Determination and Discrimination of Intraspecific Diversity of Astragalus gossypinus by Eco-Phytosociological Method from West of Iran

MATERIALS AND METHODS

Plant materials: At the first phase, different stations of Astragalus gossypinus were determined in the west of Iran by using the accessible references, Herbaria and existence information. Then we referred to the different stations in study area, along 2004-2006 years, in growth season for collecting floristic-ecologic data. Totally between studied stations, 29 stations selected for investigation in Hamadan, Kermanshah, Kordestan and Markazi provinces from west of Iran (Table 1 and Fig. 1). Data collecting from 29 selected station carried out by using the unit of study in Eco-phytosociological method (Atri, 1996, 1999; Atri and Asgari Nematian, 2006) that is named Endogenous milieu (special station). In each station, location of establishment for each relive (stand) determined on base of presence of individual study species.

Fig. 1:	The distribution map of Astragalus gossypinus in Iran

Table 1:	The different studied stations for Astragalus gossypinus from west of Iran

Then for determination of special station of individual study species, minimal area determined by using the area-species method with area-species curve and Cain method (Cain et al., 1959). All ecologic-floristic data (the studied species and its companion species as floristical markers) were collected of each special station. Plant specimens deposited in the Herbarium, of Bu-Ali Sina University in Hamadan, Iran. Studied ecological factors included (elevation, pH, EC, texture of soil, slop direction and slop percent) in each special station.

Flavonoid aglycone study: The plant leaflet of different individuals of Astragalus gossypinus, that collected from different special stations in west of Iran, were separated and then ground in a grinder. Flavonoid aglycone analysis was taken on all individuals of Astragalus gossypinus listed in (Table 1). Briefly, 2 g dried powder of leaves boiled in 50 mL 2 M HCL for 45 min. Hydrolyzed leaf extracts were allowed to cool to room temperature. The extracts were then washed 3 times with equal volumes of ethyl acetate. The pooled ethyl acetate fractions were evaporated to dryness in a fume hood. The residue of each plant sample was taken up in an equal volume of 95% ethanol (Joseph et al., 2003). The analysis was performed on Silica gel plates 25 Fuelled aluminum CCM (20x20), Gel de silica 60 F₂₅₄ (Merck). Replicate plates were developed in BAW (n-butanol: acetic acid: water, 4:1:5, top layer used). Quercetin, flavone and rutin used as standards. The plates were developed at room temperature in a vertical separating chamber to the height of approximately 14 cm from the start. After drying, visualization was performed by:

Chromatograms were interpreted in long wave UV light (366 nm), then were measured R_f of each bands (Medica-Saric et al., 2004).

Data analysis: For determination and discrimination of intraspecific diversity of Astragalus gossypinus were applied correspondence, cluster, classification and discriminate analysis. Floristical composition data (as floristical marker) analyzed by using Anaphyto software version 95 (Briane, 1995) by means FCA (Factorielle Correspondence Analysis), AHC (Ascendant Hierarchical Classification), BO (Boules Optimisees) and Marquage methods. In studying phytochemical data, once presence and absence of different bands were determined on chromatogram for different individuals of Astragalus gossypinus. Then phytochemical data analysis was taken by SPSS (version 10) and MVSP softwares by means PCA, UPGMA and CCA methods. Ecological data analyzed by MVSP software with CCA method.

RESULTS

Floristical results: Obtained results base on floristical composition analyses of 29 special station showed seven main groups by using FCA method (Fig. 2). Group (1) include special station number 0009, group (2) number 0010, group (3) numbers 0039, 0033, 0026, 0025, 0024, 0023, 0022, 0021, 0020, 0019, 0018, 0016, 0015, 0014, 0013, 0012, 0011, group (4) number 0032, group (5) numbers 0041, 0040, 0036, 0034, 0030, group (6) number 0035, group (7) numbers 0029, 0028 0027. It must indicate that the mentioned 7 groups obtained base on similarity and dissimilarity of their floristic composition (as floristical marker). The obtained results from FCA method completed by AHC, BO and Marquage methods (Fig. 3-5). These 7 main groups evidence existence of intraspecific diversity for Astragalus gossypinus in study area. As seen in (Fig. 2-5), the obtained groups by FCA method are according to defined groups of AHC, BO and Marquage methods and confirm them.

Flavonoid results: Determination of level and kind of intraspecific diversity were used by flavonoid studies. Prepared chromatograms by TLC method showed different flavonoid bands and also different quantity of bands in different individuals of Astragalus gossypinus in study area. Different bands and their R_f measured that their results illustrated in Table 2. Analyzing of flavonoid data separate some groups (Fig. 7). The obtained groups of flavonoid results (Fig. 6 and 7) had a good correlation with floristical composition groups (Fig. 2-5) that confirm them and showed intraspecific diversity in chemotype level. According to these results can introduce seven different kinds of chemotypes for Astragalus gossypinus from west of Iran. These 7 chemotypes are different regarding quality, quantity and R_f of flavonoid bands.

Ecological results: Ecological factors data that were collected by applied method analyzed by MVSP software with CCA method. The obtained results showed between studied ecological factors (elevation, pH, EC, texture of soil, slop direction and slop percent) elevation factor has the most important role in separating different determined groups (Fig. 8).

Fig. 2:	A and B) Results of floristical composition data analysis by FCA method on 2,3 and 3,4 axes. These 7 groups are the same to grouping resulted by AHC, BO and Marquage methods (Fig. 3-5)

Fig. 3:	Results of floristical composition data analysis by AHC method. As seen these groups are the same as groups from FCA

Fig. 4:	Results of floristical composition data analysis by BO method, which these seven groups have good correlation with resulted groups of FCA and AHC methods

Fig. 5:	Results of floristical composition data analysis by Marquage method. Grouping results were confirmed grouping resulted by methods of AHC and FCA and BO

Table 2:	The table of Presence and absence flavonoid bands in one dimensional TLC related to different individuals of A. gossypinus in the study area

Fig. 6:	Flavonoid data analysis of Astragalus gossypinus individuals by PCA method

Fig. 7:	Resulted cluster of aglycoside flavonoids studies by UPGMA method that has good correlation with floristical composition groupment

DISCUSSION

Biodiversity-that is result of different ecological factors existence in various stations-shows the biological capacity and ability of each area. One of the most important reserve that led to biodiversity is inter and intraspecific diversity. Creation of inter and intraspecific diversity are the main origin and storage of speciation. In this order, creation inter and intraspecific diversity in different levels cause to richness of taxa in an area. So identification of inter and intraspecific diversity in order to knowing biodiversity is very important. For determination and discrimination of inter and intraspecific diversity, should applicate an suitable method that the obtained results of applied method be correct, precision and spends lower expenses (Atri and Asgari nematian, 2006).

Fig. 8:	Results of ecological factors studies by CCA method that show many populations has influenced by elevation factor

Many studies carried out for determination intraspecific diversity (Turesson, 1922; Mooney and Billings, 1959; Perez-Alonso et al., 2003; Koch and Bernhardt, 2004; Semmar et al., 2005; Telascrea et al., 2006), but most of them do not apply a special method in plant data collecting process and carry out by time consumer and expensive experiments. While, application the floristical marker and the unite of study in Eco-phytosociological method (special station) in this kind of studies led to correct and precision results because that is according to factors governing nature. In the other hand, it prevents of more expenses and time consumer experiments (Fakhre-Tabatabaei et al., 2000; Sefidkon et al., 2003; Kalvandi et al., 2004; Sefidkon et al., 2005; Ebrahimzadeh et al., 2006; Atri and Asgari Nematian, 2006).

In regard to applied principles in this method for data collecting, we can certainly declare that floristic markers without application other markers can determine intraspecific diversity existence. Then for determinating kind and level of intraspecific diversity (Ecophene, Chemotype, Cytotype, Ecotype …), between obtained floristical groups, we can use other studies such as: morphology, anatomy, phytochemistry, cytology and etc.

Present results show that Astragalus gossypinus has high diversity in the west of Iran. According to our results of floristical analyses, there are 7 distinctive different groups of Astragalus gossypinus individuals in study region. At second phase, phytochemical studies create seven kinds of chemotypes which conform and affirm the obtained results of floristical studies. Between studied ecological factors, elevation is the most important ecological factor in creation intraspecific diversity.

So this study and other studies that done base on this method until to now, show the high efficiency of it in determination and discrimination of inter and intraspecific diversity existence. By using this method after determinating floristic groups, we should characterize kind and level of intraspecific diversity only between obtained floristic-ecologic groups and this in require us of testing all of the individuals of studied species and expending long time and much money in this way.