Molecular Phylogenetics of the genus Hexachlamys (Myrtaceae)using chloroplast and nuclear markers
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KeywordsLeaf Material CTAB Method High Bootstrap Nuclear Marker Ecological Significance
Myrtaceae family includes more than 3,800 species of trees and shrubs, distributed mainly in tropical and subtropical regions of the world. Representatives of this family have great ecological significance for forest ecosystems, and are economically important species in the pharmaceutical, food, cosmetic and perfumery industry . The genus Hexachlamys Berg. (Myrtaceae) has about 10 species distributed from the Southern to the Southeast of Brazil, and it also occurs in Paraguay, Argentina, Bolivia and Uruguay [2, 3]. Since 1968 it has been considered an independent genus. It is distinguished morphologically from the genus Eugenia by pentamerous or hexamerous flowers and exserta radicle, and as many genera and species of the Myrtaceae family, it has a complex taxonomic classification [2, 4, 5]. The goal of this study was to conduct a molecular phylogenetic analysis among species of the genus Hexachlamys (Myrtaceae) using chloroplast (cpDNA) and nuclear (nrDNA) markers, and to verify its phylogenetic relationships with the genus Eugenia in order to contribute to the systematics and taxonomy of it.
The samples were collected as leaf material(from herbaria species and samples collected in the field) of the genus Hexachlamys representing all described species, as indicated in herbariums and species of the Eugenia genus that occur in the Rio Grande do Sul state. Total genomic DNA was extracted using the CTAB method based on the protocol already described  and used for the PCR reactions. PCR products were sequenced on ABI PRISM 3100 sequencer (Applied Biosystem). Nucleotide sequences were aligned using CLUSTALW  implemented in MEGA5 (Molecular Evolutionary Genetics Analysis) version 5.0 , then checked visually and carefully improved manually before analysis. The phylogenetic analysis was reconstructed after nucleotide sequence alignments using three different approaches: the neighbor-joining (NJ), the Bayesian and the Maximum-Likelihood (ML) methods. The NJ, Bayesian and ML analysis were performed in MEGA 5.0, MrBayes  and PhyML , respectively.
Results and conclusions
To date we obtained DNA sequences of 21 species of the genus Eugenia and seven species of the genus Hexachlamys. Universal primers that amplify chloroplast region (trnL-trnF, trnL-intron, ycf5, accD, rbcLa, psbA-trnH, rpoB, rpoC1, ndhJ, matK and rps16) and nuclear (ITS) were tested. The regions used for the phylogenetic analisys were chloroplast accD, rpoB, rpoC1genes and the nuclear ITS, since the other chloroplast regions were not possible to amplify and sequence in all sampled species. These cpDNA and nrDNA regions presented polymorphisms among species studied. Results from Bayesian, NJ and ML tree analysis produced similar topologies and revealed that Hexachlamys species did not form a monophyletic clade. The Hexachlamys species have grouped together Eugenia species with high bootstrap values, indicating that Hexachlamys can be a synonymous of genus Eugenia. These results corroborate with morphological data . To confirm these results, other cpDNA and nrDNA markers will be tested and Hexachlamys species will be collected to substitute the species obtained in herbarium, since we found difficulties in the amplification of these species with all markers previously tested. This study can contribute to taxonomic classification of this group, as well as the field of conservation, since these species have an important economically and ecologically role.
Financial support: CNPq and FAPERGS
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