Patterns of leaf epicuticular waxes in species of Clusia: taxonomical implications

 

Ernesto Medina, Guillermina Aguiar, Matilde Gómez and José D. Medina

Ernesto Medina. Biologist, Universidad Central de Venezuela (UCV). Doctor in Agronomy, University of Hohemheim, Stuttgart Germany. Professor, UCV and Researcher, Instituto Venezolano de Investigaciones Científicas (IVIC). Address: Centro de Ecología. IVIC. Apartado 21827. Caracas 1020-A. Venezuela. e-mail: emedina@ivic.ve

Guillermina Aguiar. Chemist, Instituto Pedagógico de Caracas, Venezuela. Associate Research Professional, Center for Ecology, IVIC, Venezuela.

Matilde Gómez. Chemist, Universidad Simón Bolívar, Venezuela. Associate Research Professional, Center for Chemistry, IVIC, Venezuela.

José D. Medina. Chemist, UCV, Venezuela. Doctor in Chemistry, University of Laval, Canada. Researcher, Center for Chemistry, IVIC, Venezuela.

Resumen

El género Clusia L. (Clusiaceae) comprende unas 300 especies que ocurren desde México y el sur de EEUU hasta Bolivia y el sur de Brasil. Entre ellas se incluyen árboles y arbustos, hemiepifitas, epifitas y lianas. El análisis taxonómico del género se dificulta por la pobre preservación de las flores al ser secadas. Este trabajo explora la composición de ceras epicuticulares para caracterizar especies mediante marcadores químicos. Se analizó la composición del extracto obtenido de seis especies mediante lavado de la superficie foliar con hexano. Las especies pudieron separarse en base a la proporción de alcanos, >90% del total en Clusia rosea, C. orthoneura y C. minor, a la presencia de los triterpenos a-amirina y lupeol en C. multiflora, y de friedelina y taraxerol, conjuntamente con C33 y C35 en C. grandiflora y C. schomburgkiana. Los resultados sugieren que la proporción de alcanos y triterpenoides de ceras epicuticulares tiene importancia taxonómica y puede ser utilizada para separar especies o secciones infragenéricas.

Summary

The genus Clusia L. (Clusiaceae) encompasses ca. 300 species and occurs from southern USA and Mexico, to southern Brazil and Bolivia. It includes free-standing trees and shrubs, hemiepiphytes, epiphytes, and lianas. Taxonomic analysis of this genus is difficult because of the poor preservation of floral material after drying. This work explores the composition of epicuticular waxes in order to allow characterization, at the species level, using chemical markers. The six species analyzed could be separated using the relative quantity of hexane-soluble compounds extractable from the leaf surface, which amount to >90% in Clusia rosea, C. orthoneura, and C. minor, the presence of the triterpenes a-amyrin and lupeol in C. multiflora, and of friedelin and taraxerol, together with C33 and C35, in C. grandiflora and C. schomburgkiana. The results suggest that the relative proportions of alkanes and triterpenoids in epicuticular waxes may have taxonomic significance for separating species or infrageneric sections.

Resumo

O gênero Clusia L. (Clusiaceae) compreende umas 300 espécies que ocorrem desde México e o sul dos EE.UU. até Bolívia e o sul do Brasil. Entre elas se incluem árvores e arbustos, hemiepífitas, epífitas e lianas. A análise taxonômica do gênero se dificulta pela pobre preservação das flores ao ser secadas. Este trabalho explora a composição de ceras epicuticulares para caracterizar espécies mediante marcadores químicos. Se analisou a composição do extrato obtido de seis espécies mediante lavado da superfície foliar com hexano. As espécies puderam separar-se em base à proporção de alcanos, >90% do total em Clusia rosea, C. orthoneura e C. minor, à presença dos triterpenos a-amirina e lupeol em C. multiflora, e de friedelina e taraxerol, conjuntamente com C33 e C35 em C. grandiflora e C. schomburgkiana. Os resultados sugerem que a proporção de alcanos e triterpenóides de ceras epicuticulares tem importância taxonômica e pode ser utilizada para separar espécies ou seções infragenéricas.

KEYWORDS / Alkanes / Chemotaxonomy / Clusia / Epicuticular Waxes / Triterpenes /

Received: 04/12/2004. Modified: 08/23/2004. Accepted: 08/26/2004.

Introduction

The genus Clusia includes around 300 species that occur throughout the interneotropical realm, from southern USA and Mexico to southern Brazil and Bolivia (Pipoly et al., 1998). It includes trees and shrubs that grow as free-standing individuals on a variety of substrates (shallow clay or deep sandy soils, calcareous or serpentinitic soils), or as hemiepiphytes, epiphytes and lianas.

All species produce latex varying in abundance, density, and color (Engler, 1925; Pipoly et al., 1998). Numerous Clusia species possess characteristics possibly associated with tolerance to dry conditions (succulent and/or leathery leaves, low leaf conductance; Lüttge, 1996; Pipoly et al., 1998). Several species have been shown to be constitutive CAM, or switching from C3 to CAM metabolism under drought conditions (Franco et al., 1990; Lüttge, 1996).

Many Clusia species produce resiniferous waxes in the staminate, and/or the pistillate flowers, providing material for honey bees nest construction (Cuesta-Rubio, 2002). Production of these resins is significant as a pollinator attractant (Bittrich and Amaral, 1996; Marsaioli et al., 1999).

Epicuticular waxes play a role in plant biology in water loss regulation, because cuticular transpiration is related to the permeability of the cuticle to water vapor and to the habitat in which plants grow (Schreiber and Riederer, 1996). Shady and/or humid habitats frequently produce leaves more permeable to water vapor than leaves from dry and/or sunny exposed habitats (Bondada et al., 1996). In addition, epicuticular waxes act as insulators from excess environmental humidity, preventing the penetration of liquid water into intercellular spaces, and avoiding the establishment of epiphyllic organisms (Neinhuis and Barthlott, 1997).

The composition of epicuticular waxes is dominated by long- (>C21), frequently odd-chain alkanes (Barthlott, 1989). Other related compounds are very long chain alcohols and acids, and in many species triterpenoids appear in amounts usually small. The amount of epicuticular waxes in a given plant population may be used to identify ecological conditions (drought and sun exposure), while its composition may help in the identification of ecologic and taxonomic groups. The latter is possible as the result of the conservative biochemical pathways that lead to wax synthesis (Bianchi, 1987; Gülz, 1994). Extraction and identification of wax compounds is relatively straightforward using volatile low polarity organic solvents, gas chromatography and mass spectrometry. This paper reports on epicuticular wax profiles of five Clusia species in order to test the feasability to a) separate species using the relative proportions of alkanes and presence/absence of triterpenoids; b) evaluate variations of wax load and composition in relation to leaf age and leaf side, and the influence of ecological conditions such as sun exposure.

Material and methods

Clusia multiflora HBK (Section Anandrogyne) and C. minor L. (Section Retinostemon) shrubs were sampled in a disturbed cloud forest (1500m elevation) in the interior coastal range south of Caracas, Venezuela. Leaves from C. schomburgkiana Planch. & Triana ex Engler (Section Omphalanthera; female plant), C. grandiflora Splitg. (Section Chlamydoclusia; female plant), C. orthoneura Standl. (Section Cochlanthera; male plant) and C. rosea Jacq. (Section Chlamidoclusia; sterile trees) were sampled from trees (one tree per species) planted at the Botanical Garden grounds in Caracas.

Epicuticular waxes were extracted from at least three fresh leaf replicates, separating adaxial and abaxial sides of young and mature, healthy leaves. Leaf area was measured before extraction (Licor LI-3100 area meter), and leaf dry weight was determined after extraction (ventilated oven at 60ºC until constant weight). Leaves were washed with 50ml hexane (GC grade) for 90sec at room temperature using a pizette. The extract was evaporated to dryness at room temperature. This procedure extracts only surface hexane-soluble compounds without disturbing the leaf interior. The dry extract was weighed (10µg precision), redissolved in hexane and subsequently injected into a gas chromatograph (Hewlett-Packard 6890, flame ionization detector, HP5 column 30m·0.25mm·0.2µm, using He as transport gas). Temperature was increased at 10ºC/min from 160º to 240ºC, and then 4ºC/min up to 320ºC and maintained for 10min. The injector was maintained at 250ºC and the detector at 320ºC. Results are reported as amount of extracted waxes per unit leaf area, and as relative proportions as indicated by the average percentage of chromatogram area for each chemical species. Compound identification was done using commercial standards, and analyzing a subsample in GC/MS Varian 3400CX with a Varian Saturn 2000 mass detector. Mass spectra were identified using commercial libraries (Wiley Registry of Mass Spectral Data and NIST 98 Standard Reference Database).

Total amount of extracts per leaf side per species were compared using a one-way ANOVA (or a Welch ANOVA for unequal variances); means were compared using a Student’s individual pair test (SAS, 2002).

Results

Amount of hexane-soluble compounds extracted

The average load of hexane-soluble compounds (HSC; Figure 1 for adult leaves) was relatively higher in C. schomburgkiana, C. grandiflora, and C. orthoneura (³20µg·cm^-2), than in C. multiflora and C. minor (<20µg·cm^-2). On the abaxial side only C. orthoneura and C. schomburgkiana had values ³20µg·cm^-2. The amount of HSC on the adaxial leaf side of adult leaves was larger than on the abaxial side in C. rosea and C. grandiflora, while the opposite occurred in C. schomburgkiana and C. orthoneura. In C. multiflora and C. minor differences in HSC content on both sides were small. In young leaves these relationships were similar. No statistical differences could be established between leaves of different ages, or between leaf sides, due to the variability in gravimetric HSC estimations. Therefore, the relationship between extract weights and chromatogram areas, as expression of total HSCs injected, was positive and significant but noisy (data not shown) and could not be used for quantification.

 

Composition of the hexane-soluble compounds

The GC-MS analyses allowed the separation of two groups of species (Table I). In Group I, HSCs were dominated by alkanes (>90% on both leaf sides), and it included C. orthoneura, C. rosea, and C. minor. In Group II alkanes from the adaxial side constituted <70% of total HSCs, and it included C. multiflora, C. schomburgkiana, and C. grandiflora.

Linear alkanes in Group I were dominated by the compounds C29 and C31, their ratio being usually >1 in both leaf sides.

Group II was more heterogeneous. C29 was the dominant compound in C. multiflora, particularly on the abaxial side. The C29/C31 ratio on the abaxial side was consistently >1 in the male plants and <1 in female plants. This species was also characterized by the lack of C35 and the presenc e of C24, C25 and C26 on the adaxial side. In C. schomburgkiana the predominant alkanes were C31, C33 and C35 on both leaf sides. C. grandiflora differed in alkane composition depending on leaf side. On the adaxial side the most abundant compound was C35, whereas on the abaxial sides the compounds C31 and C33 predominated, showing similar proportions.

On the adaxial side the Group I species showed levels of triterpenoids below 1.5%, whereas the species of Group II were characterized by abundance levels above 20% (Table II). The triterpenes identified were a-amyrin, friedelin, lupeol, taraxerol and three unidentifed cholestans. In addition, the triterpene precursor squalene was detected in small amounts in practically all samples. The comparison of the triperpene composition of the adaxial leaf side within Group II showed distinct patterns. C. multiflora was characterized by the presence of lupeol and a-amyrin, while C. grandiflora and C. schomburgkiana were distinguished by the predominance of the compound friedelin (Table II).

 

Discussion and Conclusions

The chemical composition of epicuticular waxes has been used as an aid to solve taxonomical problems in higher plants (Salatino et al., 1989; Mimura et al., 1998). The study of Maffei (1996), who separated several tribes within the Poaceae using the alkane composition of hexane extracts of epicuticular waxes is a good example in this direction. However, several authors do not encourage this type of approach because of the predominant alkane molecules (C29 and C31) are ubiquitous for the whole set of higher plants (Barthlott and Wollenweber, 1981). Genetic analyses of the biosynthetic processes involved will provide a better understanding on how conservative are the biochemical pathways leading to epicuticular wax components, and settle the question of specificity of wax composition spectra (Bianchi, 1987; Lemieux, 1996; Kunst and Samuels, 2003).

One of the problems for using epicuticular wax composition as a taxonomic tool is the variability in the extraction power of different organic solvents reported in the literature, and the use of fresh or dried leaves (Stammiti et al., 1996). Differences in penetration depth of the solvent into the cuticula results in uncertainty about the actual localization of the extracted substances. Jetter and Schäffer (2001) showed that in Prunus laurocerasus triterpenoids are restricted to the intracuticular matrix, while the alyphatic compounds were the only truly epicuticular components. However, triterpenes could be transported to the superficial wax layer, if the mechanism for movement and regeneration of epicuticular waxes through plant cuticles described by Neinhuis et al. (2001) is of general occurrence in higher plants. These authors concluded that wax moves through the cuticle in a process analogous to steam distillation.

The Clusia species analyzed had a simple and similar form of wax deposition. The gentle extraction of the leaf surface waxes using n-hexane allowed their separation according to the proportion of alkanes and the presence of specific triterpenes into two well defined groups. Three species (Group I) showed absolute predominance of alkanes (C. minor, C. orthoneura and C. rosea), while in the rest (Group II) triterpenoids constituted 20-30% of the total HSC detected in the adaxial leaf side. Triterpene compounds such as lupeol and a-amyrin occurred only in C. multiflora, while taraxerol and friedelin characterized C. grandiflora and C. schomburgkiana. Analyses of C. multiflora waxes suggest that it may be possible to identify the sex of the sampled plant using the C29/C31 alkane ratios from HSC extracted from the abaxial leaf side. Since Clusia is a genus constituted predominantly by dioecious species, this approach opens interesting possibilities. The species studied here may be considered as intermediate or low wax accumulators on a per unit leaf area, compared to species from other environments (Oliveira and Salatino, 2000). We did not detect differences in the amount or composition of alkanes between sun and shade leaves of C. rosea; however, the shade samples of this species were richer in triterpenoids. These results may be associated with the mild conditions experienced during development of the harvested leaves.

The measured differences in composition and proportions of epicuticular waxes within this small set of species, representing large sections within the genus, suggest the potential chimio-taxonomic value of this approach, which deserves further investigation.

ACKNOWLEDGEMENTS

Silvia Llamozas (formerly at the Fundación Instituto Botánico in Caracas) helped in the obtention of leaf samples and John Pipoly (Fairchild Botanical Garden, Miami, Fl.) identified one of the cultivated species.

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