Stachys lavandulifolia Populations: Volatile Oil Profile and Morphological Diversity

The morphological and essential oil diversity of Stachys lavandulifolia populations from the west and northwest of Iran were evaluated. The results showed a significant difference between the populations for nearly all the evaluated traits. The broadest variation ranges were recorded for the auxiliary shoot length, leaf length in the main branch, and the number of flowers in the inflorescences. Furthermore, cluster analysis divided 13 populations into four separate groups. GC/MS analysis verified the presence of 28 components comprising up to 94/4% of the oils. The dominant constituents were α-pinene (1.07–34.87%), (E)-caryophyllene (0.45–25.99%), germacrene D (3.36–20.61%), ∆-cadinene (2.82–19.90%), bicyclogermacrene (1.72–12.08%) α-terpineol (0–11.86%), α-muurolol (0.31–11.50%), p-cymene (0.67–9.67%), β-elemene (0.63–9.31%), and sabinene (0.32–6.29%). The results revealed that natural habitats and the related geo-climatological cues influenced morphological traits and oil composition. Considering the substantial environmental variations and the broad diversity, there would be a rich selection pool for the traits of interest. The populations are a step forward in the breeding programs for the highlighted essential oil constituents needed by the pharmaceutical and related industries. Furthermore, with the future comparative study of the populations from all Iranian territories and the neighboring countries, we will have a realistic idea of the coming conservational and exploitation programs.


Introduction
The increasing demand for medicinal plants and their products has highlighted the role of these plants in the global economic market. Millions of people worldwide use herbs and essential oils for various therapeutic purposes [1]. Essential oils are a complex mixture of volatile low molecular weight and hydrophobic compounds found in different parts of aromatic plants, including leaves, flowers, seeds, buds, and branches [2].
The genus Stachys includes 300 species of annual and perennial plants. One of the species of this genus is Stachys lavandulifolia Vahal, which grows in different parts of Iran [3]. Stachys lavandulifolia Vahl, also known as lamb's ears or wood betony, an herbaceous perennial plant of the family Lamiaceae, is native to Iran, Iraq, Transcaucasus, Turkey, and

Morphological Traits Analysis
To evaluate the morphological traits diversity among populations, up to 15 plant samples were taken from each habitat depending on the size of the population while considering the distribution area and at a suitable distance to have a homogenous realistic sample of the habitat. Attempts were taken to prevent the harvesting of close relatives. Morphological assays were performed on plants at the full-bloom stage. Plant samples were revisited and verified by a taxonomist and herbarium specimens were stored in the Horticulture Department of the University of Maragheh, Iran.
The evaluated traits were: petal length, stem diameter, collar diameter, sepal width, sepal length, leaf width, leaf length in the sub-branches, leaf length of the main branch, the distance between internodes, petiole diameter, petiole length, inflorescence length, branch length, plant height, the number of flowering branches, and the number of flowers per inflorescence, which were measured using a digital caliper and ruler. Aerial parts were dried under shade conditions at the ambient laboratory temperature to reach the constant weight.

Essential Oil (EO) Extraction
To extract the Stachys lavandulifolia essential oils, 50 g of the shade-dried aerial parts was ground and subjected to hydro-distillation for three hours using a Clevenger-type apparatus (British Pharmacopoeia model). To remove the possible water drops in the essential oils, the samples were dehydrated over anhydrous sodium sulfate (0.5−1 g) and then kept at 4 • C until gas chromatography-mass spectrometry. The essential oil content (EO %) was calculated considering dry mass.

GS-MS Analysis
The essential oils were analyzed using GC-FID and GC-MS. Briefly, the analysis was conducted using an Agilent 7990 B gas chromatograph equipped with a 5988A mass spectrometer and an HP-5MS (0.25 mm i.d., 30 m l., 0.25 µm f.t., 5% phenyl methylpolysiloxane). The following oven temperature was used: 5 min at 60 • C, then up to 240 • C with the rate of 3 • C/min, held for 10 min. Helium (carrier gas) flow rate was 1 mL/min; the injector split ratio was 1:30; and the mass range and electron impact (EI) were 40-400 m/z and 70 eV, respectively. The identification of constituents was performed using the procedure explained by Morshedloo et al. [13], which is based on the interactive combination of linear retention indices (RIs), calculated against a homologous series of n-alkanes (C8-C40, Supelco, Bellefonte, CA, USA), and the mass spectrum (MS) matching with commercial libraries (ADAMS, WILEY 275, and NIST 17). GC-FID analysis was performed using an Agilent 7990 B gas chromatograph equipped with a flame ionization detector (FID) and capillary column VF 5MS (30 m l., 0.25 mm i.d., 0.50 µm f.t., 5% phenyl methylpolysiloxane). The same oven temperature reported for GC-MS was used. The injection volume was 1 µL of essential oil sample in n-hexane (1:100). Quantification of the constituents was performed by peak area normalization without using correction factors [14].

Statistical Analysis
ANOVA was performed using MSTAT-C ver. 2.1 software. The mean comparisons of the data were analyzed using the least significant difference (LSD) test at a 1 and 5% probability level. Pearson's correlation coefficient and cluster analysis dendrograms were drawn by XLSTAT software using the Ward method and Euclidean square distance.

Morphological Traits Diversity
The results from Table 2 show that the morphological traits (with the exclusion of sepal and petal length) had significant differences in the studied ecotypes of S. lavandulifolia (p < 0.01). ns and ** indicate no significant difference and significant at 1% probability level, respectively; # describes the statistical differences of ecotypes for the trait. Table 3 reports the mean values of the morphological traits for 13 S. lavandulifolia populations from different localities. Indeed, there was no harmonized pattern for the variations. Still, due to diverse geo-climatological and edaphic differences, the traits measured showed correspondent diversity in their range between the various locations. These variations could be ascribed to the relevant environmental cues and as an epigenetic signaling response. The results showed that the Oshnavieh, Azarbaijan population had the highest leaf width (64.3 mm), and the lowest value was related to the population of Shahin Dej (1.65 mm). Also, the plant height was variable in Baneh (20.19 cm), Oshnavieh (18.07 cm), and Shahin Dej (7.53 cm), respectively (Table 3).

Essential Oil Content
According to ANOVA, significant differences were observed in the essential oil content of S. lavandulifolia populations (p < 0.05). As depicted in Figure 2, the essential oil content of the studied population varied between 0.07 and 0.15%. The ecotypes related to Marivan Wisser, Kordestan and Shahin Dej, West Azarbaijan had lower oil content than other ecotypes. Otherwise, the highest essential oil content (0.15%) was obtained in the population of Oshnavieh, West Azarbaijan (0.06%). The results clearly showed that the essential oil content was strongly affected by environmental conditions, such as morphological characteristics. Ecotypes at lower altitudes produced less essential oil than those at higher altitudes. In addition to altitude, factors such as light, temperature, and humidity also affect essential oil production. The ecotype of Marivan Wisser, Kordestan with 700-800 mm rainfall per year attained the least essential oil content. However, this ecotype had no significant difference with Shahin Dej, West Azarbaijan's ecotype with 323 mm rainfall. This similar essential oil content may be due to the effects of other environmental factors and diverse edaphic conditions besides different intrinsic genetic responses. As is previously known, secondary metabolites biosynthesis is stimulated in response to the variable environmental factors. In our populations, the various climatic and geographic criteria may induce the biosynthesis and accumulation of a series of defined constituents to guarantee the survival of plant species [15].

Principal Component Analysis (PCA)
PCA revealed the role of each trait in the variation between the studied S. lavandulifolia populations. Eigenvalues, the percentage of explained variation, and the cumulative percentage of variance are presented in Table 6. The analysis showed that the inflorescence length, number of flowers per inflorescence, internode distance, and petal length played the most critical role in the differentiation of ecotypes. Furthermore, eigenvalues, the percentage of explained variation, and the cumulative percentage of the variance of essential oil constituents are presented in Table 7. Four components described the variations between plant samples. α-Thujene, β-pinene, myrcene, dodecanal, (E)-caryophyllene, germacrene D, and α-cadinol were the most important oil constituents describing the variations.

Discussion
Localities drastically influenced the morphological and chemical diversity of populations. Compared to others, west Azarbaijan (Oshnavieh) is located in a semi-arid mountainous habitat, so the mentioned climatic and geographical conditions are suitable for producing high EO content. The high number of leaves and flowers is essential for producing the appropriate essential oil content in plants such as S. lavandulifolia [16]. In a study performed on different species of Salvia, significant differences were observed in the measured traits such as plant height and leaf width [17]. Also, in another research on the morphological characteristics of Origanum vulgare L., a highly significant difference was recorded between the evaluated characteristics [18]. Khadivi-Khub et al. [19] reported that the most variable morphological characteristics among the studied populations of S. lavandulifolia were inflorescence length, internode length, leaf shape, crown color, and flower bowl shape. Also, Mirzaie-Nodoushan [20] reported significant differences in morphological characteristics (plant height, stem diameter, number of branches, leaf length, and width) on different species of mint (Mentha spp.), quite similar to our findings. Yousefi et al. [21], in their study on Salvia leriifolia Benth, reported a high diversity of morphological traits and essential oil composition, which was consistent with the present study.
Traits with a high coefficient of variation have a broad selection potential for future programs. The range of deviation and the mean of traits measured for the ecotypes are shown in Table 2. The coefficient of variation varied depending on the trait. Among the studied features, the length of the sub-branch had the highest range (0.90) and then the number of flowers per inflorescence with 0.70 attained the highest range. Also, other traits such as petiole diameter (0.66) and leaf width had a high coefficient of variation.
In contrast, the least coefficient variation was related to sepal length (0.17) and petal length (0.21). In an earlier study on mint, the most critical traits such as flowering branch length, the distance between nodes, leaf length and width, the number of sub-stems, the number of leaves per sub-stem, and the height of the sub-branch had the highest coefficient of variation compared to other traits [22]. In contrast, other characteristics such as flower cup length and seed diameter had the lowest coefficients [23]. Hadian et al. [24] studied 30 populations of summer savory and reported that the highest coefficient of variation was related to the leaf width, petiole length, and leaf area. The broad coefficient variations in the phenotypic and morphological traits reflect the possible genetic diversity in a given plant species in different regions. Hosseini et al. [25], in their study on morphological traits of Mentha longifolia L. at different altitudes, reported that at high elevations, the higher light intensity and ultraviolet radiation resulted in the chlorophyll photo-oxidation and the reduced photosynthetically active radiation, and hence the limited plant growth potential. They have noted that climatic factors can be the predominant source of chemical and morphological variations by inducing massive biochemical, physiological, and metabolic changes.
The climatic and genetic parameters can influence the essential oil content of different species. In addition, local species show genetic diversities due to their adaptation to the environment, which may affect the biochemical composition, biological activity, and the secondary metabolite profile of plants [26]. The essential oil content of the studied populations varied from 0.07 to 0.15 % of dry weight. Essential oil production is an adaptation response to environmental cues. Cho et al. [27] showed that altitude was the most important environmental factor affecting essential oil content because higher essential oil contents were recorded at low altitudes. Hence, the higher essential oil content in the Saqez and Oshnavieh populations is possibly related to the lower altitude and less annual rainfall.
As is previously known, besides genetic nature, the environmental signals are the most influential criteria that influence the activity of genes involved in secondary metabolites biosynthesis in medicinal plants. Numerous factors such as altitude, water availability, temperature, and other geographical and seasonal conditions, plus plant growth stage, harvest time, postharvest handling, and extraction procedure, have a noticeable impact on essential oils' quantity and chemical composition [28]. Environmental and geographical indices determine the habitat characteristics of plants and play a crucial role in the distribution pattern of plants; so, by the assessment of the ecological conditions and requirements of a defined species, they will be able to characterize and manage the naturalization and agricultural large-scale production of high-valued species [29].
Skaltsa et al. [15] assessed the antimicrobial activity of EOs from eight Greek Stachys species in which all taxa displayed a relatively low content of aliphatics and phenylpropanoids. All evaluated EOs exhibited improved anti-bacterial behaviors. In another report from Turkey, the essential oil of 23 Stachys species contained germacrene D, βcaryophyllene, caryophyllene oxide, spathulenol, and α-cadinene as the main constituents of the oils [33]. In our study, a total of 28 constituents were identified, comprising 94.5% of the oil. Environmental and genetic factors control the essential oils' biosynthesis and the oils' proportional percentage. Plant populations and/or ecotypes from different ecological conditions inevitably have divergent essential oil compositions. It seems that the species' origin affects the content and composition of the essential oil. Environmental, genetic, and physiological factors, habitat management, grazing behavior, and agricultural practices hugely impress the secondary metabolites biosynthesis, accumulation, and profile. Geoclimatological factors such as climatic and edaphic conditions affect the genes expression and metabolic pathways, and hence the biosynthesis of active substances in the species. As a result, various secondary metabolites are synthesized under different environmental conditions [34]. The biological properties of essential oils from plants are strongly influenced by their constituents. The high diversity of essential oils of S. lavandulifolia observed in the current study might focus further studies on the bioassay evaluations. This makes possible the selection of local populations of S. lavandulifolia with special biological activities for use in the pharmaceutical and cosmetic industries and reveals the economic importance of this plant in advance.
Hadian et al. [23], via cluster analysis of the morphological diversity of Khuzestan savory populations, reported a high diversity for the studied traits. The populations were divided into three groups. In another morphological study, seven populations of thyme were divided into two separate groups. The populations from Kerman and Isfahan provinces were in the first, and the populations of Semnan province were in another group. The temperature variations have been defined as the predominant cause of this grouping [35].
Correlation estimation between the traits is another major procedure in cultivar improvement programs. In addition, estimating the relationships between morphological traits can provide information for the breeding studies which determine the most efficient design for genotypic evaluations [36]. In a study on S. lavandulifolia, the Pearson's correlation showed that flower length, the number of sub-stems, the number of leaves per sub-stem, the length of the sub-stem, stem diameter, plant height, collar diameter, leaf length, and leaf length-to-width ratio were positively correlated with the production and storage of essential oils. Thus, the biomass production of plants has a significant impact on the yield and productivity of essential oils, perhaps with the variations in the essential oil biosynthesis and accumulation potential [22]. In line with this, traits correlation studies are essential for plant breeding programs to produce the desired cultivar with an appropriate productivity and chemical profile [37]. The PCA analysis clarifies the main differences between the studied traits. The relative variance of each component indicates its percent importance in the variance of the studied traits. Moreover, PCA has been frequently employed to investigate the relationship between morphological traits and evaluate the different germplasms [18,38]. Furthermore, PCA is widely used to elucidate the morphological, genetic, and biochemical traits variations within and between species and populations [39].

Conclusions
When plants are exposed to different ecological conditions, the quantity and quality of their active ingredients change to adapt to these environments. As previously understood, the ecological diversity leads to differences in the range of the biological activities of plants. In our study, the different geographical areas influenced the morphological, physiological, and chemical characteristics of plants. As is known, for the exploitation, domestication, and/or breeding programs, we need to study the phenotypic and genetic diversity of plants from natural habitats. Eventually, it will be necessary to cultivate the selected ecotypes in agricultural systems for the mass production of the desired secondary metabolites.