Metabolomic Variability in the Volatile Composition of Essential Oils from Pinus pinea and P. pinaster ^†

Abstract

In the Mediterranean basin, Pinus pinaster (maritime pine) and P. pinea (stone pine) are highly economically important pine species. These species provide raw material for forest-based industries (e.g., wood, paper, and resin), as well as of other economically relevant products, such as pine nuts (stone pine) and essential oils (EOs). Previous studies described a large genetic and phenotypic intra-species variability that ultimately hinders the comparison between reports. The present work reviews the available literature on P. pinaster and P. pinea EO composition and pinpoints the compounds that showed the highest variation. The chemical profiles of EOs extracted from the aerial parts were obtained from a total of 30 publications. Cluster analysis revealed a higher influence of geographic location on P. pinaster EO composition, than on P. pinea. A high degree of chemical variability was detected for these species. Specifically, the EO components that showed the highest variations were limonene, α-pinene, β-pinene, trans-β-caryophyllene, germacrene D, and β-myrcene for P. pinea, and α-pinene, β-pinene, trans-β-caryophyllene, germacrene D, and β-myrcene for P. pinaster. Thus, it is highly recommended that research performed in field or greenhouse conditions should first ascertain pine chemical variability.

1. Introduction

Pinus is the largest genus of the Pinaceae family. These gymnosperms are evergreen resinous trees commonly found in temperate mountainous areas of the Northern Hemisphere, but can also occur in some semi-arid desert and tropical rainforest ecosystems of the Southern Hemisphere [1]. Pines are largely responsible for shaping the Mediterranean forest ecosystems and are economically important species, providing raw material for several forest-based industries (e.g., timber, wood pulp, and resin), used as ornamentals, and as a source of edible pine nuts and essential oils (EOs) [2]. Pine EOs are obtained exclusively by hydro-, steam, or dry distillation, and consist mostly of terpenes (mono-, sesqui-, and diterpenes) and aromatic compounds, usually with one to three major components at relatively high concentrations (20–70%). The terpene compounds occurring in pine EOs have high-value commercial uses, for example in industrial and household cleaning products, disinfectants, solvents, fragrances, cosmetics, and pharmaceutical applications [3,4].

A high genetic variability has been described among populations of P. pinaster from different provenances. This variability was further linked to intraspecific variation in response to environmental stress; namely, drought, and resistance to biotic stress factors [5,6,7,8,9]. The natural variation found in P. pinaster allows it to be explored as a potential species for reforestation, in the context of the current climate change scenario (e.g., increasing temperatures, prolonged drought events), using more tolerant varieties [10]. In contrast, P. pinea is reported to display very low genetic variation but a remarkable degree of phenotypic plasticity; namely, in response to heat and drought stress [11,12]. The variability and/or phenotypic plasticity appears to be the reason for the high colonization ability of these Pinus species and their taking a central role in Mediterranean ecosystems [13].

The occurrence of EO chemotypes (i.e., qualitative and quantitative differences in the chemical composition among populations of the same species) is fairly common, namely in forest trees. EO chemotypes are a source of variation among Pinus species and have been strongly linked with geographical variation, being able to reflect the diverse environmental conditions to which pines are exposed [4,14,15,16]. The chemical diversity in pines can lead to a higher variability in research results, and ultimately hinder the comparison of different studies.

The present work explores the chemical variation found in P. pinaster and P. pinea EOs reported from different geographical locations, according to the available literature. The main EO components responsible for this chemical variation were identified.

2. Bibliographic Data

Research on bibliographic data was performed on the Google Scholar search engine using the keywords “essential oil” and “pinea” or “pinaster”. The retrieved publications reported the detailed EO composition of aerial parts (needles or branches) of P. pinea and P. pinaster. The publications were additionally selected based on definition of EO by the International Organization for Standardization (ISO), i.e., “a product obtained from natural raw material of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation, after separation of the aqueous phase, if any, by physical processes” [17]. From each report, the geographic location (i.e., the country of origin of the pine material used for EO extraction) and the EO compounds with amounts ≥ 1% were compiled and analyzed.

EO compounds with amounts ≥1% (main composition) were used to determine the relationship level between different samples by cluster analysis. Analysis was performed using R studio (RStudio Team, 2018 [18]; R Core Team, 2019 [19]) software, and the “dendextend” package [20]. EO compounds with the highest variability were identified by determining percentage of variation. Component variability was assessed by determining the range of reported values and determining its percentage of the maximum value {100 * [(Max − Min)/Max]}. Components with variability ≥90% were considered to be the highest variable compounds while those with values ≤10% the most constant compounds, across EO compositions. Variability in EO components was calculated only if EO sample number was above three samples from different reports.

3. Essential Oil Chemical Compositions

A total of 56 samples (26 for P. pinea and 30 for P. pinaster) were reported in 29 publications [16,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. The pine material used for EO extraction was mainly collected from countries in the Mediterranean region, namely Algeria, France, Greece, Italy, Jordan, Lebanon, Morocco, Portugal, Spain, Tunisia, and Turkey. The diversity of EO components, with amounts ≥ 1%, was 78 compounds for Pinus pinea EOs and 68 for P. pinaster EOs, respectively.

3.1. Pinus pinea

Cluster analysis was performed to determine the similarity between EO compositions from samples of P. pinea from different provenances (Figure 1a). Two main clusters were obtained, one cluster grouped exclusively EOs from Moroccan pine trees and some EOs from Turkey (Cluster I), while the remaining EOs were grouped together in a separate cluster (Cluster II). Cluster analysis revealed no separation based on geographic location of plant material used for EO extraction.

From a total of 78 components in P. pinea EOs, six showed very high variability, namely limonene (99%), β-pinene (98%), α-pinene (97%), trans-β-caryophyllene (94%), germacrene D (90%), and β-myrcene (90%) (

Table 1. Pinus pinea essential oil compounds with variability ≥ 90%, across 26 samples.

Compound	Variability (%) 1	Average (%)	Standard Deviation (%)
Limonene	99	45.1	21.4
β-Pinene	98	8.8	11.9
α-Pinene	97	10.7	10.3
trans-β-Caryophyllene	94	4.5	3.8
Germacrene D	90	3.8	2.7
β-Myrcene	90	2.6	2.3

¹ Range percentage of the maximum value {100 * [(Max − Min)/Max]}.

).

For each EO, the sum of these compounds varied between 25% and 92% of the total composition. In 70% of the EOs, these compounds comprised more than 50% of the total composition. Relative amounts of limonene ranged from 1 to 75%, β-pinene from 1 to 42%, α-pinene from 1 to 37%, trans-β-caryophyllene from 1 to 17%, germacrene D from 1 to 10% and β-myrcene from 1 to 11%. Other EO compounds present in high amounts (10–15%) were thymol (13%), guaiol (13%), β-phellandrene (14%), 9-octadecenoic acid (13%), and abienol (12%).

The monoterpenoid 1,8-cineole was the EO compound with the least variability (7%), varying between 4.0% and 4.3% over 3 EOs from 3 different reports.

3.2. Pinus pinaster

Chemical composition of P. pinaster EOs was grouped by similarity in two main clusters (Figure 1b). The EOs extracted from P. pinaster trees with origin in Morocco, Italy, and Spain were present in both groups. However, EOs from Western European countries (France, Italy, Portugal, and Spain) were mainly present in Cluster II, whereas Cluster I mainly grouped EOs from northern Africa (Algeria, Morocco, and Tunisia) and Greece. In addition, EOs extracted from P. pinaster trees from Portugal and France were exclusively present in Cluster II. Thus, for P. pinaster, geographical location seems to partly influence the EO chemical composition.

From the 68 main compounds in P. pinaster EOs, five showed a very high variability; namely β-pinene (99%), α-pinene (98%), trans-β-caryophyllene (97%), β-myrcene (92%) and germacrene D (91%) (

Table 2. Pinus pinaster essential oil compounds with variability ≥ 90%, across 30 samples.

Compound	Variability (%) 1	Average (%)	Standard Deviation (%)
β-Pinene	99	27.6	22.1
α-Pinene	98	25.2	13.6
trans-β-Caryophyllene	97	13.4	9.2
β-Myrcene	92	3.2	2.5
Germacrene D	91	6.8	6.2

¹ Range percentage of the maximum value {100 * [(Max − Min)/Max]}.

).

For each EO, the sum of these compounds varied between 13% and 97% of the total EO composition. In ca. 70% of the EOs, these compounds amounted to more than 50% of the EO composition. Relative amounts of β-pinene ranged from 1 to 67%, α-pinene from 1 to 62%, trans-β-caryophyllene from 1 to 31%, germacrene D from 2 to 20% and β-myrcene from 1 to 12%.

Other EO compounds present in high amounts (10–30%) were δ-3-carene (18%), α-amorphene (11%), abieta-8,13-diene (11%), abieta-7,13-diene (32%), sclarene (18%), abietadiene (11–15%) allo-aromadendrene (13%), β-selinene (13%) and isoabienol (20%).

4. Discussion

A high chemical variability was found in the composition of EOs from P. pinaster and P. pinea. The main compounds responsible for this chemical variability were α-pinene, β-pinene, trans-β-caryophyllene, germacrene D, and β-myrcene. Additionally, limonene was responsible for chemical variability in the EOs from P. pinea, but not for P. pinaster. The observed chemical variability may be due not only to genetic traits but also to a strong influence from different environmental conditions, known to influence pine phenotypic traits [49]. EO composition is well-known to be dependent on plant physiological parameters; environmental conditions, such as climate, pollution, type of soil, pests, and diseases; and, ultimately, to geographic location [8,50]. Geographic influence on the variability of EO composition was detected for P. pinaster. In this species, EOs from trees in Western Europe were grouped in one main cluster whereas EOs extracted from trees in Northern African countries were grouped in a separate cluster. This observation is in agreement with previous studies showing an influence of latitude on some phenotypic traits in relation to phylogenetic or environmental variations [1]. Cluster analysis of P. pinaster EOs, from different provenances, also revealed a higher dissimilarity in composition (i.e., higher variability), when compared to those from P. pinea. Nonetheless, the EO compounds responsible for this variability are, with the exception of limonene in P. pinea, the same between species, which may indicate the influence of limited biosynthetic pathways. Although mostly overlooked, the chemical composition of pine EOs can provide important information on variability and the mechanisms of adaptation used by pine species to respond to environmental changes.

5. Conclusions

The preliminary results obtained on the chemical variability of EOs from P. pinaster and P. pinea from different provenances indicate that chemical variability in P. pinaster may be more dependent on geographic localization than P. pinea. However, the compounds responsible for this variation are similar for both species, namely, β-pinene, α-pinene, trans-β-caryophyllene, β-myrcene, and germacrene D. Further studies, with a higher number of biological replicates, would allow for improved statistical analysis and for exploring the source of chemical variability in these Pinus species. Additionally, screening EOs to assess the chemical variability should be extended to other pine species. Linking the chemical variability to phenotypic traits can contribute to better understanding how different pine species respond to the current climate change scenario.

The need for standardization when performing field studies is necessary in order to produce reproductible data and metadata. Variations in environmental conditions and/or sampling procedures can deeply impact results; thus, metadata should be critically detailed to enable usage with a higher reliability. The analysis of pine EO composition is a non-destructive method that can be performed to assess the presence of chemotypes, that can compromise the homogeneity of the results.