Cuticular n-alkane in leaves of seven Neotropical species of the family Lecythidaceae : a contribution to chemotaxonomy

Biosynthesized from very long-chain fatty acid wax precursors, n-alkanes m a k e a valuable contribution to the taxonomy of plants . Th e a lk ane comp onents of foliar epicuticles of seven Neotropical s p e c i e s o f Lecythidaceae were investigated: Bertholletia excelsa, Cariniana legalis, Couroupita guianensis, Eschweilera alvimii, Eschweilera ovata, Gustavia augusta and Lecythis pisonis. Specimens were collected in the metropolitan area of Recife, Pernambuco, and their n-alkane fractions were analyzed by gas chromatography. Th e chemical relationships among the species were then evaluated using cophenetic correlation and UPGMA. Among the seven species, a total of 15 n-alkanes, with 21-35 carbon atoms, were identifi ed and formed a consistent group of B. excelsa, C. guianensis, E. ovata, G. augusta, and L. pisonis with n-C31. Th e greatest similarities were found between B. excelsa and L. pisonis, and between C. guianensis and G. augusta. Nevertheless, a phenetic analysis based on a larger number of species is needed to better understand the chemotaxonomic value of epicuticular n-alkanes within the Lecythidaceae.

Th e family Lecythidaceae is pantropically distributed, with 10 genera and 118 species, most of them native to Brazil, with the highest diversity in the Neotropical region (Smith et al. 2010).In the state of Pernambuco (Northeast Brazil), six genera and 14 species are found (Barbosa 2006), among which Gustavia augusta L., Eschweilera ovata (Cambess.)Miers, and Lecythis pisonis Camb.are highly prevalent in Atlantic Forest areas (Rocha et al. 2008;Silva & Rodal 2008).
1 Departamento de Biologia, Universidade Federal do Ceará, Campus Pici, 60451-760, Fortaleza, CE, Brazil 2 Departamento de Biologia, Universidade Federal Rural de Pernambuco, 52171-900, Recife, PE, Brazil 3 Departamento de Botânica, Universidade Federal de Pernambuco, 50670-901, Recife, PE, Brazil Among these components, alkanes from epicuticular waxes have acquired wide acceptance as indicators of taxonomic relations between different plant groups (families, genera and species) (Maffei 1996a;Medina et al. 2006;Li et al. 2012).n-alkanes are biosynthesized from very long-chain fatty acid (C > 22:0) wax precursors by the decarbonylation pathway (Kunst & Samuels 2003).However, inconsistencies of using alkanes of plant waxes as taxonomic markers have been pointed out.Some authors have observed that the alkane distribution can be strongly affected by several factors, among them the age of the plant organ (Stocker & Wanner 1975;Nordby & Nagy 1977;Jenks et al. 2001).This molecular alteration in the amount and distribution of alkanes in plant leaves can complicate the application of n-alkanes as taxonomic markers (Li et al. 2013).
To our knowledge, no chemical studies concerning the epicuticular n-alkane profile have been carried out with species of Lecythidaceae.
To obtain the cuticular wax, individual totally expanded whole fresh leaves from each species, with five replicates, underwent two successive washings during 30 s with 800 mL of dichloromethane (CH 2 Cl 2 ) (Souza et al. 2010).n-alkane fractions were separated by TLC and analyzed by GC/EIMS (Shimadzu 17A, Kyoto, Japan).The alkane peaks were identified by comparison with authentic samples of n-alkane standard solution C21-C40 (Fluka S.A, Costa Rica) and mass spectrometry (NIST05, Standard Reference Database).The analyses were performed with a DB-Wax fused silica capillary column (polyethylene glycol, 30 m × 0.25 mm, 5 % phenyl-95 % dimethylpolysiloxane) with helium at a flow rate of 1 cm 3 .min-1and split ratio 1:100.Injector and detector temperatures were 300 °C.The temperature of the column moved from 100 °C (3 min) to 230 °C at 3 °C.min - and was maintained at the final temperature under isothermal conditions for 20 min.
The n-alkane distribution was analyzed through the unweighted pair group method with arithmetic mean (UPGMA) and Euclidean taxonomical distances.The cophenetic correlation was generated (COPH algorithm) to verify the goodness of fit between the groups in the dendrogram and the similarity matrix coefficient.All analyses were carried out using the software NTSYS version 2.11X (Rohlf 2005).
A total of 15 n-alkanes with 21-35 carbon atoms were identified.Long-chain n-alkanes prevailed, particularly hentriacontane (n-C31).In two species (C.guianensis and C. legalis), tritriacontane (n-C33) was the most prevalent, and in E. alvimii, heneicosane (n-C21) represented over 60 % of the cuticular alkanes identified.The quantitative distribution of the n-alkanes from the seven Lecythidaceae species studied is shown in Table 1.
A high cophenetic correlation coefficient (rcoph = 0.964) was found, which suggests a good fit between the data matrix (Tab. 1) and the phenogram obtained (Fig. 1).The n-alkane profile of E. alvimii enabled isolating it from a large group formed by most species.The main reason for this isolation was the exceptionally high content of n-C21 in E. alvimii (over 60 %).Although this species had n-C31, the characteristic cuticular alkane of the Lecythidaceae species studied, its content (26 %) was lower than those of five species studied, including E. ovata, a species of the same genus, but with high n-C31 content (57 %).In contrast, the n-C21 content in E. ovata was very low.Thus, the n-alkane profiles of these two species are very different.It is also worth pointing out the negligible amount of n-C31 in C. legalis.Although this species was grouped with most of the species studied, C. legalis is clearly isolated within this group.Despite the very small n-C21 content, the moderate contents of pentacosane (n-C25) and heptacosane (n-C27) make the alkane profile of C. legalis quite peculiar.
The group formed by B. excelsa, C. guianensis, E. ovata, G. augusta and L. pisonis was characterized by the highest concentrations of n-C31 found (38.3 to 57.2 %).In this  group, the greater similarities between B. excelsa and L. pisonis and between C. guianensis and G. augusta were mainly due to the even distribution of n-C31 and n-C33.
All species studied h e re belong to the subfamily Lecythidoideae, which is characterized by genera with fibrous bark, simple, alternate leaves, actinomorphic or zygomorphic flowers, numerous stamens, inferous to superous ovaries, and bitegumented ovules, among other characteristics (Prance & Mori 1979).According to Huang et al. (2011), the relations within Lecythidaceae are not fully understood, particularly within Lecythidoideae.
Our findings, for instance, show strong similarity between C. guianensis and G. augusta.However, C. guianensis has zygomorphic flowers, ovules inserted along a bilamellar placenta, and indehiscent fruits with lenticular seeds, while G. augusta has actinomorphic flowers and is the only Lecythidaceae genus with poricidal anthers and plano-convex cotyledons.These characteristics, except for the indehiscent fruits in Couroupita, are synapomorphies in both genera (Mori et al. 2007).
The results also showed high similarity between B. excelsa and L. pisonis.Cladistic analyses based on anatomic and morphologic data of vegetative and reproductive organs place B. excelsa in the same clade as other Lecythis species (section Lecythis A), but L. pisonis, the species analyzed here, and other species of this genus do not belong to this section.Lecythis is not monophyletic according to Huang et al. (2011).
According to Mori et al. (2015) and Huang et al. (2015), E. alvimii and E. ovata are placed in different clades in the Lecythidaceae family (Tetrapetala and Parvifolia, respectively).These species can be differentiated by the ligule morphology, which is double coiled in E. ovata and simple in E. alvimii.The distribution of these two species in different sections matches the n-alkane profile found (Fig. 1).
Nevertheless, a phenetic analysis based on a larger number of species is needed to better understand the chemotaxonomic value of cuticular n-alkanes in Lecythidaceae.

Figure 1 .
Figure 1.Phenogram of UPGMA clustering of Euclidian distance based on the quantitative distribution of cuticular n-alkanes from Lecythidaceae species found in the Atlantic Forest (Pernambuco, Brazil).Cophenetic correlation coefficient (r = 0.964).