Do we have infraspecific taxa of Salvia multicaulis Vahl . ( Lamiaceae ) in Iran ?

Salvia multicaulis is a widespread species of Lamiaceae family in Iran. There are many discussions about its infraspecific variations. Although some varieties were definite for this species in various parts of the world, no infraspecific taxon was reported in Iran and all samples of this species were named as S. multicaulis. In this study, morphological characteristics of S. multicaulis populations, naturally growing in Iran, was examined. Twenty-two traits were examined in 94 individuals of this species to identify their phenotypic difference. Most of the investigated features were showing a high degree of variability, but it was highly pronounced for some characteristics such as basal leaf shape, basal leaf width, basal leaf length/ width ratio and basal petiole length. Significant positive/negative correlations were observed between some morphological variables. Furthermore, significant negative correlations were found between the eastern distribution of populations with basal leaf petiole length and basal leaf length/ width ratio. Based on the UPGMA cluster analysis, populations were divided into two main branches. The first branch contained four populations, while the second branch was bigger and clustered in two sub-branches. In one of them, three populations and in another one the rest populations arranged in two groups. CA joined plot confirmed that each of studied populations or group of populations had distinct morphological trait(s), which were useful in identification of them. Our findings supported population no. 13 had unique morphological traits such as the largest bracts and basal leaf petiole, highest flower number of each inflorescence cycle, widest and largest calyx. The conservation of the highly diverse populations of Iranian S. multicaulis is recommended.


Introduction
Salvia L. is the largest genus of Lamiaceae that comprises of more than 900 taxa in the world (Davis 1982).This genus has 62 species in Iran (Jamzad, 2012).Different species of the genus have been used in folk medicine for the treatment of many disorders such as diabetes (Jimenez et al. 1986) and skin diseases like psoriasis and eczema (Topcu et al. 2007).
Investigations of infraspecific morphological diversity have been performed for different species of Labiatae family (e.g., Talebi, 2014, Talebi, 2015).However, despite many reports about essential oil composition of S. multicaulis, we still do not have comprehensive information about the morphological variations of this species.Therefore, the main objective of this investigation was to identify the variability in 94 accessions of 14 populations of this species growing wild in Iran, to determine the possible correlation between the characteristics, to identify the most useful features for discrimination between the populations and to detect relationships between them.

Plant material
Six qualitative and sixteen quantitative morphological traits of fourteen populations of S. multicaulis were examined (Table 1).
These samples were elected from natural populations of the species during spring 2016 and identified based on the descriptions provided in valuable references like Flora Iranica (Rechinger, 1982) and Flora of Iran (Jamzad, 2012).On the basis of population size, up to eight individuals were selected for each population.The mean and also standard deviation was determined for quantitative features (Table 2).The studied variables were: stem length, shapes of basal and floral leaves margin, blade and their length, width, length/wide ratio and petiole length, calyx and corolla color, length, width, their length/ width ratio and pedicle length.Statistical analysis Analysis of variance was performed for morphological traits using SPSS software.The mean and standard deviation and simple correlation coefficient were calculated to determine the relationships between the studied morphological features using the Pearson correlation coefficient by SPSS.Relationships among the populations were studied by principal component analysis (PCA).The morphological similarity coefficients according to Manhattan method were calculated using the MVSP ver.3.2 program of the numerical taxonomy multivariate analysis and the dendrogram were constructed using the unweighted pair group method with arithmetic means (UPGMA).PCA and PCO scatter plots were constructed for representing morphological similarity among the studied populations.The CA-Joined plot and PCA-biplot representing morphological similarity relationships among populations, linking the morphometric traits (Podani, 2000).
Table 1.Localities address and coding of studied S. multicaulis populations.

Results
ANOVA test showed significant differences (p≤ 0.05) for all of the quantitative studied characteristics with except basal leaf width, floral leaf length/width ratio and calyx leaf length/width ratio (Table 3).Quantitative features such as floral and basal leaf blade shape and their marginal shape differed between the populations.They were oblong, ovate, obovate, lanceolate, oblanceolate, linear and linear-lanceolate.Moreover, the color of calyx was purple or violet in all of the studied populations, with except population number 13, which had lavender calyx.It means that most of the morphological traits varied significantly among the populations.PCA analyses proved that seven characteristics: stem length, basal leaf shape, basal leaf width, basal leaf length/ width ratio and basal petiole length comprises 72.47% of variations.Therefore these traits were more variable among the examined characters (Table 4).Significant positive/negative correlations were seen between some morphological traits.For example, corolla length had significant negative correlations (P ≤ 0.05) with floral leaf length and width, calyx width, flower number of each inflorescence cycle.Significant positive correlations (P ≤ 0.01) were found between calyx length and width with floral leaf length, pedicle length, and flower number per each inflorescence cycle.Basal leaf length and width had significant positive correlations (P ≤ 0.01) with stem length, floral leaf length and width and pedicle length.The studied populations separated from each other in UPGMA dendrogam (fig 1); in addition, PCA, as well as PCO plots (figs 2, 3) created similar results.So population's arrangement in the dendrogam was discussed here: it is composed of two branches (A, B).The smallest one (A) had two sub-branches.Populations number 5, 7, and 10 were grouped in the bigger sub-branch, while population no. 13 was found in the smaller one far from others.The largest branch (B) divided into two sub-branches; one of them was small (C) and composed of three populations number 4, 8, and 14.In the bigger sub-branch (D), populations constructed two groups.Populations number 1, 2, and 3 arranged in one group (E) and the rest populations were clustered in the other group (F), so that populations number 6 and 11 placed closely and the population no. 12 were far from the others.Table 3. Results on the ANOVA analysis to assess for differences in morphological traits of studied S. multicaulis populations.d.f.: degrees of freedom; F: F-statistic; P: probability.C.A joined plot showed that each of studied populations or identified group of populations had the distinct morphological variable(s), which were useful in identification of them (fig.4).For example, floral leaf shape, the ratio of basal leaf blade length/ petiole length and calyx length/ pedicle length ratio were valuable traits for populations no. 8, 14 and 4, respectively.Furthermore, petal color putts population no.14 far from populations no. 8 and 4. Stem length was a distinguishing character for branch A. Then, each of these populations had distinct features and separated from each other.Floral leaf length was a specific trait for populations no. 5 and 10.Pedicle length, calyx length and width and flower number of each inflorescence cycle separated population no. 13.Table 4. PCA analyses of the studied morphological variables between the studied populations.

Discussion
S. multicaulis has several populations in various parts of Iran, which growth under different ecological conditions.Therefore, this species can definite as an ecological generalist plant species.These plants have highly plastic genotypes and phenotypes (Sultan, 1995), but taxa whose constituent individuals express limited adaptive plasticity might be restricted to narrower, 'specialist' ecological ranges.Williams et al. (1995) stated that plasticity might facilitate the rapid distribution of introduced and also native species into new habitats without the evolutionary lag time needed to adapt to new habitats through natural selection.
Our results revealed high morphological diversity between different populations of this species.ANOVA and PCA tests showed significant differences for most of the studied morphological traits between the studied populations and different shapes and amounts of qualitative as well as qualitative characteristics were observed between the populations.For this reason, high degree of phenotypic plasticity is seen in this species.Sultan (2000) stated that a single genotype can create various phenotypes in different ecological conditions.This phenomenon is definite as phenotypic plasticity.More investigations have proved that plants are plastic for a remarkable array of environmental important factors.Comparative, quantitative genetics and molecular approaches are leading to new insights into the adaptive nature of plasticity, its underlying mechanisms and its role in the ecological distribution and evolutionary diversification of plants (Sultan, 2000).
Previous studies showed high infraspecific variations in essential oil compositions of this species in Iran.Ahmadi and Mirza (1999)  major constituents of essential oil compositions of this plant, while results of Morteza-Semnani et al ( 2005) studies showed that the major constituents of the essential oil of S. multicaulis are camphor (11.0%), 1,8-cineole (10.7%), borneol (8.6%) and αpinene (7.5%).
On the basses of mentioned essential oil researches on this species, it seems that a phenomenon beyond the phenotypic plasticity creates such morphological variations between the populations of S. multicaulis.Although, no infraspecific taxon definite for this species in Flora Iranica (Rechinger 1982), Flora of Iran (Jamzad 2012) and Flora of Turkey (Davis, 1982), in Flora of Lebanon one variety of this species, Salvia multicaulis Vahl.var.simplicifolia Boiss, is present (Senatore et al 2004).
The population no. 13 had special morphological characteristics.When morphological traits of it were compared with morphological descriptions of S. multicaulis in Flora Iranica (Rechinger, 1982), Flora of Iran (Jamzad, 2012) and Flora of Turkey (Davis, 1982), it was cleared that some main morphological traits of this population that were useful in identification of this species were different from S. multicaulis.For example, the color of calyx and corolla length of this population was not to S. multicaulis description.So these characteristics are used in identification key of this species, therefore we predict that this population was a new variety or subspecies of this species in Iran.

Fig. 1 .
Fig. 1.UPGMA dendrogam of the studied populations based on the morphometric data.Numbers indicate population's code 1-14 as in Table1.

Table 2 .
Mean and standard deviation of some examined morphological characteristics between the studied populations (all values are in cm).Numbers indicate population's code 1-14 as in Table1.