Biochemical Systematics and Ecology 45 (2012) 130–137 Contents lists available at SciVerse ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Chemical and molecular characterization of fifteen species from the Lantana (Verbenaceae) genus José Guedes de Sena Filho a, *, Allívia Rouse Carregosa Rabbani a, Tânia Regina dos Santos Silva b, Ana Veruska Cruz da Silva a, Igor Azevedo Souza a, Maria José Bryanne Araujo Santos a, Jemmyson Romário de Jesus c, Paulo Cesar de Lima Nogueira c, Jennifer M. Duringer d a Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA, Coastal Tablelands, Av. Beira, Mar 3250, 49025-040 Aracaju, SE, Brazil Herbário, Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, BR 116 Norte, Km 03, 44031-460 Feira de Santana, BA, Brazil LABORGANICS – Laboratório de Pesquisa em Química Orgânica de Sergipe, Departamento de Química – CCET, Universidade Federal de Sergipe, Av. Marechal Rondon s/n- Jd. Rosa Elze, 49100-000 São Cristóvão, SE, Brazil d Department of Environmental & Molecular Toxicology, Oregon State University, 139 Oak, Creek Building, Corvallis, OR 97331, USA b c a r t i c l e i n f o a b s t r a c t Article history: Received 28 April 2012 Accepted 7 July 2012 Available online xxx The essential oil from two Lantana species (Lantana lucida Schauer and Lantana salzmannii Schauer) were evaluated for their chemical composition by GC/MS. Results showed 17 predominant compounds for L. lucida, among which (E)-caryophyllene (19.0%) and a-humulene (33.0%) were the major components. L. salzmannii showed the presence of 58 compounds, the most abundant of which were (E)-caryophyllene (15.6%) and selin-11-en-4-ol (11.2%). Next, cluster analyses of the chemical composition of the volatile fraction of five Lantana species from our studies (Lantana radula, Lantana canescens, L. lucida, L. salzmannii and Lantana camara), as well as 10 Lantana species published in the literature (Lantana achyranthifolia, Lantana aculeata, Lantana balansae, Lantana hirta, Lantana involucrata, Lantana fucata, Lantana salviifolia, Lantana trifolia, Lantana velutina and Lantana xenica) were performed. Species fell into three main groups. A cluster analysis of (E)-caryophyllene content was also performed which resulted in the 15 Lantana species being segregated into four main groups. In addition, Inter-Simple Sequence Repeat (ISSR) was used to evaluate the genetic variation between five Lantana species collected from northeastern Brazil (L. radula, L. canescens, L. lucida, L. salzmannii and L. camara). Analysis showed a 36% similarity between, L. salzmanii and L. canescens, and a 48% similarity between L. lucida and L. canescens. Overall, results, indicate that it is possible to discriminate between groups of Lantana taxa based on both their chemical, composition and ISSR markers. In addition, this study provided further support for using (E)-caryophyllene as a chemical marker for species belonging to the Lantana genus. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Lantana Lantana lucida Lantana salzmannii (E)-caryophyllene Taxonomy ISSR 1. Introduction The Lantana genus consists of approximately 150 plant species, geographically spanning from the tropics to the subtropics of the Americas, with a few members found in tropical Asia and Africa (Ghisalberti, 2000). Lantana species are used in folk medicine for many diseases and for ornamentation in gardens (Chowdhury et al., 2007). They have a very pungent odor which * Corresponding author. Tel.: þ55 83 32354749. E-mail addresses: guedesena@yahoo.com.br, jose-guedes.sena@embrapa.br (J.G. de Sena Filho). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.024 J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 131 originates from their leaves (Ghisalberti, 2000; Walden et al., 2009). To date, many studies have described the chemical composition and pharmacological activity of a variety of Lantana species (Jimenez-Arellanes et al., 2003; Julião et al., 2009; Sena Filho et al., 2009, 2010). Taxonomically, the Lantana genus is divided into four sections: Lantana, Callioreas, Rhytidocamara and Sarcolippia (Schauer, 1847; Briquet, 1904). The divisions are based on floral and carpological characteristics, the best tools for classification available at the time they were made. In general, specimens from this genus are very difficult to classify, due to the shape of their inflorescence which changes with age and flower color. A taxonomic study of four genera from the Verbenaceae family (Lippia, Lantana, Aloysia and Phyla) proposed using iridoid glucosides as a taxonomic marker for this family (Rimpler and Sauerbier, 1986). This study contributed a great deal of information regarding the chemotaxonomy of Verbenaceae. Unfortunately, the presence and type of iridoid glucosides in plants from the morphologically similar Lippia and Lantana genera are virtually indistinguishable, and so are not very helpful in differentiating between them. Sena Filho et al. (2010) proposed a chemical marker for the Lantana genus, in which (E)-caryophyllene was the major compound detected, together with phellandendrene, cubebene and elixene as minor components in an analysis of 15 species. In the Lippia species evaluated, (E)-caryophyllene was not detected as the major compound; rather, it was suggested that species belonging to the Lippia genera would contain limonene, citral, carvacrol, b-myrcene, camphor and thymol as their main chemical markers. Recently, in addition to the phenotypic characteristics of an individual plant species, genetic characteristics have been found to be useful and oftentimes necessary in distinguishing between plant species, cultivars and/or individuals occupying different ecological niches (Santos et al., 2011; Costa et al., 2011; Silva et al., 2012). In addition, an understanding of the genetic diversity within a species is indispensable to optimally manage genetic resources for conservation and taxonomic categorization, (Azizi et al., 2009). DNA analysis based on molecular markers such as Inter-Simple Sequence Repeat (ISSR) can be taxonomically useful in phylogenetic studies to distinguish between plant species and subspecies (Khan et al., 2000; Raina et al., 2001; Monteleone et al., 2006). These markers are not affected by environmental conditions, and have become increasingly important for surveying genetic diversity and for genotype identification of medicinal plants (Nybom and Weising, 2007). Thus, the first aim of this study was to evaluate the essential oil of two endemic Lantana species from the salt marsh Atlantic forest landscape in Brazil (Lantana lucida Schauer and Lantana salzmannii Schauer). The second was to characterize the intraspecific variation of the essential oil composition in natural populations of those Lantana species, as well as 13 Lantana species published in the literature (Lantana achyranthifolia (Hernandes et al., 2005), Lantana aculeate (Saxena and Sharma, 1999), Lantana balansae (De Viana et al., 1973), Lantana camara (Rana et al., 2005), Lantana canescens (Sena Filho et al., 2010), Lantana hirta (Walden et al., 2009), Lantana involucrate (Pino et al., 2006), Lantana fucata (De Oliveira et al., 2008), Lantana radula (Sena Filho et al., 2010), Lantana salviifolia (Ouamba et al., 2006), Lantana trifolia (Juliao et al., 2009), Lantana velutina (Walden et al., 2009) and Lantana xenica (Juliani et al., 2002)) using the Weighted Pair Grouping Method (WPGM). The third was to cluster the 15 Lantana species based on their (E)-caryophyllene concentration. The last was to perform a cluster analysis using molecular characterization of five Lantana species (L. radula, L. canescens, L. lucida, L. salzmannii and L. camara) by ISSR–PCR and compare this to the (E)-caryophyllene results for chemical similarity, so that the taxonomy of this genus could be evaluated. 2. Methodology 2.1. Plant material L. salzmannii Schauer and L. lucida Schauer were collected in Itaporanga d’ajuda, Sergipe, Brazil in April 2011. Voucher specimens (J. G. de Sena Filho) were deposited at the Herbarium of the Universidade Estadual de Feira de Santana (HUEFS), Bahia, Brazil under the numbers HUEFS 178287 and HUEFS 178288, respectively. L. canescens Kunth, L. radula Sw and L. camara L. were collected in January of 2011 and identified by Dr. Rita de Cassia Pereira. Voucher specimens were deposited at the Herbarium Dárdano de Andrade Lima (IPA), in the Instituto Pernambucano de Pesquisa Agropecuaria, Pernambuco, Brazil under numbers 74,048, 70,004 and 86,846, respectively. 2.2. Oil isolation procedure The oil was obtained by hydrodistillation over 4 h using a Clevenger-type apparatus with 600 g of fresh leaves cut into pieces (Sena Filho et al., 2010). The oil was dried with anhydrous sodium sulphate and stored at 20  C in a sealed amber bottle until chemical analysis was performed. The yield afforded from L. salzmanni was 0.8% and from L. lucida was 0.6%. 2.3. Essential oil analysis Essential oil analyses of L. lucida and L. salzmannii were performed on a Shimadzu QP5050A GC/MS system equipped with an AOC-20i auto-injector. A J&W Scientific DB-5MS (coated with 5% phenyl–95% dimethylpolysiloxane) fused capillary column (30 m  0.25 mm  0.25 mm film thickness) was used as the stationary phase. Helium was used as the carrier gas, at a flow rate of 1.2 mL/min. The column temperature program was as follows: 40  C for 4 min, raised to 220  C at 4  C/min, then heated to 280  C at 20  C/min. The injector and detector temperatures were 250  C and 280  C, respectively. Samples (0.5 mL in 132 J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 CH2Cl2) were injected with a 1:20 split ratio. MS were taken at 70 eV with a scan interval of 0.5 s and fragments from 40 to 350 Da. The retention indices were obtained by mixing the oil sample with a C9–C18 linear hydrocarbon mixture (Van den Dool and Kratz, 1963). The volatile components were analyzed by GC/MS, and identification was made by comparing retention indices and mass spectra (Adams, 2007) with those in the literature, as well as by computerized matching of the acquired mass spectra with those stored in the NIST and Wiley mass spectral libraries and other published mass spectra. GC analyses were carried out using a Perkin Elmer (Shelton, CT, USA) Clarus 500 gas chromatograph fitted with a flame ionization detector (FID) and TC Navigator software. Separation of the compounds was achieved with a Perkin Elmer Elite Plot 5 capillary column (5% diphenyl–95% dimethylpolysiloxane, 30 m  0.25 mm i.d. X 0.25 mm film thickness) and N2 as the carrier gas. The other parameters (oven temperature program, injector and detector temperature and amount of sample) were the same as those used for the GC/MS analysis described above. Peak areas and retention times were measured using an electronic integrator and TC Navigator software. The percentage composition of each component was determined by dividing the area of the component by the total area of all components isolated under these conditions, without an FID response factor correction. 2.4. Cluster analyses of essential oil chemical composition For the first cluster analysis, we included chemical components of the essential oils from the two Lantana species evaluated in this study, as well as 13 species referenced in the literature (L. achyranthifolia, L. aculeate, L. balansae, L. camara, L. canescens, L. hirta, L. involucrate, L. fucata, L. radula, L. salviifolia, L. trifolia, L. velutina and L. xenica) that had at least a 2% or greater recovery of any compound (Appendix 1). Species were clustered using ranges of all volatile compounds reported in the literature: 0–2%; 2.01–5%, 5.01–10%, 10.01–15%, 15.01–20%, 20.01–25%, 25.01–30%, 30.01–35%, 35.01–40%, 40.01–45%, 45.01–50%, 50.01–55%, and 55.01–60%. Then, a second cluster analysis was performed using only (E)-caryophyllene content, with ranges of: 0–15%, 15.01–30% and 30.01–45%. Based on the presence or absence of constituents in the oil of a species, a dissimilarity coefficient matrix was calculated (Jaccard, 1908). Clustering of the matrix for both analyses was carried out using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster algorithm (Sokal and Michener, 1958). Statistical analysis was performed using the PASW Statistics v. 18 software (http://www.ibm.com/us/en/). 2.5. Genetic analysis with ISSR markers Five Lantana species (L. radula, L. canescens, L. lucida, L. salzmannii, and L. camara) were analyzed by ISSR. DNA was isolated from young leaves as previously described by Doyle (1991). Fourteen primers were used to screen for polymorphism (Table 1). The reaction volume was 20 mL, and consisted of 2 mL genomic DNA (40 ng), 1 mL of each primer (0.2 mM), and a mix composed of: 14.4 mL ultrapure sterile water, 2 mL 10X buffer (3 mM MgCl2, 100 mM MgSO4, 100 mM KCl, 80 mM (NH4)2SO4, 100 mM Tris–HCl) (Neo Taq, Koma Biotech, Korea), 0.4 mL dNTP (10 mM) and 0.2 mL Taq polymerase (5 U/mL). PCR amplification was performed in a PTC-100 thermocycler (MJ Research, Inc., Watertown, MA, USA) using a cycle of 95  C for 5 min for initial denaturation, followed by 45 cycles of denaturation at 94  C for 1 min, 51.5  C for 45 s for primer annealing, 72  C for 2 min for extension, and finally one cycle of 72  C for 10 min for final extension. For fragment visualization, a 2% agarose gel (1X TEB: 89 mM TRIS, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) was used in a horizontal electrophoresis system (Sunrise, Gibco BRL, Table 1 Primers and number of polymorphic fragments generated from five species of Lantana. Primera Sequence 50 –30 NPFb ISSR ISSR ISSR ISSR ISSR ISSR ISSR ISSR 843 807 810 823 835 841 845 CACACACACACAGG CTCTCTCTCTCTCTCTAC CACACACACACAAC GAGAGAGAGAGAGG GAGAGAGAGAGACC CACCACCACGC GAGGAGGAGGC CTCCTCCTCGC CTCTCTCTCTCTCTCTRA AGAGAGAGAGAGAGAGT GAGAGAGAGAGAGAGAT TCTCTCTCTCTCTCTCC AGAGAGAGAGAGAGAGYC GAGAGAGAGAGAGAGAYC CTCTCTCTCTCTCTCTRG 6 6 3 6 6 7 6 7 5 10 8 5 7 5 6 1 2 4 8 10 12 13 14 a Primers ISSR 1–14 were from Invitrogen (New York, NY USA); 843, 807, 810, 823, 835, 841 and 845 were from Integrated DNA Technologies (Coralville, IA, USA). b NPF – Number of polymorphic fragments generated. J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 133 USA), carried out at a constant voltage of 100 V for 90 min. The gel was then stained with ethidium bromide solution (5 mg/ mL) for 15 min. The ISSR fragment amplification products were visualized under ultraviolet light using a Gel Doc L-Pix image system (Loccus Biotecnologia, Brazil). ISSR markers were scored for the presence (1) or absence (0) of a fragment, and a data matrix of I-scores were generated and similarity coefficients calculated using Jaccard’s arithmetic complement index (Jaccard, 1908). The dendrogram was constructed using the UPGMA cluster algorithm (Sokal and Michener, 1958) to determine the robustness of the dendrogram; the data was bootstrapped with 10,000 replications using FreeTree software (http://web.natur.cuni.cz/flegr/programs/ freetree.htm) and, for visualization of the cluster, we used XLSTAT software (www.xlstat.com). 3. Results and discussion Species of the Lantana genus were last subdivided over 100 years ago using only morphological characteristics (Schauer, 1847; Briquet, 1904). With the advent of more advanced tools for both chemical and genomic analysis, our objective in this study was to refine the classification of species in this genus, especially in regards to testing the proposal of using (E)-caryophyllene as a chemical marker. First, essential oils from L. lucida and L. salzmannii were evaluated for their chemical composition. Seventeen volatile compounds from L. lucida were identified which amounted to 94.9% of all peaks in the essential oil (Table 2). (E)-caryophyllene (19.0%), a-humulene (33.0%), d-cadinene (11.3%), a-copaene (6.9%), bicyclogermacrene (4.9%) and b-cubebene (4.4%) were the major compounds detected. In the L. salzmannii essential oil, 58 compounds were identified, representing 92.9% of all peaks. The most abundant compounds were (E)-caryophyllene (15.6%), selin-11-en-4a-ol (11.2%), trans-calamenene (6.6%), b-selinene (5.8%) and trans-cadina-1,4-diene (4.5%) (Table 2). A complete listing of the volatile components of L. lucida and L. salzmannii essential oil extracts and their percentages are presented in Table 2. Notably, monoterpenes were only present in L. salzmannii (12.4%); none were detected in the L. lucida essential oil used in this study. In general, terpenoids have been used effectively as chemotaxonomy markers; chemical variation in this group of compounds has been used to define intra- and inter-specific variability in a variety of plant species (Sena Filho et al., 2007; Adams et al., 2003). In this context, our research group performed a clustering analysis of 13 Lantana species from the literature (Appendix 1), as well as two Lantana species, endemic to Brazil, whose volatile components had not been previously characterized, for the variation of 77 mono- and sesquiterpenes in their essential oils. Plant species came from the following Lantana genus subdivisions: Lantana (L. camara, L. lucida and L. aculeate), Rhytidocamara (L. achyranthifolia) and Callioreas (L. xenica, L. canescens, L. balansae, L. hirta, L. involucrata, L. fucata, L. salviifolia, L. salzmannii, L. radula, L. trifolia, and L. velutina). The cluster analysis found three major groupings using a level of 23% dissimilarity (73% similarity): 1) L. radula, L. canescens, L. salviifolia, L. involucrate, L. trifolia, L. salzmannii, L. camara, L. fucata; 2) L. balansae, L. velutina; 3) L. achyranthifolia, L. aculeate; 4) L. lucida, L. xenica; and 5) L. hirta (Fig. 1A). In order to support using (E)-caryophyllene as a chemical marker for the Lantana genus, we performed a second clustering analysis grouping each species based on (E)-caryophyllene content which resulted in four main groups: 1) L. trifolia, L. velutina, L. salzmannii, L. radula, L. camara, L. lucida; 2) L. fucata, L. salvifolia, L. aculeata, L. hirta, L. involucrata, L. balansae; 3) L. achyranthifolia; and 4). L. canescens, L. xenica (Fig. 1B). The group Rhytidocamara was distinguished from the other species, as suggested by Briquet (1904) and Schauer (1847). When only the five species our group had collected were clustered for (E)caryophyllene content (Fig. 1C), a total of three groups were observed: 1) L. radula, L. camara; 2) L. salzmannii, L. lucida and 3) L. canescens. We believe that the isolation of L. canescens from the other Lantana species is due to the exceedingly high amount of (E)-caryophyllene it contains (43.9% versus 15.6–23.3% in the other four species). In addition, the five Lantana species that we collected from northeastern Brazil (L. radula, L. canescens, L. lucida, L. salzmannii and L. camara) were subjected to a cluster analysis using molecular characterization by ISSR markers. ISSR is a powerful technique that has been used to resolve species/subspecies differences in a genus or to differentiate between genera in a family. For example, ISSR was used to distinguish between genera for Lolium, Festuca (Pasakinskiene et al., 2000) and Diplotaxis (Martin and Sanchez-Yelamo, 2000), as well as several aromatic and medicinal plant groupings (Farajpour et al., 2011; Pezhmanmehr et al., 2009; Manica-Cattani et al., 2009; Suárez González et al., 2007; Fracaro et al., 2005). In our study, ISSR fingerprints clearly distinguished all five tested species (Fig. 2). The 14 ISSR primers generated a total of 93 fragments, 100% of which were polymorphic. The primer with the highest number of fragments was 807 (10 fragments), while the lowest was ISSR 4 (3 fragments). Genotypes of the five species of Lantana selected for this study were clustered by UPGMA using the Jaccard coefficient (JC), estimated from the binary data (Fig. 2). The similarity mean was 0.17 JC (0.10–0.23 JC, Table 3). We observed a clear separation of two groups, with L. camara being the most isolated of the five species. Interestingly, L. canescens and L. salzmannii contain a large variety of mono- and sesquiterpenes; the ISSR results corroborate the cluster analysis performed on essential oil components which found a 36% similarity between the two species. Our results suggest that the genetically directed production of volatile compounds is correlated in the Lantana species we studied, and could be a possible alternative for grouping the species taxonomically. Combining the chemical results with those using ISSR provided additional information on the similarity of the Lantana species evaluated in this study. We suggest that species in the same grouping may have similar routes of biosynthesizing secondary compounds, which result in activation of similar genes. However, the presence or absence of insect and other parasites and other environmental factors could affect the metabolic routes that are activated, (Pichersky and Gershenzon, 2002; Paolini et al., 2010) which must be taken into consideration when making associations between genes and 134 J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 Table 2 Essential oil composition of Lantana salzmannii and Lantana lucida leaves collected in Sergipe, Brazil. Compounds RIa RIb a-thujene a-pinene 924 930 970 980 989 1005 1006 1015 1018 1023 1028 1030 1046 1057 1080 1084 1099 1179 1335 1346 1370 1376 1389 1387 1406 1421 1424 1430 1435 1441 1449 1456 1460 1482 1483 1490 1492 1495 1496 1497 1503 1507 1513 1518 1522 1523 1533 1541 1560 1576 1582 1586 1595 1605 1610 1628 1638 1642 1647 1655 1660 1667 1671 1679 924 932 969 974 988 1002 1008 1014 1020 1022 1024 1026 1044 1054 1085 1086 1095 1174 1335 1345 1373 1374 1389 1389 1409 1417 1434 1430 1437 1442 1448 1452 1458 1484 1484 1489 1493 1500 1498 1500 1505 1505 1513 1522 1521 1528 1533 1544 1561 1577 1582 1586 1592 1602 1608 1627 1639 1645 1644 1658 1658 1655 1675 1687 % Peak area L. salzmannii sabinene oct-1-en-3-ol myrcene a-phellandrene d-3-carene a-terpinene p-cymene o-cymene limonene 1,8-cineole (E)-b-ocimene g-terpinene p-mentha-2,4(8)-diene terpinolene linalool terpinen-4-ol d-elemene a-cubebene a-ylangene a-copaene b-elemene b-cubebene a-gurjunene (E)-caryophyllene g-elemene b-copaene a-guaiene guaia-6,9-diene cis-muurola-3,5-diene a-humulene allo-aromadendrene germacrene D b-chamigrene b-selinene trans-muurola-4(14),5-diene bicyclogermacrene a-selinene a-muurolene (E,E)-a-farnesene b-bisabolene g-cadinene d-cadinene trans-calamenene zonarene trans-cadina-1,4-diene a-calacorene (E)-nerolidol spathulenol caryophyllene oxide gleenol viridiflorol ledol humulene epoxide II epi-cuben-1-ol caryophylla-4(12),8(13)-dien-5-(a/b)-ol cubenol a-muurolol neo-intermedeol selin-11-en-4a-ol humulane-1,6-dien-3-ol cadalene eudesma-4(15),7-dien-1b-ol Total peaks identified Retention indices obtained from a DB-5 MS column and calculated according to a 0.1 0.1 0.1 0.1 0.2 0.3 3.7 0.2 0.5 1.3 2.8 0.3 0.2 1.7 0.1 0.9 0.1 0.2 0.6 0.3 0.1 3.2 2.4 – – – – – – – – – – – – – – – – – – – – – 6.9 4.4z z 0.7 19.0 – 2.5 – – – 33.0 0.9 3.5 1.8 0.6 0.8 4.9 – 2.8 – – – 11.3 – 0.5 – – – – – – – – 1.3 – – – – – – – – – 94.9 – 0.6 15.6 0.2 0.1 0.2 0.1 0.8 3.2 1.2 3.0 – 5.8 – – 3.7 – 0.1 0.9 0.1 1.6 6.6 – 4.5 0.2 2.0 0.3 2.0 0.4 0.3 0.9 0.6 2.6 0.3 1.0 0.4 1.4 11.2 0.7 0.2 0.6 92.9 Van den Dool and Kratz (1963) or L. lucida b Adams (2007). zCo-eluting peaks. J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 A Dissimilarity coefficient (%) C B 135 Dissimilarity coefficient (%) Dissimilarity coefficient (%) Fig. 1. Dissimilarity phenograms derived from the: (A) chemical variation of 77 compounds observed in essential oil extracts from Lantana species; (B) amount of (E)-caryophyllene found in the 15 Lantana species used in this study; and (C) amount of (E)-caryophyllene evaluated in the five Lantana species which have been collected by our group. component concentration. For example, gene expression of three monoterpene synthase genes (LaTPS12, LaTPS 23 and LaTPS 25) in Lippia alba (Verbenaceae) were recently evaluated; results showed that the production of essential oils was higher in young versus older leaves (Pandelo et al., 2012). Thus, further research is necessary to more thoroughly evaluate the genetic and chemical correlations regarding essential oil production and its components in Lantana species. Fig. 2. Dendrogram of genetic similarity from ISSR using the Jaccard coefficient and the Unweighted Pair Group with Arithmetic Mean method with bootstrap analysis for five species of Lantana. 136 J.G. de Sena Filho et al. / Biochemical Systematics and Ecology 45 (2012) 130–137 Table 3 Genetic similarity among five species of Lantana using the Jaccard coefficient. Lantana Lantana Lantana Lantana Lantana radula canescens salzmannii camara lucida L. radula L. canescens L. salzmannii L. camara L. lucida 1.00 0.23 0.19 0.11 0.22 1.00 0.23 0.16 0.10 1.00 0.21 0.15 1.00 0.13 1.00 In summary, we characterized the essential oils from two Brazilian species of Lantana which had not been previously reported, as well as differentiated the chemical and genetic characteristics of 15 Lantana species through cluster analyses. The idea of combining chemical, genetic and morphological evaluations and synthesizing taxonomic relationships using currently available statistical software has the potential to greatly refine botanical taxonomy and aid in the most accurate identification/ separation of species to date. The compilation of the three different analyses performed here provides an example of classifying plants using a multidisciplinary approach. This work will support future studies on the genetic and chemical evaluation of Lantana species and other genera belonging to the Verbenaceae family which should include a larger number of species so that a more complete study of the chemical, genetic and taxonomic diversity can be performed. Acknowledgments The authors would like to thank Dr. Rita de Cassia Perreira, and Dr. Olivia Cano for their great contributions in species identification. The authors are grateful to CAPES and CNPq for financial support. Appendix A. 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