativa (10.4). The main alkanes, C29 and C31, were found in very high percentages in S. perfoliatum (85.7) and L. slier (77.3). The C3o and C32 percentages of B. fruticosum leaf wax alkanes were particularly high (35.1 and 38.6, respectively), while the highest percentages of the

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Torclylieae

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FIG. 1. CLUSTER ANALYSIS OF THE UMBELLIFERAE. A clear distinction is evident between the Saniculeae and the other tribes. The distance metric was 1 -Pearson correlation coefficient, single linkage method.

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long-chain alkanes (C33-C36) were found in E amethystinum (49.5), P. sativa (32.1), E. maritimum (12.4) and D. ammoniacum (5.8). Table 1 also reports the mean alkane values for each tribe. In the subfamily Apioideae, the Angeliceae possessed the highest percentages of C18, C2o, C21, C22, C23, C24, C25, C26 and C2s (8.1, 1.2, 6.0, 3.8, 7.2, 8.0, 10.9, 8.9 and 7.8, respectively), while the Scandiceae had the highest values for C~ 9 and C27 (4.2 and 11.1, respectively). The Smyrnieae possessed the highest C29 percentages (45.9), while the Laserpitieae had the highest percentages of C3~ (36.4). The Tordylieae had high percentages of C3o, C32 and C34 (10.5, 5.5 and 8.6, respectively). In the Apioideae, the highest alkane content was found for the Smyrnieae (0.71 mg g-1 fresh wt). The Saniculeae had the highest percentages of C33 and C3s (22.8 and 3.4, respectively).

Figure 1 shows the tree diagram obtained from Cluster Analysis (CA) calculated on the data matrix of Table 1, by considering the content and the percentages of alkanes of the individual tribes. The distance metric used was 1-Pearson correlation coefficient with the single linkage method. Two main clusters are present, the first made by the Saniculeae (subf. Saniculoideae) and the second gathering all the Apioideae (Fig. 1 ). In the latter cluster, several sub-clusters are evident. A close statistical linkage was found between the Smyrnieae and the Caucalideae, and between the Apieae and the Scandiceae. These tribes showed a statistical linkage to the Coriandreae and the Peucedaneae. A lower statistical linkage with the above tribes was found for the Tordylieae, the Laserpitieae and the Angeliceae, respectively.

According to Pimenov and Leonov (1993), the mode of evolution of the Umbelliferae makes its generic delimitation very difficult. As a result different approaches made by using different sets of characters may lead to different results. The clear partition between the Saniculoideae and the Apioideae confirms the ability of wax alkanes to discriminate between taxa at the subfamilial level (see also Maffei, 1 994a; Maffei, 1996).

Leaf wax alkanes of the Cruciferae According to Heywood (1978), among the various classifications of the Cruci-

ferae, the most widely used is that proposed by Schulz in 1936. In this classification, the tribes usually recognized are 13. The Cruciferae considered for this study (35 genera and 61 species) belong to the tribes Alysseae, Arabideae, Bras- siceae, Hesperideae, Lepidieae and Sisymbrieae. The species with the highest leaf wax alkane content was H. matronalis (8.19 mg g-1 fresh wt), which also had the highest percentages of ClS, C19 and C2o. The highest percentages of C21, C22, C23, C24, C2s and C2s were found in A. ciliata, B. orientalis, A. turrita and L. latifolium. Crambe cordifolia (16.3) and L. annua (7.9) had the highest percentages of C27 and C2s, respectively, while very high percentages of C2s were found in A. utri- culata (89.5).A. brassica (44.3), A. montanum (57.5) andA. saxatile (34.4) had the highest percentages of C3o, C31 and C33, respectively, while C. hirsuta, A. ciliata, and D. dubia possessed the highest percentages of C32, C34, C3s and C36 (Table 1 ). The mean values expressed by considering the six tribes indicated the highest leaf wax alkane content in the Hesperideae (2.78 mg g-1 fresh wt.), which also had the highest percentages of Cls, Cls, C2O, C31 and C32 (18.4, 6.3, 4.1,29.0 and 3.5, respectively). The Alysseae had the highest percentages of C21, C33, C35 and C3s (5.6, 9.3, 1.3 and 0.5, respectively), while the Arabideae had high percentages of C3o and C34 (5.2 and 0.8, respectively). High percentages of C2e were found for the Brassiceae (28.8), while the Lepidieae possessed high percentages of C25, C2s and C27 (5.3, 4.7 and 7.6, respectively). Finally the Sisymbrieae had the highest percentages of C22, C23, C24 and C2s (3.9, 6.1,5.4 and 5.5, respectively).

542 M. MAFFEI

High contents of C26"-C31 have already been reported for the genus Brassica (Vioque et al., 1994 and refs cited therein).

The CA calculated by using an Euclidean distance metric with the single linkage method shows two clusters (Fig. 2). The first is made by the Hesperideae which showed a low statistical linkage with the other tribes. In the second cluster, the Arabideae and the Lepidieae showed a close statistical linkage, these two tribes were progressively statistically linked to the Brassiceae and the Sisymbrieae. The Alysseae showed a lower statistical linkage with the latter tribes.

According to Heywood (1978), only two of the tribes, the Brassiceae and the Lepidieae, can be regarded as natural. In the CA of Fig. 2 these two tribes indicate a close chemotaxonomic relation to the Arabideae.

Leaf wax alkanes of the Leguminosae (Subf. Papilionoideae) Of the 33 tribes present in the subf. Papilionoideae, 14 were analysed in the

present study (Table 1 ). The highest alkane content was found in T. alpinum (5.80 mg g-1 fresh wt), while the percentages of C18, C19, C2o, C21, C22 and C23 were found to be high in L. alpinum (20.4), T. campestre (13.2 and 14.7), L. bicolor (22.9) and C. varia (8.5 and 9.7), respectively. Amorpha fruticosa (9.5 and 17.9) had the highest percentages of C24 and C25, while L. alpinum (24.8), T. campestre (49.1) and V. pisiformis (15.3 and 8.9) had high percentages of C2s, C27, C28 and C3o, respectively. T. montanum (78.3) had very high percentages of C29, while C31 was very high in S. japonica (83.4). A. cicer (19.1 ), W. sinensis (28.0), T. fabacea (26.0 and 1 7.5) and A. neglectus (5.2) had the highest percentages for C32 , C33 , C34, C35 and C36, respectively (Table 1 ). At the tribal level, the highest leaf wax alkane content was found for the Loteae (1.1 0 mg g-1 fresh wt), which also had the highest percentages of C33 (8.1). The Amorpheae had the highest percentages of C24, C25 and C27 (9.5, 1 7.9 and 20.0, respectively), while the Coronilleae had the highest percentages of C19 C2o, C21 and C22 (2.9, 4.9, 9.9 and 4.4, respectively). The Hedisareae had high percentages of C18 and C26 (9.7 and 13.5, respectively), while the Desmodieae (11.3) and the Phaseoleae (7.1) had high percentages of C32 and C28, respectively. The highest percentage of C34 (26.0) and C35 (17.5) were found for the Sophoreae, while C29 (42.6), C31 (44.5) and C36 (0.6) were particularly high in the Thermopsideae, the Vicieae and the Galegeae, respectively. Finally, the Tephrosieae had the highest percentage of C23 (7.0) and C29 (4.7).

The CA calculated using a 1-Pearson correlation coefficient and a median linkage method (Fig. 3) shows a clear separation of the Sophoreae from the other tribes. The Amorpheae represents the second cluster, while the third cluster

Hesperideae

Alysseae

Brassiceae

Arabideae

Lepidieae

Sisymbrieae

0.00 Dis tance 10.00

FIG. 2. CLUSTER ANALYSIS OF THE CRUCIFERAE. The Hesperideae show a low statistical linkage to the other tribes. The distance metric was Euclidean, single linkage method.

LEAF WAX ALKANES OF UMBELLIFERAE, CRUCIFERAE AND LEGUM INOSAE 543

Amorpheae

Thermopsideae

Trifolieae

Desmodieae

Coronilleae

Hedisareae

0.00 Distances 1.00

Tephrosieae

Robinleae

Galegeae

Genisteae

Loteae 1

Vicieae

Phaseoleae

Sophoreae

FIG. 3. CLUSTER ANALYSIS OF THE LEGUMINOSAE (SUBF. PAPILIONOIDEAE). Four main clusters are evident, the first made by the Sophoreae. the second by the Amorpheae and the other two clusters by the remaining tribes. The distance metric was 1 -Pearson correlation coefficient, median linkage method.

includes the Thermopsideae, the Trifolieae, the Desmodieae, the Coronilleae and the Hedisareae. The last cluster consists of the Phaseoleae and a subcluster in which a close statistical linkage is observed between the Tephrosieae and the Robinieae, between the Galegeae and the Genisteae, and between the Loteae and the Vicieae.

The Papilionoideae (or Faboideae) is a huge subfamily which comprises 440 genera and 1 2,000 species widely distributed throughout the world (Polhil, 1981 ). Several attempts have been made in order to provide a chemosystematic classification of this subfamily. Among the low molecular weight markers, flavonoids, isoflavonoids, quinolizidine alkaloids and non-protein amino acids have been used, giving results comparable to those obtained from morphological data (Gomes et al., 1981 ). Seed polysaccharides (starch, galactomannans and amyloid) have also been successfully used (Hegnauer and Grayer-Barkmeijer, 1993). The CA obtained from the data matrix of leaf wax alkanes of Table 1 indicates a clear separation of the Sophoreae, as was found in the subfamily Papilionoideae when quinolizidine alkaloids were considered (Polhil, 1981 ; Gomes et al., 1 981, and refs cited therein). The leaf wax composition of the Amorpheae show no statistical linkage to the Loteae, and to the Coronilleae, as found based on the distribution of flavonoids, isoflavonoids and bases reported by Gomes et al. (1981 ). The leaf wax alkane profile of the Amorpheae results in a statistical linkage closer to the Sophoreae than to the other tribes. According to Polhil (1981), while a division is apparent between the Amorpheae and the Desmodieae and the Phaseoleae, the genus Apoplanesia, which belongs to the Amorpheae, is reminiscent of the Myr- oxylon group of the Sophoreae. There is no reference on the linkage between the

544 M. MAFFEI

Thermopsideae and the Trifolieae, as obtained in the CA performed on leaf wax alkanes. Based on quinolizidine alkaloids, isoflavonoids, and flavonoids the Ther- mopsideae appear to be related to the Genisteae, and the Trifolieae to the Gen- isteae (Gomes et al., 1981 ). The third cluster finds more common points to other chemical and morphological criteria of classification. The close statistical linkage between the leaf wax alkanes of the Tephrosieae and the Robinieae agrees with a considerable similarity observed by analysing their woody components (Polhil, 1 981 ). The Phaseoleae are the largest tribe and the distinctions from the Tephro- sieae are only grade characters (i.e. condensation of leaves and inflorescences, etc.) (Polhil, 1981). The leaf wax alkane profiles of these two tribes are quite similar; however, a better correlation was observed between these two tribes when phenolic compounds were used as chemotaxonomic markers (Gomes etal., 1981 ). The very close relationship between the Loteae and the Vicieae finds no comparison with other morphological or chemical data, with the only exception of the common reduction of the pulvinus (Polhil, 1 981 ). The Galegeae are the Old World temperate offshoot of Tephrosieae, and are present in the same cluster in Fig. 3. However, besides leaf wax alkanes, no morphological or chemical data support their close relationship to the Genisteae.

Leaf wax alkanes of the Umbelliferae, Cruciferae and Leguminosae (Subf Papilionoideae)

The CA, calculated with an Euclidean distance and the single linkage method, performed on the mean family values of Table 1, shows a close statistical linkage between the Umbelliferae and the Leguminosae (Fig. 4). These two families, which belong to the Apiales and the Rosales, respectively, are gathered in the subclass Rosidae, while the Cruciferae belong to the subclass Magnoliidae. In the Leguminosae, even though the closest families are probably the Sapindaceae and the Connaraceae (Dickison, 1981), a close chemical relationship to the Umbelli- ferae has also been observed in a preliminary comparison with eight other families (Maffei, 1994b).

Concluding Remarks The results of this report confirm the chemotaxonomic significance of leaf wax alkanes both at the familial, subfamilial and tribal levels. The composition of wax components enables leaf alkane patterns to be utilized with success as con- firmatory taxonomic criteria in plant classification even when a limited number of genera is considered (Maffei, 1994ab, 1996, and refs cited therein). The ease of extraction and analysis of such molecules (such as the leaf wax alkanes) makes their utilization available for large data set studies (Kellogg and Watson, 1993). Furthermore, the composition of leaf wax alkanes is well defined genetically and provides an important tool for selection and breeding programs for plant resistance to pathogens and adverse environmental conditions.

0.0 Distances 1.8

Umbel l i fe rae

Leguminosae I

Cruc i ferae

FIG. 4. CLUSTER ANALYSIS OF THE UMBELLIFERAE (SUBF. PAPILIONOIDEAE), CRUCIFERAE AND LEGUMINOSAE. The Umbelliferae and the Leguminosae show a close statistical linkage. The distance metric was Euclidean, single linkage method.


Acknowledgements - -The author is grateful to F. Bianco for technical assistance during GC analyses. This work was supported by a M.U.R.S.T. grant (quota 60%).

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