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Leaf cuticular alkanes of Solanum subg. Leptostemonum Dunal (Bitter) of some northeast Brazilian species: Composition and taxonomic significance
Biochemical Systematics and Ecology 44 (2012) 48–52
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Leaf cuticular alkanes of Solanum subg. Leptostemonum Dunal (Bitter) of some northeast Brazilian species: Composition and taxonomic significance Keylla Michelline Miranda da Silva a, Maria de Fátima Agra b, Deborah Yara Alves Cursino dos Santos c, Antonio Fernando Morais de Oliveira a, * a b c
Department of Botany, Federal University of Pernambuco, 50670-901 Recife, PE, Brazil LTF, Federal University of Paraíba, 58051-970 João Pessoa, PB, Brazil Department of Botany, University of São Paulo, 05422-970 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2011 Accepted 7 April 2012 Available online
Solanum subgenus Leptostemonum (Dunal) Bitter comprises approximately 450 species, of which 110, including 58 endemics, occur in Brazil, which is recognized as one of the centers of diversity of the group. Due the great morphological plasticity and its large number of species, several taxonomic treatments have been proposed for the genus Solanum, but its infrageneric classification is problematic. The aim of this study was to analyze the alkane composition of the leaf epicuticular waxes of nine species of the subgenus Leptostemonum to evaluate the chemotaxonomic potential of the alkanes. As results, were identified in nine species thirty-one alkanes, including iso- and anteisoalkanes. The major constituent of wax in most species was tritriacontane. Hentriacontane was the main constituent of Solanum paraibanum and Solanum torvum, and pentatriacontane was predominant in different populations of Solanum stramonifolium. The phenetic analysis of nine species based on the distribution profile of alkanes (Euclidean distance and UPGMA method) show three clusters with distinct main homolog. The profiles of alkanes showed some qualitative taxonomic value for species analyzed, although, a larger number of representative samples of this subgenus must be investigated. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Alkanes Chemotaxonomy Spiny solanums Solanaceae Waxes
1. Introduction Solanum L. is the largest and most complex genus of the Solanaceae. It is widely distributed in South America, Africa and Australia. It is among the ten largest genera of flowering plants, with approximately 1500 species (Bohs, 2007). The species of Solanum are characterized by poricidal anther dehiscence, a synapomorphy of the genus. This characteristic is only shared with Lycianthes (Dunal) Hassl., a genus very close to and previously considered part of Solanum (Bohs, 2005). The understanding of the infrageneric relationships of the genus Solanum has not been fully elucidated because the genus is morphologically extremely diverse. Solanum is divided into seven subgenera (Archaesolanum Marzell, Bassovia (Aubl.) Bitter, Leptostemonum (Dunal) Bitter, Lyciosolanum Bitter, Minon Raf., Brevantherum (Seithe) D’Arcy, Potatoe (G. Don) D’Arcy, and Solanum Seithe) and 70 sections (D’Arcy, 1972, 1991).
* Corresponding author. Tel.: þ55 81 21267813; fax: þ55 81 21267803. E-mail address: [email protected] (A.F.M. Oliveira). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.04.010
K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
49
Approximately one-third of Solanum species belong to the subgenus Leptostemonum (Levin et al., 2006). In Brazil, this subgenus is the second-largest and includes approximately 110 species, of which 58 are endemic. With about 44 species in the Brazilian Northeast, it is most representative of this region. According to Agra (2007), the species of the subgenus Leptostemonum occurring in Brazil belong to sections Acanthophora, Crinitum, Melongena, Erythrotrichum, Herposolanum, Lasiocarpa, Micracantha, Persicariae, Polytrichum, and Torva. Solanum subg. Leptostemonum is particularly interesting for study because of its reproductive strategies and phenotypic plasticity (Chiarini, 2004). Species of this subgenus is easily distinguished morphologically; however, they exhibit great phenotypic plasticity and variability in their habit, floral traits, sexual system and dispersal syndrome (Chiarini and Bernardello, 2006). Several species of this subgenus have been the subject of numerous molecular analyses. These analyses have contributed greatly to the current understanding of the phylogeny of Solanum (Olmstead and Palmer, 1997; Levin et al., 2006; Weese and Bohs, 2007; Poczai et al., 2008; Stern et al., 2010). Chemotaxonomic studies of the subgenus Leptostemonum are scarce. Nevertheless, the studies of Zydgalo et al. (1994) have indicated that the epicuticular alkanes were similar in some species of this subgenus, and Silva et al. (2004) have found that the flavonoids in five species of the subgenus Leptostemonum supported the traditional infrageneric classification. The chemotaxonomic significance of leaf wax alkanes has also been investigated in other subgenera of Solanum (Mecklenburg, 1966; Maxzud and Zydgalo, 1991; Mohy-Ud-Din et al., 2010). The aim of this study was to evaluate the composition of leaf wax alkanes in some Solanum species belonging to eight sections of the subgenus Leptostemonum of wild and cultivated populations in northeastern Brazil. Possible chemotaxonomic significance is discussed. 2. Materials and methods 2.1. Plant material Samples of nine species of Solanum subg. Leptostemonum were collected in Paraíba and Pernambuco States (Northeastern Brazil). The material was identified and the voucher samples were deposited at Herbarium Geraldo Mariz (UFP) and Herbarium Lauro Pires Xavier (JPB) of Federal University of Pernambuco and Federal University of Paraíba, respectively (Table 1). 2.2. Extraction and analysis of cuticular alkanes The waxes from fully expanded, fresh, intact leaves of each species were extracted by means of two rapid washes with dichloromethane (30 s). The cuticular extracts were fractionated by preparative thin layer chromatography and alkane fractions were analyzed in a gas chromatograph (GC-FID, Shimadzu, model 17A, Kyoto, Japan) according Souza et al. (2010). GC was performed using a DB-5 capillary column (30 m, 0.32 mm, 5% phenyl-95% dimethylpolysiloxane), helium as carrier gas at a flow rate of 1 cm3 min1 and split ratio 1:100. Injector and detector temperatures were 300 C. The temperature of the column moved from 150 C (3 min) to 280 C at 10 C min1 and was maintained at the final temperature under isothermal conditions for 34 min. The identification of the compounds was performed through the comparison of retention times with authentic samples n-alkane standard solution C21-C40 (Fluka S.A, Costa Rica). The anteiso and iso-alkanes were identified by their mass fragmentation patterns (GC-EIMS QP5050, Shimadzu) and by comparison with data of literature (Kavouras et al., 1998). The MS ionization was performed at 70 eV. 2.3. Cluster analysis The chemical relationships between species were evaluated using the alkane profiles. The quantitative distribution of the alkanes was examined using cluster analysis with Euclidean distance and UPGMA. The analysis was made using the NTSYS Ver. 2.11 software (Rohlf, 2005). Alkane concentrations below 0.5% were not included in the analysis.
Table 1 Species and sections of Solanum subg. Leptostemonum collected in Paraíba (PB) and Pernambuco (PE) states, Northeast of Brazil. Section
Species
Locality
Status
Voucher
Acanthophora Crinitum Erytrothrichum
S. S. S. S.
Lasiocarpa
S. rhytidoandrum Sendtn S. stramoniifolium Jacq.
Micracantha
S. paraibanum Agra
Persicariae Torva
S. gardneri Sendtn. S. torvum Sw.
PB, João Pessoa, Federal University of Paraíba PB, João Pessoa, Conde PB, João Pessoa, Conde PB, João Pessoa, Federal University of Paraíba PE, Igarassu, Usina São José, Fragmento Pezinho PB, João Pessoa, Alto do Mateus PB, João Pessoa, Benjamim Maranhão Botanical Garden PE, Recife, Dois Irmãos State Park PB, João Pessoa, Federal University of Paraíba PE, Parnamirim, BR 316, KM 151 PE, Parnamirim, Fragmento Olho d’água PB, João Pessoa, Federal University of Paraíba
Cultivated Native Native Native Native Cultivated Native Native Native Native Native Cultivated
64,326 59,188 42,185 59,190 59,175 59,177 59,176 59,174 59,183 59,181 59,180 59,185
mammosum L. palinacanthum Dunal crinitum Lam. paludosum Moric.
50
Species
Alkanes (%) n-C24
n-C25
i-C26
n-C27
n-C28
n-C29
a-C30
n-C30
i-C31
n-C31
i-C32
a-C32
n-C32
i-C33
a-C33
n-C33
i-C34
a-C34
n-C34
i-C35
a-C35
n-C35
S. S. S. S. S. S. S. S. S. S. S. S.
tr tr tr – tr tr 0.69 tr – – tr tr
0.54 tr tr – – tr tr tr – – tr tr
tr tr tr tr 0.65 tr 7.08 1.21 2.99 4.59 2.09 tr
3.05 tr 0.63 0.91 2.72 1.01 2.25 4.35 tr tr 1.19 0.71
tr tr tr tr tr tr 0.96 2.62 tr tr tr tr
5.74 tr 0.51 10.33 5.76 1.56 2.05 23.98 0.89 1.02 1.40 2.85
– tr tr 0.69 tr tr – – – – – tr
0.67 tr tr 0.89 0.53 tr 1.02 2.83 – tr tr 0.92
1.35 tr tr 1.80 3.21 3.19 2.04 0.90 0.71 0.79 1.55 3.11
32.10 3.56 7.34 22.08 13.92 16.92 16.50 27.32 6.52 3.76 5.48 31.31
tr tr tr – – 0.68 tr – – – tr 0.67
– 1.78 1.63 6.10 3.79 tr – 4.50 tr – – tr
2.96 1.34 1.79 5.98 2.51 3.62 5.82 3.85 1.64 1.75 2.55 9.12
1.71 9.33 5.38 7.68 15.20 11.28 12.33 3.07 8.81 7.18 8.48 14.60
– 0.54 0.87 0.95 – 0.89 – – – – – –
46.11 68.07 64.84 37.47 30.27 53.38 44.12 19.63 49.32 31.43 34.36 20.03
– 0.61 tr – – tr 0.53 – 0.98 0.83 0.78 1.11
– 6.71 6.56 1.73 5.73 tr – – 2.04 – – 0.75
0.68 0.89 1.50 1.03 2.14 1.46 1.33 1.51 3.19 4.36 3.06 3.23
tr 1.86 1.17 1.06 3.97 1.09 0.68 0.62 3.54 5.33 4.33 2.86
– 0.53 0.63 – – tr – – – – – –
2.50 3.79 5.01 tr 5.22 1.50 0.75 2.36 18.34 37.22 32.36 7.36
crinitum gardneri* gardneri* mammosum palinacanthum paludosum* paludosum* paraibanum rhytidoandrum stramoniifolium* stramoniifolium* torvum
– ¼ not detected, n ¼ n-alkane, i ¼ iso-alkane, a ¼ anteiso-alkane, tr ¼ trace < 0.5%, * ¼ indicate different populations (See Table 1 for the voucher data).
K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
Table 2 Patterns of distribution of cuticular wax alkanes in species of Solanum subg. Leptostemonum of Paraíba and Pernambuco states (Northeast Brazil).
K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
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3. Results and discussion Thirty-one alkanes between C21 and C35 carbon atoms in length, including iso and anteiso-alkanes, were identified in nine species of Solanum subg. Leptostemonum. High values for tritriacontane (n-C33) were detected in most species, notably in S. gardneri, in which it represented more than 60% of the total alkane content. Hentriacontane (n-C31) was the main constituent of the cuticular wax of Solanum paraibanum and Solanum torvum, 27.3 and 31.31%, respectively, whereas Solanum stramonifolium was the only species found to have a high content of pentatriacontane (n-C35), 34.79% on average (Table 2). The distribution pattern of the n-alkanes of the species of Solanum subg. Leptostemonum examined is similar to that already reported by other studies in the genus Solanum (Maxzud and Zydgalo, 1991), including the subgenus Leptostemonum ski et al., 2011). According to Zydgalo et al. (1994), the subgenus Leptostemonum can be distin(Zydgalo et al., 1994; Halin guished from the subgenus Solanum by the high amounts of n-C33 found in the former. This n-alkane was also the major component of the cuticular wax of three other species placed in the subgenus Leptostemonum: Solanum melongena L., Solanum ski et al., 2011). In our study, n-C33 was found at high levels in six of the nine species incanum L. and Solanum insanum L. (Halin analyzed. However, in those species where this n-alkane was not found as a major constituent, for example, in S. paraibanum and S. torvum, its presence should not be neglected (approximately 20%, Table 2). We believe that future studies with larger number of species may contribute to distinction of subgenus Leptostemonum from subgenus Solanum based on n-C33 as major component of n-alkanes on cuticular waxes. Branched iso- (2-methyl) and anteiso- (3-methyl) alkanes were conjointly identified in all nine species of the subgenus Leptostemonum. The 2-methyl tritriacontane (i-C33) was the predominant branched alkane, followed by 2-methyl pentatriacontane (i-C35) (Table 2). In angiosperms, branched alkanes are less frequent than n-alkanes. This difference may indicate that the biosynthesis of methylalkanes is restricted to some taxa and is not as widespread as the biosynthetic pathway of nalkanes. According to some authors, n-alkanes and methylalkanes may possibly originate from distinct biosynthetic pathways (Kolattukudy, 1970; Kroumova and Wagner, 1999). The iso- and anteiso-alkanes are commonly found in Solanaceae (Mecklenburg, 1966; Maxzud and Zydgalo, 1991; Rogge et al., 1994; Zydgalo et al., 1994), but they are not usually found in other families. It is possible that the branched alkanes can be useful as diagnostic character for Solanaceae. However, ski et al. (2011), methylalkanes are more subject to variations during plant growth than the n-alkanes, and according to Halin the latter are more suitable for chemosystematic studies. A numerical analysis based on the quantitative distribution of the cuticular alkanes in Solanum subg. Leptostemonum is shown in Fig. 1. Three clusters were identified. The first cluster, formed by different populations of S. stramoniifolium, was characterized by similar high levels of n-C35 and n-C33. The second cluster included most species and was marked by n-C33 percentage above 30%. The third cluster, comprising only two species (S. paraibanum and S. torvum), was characterized by high percentage of n-C31 in relations to the other species. A higher population similarity was found between the accessions of S. gardneri, S. stramoniifolium and S. paludosum. This finding indicates a high genetic homogeneity in the biosynthesis of alkanes for these species. Similar results have been
Fig. 1. UPGMA dendrogram of nine species of Solanum subg. Leptostemonum based on patterns cuticular alkanes. Indications above branches correspond to the main n-alkane. Legends: n-C31 ¼ hentriacontane, n-C33 ¼ tritriacontane, n-C35 ¼ pentatriacontane. Asterisks indicate different populations.
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K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
reported by Zydgalo et al. (1994) for the n-alkane profiles of the epicuticular wax of the Tribe Nicotianeae from different phytogeographical areas. These data underscore the potential of cuticular alkanes for use in taxonomic studies. One of the most important phylogenetic studies of Solanum subgenus Leptostemonum has been conducted by Levin et al. (2006). The phylogenetic analysis performed by these authors includes 24 species of the Solanum subg. Leptostemonum, five of which (S. torvum, Solanum crinitum, Solanum mammosum, Solanum palinacanthum, and S. stramonifolium) were included in our study. More recently, Miz et al. (2008) also investigated the phylogenetic relationships of this subgenus with some southern Brazilian species from section Acanthophora, Lasiocarpa, and Torva. The molecular data for Brazilian species showed more resolution among species than that including Old World species. Despite the reduced number of species from our study some interesting suggestions can be made. As already pointed out, most species analyzed in the present research presented n-C33 as main n-alkane. Among then, only S. mammosum and S. palinacanthum have been sampled at Levin et al. (2006) study. Interestingly, these two species are placed at the same cluster based on chemical data (Fig. 1) and are part of the same clade at Levin et al. (2006) study. On the other hand, S. torvum, S. crinitum, and S. stramonifolium, all of which with other main homolog than n-C33, do not share either same cluster (Fig. 1) or clade (Levin et al., 2006). Although very few species have been sampled for n-alkanes analyzes, putting together our results with those previously published (Zydgalo et al., 1994), future investigations deserve to be taken forward. Sampling more species of Solanum is very important to verify if the n-C33 is the main component of cuticular alkane for the genus or for some subgenus. Other important point is to verify whether iso- and anteiso-alkanes are important features for the whole Solanaceae family or only for Solanum. Acknowledgements The authors are grateful to Kiriaki Nurit and Ionaldo Basílio for their technical assistance. This work was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) with grants to KMM Silva and MF Agra. References Agra, M.F., 2007. Diversity and distribution of Solanum subg. Leptostemonum in Brazil. Acta Hortic. 745, 31–42. Bohs, L., 2005. Major clades in Solanum based on ndhF sequence data. In: Hollowell, V., Keating, T., Lewis, W., Croat, T. (Eds.), A Festschrift for William G. D’Arcy: The Legacy of a Taxonomist. Monographs in Systematic Botany from the Missouri Botanical Garden, vol. 104. Missouri Botanical Garden Press, St. Louis, pp. 29–49. Bohs, L., 2007. Phylogeny of the Cyphomandra clade of the genus Solanum (Solanaceae) based on ITS sequence data. Taxon 56, 1012–1026. Chiarini, F.E., 2004. A new species of Solanum subgen. Leptostemonum (Solanaceae) from Argentina. Brittonia 56, 284–287. Chiarini, F., Bernardello, G., 2006. Karyotype studies in South American species of Solanum subgen. Leptostemonum (Solanaceae). Plant Biol. 8, 486–493. D’Arcy, W.G., 1991. The Solanaceae since 1976, with a review of its biogeography. In: Hawkes, J.G., Lester, R.N., Nee, M., Estrada, N. (Eds.), Solanaceae III: Taxonomy, Chemistry, Evolution. Royal Botanic Gardens, Kew, pp. 75–137. D’Arcy, W.G., 1972. Solanaceae studies II: typification of subdivisions of Solanum. Ann. Mo. Bot. Garden 59, 262–278. ski, q.P., Szafranek, J., Stepnowski, P., 2011. Leaf cuticular n-alkanes as markers in the chemotaxonomy of the eggplant (Solanum melongena L.) and Halin related species. Plant Biol. 13, 932–939. Kavouras, I.G., Stratigakis, N., Stephanou, E.G., 1998. Iso- and anteiso-alkanes: specific tracers of environmental tobacco smoke in indoor and outdoor particle-size distributed urban aerosols. Environ. Sci. Technol. 32, 1369–1377. Kolattukudy, P.E., 1970. Biosynthesis of cuticular lipids. Annu. Rev. Plant Physiol. 21, 163–192. Kroumova, A.B., Wagner, G.J., 1999. Mechanism for elongation in the biosynthesis of fatty acid components of epicuticular waxes. Phytochemistry 50, 1341–1345. Levin, R.A., Myers, N.R., Bohs, L., 2006. Phylogenetic relationships among the “spiny solanums” (Solanum subgenus Leptostemonum, Solanaceae). Am. J. Bot. 93, 157–169. Maxzud, M.K., Zydgalo, J.A., 1991. Variación de alcanos foliares en algunas especies argentinas de Solanum (Solanaceae). Anales de Biología 17, 137–140. Mecklenburg, H.C., 1966. Inflorescence hydrocarbons of some species of Solanum L. and their possible taxonomic significance. Phytochemistry 5, 1201–1209. Miz, R.B., Mentz, L.A., Souza-Chies, T.T., 2008. Overview of the phylogenetic relationships of some southern Brazilian species from section Torva and related sections of “spiny Solanum” (Solanum subgenus Leptostemonum, Solanaceae). Genetica 132, 143–158. Mohy-Ud-Din, A., Khan, Z., Ahmad, M., Kashmiri, M.A., Yasmin, S., Asghar, M.N., Ahmad, S.R., 2010. Epicuticular waxes from Solanum nigrum complex: chemotaxonomic implications. Asian J. Chem. 22, 2919–2927. Olmstead, R.G., Palmer, J.D., 1997. Solanum: implications for phylogeny, classification, and biogeography from cpDNA restriction site variation. Syst. Bot. 22, 19–29. Poczai, P., Taller, J., Szabó, I., 2008. Analysis of phylogenetic relationships in the genus Solanum (Solanaceae) as revaled by RAPD markers. Plant Systematics Evol. 275, 59–67. Rogge, W.F., Hildemann, L.M., Mazurek, M.A., Cass, G.R., Simoneit, B.R.T., 1994. Sources of fine organic aerosol 6. Cigarette smoke in the urban atmosphere. Environ. Sci. Technol. 28, 1375–1388. Rohlf, F.J., 2005. NTSYSpc: Numerical Taxonomy System. Exeter Publishing Ltd, Setauket, NY. Silva, T.M.S., Nascimento, R.J.B., Camara, C.A., Castro, R.N., Braz-Filho, R., Agra, M.F., Cavalho, M.G., 2004. Distribution of flavonoids and N-trans-caffeoyltyramine in Solanum sub-genus Leptostemonum. Biochem. Syst. Ecol. 32, 513–516. Souza, R.J.C., Silva, S.I., Oliveira, A.F.M., 2010. Chemical similarity among domesticated and wild genotypes of peanut based on n-alkanes profiles. Pesquisa Agropecuária Brasileira 45, 1321–1323. Stern, S.R., Weese, T., Bohs, L.A., 2010. Phylogenetic relationships in Solanum section Androceras (Solanaceae). Syst. Bot. 35, 885–893. Weese, T.L., Bohs, L., 2007. A three-gene phylogeny of the genus Solanum (Solanaceae). Syst. Bot. 32, 445–463. Zydgalo, J.A., Maestri, D.M., Grosso, N.R., 1994. Alkane distribution in epicuticular wax of some Solanaceae species. Biochem. Syst. Ecol. 22, 203–209.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Leaf cuticular alkanes of Solanum subg. Leptostemonum Dunal (Bitter) of some northeast Brazilian species: Composition and taxonomic significance Keylla Michelline Miranda da Silva a, Maria de Fátima Agra b, Deborah Yara Alves Cursino dos Santos c, Antonio Fernando Morais de Oliveira a, * a b c
Department of Botany, Federal University of Pernambuco, 50670-901 Recife, PE, Brazil LTF, Federal University of Paraíba, 58051-970 João Pessoa, PB, Brazil Department of Botany, University of São Paulo, 05422-970 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2011 Accepted 7 April 2012 Available online
Solanum subgenus Leptostemonum (Dunal) Bitter comprises approximately 450 species, of which 110, including 58 endemics, occur in Brazil, which is recognized as one of the centers of diversity of the group. Due the great morphological plasticity and its large number of species, several taxonomic treatments have been proposed for the genus Solanum, but its infrageneric classification is problematic. The aim of this study was to analyze the alkane composition of the leaf epicuticular waxes of nine species of the subgenus Leptostemonum to evaluate the chemotaxonomic potential of the alkanes. As results, were identified in nine species thirty-one alkanes, including iso- and anteisoalkanes. The major constituent of wax in most species was tritriacontane. Hentriacontane was the main constituent of Solanum paraibanum and Solanum torvum, and pentatriacontane was predominant in different populations of Solanum stramonifolium. The phenetic analysis of nine species based on the distribution profile of alkanes (Euclidean distance and UPGMA method) show three clusters with distinct main homolog. The profiles of alkanes showed some qualitative taxonomic value for species analyzed, although, a larger number of representative samples of this subgenus must be investigated. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Alkanes Chemotaxonomy Spiny solanums Solanaceae Waxes
1. Introduction Solanum L. is the largest and most complex genus of the Solanaceae. It is widely distributed in South America, Africa and Australia. It is among the ten largest genera of flowering plants, with approximately 1500 species (Bohs, 2007). The species of Solanum are characterized by poricidal anther dehiscence, a synapomorphy of the genus. This characteristic is only shared with Lycianthes (Dunal) Hassl., a genus very close to and previously considered part of Solanum (Bohs, 2005). The understanding of the infrageneric relationships of the genus Solanum has not been fully elucidated because the genus is morphologically extremely diverse. Solanum is divided into seven subgenera (Archaesolanum Marzell, Bassovia (Aubl.) Bitter, Leptostemonum (Dunal) Bitter, Lyciosolanum Bitter, Minon Raf., Brevantherum (Seithe) D’Arcy, Potatoe (G. Don) D’Arcy, and Solanum Seithe) and 70 sections (D’Arcy, 1972, 1991).
* Corresponding author. Tel.: þ55 81 21267813; fax: þ55 81 21267803. E-mail address: [email protected] (A.F.M. Oliveira). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.04.010
K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
49
Approximately one-third of Solanum species belong to the subgenus Leptostemonum (Levin et al., 2006). In Brazil, this subgenus is the second-largest and includes approximately 110 species, of which 58 are endemic. With about 44 species in the Brazilian Northeast, it is most representative of this region. According to Agra (2007), the species of the subgenus Leptostemonum occurring in Brazil belong to sections Acanthophora, Crinitum, Melongena, Erythrotrichum, Herposolanum, Lasiocarpa, Micracantha, Persicariae, Polytrichum, and Torva. Solanum subg. Leptostemonum is particularly interesting for study because of its reproductive strategies and phenotypic plasticity (Chiarini, 2004). Species of this subgenus is easily distinguished morphologically; however, they exhibit great phenotypic plasticity and variability in their habit, floral traits, sexual system and dispersal syndrome (Chiarini and Bernardello, 2006). Several species of this subgenus have been the subject of numerous molecular analyses. These analyses have contributed greatly to the current understanding of the phylogeny of Solanum (Olmstead and Palmer, 1997; Levin et al., 2006; Weese and Bohs, 2007; Poczai et al., 2008; Stern et al., 2010). Chemotaxonomic studies of the subgenus Leptostemonum are scarce. Nevertheless, the studies of Zydgalo et al. (1994) have indicated that the epicuticular alkanes were similar in some species of this subgenus, and Silva et al. (2004) have found that the flavonoids in five species of the subgenus Leptostemonum supported the traditional infrageneric classification. The chemotaxonomic significance of leaf wax alkanes has also been investigated in other subgenera of Solanum (Mecklenburg, 1966; Maxzud and Zydgalo, 1991; Mohy-Ud-Din et al., 2010). The aim of this study was to evaluate the composition of leaf wax alkanes in some Solanum species belonging to eight sections of the subgenus Leptostemonum of wild and cultivated populations in northeastern Brazil. Possible chemotaxonomic significance is discussed. 2. Materials and methods 2.1. Plant material Samples of nine species of Solanum subg. Leptostemonum were collected in Paraíba and Pernambuco States (Northeastern Brazil). The material was identified and the voucher samples were deposited at Herbarium Geraldo Mariz (UFP) and Herbarium Lauro Pires Xavier (JPB) of Federal University of Pernambuco and Federal University of Paraíba, respectively (Table 1). 2.2. Extraction and analysis of cuticular alkanes The waxes from fully expanded, fresh, intact leaves of each species were extracted by means of two rapid washes with dichloromethane (30 s). The cuticular extracts were fractionated by preparative thin layer chromatography and alkane fractions were analyzed in a gas chromatograph (GC-FID, Shimadzu, model 17A, Kyoto, Japan) according Souza et al. (2010). GC was performed using a DB-5 capillary column (30 m, 0.32 mm, 5% phenyl-95% dimethylpolysiloxane), helium as carrier gas at a flow rate of 1 cm3 min1 and split ratio 1:100. Injector and detector temperatures were 300 C. The temperature of the column moved from 150 C (3 min) to 280 C at 10 C min1 and was maintained at the final temperature under isothermal conditions for 34 min. The identification of the compounds was performed through the comparison of retention times with authentic samples n-alkane standard solution C21-C40 (Fluka S.A, Costa Rica). The anteiso and iso-alkanes were identified by their mass fragmentation patterns (GC-EIMS QP5050, Shimadzu) and by comparison with data of literature (Kavouras et al., 1998). The MS ionization was performed at 70 eV. 2.3. Cluster analysis The chemical relationships between species were evaluated using the alkane profiles. The quantitative distribution of the alkanes was examined using cluster analysis with Euclidean distance and UPGMA. The analysis was made using the NTSYS Ver. 2.11 software (Rohlf, 2005). Alkane concentrations below 0.5% were not included in the analysis.
Table 1 Species and sections of Solanum subg. Leptostemonum collected in Paraíba (PB) and Pernambuco (PE) states, Northeast of Brazil. Section
Species
Locality
Status
Voucher
Acanthophora Crinitum Erytrothrichum
S. S. S. S.
Lasiocarpa
S. rhytidoandrum Sendtn S. stramoniifolium Jacq.
Micracantha
S. paraibanum Agra
Persicariae Torva
S. gardneri Sendtn. S. torvum Sw.
PB, João Pessoa, Federal University of Paraíba PB, João Pessoa, Conde PB, João Pessoa, Conde PB, João Pessoa, Federal University of Paraíba PE, Igarassu, Usina São José, Fragmento Pezinho PB, João Pessoa, Alto do Mateus PB, João Pessoa, Benjamim Maranhão Botanical Garden PE, Recife, Dois Irmãos State Park PB, João Pessoa, Federal University of Paraíba PE, Parnamirim, BR 316, KM 151 PE, Parnamirim, Fragmento Olho d’água PB, João Pessoa, Federal University of Paraíba
Cultivated Native Native Native Native Cultivated Native Native Native Native Native Cultivated
64,326 59,188 42,185 59,190 59,175 59,177 59,176 59,174 59,183 59,181 59,180 59,185
mammosum L. palinacanthum Dunal crinitum Lam. paludosum Moric.
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Species
Alkanes (%) n-C24
n-C25
i-C26
n-C27
n-C28
n-C29
a-C30
n-C30
i-C31
n-C31
i-C32
a-C32
n-C32
i-C33
a-C33
n-C33
i-C34
a-C34
n-C34
i-C35
a-C35
n-C35
S. S. S. S. S. S. S. S. S. S. S. S.
tr tr tr – tr tr 0.69 tr – – tr tr
0.54 tr tr – – tr tr tr – – tr tr
tr tr tr tr 0.65 tr 7.08 1.21 2.99 4.59 2.09 tr
3.05 tr 0.63 0.91 2.72 1.01 2.25 4.35 tr tr 1.19 0.71
tr tr tr tr tr tr 0.96 2.62 tr tr tr tr
5.74 tr 0.51 10.33 5.76 1.56 2.05 23.98 0.89 1.02 1.40 2.85
– tr tr 0.69 tr tr – – – – – tr
0.67 tr tr 0.89 0.53 tr 1.02 2.83 – tr tr 0.92
1.35 tr tr 1.80 3.21 3.19 2.04 0.90 0.71 0.79 1.55 3.11
32.10 3.56 7.34 22.08 13.92 16.92 16.50 27.32 6.52 3.76 5.48 31.31
tr tr tr – – 0.68 tr – – – tr 0.67
– 1.78 1.63 6.10 3.79 tr – 4.50 tr – – tr
2.96 1.34 1.79 5.98 2.51 3.62 5.82 3.85 1.64 1.75 2.55 9.12
1.71 9.33 5.38 7.68 15.20 11.28 12.33 3.07 8.81 7.18 8.48 14.60
– 0.54 0.87 0.95 – 0.89 – – – – – –
46.11 68.07 64.84 37.47 30.27 53.38 44.12 19.63 49.32 31.43 34.36 20.03
– 0.61 tr – – tr 0.53 – 0.98 0.83 0.78 1.11
– 6.71 6.56 1.73 5.73 tr – – 2.04 – – 0.75
0.68 0.89 1.50 1.03 2.14 1.46 1.33 1.51 3.19 4.36 3.06 3.23
tr 1.86 1.17 1.06 3.97 1.09 0.68 0.62 3.54 5.33 4.33 2.86
– 0.53 0.63 – – tr – – – – – –
2.50 3.79 5.01 tr 5.22 1.50 0.75 2.36 18.34 37.22 32.36 7.36
crinitum gardneri* gardneri* mammosum palinacanthum paludosum* paludosum* paraibanum rhytidoandrum stramoniifolium* stramoniifolium* torvum
– ¼ not detected, n ¼ n-alkane, i ¼ iso-alkane, a ¼ anteiso-alkane, tr ¼ trace < 0.5%, * ¼ indicate different populations (See Table 1 for the voucher data).
K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
Table 2 Patterns of distribution of cuticular wax alkanes in species of Solanum subg. Leptostemonum of Paraíba and Pernambuco states (Northeast Brazil).
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3. Results and discussion Thirty-one alkanes between C21 and C35 carbon atoms in length, including iso and anteiso-alkanes, were identified in nine species of Solanum subg. Leptostemonum. High values for tritriacontane (n-C33) were detected in most species, notably in S. gardneri, in which it represented more than 60% of the total alkane content. Hentriacontane (n-C31) was the main constituent of the cuticular wax of Solanum paraibanum and Solanum torvum, 27.3 and 31.31%, respectively, whereas Solanum stramonifolium was the only species found to have a high content of pentatriacontane (n-C35), 34.79% on average (Table 2). The distribution pattern of the n-alkanes of the species of Solanum subg. Leptostemonum examined is similar to that already reported by other studies in the genus Solanum (Maxzud and Zydgalo, 1991), including the subgenus Leptostemonum ski et al., 2011). According to Zydgalo et al. (1994), the subgenus Leptostemonum can be distin(Zydgalo et al., 1994; Halin guished from the subgenus Solanum by the high amounts of n-C33 found in the former. This n-alkane was also the major component of the cuticular wax of three other species placed in the subgenus Leptostemonum: Solanum melongena L., Solanum ski et al., 2011). In our study, n-C33 was found at high levels in six of the nine species incanum L. and Solanum insanum L. (Halin analyzed. However, in those species where this n-alkane was not found as a major constituent, for example, in S. paraibanum and S. torvum, its presence should not be neglected (approximately 20%, Table 2). We believe that future studies with larger number of species may contribute to distinction of subgenus Leptostemonum from subgenus Solanum based on n-C33 as major component of n-alkanes on cuticular waxes. Branched iso- (2-methyl) and anteiso- (3-methyl) alkanes were conjointly identified in all nine species of the subgenus Leptostemonum. The 2-methyl tritriacontane (i-C33) was the predominant branched alkane, followed by 2-methyl pentatriacontane (i-C35) (Table 2). In angiosperms, branched alkanes are less frequent than n-alkanes. This difference may indicate that the biosynthesis of methylalkanes is restricted to some taxa and is not as widespread as the biosynthetic pathway of nalkanes. According to some authors, n-alkanes and methylalkanes may possibly originate from distinct biosynthetic pathways (Kolattukudy, 1970; Kroumova and Wagner, 1999). The iso- and anteiso-alkanes are commonly found in Solanaceae (Mecklenburg, 1966; Maxzud and Zydgalo, 1991; Rogge et al., 1994; Zydgalo et al., 1994), but they are not usually found in other families. It is possible that the branched alkanes can be useful as diagnostic character for Solanaceae. However, ski et al. (2011), methylalkanes are more subject to variations during plant growth than the n-alkanes, and according to Halin the latter are more suitable for chemosystematic studies. A numerical analysis based on the quantitative distribution of the cuticular alkanes in Solanum subg. Leptostemonum is shown in Fig. 1. Three clusters were identified. The first cluster, formed by different populations of S. stramoniifolium, was characterized by similar high levels of n-C35 and n-C33. The second cluster included most species and was marked by n-C33 percentage above 30%. The third cluster, comprising only two species (S. paraibanum and S. torvum), was characterized by high percentage of n-C31 in relations to the other species. A higher population similarity was found between the accessions of S. gardneri, S. stramoniifolium and S. paludosum. This finding indicates a high genetic homogeneity in the biosynthesis of alkanes for these species. Similar results have been
Fig. 1. UPGMA dendrogram of nine species of Solanum subg. Leptostemonum based on patterns cuticular alkanes. Indications above branches correspond to the main n-alkane. Legends: n-C31 ¼ hentriacontane, n-C33 ¼ tritriacontane, n-C35 ¼ pentatriacontane. Asterisks indicate different populations.
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K.M.M. Silva / Biochemical Systematics and Ecology 44 (2012) 48–52
reported by Zydgalo et al. (1994) for the n-alkane profiles of the epicuticular wax of the Tribe Nicotianeae from different phytogeographical areas. These data underscore the potential of cuticular alkanes for use in taxonomic studies. One of the most important phylogenetic studies of Solanum subgenus Leptostemonum has been conducted by Levin et al. (2006). The phylogenetic analysis performed by these authors includes 24 species of the Solanum subg. Leptostemonum, five of which (S. torvum, Solanum crinitum, Solanum mammosum, Solanum palinacanthum, and S. stramonifolium) were included in our study. More recently, Miz et al. (2008) also investigated the phylogenetic relationships of this subgenus with some southern Brazilian species from section Acanthophora, Lasiocarpa, and Torva. The molecular data for Brazilian species showed more resolution among species than that including Old World species. Despite the reduced number of species from our study some interesting suggestions can be made. As already pointed out, most species analyzed in the present research presented n-C33 as main n-alkane. Among then, only S. mammosum and S. palinacanthum have been sampled at Levin et al. (2006) study. Interestingly, these two species are placed at the same cluster based on chemical data (Fig. 1) and are part of the same clade at Levin et al. (2006) study. On the other hand, S. torvum, S. crinitum, and S. stramonifolium, all of which with other main homolog than n-C33, do not share either same cluster (Fig. 1) or clade (Levin et al., 2006). Although very few species have been sampled for n-alkanes analyzes, putting together our results with those previously published (Zydgalo et al., 1994), future investigations deserve to be taken forward. Sampling more species of Solanum is very important to verify if the n-C33 is the main component of cuticular alkane for the genus or for some subgenus. Other important point is to verify whether iso- and anteiso-alkanes are important features for the whole Solanaceae family or only for Solanum. Acknowledgements The authors are grateful to Kiriaki Nurit and Ionaldo Basílio for their technical assistance. This work was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) with grants to KMM Silva and MF Agra. References Agra, M.F., 2007. Diversity and distribution of Solanum subg. Leptostemonum in Brazil. Acta Hortic. 745, 31–42. Bohs, L., 2005. Major clades in Solanum based on ndhF sequence data. In: Hollowell, V., Keating, T., Lewis, W., Croat, T. (Eds.), A Festschrift for William G. D’Arcy: The Legacy of a Taxonomist. Monographs in Systematic Botany from the Missouri Botanical Garden, vol. 104. Missouri Botanical Garden Press, St. Louis, pp. 29–49. Bohs, L., 2007. Phylogeny of the Cyphomandra clade of the genus Solanum (Solanaceae) based on ITS sequence data. Taxon 56, 1012–1026. 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