1. Introduction
The genus Lavandula of the Lamiaceae family comprises about 39 species [
1]. This genus is made up of small perennial green shrubs, with aromatic flowers and forage from which essential oil (EO) can be obtained. The main species of the genus from which commercial EO is obtained are Lavandula latifolia Medik. with an estimated average annual production of 200 t,
Lavandula angustifolia Mill. with another 200 t, and the hybrid of the two previous species, called Lavandin (L x intermedia Emerik ex Loisel) with about 1000 t [
2]. In France, in 2018, 4662 ha of
L. angustifolia Mill were cultivated with a production of 116.62 t of EO and 20,770 ha of Lavandin with a production of 1646.13 t of EO [
3], which represents slightly more than 3 times kg/ha yield than lavender.
The spike lavender (
L. latifolia Medik.) is native to the Mediterranean region, growing wild mainly in the former Yugoslavia, Italy, France, Spain, and Portugal [
1]. It grows in forest clearings, especially in limestone rocky or dry pastures on sunny slopes, in basic substrates and alluvial sands [
1]. It prefers areas between 600–1000 m of altitude [
4]. It is collected in the field and cultivated for its EO, to which antibacterial and antifungal, sedative, and antidepressant properties have been attributed, highly appreciated in aromatherapy and phytotherapy [
5,
6,
7], and is a source of natural antioxidants [
8,
9]. The spike lavender has been in the past in Spanish regions the species with the highest incidence in its spontaneous collection for the perfume industry [
4].
Due to its economic importance, the composition of the spike lavender EO has been widely studied (bibliographic reviews by Boelens [
10], García-Vallejo [
11], Lis-Balchin [
12]). Spike lavender EO consists mainly of monoterpenes and is produced and stored in the glandular trichomes that cover the surface of the aerial parts of the plant, although its production and composition are different in the flower than in other parts of the plant [
13]. The main monoterpenes are 1,8-cineole, linalool, and camphor, which determine the olfactory body of EO and comprise about 80% of EO [
10,
12]. The commercial value of an aromatic plant is determined by the EO yield and composition. Higher levels of linalool and lower amounts of 1,8-cineole and camphor in
L. latifolia Medik. are positive factors for the pharmaceutical and cosmetic industry and are considered higher quality EO [
10,
14]. EO with high proportions of camphor is used in the phytosanitary industry [
7,
15].
The EO of spike lavender, like that of other EOs, is the end product of a complex biological process and its production and composition can vary considerably at the intraspecific level depending on the genotype, on the part of the plant that is used for extraction [
10,
13], on environmental factors such as climatic conditions and soil composition, geographic location [
16], and date of collection [
17]. Despite the commercial importance of spike lavender EO, the influence of some environmental factors on its production and quality has been poorly characterized.
Spain is the largest producer of spike lavender EO with 150–200 t per year [
2] and its EO is the most important of the essential oils commercially produced in Spain [
12]. The Spanish Ministry of Agriculture publishes joint data on lavender and lavandin on acreage and biomass production. In 2018, there were 4725 ha and 15,844 t of biomass [
18]. Most studies on the production and chemical composition of EO have been carried out in wild populations in different Spanish regions. The most extensive study of populations has been carried out by Herraiz-Peñalver et al. [
16], analyzing 194 samples from 6 different biogeographic Spanish regions. Muñoz-Bertomeu et al. [
13] analyzed the differences in production and quality in 7 populations of the Valencian region. Salido et al. [
17] analyzed the seasonal behavior in samples from 3 different localities in Andalucía. Its behavior has also been analyzed under cultivation conditions in Spanish regions adapted to the species [
4,
7].
The quality of an aromatic plant is determined by its secondary metabolite content and its biomass yield. The prospecting and chemical characterization of wild populations and the analysis of the factors that influence their quantity and quality provide essential starting information for the conservation programs and for the selection of parental lines that allow obtaining in future adapted cultivars. Productions from wild collections carry products that are difficult to trace and can pose environmental problems due to overexploitation of natural populations. It is more convenient to collect the species plants in culture.
The objective of this study is to estimate the environmental effects on the production and composition of spike lavender EO in wild populations of various Spanish regions of the North, Center, and Southeast with the dual purpose of conserving natural populations and selecting populations in order to obtain suitable genotypes for spike lavender cultivation and as progenitors of new Lavandin varieties in crosses with L. angustifolia.
2. Materials and Methods
2.1. Vegetable Samples
Plant samples were collected in 34 wild populations of
L. latifolia Medik. for three consecutive years in 2011, 2012, and 2013 in three Biogeographic Regions: Atlantic European 4, Cévenno-Pyrenean 7, Mediterranean Central Iberian 18, defined by Rivas-Martínez and Rivas-Saenz [
19]. The populations were distributed in four bioregions dividing the Mediterranean Biogeographic region in two due to its extension. The Cantabroatlantic bioregion 4a (C-4a) with 9 populations, the Prepyrenean 7a (P-7a) with 9 populations, the Mediterranean Castillian 18a (MC-18a) with 7 populations, and the Mediterranean Oroiberian 18b (MO-18b) with 9 populations were the four bioregions (
Figure 1). Biogeographic regions layer source Ministerio para la Transición Ecológica (MITECO) (10 February 2018). Software Diva-GIS is available at
https://www.diva-gis.org/download.
Table S1 shows the latitude and longitude coordinates and collection date for three years of the 34 populations studied.
The plant samples collected consisted of flowers and leaves of about 25 plants per population until reaching an approximate weight of 500 g. The collections were made at the time of full flowering, with more than 50% of the plants with open flowers [
20] at the end of August–September, depending on the geographical location. The samples from each population were dried at room temperature in shade, and they reached a constant weight in a week.
2.2. Soils
To establish the edaphic characteristics, soil samples were collected from each population in which the plant material was collected. By removing the first centimeters of soil to eliminate the vegetation cover, a kg of soil was collected from the first 20 cm or until reaching bedrock. The characteristics that were analyzed are: pH using a CRISON BASIC 20 (CRISON INSTRUMENTS, S.A., Alella, Spain) [
21] model pH meter, % fines, percentage of soil less than 2 mm in diameter, electrical conductivity (EC) using a model conductivity meter micro CM 2200 CRISON (CRISON INSTRUMENTS, S.A., Alella, Spain) [
22], the oxidizable carbon content in the soil (SOC) according to Walkley and Black [
23], the total nitrogen content of the soil (SN) according to the Kjeldahl method, the assimilable phosphorus (P) content [
24] using a spectrophotometer Genesys 10S UV-VIS (Thermo Scientific, Madison, WI, USA), and the bioavailable potassium (K) content [
25] using an inductively coupled plasma atomic emission spectrometer ICP-AES Óptima 5300 DV (Perkin Elmer, Wellesley, MA, USA).
2.3. Climatic Data
The climatic variables were obtained from the information provided by the State Meteorological Agency (AEMET) Ministry of Agriculture, Food, and Environment.
For the meteorological data, the three campaigns from August 2010 to August 2013 were considered, the end of the agricultural year coinciding with the collection of the material to be distilled. From the data of extreme temperatures by months and monthly precipitation, the quarterly values were calculated taking the agricultural years: Quarter 1: from September to November; Quarter 2: from December to February; Quarter 3: from March to May; Quarter 4: from June to August.
A Gaussen climogram was constructed [
26] to represent the climatic variability to which the sampled populations were subjected.
2.4. Analysis and Quantification of Chemical Parameters
The EO was obtained following the methodology proposed by the European Pharmacopoeia with simple hydrodistillation, in Clevenger [
27]. Each sample (180 g of leaves and flowers of dry material) is introduced so as to form a fixed bed in an alembic with water (2L) and boiled for 150 min. The identification of the volatile active principles that make up the EO is analyzed with Gas–Liquid chromatography. The analyses were performed on a Hewlett-Packard Agilent HP 6890N GC system equipped with a quadrupole mass spectrometer Agilent 5973N (Agilent Technologies, S.L., Palo Alto, CA, USA) and DB-5 capillary column with stationary phase phenyl methyl silicone (non-polar) of 30 m long (0.25 mm in diameter and 0.25 µm in film thickness), applying a temperature gradient from 343.15 K to 513.15 K, with an increase of 276.15 K per minute, maintaining the final temperature for two minutes. Additionally, an Agilent 5975 B (Agilent Technologies, S.L., Santa Clara, CA, USA) model gas chromatograph (GC/MS) was used, coupled to an electronic impact mass spectrometer (70 eV) with a column equal to that used in the FID gas chromatograph, to check the active principles.
For the identification of the EO components, n-alkanes standards from C6 (hexane) to C25 (pentacosane) have been injected into the GC/MS column under the same conditions as the samples, the relative retention times of pure substances (standards) and the corresponding Kovats retention indices (RI) were used. The quantification of the percentages of the components is performed according to the areas of the chromatographic peaks. With this methodology, there are several samples in which some active principles do not separate well, so they are considered together. These active principles are: sabinene + β pinene and limonene + 1,8-cineole.
The EO yield and the active principles that were detected with a percentage higher than 1% that appear in the ISO-4719 [
28] were EO yield, α pinene, camphene, sabinene + β pinene, limonene + 1,8-cineole, linalool, camphor, borneol, and α-terpineol. Given the low percentage of limonene, around a mean value of 1% [
13,
16,
17,
29], the result tables will show only 1-8-cineole instead of limonene + 1,8-cineole, so the percentage of 1,8-cineole will be somewhat overrated.
2.5. Statistical Analysis
In order to compare the different variables, they were standardized. The means are presented with their standard differences (±S.D). One-way analysis of variance (ANOVA) was used to test the effect of experimental years and different geographical origins on the variables EO yield, 1,8-cineole, linalool, camphor, and rainfall. Tukey’s test was performed to determine differences between treatment means. The Pearson correlation coefficients between EO yield, 1,8-cineole, linalool, and camphor were also determined. Linear regressions of the dependent variables, EO yield, 1,8-cineole, linalool, and camphor, were made on edaphic and location characteristics of the populations. The level of statistical significance was set at p < 0.05 and p < 0.01 (**). All statistical analyses were performed with the InfoStat statistical package (InfoStat v2016; Grupo InfoStat, FCA, Universidad Nacional de Córdoba, Argentina, 2016).