Differentiation of Five Pine Species Cultivated in Brazil Based on Chemometric Analysis of their Volatiles Identified by Gas Chromatography-Mass Spectrometry

e espectrometria de massas (GC-MS) com analisador de quadrupolo. A composição relativa foi usada para análises de componentes principais (PCA) e de agrupamento hierárquico (HCA) para a discriminação das cinco espécies. The composition of the volatiles emitted by different trees has been successfully used as a marker in a wide variety of studies, such as chemotaxonomy. Characterization of the volatiles emitted by different species of Pinus has proven to be an important tool to understand the process of host-tree selection by herbivorous insects. The volatiles present in samples of the branches of five species of Pinus ( P. caribaea, P. elliottii , P. maximinoi , P. patula and P. taeda ) were collected by aeration, and the contents analyzed by gas chromatography using a flame ionization detector (GC-FID), applying non-polar and chiral column phases, and mass spectrometry (GC-MS) using a quadrupole mass analyzer. The relative composition of the different volatiles was used to perform a discriminant analysis among the five pine species, by means of cluster (HCA) and principal component (PCA) analyses.


Introduction
The genus Pinus (Pinaceae) comprises 105 tree species, which are important and often dominant vegetation in large land areas of the Northern Hemisphere. 1 In addition to their wood and other products of high economic value, trees of Pinus influence ecosystems in various ways as they affect biochemical processes and hydrological regimes, and provide food and create habitat for animals. Pinus trees are cultivated in many countries, both inside and outside their natural habitats. 2 In Brazil, pine trees, mainly Pinus taeda L. and Pinus elliottii Engelmann, have been cultivated on a commercial scale for over 30 years. 3 Currently there are about 2 million hectares of commercial pine plantations, in large continuous areas and generally in stands with a narrow genetic base, mainly in the South and Southeast Regions. 4 The product of these plantations is mostly destined for the timber and cellulose industries. 5 Since 2001, the banded pine weevil Pissodes castaneus (De Geer, 1775), which attacks preferentially P. taeda, has become an important pest of pine trees in Brazil. In 2002, 7.6% of the trees of a Pinus taeda plantation in Cambará do Sul City (Rio Grande do Sul State, Brazil) was attacked by P. castaneus; while in São Joaquim City (Santa Catarina State, Brazil), 16.53% of the trees was attacked. 4 A recent study found that P. castaneus adults are attracted to the volatiles emitted by P. taeda. 6 Chemometric and bioinformatic methodologies are being extensively applied in taxonomic and metabolomic studies. [7][8][9][10][11][12][13][14] For instance, Scrivant et al. 15 succeeded in delimiting the species of Bothriochloa, by studying the essential oil composition by means of GC-MS (gas chromatography-mass spectrometry). Fischedick et al. 16 discriminated varieties of Cannabis by GC, using the quantitative data for monoterpenoids, sesquiterpenoids and cannabinoids. Similar studies involving Pinus were reported previously, for different species than those studied here, based on chemical analysis of the volatiles produced by different tissues including needles, [17][18][19] seeds, 20 cones, 21 xylem 22,23 and phloem. 24 The application of chemometry in studies in Brazil was recently reviewed by Bruns et al. 25 In the present study, it was collected and characterized the volatiles emitted by branches of five pine species cultivated in Brazil, P. elliottii, P. taeda, Pinus caribaea Morelet, Pinus maximinoi H. E. Moore and Pinus patula Schiede ex Schlechtendal et Chamisso, by means of aeration, GC-FID (gas chromatography using a flame ionization detector), GC-MS and hierarchical cluster (HCA) and principal component (PCA) analyses, as potential methods to be used in the differentiation of these species.

Research materials
The volatiles were collected using aeration of branches from one-year-old trees of P. caribaea, P. elliottii, P. maximinoi, P. patula and P. taeda collected from stands located in Southern Brazil (Table 1).

Sample preparation
Branches from the five species of Pinus were aerated in a 1 L glass chamber. Aeration was carried out under controlled conditions at 23 ± 2 °C, relative humidity 70 ± 10% and a photoperiod of 12L:12D (12 h light:12 h darkness). Volatiles were trapped on a 0.4 cm long bed of Super Q resin (Alltech, Deerfield, Illinois, USA) held in place by glass-wool plugs in a glass tube (4 mm ID). Volatiles were collected for two days (flow 1.0 L min -1 ), then eluted with hexane (3 × 0.5 mL). Extracts were concentrated as required (ca. 100 µL) under argon. All procedures were carried out in triplicate.

GC-FID analysis
Extracts and n-alkane standards were analyzed by GC in the splitless mode with a Varian 3800 instrument equipped with a VA-5 capillary column (30 m × 0.25 mm × 0.25 µm), using helium as carrier gas, at flow of 1.02 mL min -1 . Oven temperature was initially held at 60 °C for 1 min and increased at a rate of 3 °C min -1 to 180 °C, and the final temperature was held for 25 min.
The enantiomers were detected by co-injection of standards.

Identification of the terpenes
Extracts and n-alkane standards were also analyzed by coupled GC-MS with a Varian Saturn 2000 system equipped with a quadrupole detector using a CP-Sil 8 CB (Crossbond 5% phenyl/95% dimethylpolysiloxane) low-bleed column (30 m × 0.25 mm × 0.25 µm), using helium as carrier gas, at flow of 1.02 mL min. Oven temperature was initially held at 60 °C for 1 min and increased at a rate of 3 °C min -1 to 250 °C. The mass spectrometer operated in electron impact mode (70 eV) with a mass range set from 40 to 350 m/z. The interface and source temperatures were set at 250 °C.

Peak identification
The Kovats retention index (K.I.) was calculated for the compounds, and their mass spectra were compared with the literature. 26

Principal component analysis and hierarchical clustering analysis
The PCA and HCA analyses were performed using the relative area percentages of the terpenes. 27,28 Both the racemic and the enantiomeric compositions were used as variables for the sample description. These two exploratory data analyses were applied to a data matrix of 15 × 18 (15 being the samples, 18 being the volatile compounds), by using PLS-Toolbox 3.0 (Eigenvector Research, Inc.) operating in MatLab 6.5 (Math Work Inc.). 29

Identification of the volatiles
The chromatogram profiles of the volatiles produced by the five species of Pinus analyzed are exemplified in Figure 1. It was identified 14 monoterpenes and 4 sesquiterpenes in the volatiles of all five species. The enantiomeric compositions of αand β-pinene for the species are shown in Table 2.

Principal component analysis
The PCA analysis was performed using the 18 terpenes (the two enantiomers of αand β-pinene were considered as two separate components) identified as sample descriptors for all the Pinus species ( Table 2).
The volatiles p-mentha-2,4(8)-diene (12) and thymol methyl ether (14) were found only in P. patula samples, while methyl octanoate (13) was found only in P. taeda  (Table 2). If these compounds are in fact being produced only by these respective species, they could be used as chemical markers; however, the design of the present study does not permit this conclusion.

Hierarchical cluster analysis
The data matrix was autoscaled because of the different magnitudes of the relative compositions of the volatile compounds found. The Euclidean distance metric was used to measure the similarity, and k-nearest neighbor clustering was applied to obtain the dendrogram ( Figure 4).
As seen in the dendrogram (Figure 4), P. elliottii was the most homogeneous group, while P. maximinoi showed greater heterogeneity than the other species studied. P. caribaea, P. elliottii and P. taeda were grouped together, while P. patula and P. maximinoi were isolated. This is consistent with the morphological classification 2   Table 2).  ( Figure 5), in which P. caribaea, P. elliottii and P. taeda belong to the subsection Australes, while P. patula belongs to Oocarpae, and P. maximinoi to Ponderosae.

Conclusions
Our results indicate that it is possible to discriminate Pinus species based on the terpenes emitted, using GC-FID and GC-MS followed by analysis of chemometrics. The discrimination among all five species using HCA reflected the morphological classification of this genus, and proved to be a powerful method to classify Pinus trees.
The preference of adults of P. castaneus to attack P. taeda and P. elliottii in Brazil makes it likely that this pest locates the host plants based on the specific volatiles that they emit. This study will be very helpful in further behavioral and electrophysiological studies to determine the role that each volatile plays in these insect-plant chemicalmediated communications.

Supplementary Information
The chromatograms in achiral and chiral columns, and the mass spectra of the identified compounds are available free of charge at http://jbcs.sbq.org.br as a PDF file.