Influence of climatic factors on essential oil content and composition of 20 populations of Nepeta binaludensis Jamzad from Iran

Nepeta binaludensis Jamzad is an endemic and rare perennial plant belonging to the Lamiaceae family, which grows in a limited area in Binaloud Mountain in northeast of Iran. In this study to evaluate the diversity of 20 populations (localities) of N. binaludensis and the influence of environmental factors on essential oil (EO) content and composition, the plant aerial parts were collected at the full flowering stage. The plant materials dried in shade and subjected to hydro-distillation for obtaining their EOs. Analysis of the EO was carried out using GC and GC/MS. The oil yields were varied from 1.2 to 4.9%. Classification of populations was made based on EO compounds. The results of mean comparison between populations belong to different habitats showed that the populations of Darood and Friezy with average values of 4.91 and 1.2% had the highest and lowest EO yield, respectively. Twenty-two compounds were identified in the oils with 1,8-cineol (25.4–59.0%), 4aα,7α,7aα-nepetalactone (13.8–55.1%), myrcene (2.3–5.5%) and p-cymene (1.1–5.7%) as the main components. Result of correlation analysis showed that the oil yield was positively correlated with precipitation and negatively with temperature. In addition, 1,8-cineole was positively and nepetalactone was negatively correlated with altitude. Cluster analysis by Ward method categorized the populations into two groups. The major compound of the oils in cluster 1 was nepetalactone (with an average of 37.9%), while the oils in cluster 2, contained higher percentage of 1,8-cineole (52–59%). Most of the populations in cluster 2, were originated from high, cold, rainy, and steep areas. By increasing the altitude, changes in growth and essential oil composition of the plant are observed Result of correlation analysis showed that essential oil yield was positively correlated with precipitation, and negatively with temperature. In the present study the populations with high-amount of nepetalactone and 1,8 cineole were identified, The populations of cluster 2 with higher amount of 1,8-cineole, were growing in the high altitude, cold, rainy and sloping areas By increasing the altitude, changes in growth and essential oil composition of the plant are observed Result of correlation analysis showed that essential oil yield was positively correlated with precipitation, and negatively with temperature. In the present study the populations with high-amount of nepetalactone and 1,8 cineole were identified, The populations of cluster 2 with higher amount of 1,8-cineole, were growing in the high altitude, cold, rainy and sloping areas


Introduction
The genus Nepeta L. comprises approximately 300 species, widely distributed in Eurasia. It is one of the largest genera of subfamily Nepetoideae, family Lamiaceae in southwestern Asia. Iran is one of the main origins of this genus with 79 endemic species (ca. 77%) (Jamzad et al. [11]).
Nepeta binaludensis Jamzad is an endemic and rare perennial aromatic herb which is distributed in a limited area in the Binalud mountains in northeast of Iran [18]. It grows wild in habitats with 2300 to 2700 m altitude, 350-370 mm annual rainfall and annual mean temperature of 6-7 °C [17,18].
The medicinal properties of Nepeta species are usually attributed to their essential oil (EO) and flavonoids. Literature review showed medicinal properties of several Nepeta species in Iran such as, N. ispahanica, N. binaloudensis, N. bracteata, N. pogonosperma, and N. pungens. Some other species like N. crispa are used as culinary herbs [11]. There are some studies showing the effects of environmental parameters such as temperature, moisture and soil on EO content and composition of aromatic plants (Karousou [12]).
Most of mentioned reports indicate that the major constituents of EO of N. Binaluednsis are more or less the same in a different area, but the concentrations of EOs may be varied in different habitats, depending on genetic factors and their growing habitat. For other Nepeta species, Amirmohammadi et al. [2] obtained relatively high nepetalactone (73.9% and 55.5) as the main constituents N. cataria and N. assurgens, respectively. Similarly, Srivastava et al. [21] and Ashrafi et al. [5] obtained high nepetalactone (57. 3 and 53.8) in N. cataria, respectively.
For EO compounds the trends were partly similar as the EO yield. Some reports indicating negative relationships between altitude and some oil components, like carvacrol in Coridothymus capitatus and Satureja thymbra [12] and 1,8-cineole in T. vulgaris [23]. In contrast, there were positive relationships between altitude and some other EO constituents, such as thymol in C. capitatus and S. thymbra [12], linalool in T. vulgaris [23]) and T. kotschyanus [10].
The assessment of EO composition related to environmental conditions can provide important insight into the factors that determine chemical polymorphism in EO. Therefore, the aim of this study was to identify the distribution areas, and evaluate the EO variation and effects of climatic conditions on the EO yield and composition of 20 populations of N. binaludensis, as an endemic aromatic and medicinal plant in Iran, to introduce the best germplasm to domestication and improve breeding varieties.

Materials and Methods
The germplasm used in this study included 19 wild populations of N. binaludensis, collected from the elevations of Binalud Mountain and one cultivated population in the field of Mashhad's Agricultural Research Center, Iran (altitude 1100 m). The samples of flowering shoots of all populations were collected in the range of 2100 to 2600 m altitude in early July to late August, 2018 (Fig. 1). The localities were Gerineh, Dizabad, Shandiz, Shirabad, Ferizi, Galamkan, Dowlat abad, Kang, Dehbar, kordine, Jaghargh, Feleske, Azghad, Moghan1 and Moghan2. Geographical coordinates of these localities climatic conditions are shown in Table 1.
For each locality, three replications were collected. The plant materials were dried in shade. Then, 60-80 g of the dried flowering shoots of each sample was subjected to hydro-distillation for 3 h using a Clevenger-type apparatus. The EOs analysed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS). GC analysis was performed using a Thermo-UFM gas chromatograph, equipped with Ph-5 fused silica column (10 m × 0.1 mm i.d., film thickness 0.40 µm). Then a Varian 3400 GC-MS system equipped with DB-5 column, 30 m in length, 0.25 mm in diameter and 0.25 μm in the thickness were used for EO identification, [1] and [20]. The data of EO yield were statistically analyzed using one-way ANOVA. Mean comparisons were made using Tukey's test at p ≤ 0.05 levels. Since the EO components were measured in only one replication, therefore, the overall means and standard error of all EO constituents were computed over 20 populations. Relationships among climatic conditions with EO components were determined using correlation analysis. Data were also subjected to principle component analysis (PCA) and cluster analysis (Ward method). The Minitab16 software was used for all of the analysis.

Correlation between climatic factors and oil compound
Result of correlation analysis showed that EO yield was positively correlated with precipitation and negatively with temperature (p < 0.05) ( Table 3). The higher value of EO was obtained in high altitude, cold and rainy areas and its value decreased with increasing habitat temperature. Imedicating that domestication of this species in the lowland areas with high temperature may lead to decrease its EO yield. Based on our results, the highest EO yield, was obtained in altitude 2480 m.
Result of correlation analysis showed that many EO compound such as α-pinene, sabinene, myrcene, p-cymene, 1,8-cineole and δ-tepineol were positively correlated with altitude, indicating that the percentage of these compounds increased at higher altitude (Table 3). In contrast, Nepetalactone was negatively correlated with altitude.
Both p-cymene and Tepinen-4-ol were negatively correlated with precipitation and positively correlated with temperature. γ-terpinene, α-pinene and myrcene were   The means oil yield (in the last row) followed by the same letters has no significant differences at 5% probability by Tukey test    positively correlated with slope%, indicating that the higher values of this compound could be obtained in steep areas (Table 3).

Principle component analysis (PCA)
The first four main components accounted for 81% of the total observed variation. Results showed the, 8-cineole, myrcene, p-cymene positively and nepetalactone were negatively correlated with PC1. The climatic factors as: temperature, precipitation, altitude and slope correlated with PC2. The compounds of δ-tepineol, γ-terpinene and α-terpinene correlated with PC3 and finally the compounds of tepinene-4-ol, trans-sabinene hydrate, sabinene and EO yield correlated with PC4 ( Table 5).
The 20 localities of N. binaludensis for were scattered on PCA1 and PCA 2 ( Fig. 2 and Table 5). The first component well separated localities similar to cluster separation. The localities in the right side of the diagram had higher values of 1, 8-cineole, myrcene and p-cymene. The localities in the left side had higher value of nepetalactone. The second component was associated with climatic condition and localities in upper side grown in high temperature area. For example in cluster 2 the localities of Dizbad and Cultivated were grown in warmer area coupled with low precipitation and localities of Gerine, Shirbad and Zoshk in lower side of the diagram are grown in cold temperature coupled with high precipitation (Fig. 2). This result indicated that the distribution of localities based on the first two component scores were in agreement with cluster analysis (Figs. 2, 3).

Discussion
There was significant difference among localities for EO yields (p < 0.05). The seven localities with range of 2.8 to 4.9% had the higher EO yields than the other localities. These values were much higher than the oil yield (0.8%) that reported by Rustaiyan and Nadji [19] for N. binaluednsis.
EO yield was positively correlated with precipitation, and negatively with temperature and the higher EO yields were obtained in high altitude, cold and rainy areas and its value decreased with increasing habitat temperature. However, this trend was not similar in all localities, and there were some exceptions. The published data for some Lamiaceae species species were also different. Layegh Haghighi et al. [15] in N. pogonosperma, Torras et al. [23] in Thymus vulgaris, Ghasemi Pirbalouti et al. [7] in both T. vulgaris and T. daenensis found positive correlation between altitude and EO yield, indicating that domestication of this species in the lowland areas with high temperature may lead to decrease its EO content. Similarly, we obtained the lower EO value (1.8%) from a population that was cultivated in the field in low altitude in Mashad (1100 m) that the value was much lower than mention localities. In contrast, Figueiredo et al. [6] in T. vulgaris, Yavari et al. [25] in T. migricus, Habibi et al. [10] in T. kotschyanus found negative correlation between altitude and EO yield [10].
Result of correlation analysis showed that many EO compound such as α-pinene, sabinene, myrcene, p-cymene, 1,8-cineole and δ-tepineol were positively correlated with altitude, indicating that the percentage of these compounds increased at higher altitude (Table 3). In contrast, Nepetalactone was negatively correlated with altitude. Accordingly, Kofidis & Bosabalidis [14], found that altitude significantly affected the chemical  composition of essential oil in Lamiaceae taxa. Result of correlation analysis showed that myrcene, p-cymene, 1,8-cineole and δ-tepineol were positively correlated with altitude, indicating that the percentage of these compounds increased at higher altitude. Such positive relationships between altitude and some EO components were reported [10,12,15,23]. In contrast, Aćimović et al. [3] in Serbia reported that temperature positively and precipitation negatively were effective on accumulation of 1,8-cineole in Nepeta nuda L. in Rtanj Mountain. It seems their result was obtained in a low land in Serbia.
Nepetalactone was negatively correlated with altitude, indicating that the higher values of this compound could be obtained in low altitude and steep areas.
Result of cluster analysis, showed that nepetalactone was the main component with an average value of 37.9% in the cluster 1, while 1, 8-cineole was the main constituent of cluster 2 with an average value of 55.6%. Results of PCA showed that the first PC was positively correlated with 1, 8-cineole and negatively correlated with nepetalactone. Scatter of 20 localities based on PCA1 and PCA2 showed, in the first axe the localities in the left side had higher values of nepetalactone and localities in the right  In this study the localities with high-amount of nepetalactone and 1, 8 cineole were identified, which can be exploited depending on the purpose of breeding and cultivation. The high phytochemical differences in EO composition among N. binaludensis localities could provide useful information to improve cultivar with high yield and biological activity.

Conclusion
The localities of Dowlatabad, Jagharg2, Jagharg1, Moghan2 and Darrod with average values of 3.3, 3.7, 3.8, 4.2 and 4.9%, respectively had higher EO than other ones. The overall means values of 32.7 and 42.8% of were obtained for nepetalactone and 1, 8-cineole, respectively. The localities Gerine, Shirbad and Zoshk had higher value of 1, 8-cineole in the cold area and two localities Dizbad and Cultivated had higher value of 1, 8-cineole in the warm area. For nepetalactone, localities of Ferizi1, Ferezi2, Jagharg2, Moghan2, Zoshk2 and Kordine with a range of 40.6 to 50.4% had higher values than other ones. This variation was related to habitats with different altitudes, slopes and slopes, and coupled with genetic variation. Therefore, in order to introduce the superior cultivar, it is necessary to identify the elite localities and evaluating them in the similar environment in several locations/environments in order to domesticate and introduce high-yielding localities.