Infraspecific morphological variations of Salvia limbata in Iran

Salvia limbata is an aromatic herb of Lamiaceae, which distributed in different parts of Iran. Studies have revealed that this species has several benefit compounds for human health. In this study, we investigated infra‒specific morphological difference in this species. In total, 21 inflorescence morphological features were evaluated in eight populations; ten individuals were evaluated per each population. Data were analysed using SPSS and MVSP softwares. Morphological traits highly differed among the studied populations and ANOVA test revealed significant differences among most of them. According to UPGMA tree, PCA and PCO plots of morphological characteristics, we reported four distinct phenotypes among the studied populations. CA. Joined plot revealed each of these group were characterized by special traits, which were useful in identification of them. Two phenotypes were monotypic, while the rest were composed of two to four populations. This species has a relative large geographical distribution, the distribution of its populations in diverse area will prevent the focus of natural selection in a given direct. Therefore different phenotypes were found in its different populations.


Introduction
In nature, the creation of infra-specific difference can occur through a wide range of mechanisms such as local adaptation, phenotypic plasticity, parental conditions and artificial selection (Violle et al., 2012).West-Eberhard (1989) has believed that when it is resulted from plasticity, features can differ rapidly within generations and vary drastically across populations in dissimilar habitats.Recently, studies have broadened in the identification of variation to incorporate the considerable phenotypic difference within and among populations of the same species (Violle et al., 2012).
Investigations have showed that difference of phenotypic feature within species can be as extreme as the characteristic difference across species (Albert et al., 2010).Moreover, infra-specific difference may influence structure of community and function of ecosystem as much as difference among species (Govaert, Pantel, & De Meester, 2016;Palkovacs & Post, 2009).
In current study, we studied morphological characteristics of inflorescence in eight populations of S. limbata, in order to evaluate pattern of morphological variations among the populations and also find possible phenotype(s) among the studied populations.

Morphological studies
In current study, eight natural populations of S. limbata were harvested from different regions of the country (Table 1), and were identified according to descriptions provided in Flora of Iran (Jamzad, 2012).Morphological differences were carefully evaluated for reproductive organs from collected samples of the studied populations.The morphological characteristics of flowers were studied under dissecting stereo microscopes.For morphometric analysis, 80 plant specimens from 8 populations were used.Voucher specimens were deposited at Herbarium of Arak University.Character measurements Measurements of morphological traits were performed on each plant, including its flowering stem with flowers in bloom.In total, 21 qualitative features were measured on each specimen.Each character was measured four times per each plant sample, and their average determined.The studied characteristics were: calyx width, length and calyx length / width ratio, petal length, width and petal length / wide ratio, calyx short teeth length, calyx short teeth width, calyx short teeth length / width ratio, style length, stigma length, calyx long teeth length, calyx long teeth width, calyx long teeth length / width ratio, anther length, anther width, anther length /width ratio, pedicle length, long filament length, short filament length, long /short filament length ratio.

Statistical analyses
Morphometric data was subjected to one-way analysis of variance (ANOVA) to determine if significant difference existed among populations for each feature measured.Mean and standard deviations of characteristics were tacalculated.These analyses were performed using SPSS ver.17.Cluster analysis was carried out based on quantitative features using UPGMA, PCO, PCA and C.A-Joined plots clustering in Multivariate Statistical Package (MVSP) program (Podani, 2000).

Results
The mean and standard deviation of the studied morphological variables were presented in Table 2. Morphological traits differed among the studied populations.Biggest and smallest anther lengths were recorded in population no. 5 and 1, respectively.While, reverse pattern were recorded for long filament length.Therefore, largest and shortest long filament lengths were reported for populations no. 1 and 5, respectively.Maximum and minimum lengths of pedicle were belonged to population's no. 3 and 2, respectively.Largest stigma was observed in population no. 1, but population no. 4 had smallest stigma.Longest style was found in population no. 4, and shortest in population no. 5. Maximum and minimum petal lengths were registered in populations no. 5 and 2, respectively.Population no. 1 had longest calyx and widest calyx short teeth; however, shortest calyx and narrowest calyx short teeth were belonged to population no. 7. Largest and smallest short filament length was found in populations no.6 and 5, respectively (Fig. 1).Furthermore, the ANOVA test confirmed significant variations (p ≤ 0.01) for all studied variables, except for anther width (Table 3).The studied populations were clustered separately in UPGMA tree (Fig. 2); moreover, PCA and PCO (Fig. 3, 4) plots produced similar outputs.Therefore, population's arrangement in the tree was discussed here.The tree had two branches: we found population no. 1 in small branch, while other populations were observed in large branch, which was divided into two subbranches.Population no. 5 placed far from others in a sub-branch and the rest populations were registered in another sub-branch into two groups.Populations no. 2 and 4 were observed as a pair.Eventually, in the other sub-group populations no. 3 and 6 were close together and populations no.7 and 8 gradually joined them.Therefore, we had four distinct morphological groups among the studied populations.CA. joined plot (Fig. 5) revealed each of these groups were characterized by special features that were useful in identification of them.For example, calyx length, calyx short teeth length and calyx long teeth length were prominent variables for populations no.1.Moreover, populations no. 2 and 4 were characterized by style length, calyx short teeth length/width ratio and calyx long teeth length/width ratio.Populations no. 5 was identified according to calyx length/width ratio.

Discussion
In this research, we evaluated morphological variables of inflorescence in the selected populations of S. limbata.
Because, these characteristics are more stable than vegetative characteristics (Stace, 1989), in addition, Jamzad (2012) used inflorescence characteristics along with palynological as well as ITS data in her valuable work on Iranian Lamiaceae species, Flora of Iran.
Findings of these investigations showed high level of infra-specific variability in S. limbata.There are some reasons for morphological variations among these populations.It seems that genetic variations and ecological conditions of habitat are more important than others are.Several studies (Talebi, Atri, Sheidai, Sharifnia, & Noormohammadi, 2014, Sheidai et al., 2014) have revealed that infra-specific genetic variation among populations leads to morphological difference.In addition, ecological factors have strong effect on morphological characteristics and through phenotype plasticity process lead to infraspecific morphological variations (Talebi, Rezakhanlou, & Matsyura, 2017).
ISSN 2412-1908; http://journal.asu.ru/biol/So, it seems that in the case of our studied populations, ecological factors have a very strong effect on population's similarity or difference.This condition creates morphological polymorphism among populations.Of course, this situation gives the species that has the genetic background to occupy a variety of environments and emergence of morphological polymorphism.

Conclusion
We evaluated inflorescence morphological characteristics among eight populations of S. limbata.Morphological traits highly differed among the populations and ANOVA test revealed significant difference among the populations.According to UPGMA tree, PCA and PCO plots, we found four primitive phenotypes, which were characterized by special morphological features.Two phenotypes were monotypic, while one phenotype consisted of two, and another one has four populations.The distribution domain of a species is not composed exactly identical ecological environments, but also a set of different environment with different ecological conditions.So species must adapt its phenotype structure in accordance with those conditions in order to be able to live under those conditions.

Fig. 1 .
Fig. 1.Image of Salvia limbata and its reproductive organs.A) Flowering stem, B) inflorescence cycle, C) lateral view of flower, D) calyx, E) fertile and sterile anther, F) stigma, style and ovary.

Fig. 2 .
Fig. 2. UPGMA tree of the studied populations based on morphological variables.

Fig. 4 .
Fig.4.PCA plot of the studied populations based on morphological characteristics.

Table 1 .
Locality address of the studied populations of S. limbata

Table 2 .
Mean and standard deviations of some morphological characteristics of studied populations (all values are in mm).