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Verbenaceae) Milene C Almeida et al. BMC Res Notes (2018) 11:725 https://doi.org/10.1186/s13104-018-3839-y BMC Research Notes RESEARCH NOTE Open Access Genetic diversity and chemical variability of Lippia spp. (Verbenaceae) Milene C. Almeida1, Ediedia S. Pina1, Camila Hernandes2, Sonia M. Zingaretti1, Silvia H. Taleb‑Contini1, Fátima R. G. Salimena3, Svetoslav N. Slavov4, Simone K. Haddad4, Suzelei C. França1, Ana M. S. Pereira1 and Bianca W. Bertoni1* Abstract Background: The genus Lippia comprises 150 species, most of which have interesting medicinal properties. Lip- pia sidoides (syn. L. origanoides) exhibits strong antimicrobial activity and is included in the phytotherapy program implemented by the Brazilian Ministry of Health. Since species of Lippia are morphologically very similar, conventional taxonomic methods are sometimes insufcient for the unambiguous identifcation of plant material that is required for the production of certifed phytomedicines. Therefore, genetic and chemical analysis with chemotype identifca‑ tion will contribute to a better characterization of Lippia species. Methods: Amplifed Length Polymorphism and Internal Transcribed Spacer molecular markers were applied to determine the plants’ genetic variability, and the chemical variability of Lippia spp. was determined by essential oil composition. Results: Amplifed Length Polymorphism markers were efcient in demonstrating the intra and inter-specifc genetic variability of the genus and in separating the species L. alba, L. lupulina and L. origanoides into distinct groups. Phy‑ logenetic analysis using Amplifed Length Polymorphism and markers produced similar results and confrmed that L. alba and L. lupulina shared a common ancestor that difer from L. origanoides. Carvacrol, endo-fenchol and thymol were the most relevant chemical descriptors. Conclusion: Based on the phylogenetic analysis it is proposed that L. grata should be grouped within L. origanoides due to its signifcant genetic similarity. Although Amplifed Length Polymorphism and Internal Transcribed Spacer markers enabled the diferentiation of individuals, the genotype selection for the production of certifed phytomedi‑ cines must also consider the chemotype classifcation that refects their real medicinal properties. Keywords: Lippia origanoides, Phytomedicine, AFLP, ITS2, CG/MS, Phylogenetic relationships Background are considered rare or endangered, and nine are under Te genus Lippia comprises 150 species, most of which threat of extinction due to the destruction of their natu- are distributed in the Neotropical ecozone [1]. Brazil ral environments in the Cerrado region (Brazilian type of stands out as the centre of diversity of the genus with 98 Savana) [3]. species presenting high degrees of endemism. More than Te Brazilian Ministry of Health has developed an half of these species are concentrated in the Espinhaço extensive phytotherapy program over the last decade Range, which stretches 1000 km through the Brazilian with the aim of providing access to herbal medicines states of Minas Gerais and Bahia [2]. However, 18 species for the entire population. One of the target species of this program is Lippia sidoides Cham. (syn. L. origa- noides) (Verbenaceae), a plant that was included in the *Correspondence: [email protected] Formulário de Fitoterápicos da Farmacopéia Brasileira 1 Departamento de Biotecnologia, Universidade de Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil [4, 5] based on its strong antimicrobial activity, against Full list of author information is available at the end of the article Candida albicans [6, 7], Staphylococcus aureus, and © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Almeida et al. BMC Res Notes (2018) 11:725 Page 2 of 14 Escherichia coli [8] were included due to the presence of we consider the interaction between the genotype and terpenoids in the essential oil. It is well known that ter- the environment [19]. In this context, studies aimed at penoids are produced as part of the plant defense system evaluating the genetic structure of the genus through and have been considered a promising source of biologi- analysis of molecular markers could be useful in clas- cal compounds [9–12]. Several essential oil compounds sifying species into clusters according to their genetic such as linalool, eugenol, carvone, vanillin, carvacrol, and similarities. thymol have been accepted by the European Commission A number of reports confrm that the association of to be used in food preservation or favorings [13]. molecular markers such as amplifed fragment length Te morphological similarities between this and other polymorphism (AFLP) and internal transcribed spacer species within the genus tend to complicate the accurate 2 (ITS2) can contribute signifcantly to the analysis of botanical identifcation, leading to difculties in the pro- genetic variability and phylogenetic inferences [20, 21]. duction of certifed herbal medicines. Besides molecular markers, chemical markers can also Based on the diferential morphological characteris- be used to help the correct plant characterization. WinK tics, the genus Lippia was classifed in seven sections [22] developed a phylogenetic classifcation based on [14]. Te Zapania Schauer section is the most com- the secondary metabolites produced by Fabaceae, Sola- plex and exhibits inforescences with fat bracts, spirally naceae and Lamiacea families. Te author considered arranged, globose or hemispheric type, capituliform, with that the ability or inability to produce a specifc metabo- varying numbers of chromosomes (2n = 10–28). L. alba lite—shown by diferent members of related phylogenetic (Mill.) N.E.Br., L. aristata Schauer, L. brasiliensis (Link) groups, are the result of diferential expression patterns T.R.S. Silva, L. corymbosa Cham., L. diamantinensis that refect specifc plant strategies for adaptation that Glaz., L. duartei Moldenke, L. flifolia Mart. & Schauer, were incorporated into the phylogenetic structure. L. hermannioides Cham., L. lacunosa Mart. & Schauer, Terefore, the aim of the present study was to assess L. rotundifolia Cham. and L. rubella (Moldenke) T.R.S. the genetic and chemical variability of species of Lip- Silva & Salimena [15, 16] are among the representatives pia spp. using molecular and chemical markers, to draw of this section in the Brazilian fora. inferences regarding the phylogenetic relationships Te Goniostachyum Schauer section presents tetras- within the genus, and to identify inconsistencies in the tic inforescences formed by four series of keeled bracts current taxonomic classifcation for the correct use of aligned in rows. Tis section is considered monophy- those plants in phytomedicine. letic and is characterized by small variations (2n = 12) in the number of chromosomes [15, 17]. A recent revi- Methods sion of the species belonging to Goniostachyum resulted Plant materials, DNA extractions, PCR amplifcations in the validation of only four representatives, namely: L. and sequencing grata Schauer, L. origanoides Kunth, L. sericea Cham. We used 141 accessions (Table 1) comprising six Lip- and L. stachyoides Cham. [17]. Tus, some nominations pia species; although L. sidoides and L. origanoides are of species or varieties must be considered synonyms of synonymous, they were considered, for the purposes L. origanoides including, amongst others, L. sidoides, L. of this study, as they were classifed. Tirty-seven of graveolens Kunth, L. microphylla Cham., L. salviifolia these accessions were from the medicinal plants germ- Cham., L. velutina Schauer, and Lantana origanoides plasm bank (Ribeirão Preto University, Brazil) and 104 Martens & Galeotti. Additionally, L. dumetorum Herzog, were collected in the medicinal botanical garden of L. gracilis Schauer ex DC, L. hickenii Tronc., L. laxibrac- Nature Pharmacy, Brazil, with voucher numbers; 1340; teata Herzog, and others have received the synonym L. 1350;1351; 1353; 1355; 1359; 1360; 1362–1365; 1368– grata. [17]. Te Rhodolippia Schauer section comprises 1376; 1378–1380; 2000–2015; 2017–2112; 2114; 2471; species with numbers of chromosomes that are inter- 2473–2475. Sampling permission, for both locations, mediate between those of sections Zapania and Gonio- were obtained from by the Brazilian Council for the stachyum [15, 18], including L. bradei Moldenke, L. Administration and Management of Genetic Patrimony felippei Moldenke, L. forida Cham., L. hederaefolia Mart. (CGEN) of the Brazilian Ministry of the Environment & Schauer, L. lupulina Cham., L. pseudothea Schauer, L. (MMA) by the National Council for Scientifc and Tech- rhodocnemis Mart. & Schauer, and L. rosella Moldenke. nological Development (CNPq—CGEN/MMA Process However, the taxonomic classifcation of Lippia #: 02001.005059/2011-71). Fátima R. G. Salimena (Juiz remains incoherent mainly due to the morphological de Fora Federal University, Brazil) identifed all samples. variability within the genus and the existence of a great Total genomic DNA was extracted from 0.15
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