Biochemical Systematics and Ecology 35 (2007) 757e763 www.elsevier.

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Isoenzyme variation patterns and species concept in Astragalus gossypinus and Astragalus persicus complexes (Fabaceae) in Iran
Shahin Zarre*, Zeinab Khodaei, Zahra Karamali, Vahid Niknam, Massoud Mirmasoumi
Department of Botany, School of Biology, University College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Received 20 January 2007; accepted 26 May 2007

Abstract Isoenzyme electrophoresis was employed to examine the relationships of 21 individuals representing four populations of Astragalus gossypinus complex as well as 20 individuals representing ve populations of Astragalus persicus complex. A total of 27 bands from three enzyme systems (superoxide dismutase, esterase, and peroxidase) were obtained. UPGMA clustering method resulted in two distinct clusters for different populations corresponding to the two species complexes analysed. In both species, populations distributed on Alborz mountain range in northern Iran form separated clusters from those distributed on Zagros mountain range in the West. The results also show that both species complexes exhibit a high diversity based on ShannoneWeaver diversity index (H0 ) compared with other species of Astragalus reported previously. The mean number of bands per each presumed isoenzyme ranges from 2.14 to 3.57. The value of Euclidean distance ranges from 1.251 to 3.152. Our data suggest that both species should be circumscribed wider than that treated by most taxonomists, and several taxonomic names should be reduced under synonymy of the corresponding species. 2007 Elsevier Ltd. All rights reserved.
Keywords: Astragalus gossypinus; Astragalus persicus; Fabaceae; Isoenzyme variation; Euclidean distance; Alborz mountain range; Zagros mountain range; Iran

1. Introduction Astragalus gossypinus Fisch. and Astragalus persicus Fisch. & C.A. Mey. are two important tragacanthic species of Astragalus belonging to sections Rhacophorus Bunge and Hymenostegis Bunge, respectively. Both species are widely distributed in subalpine areas of Iran and adjacent countries. Taxonomic problems regarding these species are mainly due to delimitation from their relatives and the possible conspecity with their related taxa. Different taxonomic treatments of these groups of species indicate considerable contradictions among them. The irjaev, 1939) or sepatreatments vary from considering several infraspecic taxa for these species (Boissier, 1872; S rating different species, like Astragalus xanthogossypinus Hand.-Mazz. in the case of A. gossypinus (Chamberlain and Mathews, 1970), and Astragalus bounophilus Boiss. & Hohen., Astragalus capax Maassoumi., Astragalus rubriorus
* Corresponding author. Tel.: 98 21 61112482; fax: 98 21 66405141. E-mail address: zarre@khayam.ut.ac.ir (S. Zarre). 0305-1978/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.05.012

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irj. & Rech. in the case of A. persicus (Rechinger et al., 1958; Maassoumi, 1994; Bunge. and Astragalus naftabensis S Podlech and Maassoumi, 2001). Therefore, we prefer to use the term species complex instead of species for both taxa until the taxonomic borders in these groups are claried. A. gossypinus and A. persicus are perennial, spiny, and cushion forming plants with crowdy habit, with dense and many (15e70) owered inorescences that overtop the leaves in the case of A. persicus but are attached to the stem in the case of A. gossypinus. The calyx is funnel shaped and rupturing in fruit in A. gossypinus, while tubular and slightly inating in fruit in A. persicus. A typical papilionoid corolla consisting of a pandurate banner, two wings and a keel of two attached petals are characteristic for both species. A unilocular mono-seeded legume is present in both species. These species ower from June to July. Among these species, A. gossypinus produces considerable amount of the socalled gum tragacanth, exudates of branches and roots with a wide range of application in pharmaceuticals, cosmetics, and industrial textile sizing, as well as a thickening agent in foods (Gentry, 1957). The recorded chromosome number for A. gossypinus is 2n 2x 16, but for A. persicus is 2n 4x 32 (Esmailzadeh, 2002). The electrophoretic analysis of isoenzyme variation has proved to be useful in assessing genetic diversity in legumes (Doyle and Brown, 1985; Baskauf and Snapp, 1998; Jenczewski et al., 1999; Alba et al., 2001). In fact, this strategy has been applied in the delimitation of natural groups, species and subspecic taxa in different species of Astragalus (Zarre et al., 2004; Liston, 1992). We applied this method for better understanding of genetic variation in A. gossypinus and A. persicus, and for the delimitation of taxa in these species complexes. In addition to the taxonomic problems regarding the selected species, both of them comprise populations distributed along both major Iranian mountain ranges, Alborz in the North and Zagros in the West. Thus, the isoenzyme variation pattern could provide evidence on the genetic structure of plant populations distributed on these mountains. The aims of this study were the following: (1) description of electrophoretic isoenzyme phenotypes and their variation patterns among the populations of A. gossypinus and A. persicus; (2) evaluation of isoenzyme markers useful in delimitation of taxa in these complexes; and (3) the possible effect of geographical distribution on isoenzyme variation patterns in different populations of these species. 2. Materials and methods 2.1. Plant materials and isoenzyme analysis 2.1.1. Sampling Forty-one individuals belonging to nine populations and representing eight morphs were collected. Each individual represents a patch of 20 plants about 2 m distant from each other. At least three and maximally six individuals (patches) were analysed for each population. These patches were at least 20 m apart from each other. In total, 60e 120 samples were analysed for each population. A list of voucher specimens (one voucher for each population) which are deposited in the Central Herbarium of Tehran University (TUH) and some characteristic features of the populations are presented in Table 1. These specimens were collected during MayeJune 2006 by Zarre, Karamali and Khodaei. Leaves were collected in resealable bags and transferred into the laboratory on ice. They were kept cool (4  C) for a few days until enzyme extraction. 2.1.2. Extraction Leaets (1000 mg) were homogenized with 2 ml of the following buffer: TriseHCl 0.05 M, cystein 0.02 M, magnesium sulphate 0.002 M, EDTA 0.01 M, and glycerol 10% w/v. Before extraction, each 2 ml of this stock solution was combined with 0.02 g PVP-40. Following centrifuging at 13,000 rpm for 20 min, the extracts were stored frozen at 18  C until electrophoresis in vertical polyacrylamide gel slabs (Soltis and Soltis, 1990). Seven enzyme systems were assayed, of which only three gave satisfactory activities, good band resolutions, and consistent results, and thus were selected for this study. These enzymes were peroxidase (PRX) (E.C. 1.11.1.7), superoxide dismutase (SOD) (E.C. 1.15.1.1), and esterase (EST) (E.C. 3.1.1.6). 2.1.3. Electrophoresis It was carried out using 10% polyacrylamide gels for EST and PRX and 12% polyacrylamide gels for SOD. Electrophoretic phenotypes were numbered consecutively, beginning with the band running closer to the origin.

S. Zarre et al. / Biochemical Systematics and Ecology 35 (2007) 757e763 Table 1 A list of voucher specimens including the abbreviations and morphological characteristics of the populations applied in the present analysis Names of putative taxa and population acronyms A. bounophilus (ara1) A. capax (emam) Locality and altitude Herbarium number in TUHa 36775 36666 Morphological characteristics

759

A. naftabensis (nesa) A. persicus (zarr) A. rubriorus (tuch) A. gossypinus (ara2) A. gossypinus var. nervulosus (pal1) A. gossypinus var. nervulosus (pal2) A. xanthogossypinus (west)
a

Prov. Tehran, Karaj e Chalus road, Arangeh village, 1910 m Prov. Tehran, Haraz road Ab e Ali to Polur, mts in front of emam zadeh Hashem, 2715 m Prov. Tehran, Karaj e Chalus road, mts above Nessa village, 2250 m Prov. Zangan, 50 km to Zangan from Halab, 1805 m Prov. Tehran, Tuchal mts, 2730 m Prov. Tehran, Karaj e Chalus road, Arangeh village, 1910 m Prov. Qom, Palang Darreh protected area, 1300 m Prov. Qom, Palang Darreh protected area, 1495 m Prov. Kordestan, 40 km after Kamyaran towards Sanandaj, 1415 m

Stipule 7e10 mm, bract 6e9 mm, corolla red to purple Stipule (12e)16e22 mm, bract 10e20 mm, corolla red to violet Stipule 8e10 mm, bract 9e12 mm, corolla red to violet Stipule 12e14 mm, bract 5e8 mm, corolla violet to purple Stipule 8e15 mm, bract 6e9 mm, corolla purple Corolla pink, bract 6 mm, plant 10e15 cm tall Corolla pink, bract 8e9 mm, plant 10e20 cm tall, leaets with long mucro Corolla pink, bract 8e9 mm, plant ca. 10e20 cm tall, leaets with long mucro Corolla pale yellow, bract 8e9 mm, plant 20e30 cm tall

36774 36651 36776 36685 36672 36773 36697

Tehran University Herbarium.

The front line was monitored with bromophenol blue (0.1% in ethanol). Migration was performed at 4  C and stopped when the front line was 8e10 cm away from the application point, which occurred after 2e3 h. During migration, electric voltage was checked every 25e35 min and adjusted if needed. Staining protocols generally followed Wendel and Weeden (1989). 2.2. Data analysis Band frequencies were calculated for each population and for each isoenzyme system. The intra-population variation was estimated by the ShannoneWeaver diversity index (H0 ) (Shannon and Weaver, 1949). Based on the matrix of band frequencies the variation among the populations was examined by cluster analysis, and UPGMA method using program package NTSYS-pc version 2.02k (Applied Biostatistics Inc., 1986e1998) which was utilized based on Euclidean distances. 3. Results 3.1. Isoenzyme variation and allozyme diversity Gel electrophoresis of the plant extracts resulted in clear and consistent staining for three enzymes attributed to seven putative isoenzymes: EST-1, EST-2, SOD-1, SOD-2, PRX-1, PRX-2, and PRX-3. In total, 27 putative allozymes were detected in different populations of A. gossypinus and A. persicus. The frequency values range from 0 to 1 (Table 2). Isoenzyme frequency measured within a certain individual (patch) range in most cases between 0.7 and 1, when the bands were present (probably indicating high proportion of autogamy within individuals or patches). However, this frequency shows a wider range (0.166e1) at the population level (among individuals or patches). Allozymes C and D of SOD-1, and G of SOD-2, were present at a high frequency among all populations (Fig. 1). Allozyme A of EST-1 was characteristic for A. gossypinus, while allozymes L and M of EST-2, as well as H and I of SOD-2, were characteristic for A. persicus (Table 2). Different frequencies were observed among different populations of both species for certain allozymes. For instances, the frequency of allozyme H of EST-2, and D of PRX-2 in most populations of A. persicus, was lower than those of A. gossypinus. The mean number of allozymes for each isoenzyme

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Table 2 Band frequencies for three enzyme systems among nine populations of A. gossypinus and A. persicus Isoenzymes EST-1 Band/ population A B C D E F G H I J K L M A B C D E F G H I A B C D E ara1 0.333 0.333 0.000 0.333 0.000 0.000 1.000 0.000 0.333 0.333 0.000 1.000 0.666 0.000 0.000 1.000 1.000 0.000 0.000 1.000 0.333 0333 0.000 0.666 0.666 0.666 0.000 2.28 nesa 0.000 0.166 0.166 0.666 0.500 0.833 0.166 0.166 0.666 0.333 0.166 0.666 0.666 0.166 0.333 1.000 1.000 0.166 0.166 1.000 0.333 0.333 0.166 0.666 0.666 0166 0.000 3.57 emam 0.000 0.000 0.250 0.250 0.500 0.750 0.250 0.500 1.000 0.250 0.000 1.000 1.000 0.000 0.000 1.000 1.000 0.000 0.000 1.000 0.000 0.000 0.250 0.750 0.000 0.000 0.000 2.14 tuch 0.000 0.750 0.750 0.000 0.000 1.000 0.500 0.000 0.250 0.750 0.250 0.250 0.500 0.000 0.750 1.000 1.000 0.000 0.000 1.000 0.500 0.500 0.250 1.000 0.500 0.000 0.000 2.57 zarr 0.000 0.000 0.000 0.333 0.000 0.000 0.333 0.000 0.666 0.000 0.666 0.333 0.000 0.666 0.666 1.000 1.000 0.000 0.000 1.000 0.666 0.666 0.666 1.000 0.333 0.000 0.000 2.14 ara2 1.000 0.500 0.666 0.000 0.000 0.0000 0.666 0.833 0.833 0.000 0.000 0.000 0.000 0.666 1.000 1.000 0.833 0.833 0.500 0.666 0.000 0.000 1.000 0.500 1.000 1.000 0.333 2.71 pal1 1.000 0.250 0.250 0.500 1.000 0.000 0.500 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.750 1.000 1.000 0.250 0.250 1.000 0.000 0.000 1.000 0.750 1.000 1.000 0.25 2.57 pal2 1.000 0.000 0.400 0.400 0.400 0.000 0.200 1.000 1.000 0.000 0.000 0.000 0.000 0.400 0.600 0.666 1.000 0.750 0.600 0.600 0.000 0.000 0.800 0.800 0.800 1.000 0.000 2.57 west 1.000 0.000 0.500 0.333 0.100 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.500 0.666 1.000 0.833 0.666 0.666 1.000 0.000 0.000 0.166 1.000 1.000 0.833 0.000 2.57

EST-2

SOD-1

SOD-2

PRX-1 PRX-2 PRX-3 Mean alleles per locus

ranged from 2.14 to 3.57 (Table 2). The ShannoneWeaver diversity index (H0 ) for calculating the diversity expressed by the enzyme system and populations is shown in Table 3. The average value of H0 , estimated for each population ranged from 0.406 (population emam) to 0.936 (population nesa). Similarly, the average H0 value for each enzyme system ranged from 0.082 (PRX-2 in A. gossypinus) and 0.284 (SOD-1, in A. persicus) to 1.114 (EST-1, in A. gossypinus) and 1.461 (EST-2, in A. persicus).

Fig. 1. Isoenzyme banding pattern and zymograms for superoxide dismutase enzyme in different populations of A. gossypinus and A. persicus. Upper gel and zymogram indicate A. gossypinus. Lower gel and zymogram indicate A. persicus. The abbreviation of populations below each gel corresponds to those applied in Table 1.

S. Zarre et al. / Biochemical Systematics and Ecology 35 (2007) 757e763 Table 3 The ShannoneWeaver diversity index (H0 ) for three enzyme systems observed in populations of A. gossypinus and A. persicus Isoenzymes Populations Astragalus persicus ara1 EST-1 EST-2 SOD-1 SOD-2 PRX-1 PRX-2 PRX-3 Mean 1.090 1.000 0.000 0.732 0.270 0.540 0.000 0.519 nesa 1.364 2.060 0.664 1.328 0.568 0.568 0.000 0.936 emam 1.253 1.030 0.000 0.000 0.561 0.000 0.000 0.406 tuch 0.430 1.945 0.215 0.692 0.346 0.346 0.000 0.567 zarr 0.366 1.270 0.540 0.540 0.270 0.366 0.000 0.478 Mean 0.900 1.461 0.284 0.658 0.403 0.364 0.000 0.581 Astragalus gossypinus ara2 0.616 0.602 0.422 0.768 0.346 0.000 0.366 0.446 pal1 1.038 0.346 0.215 0.692 0.215 0.000 0.346 0.407 pal2 1.090 0.321 0.942 0.827 0.357 0.178 0.000 0.530 west 1.712 0.000 0.768 0.540 0.298 0.152 0.000 0.496

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Mean 1.114 0.317 0.586 0.706 0.304 0.082 0.178 0.469

3.2. Phenetic analysis of genetic afnities q q Pn Pn 2 2 The average taxonomic distance dij 1=n P k Xki Xkj , Euclidean distance Eij k Xki Xkj and n average Manhattan (city block) distance Mij 1=n k jXki Xkj j (Sneath and Sokal, 1973) were calculated for all possible pair-wise comparisons among the nine OTUs. The maximal distance based on Euclidean distance was obtained for the populations ara2 and emam equalling 3.152. The lowest Euclidean distance was observed between population pal1 and pal2 equalling 1.251. The distance matrix was clustered by UPGMA (Fig. 2). No difference was observed between the results of all three coefcients with respect to grouping on the dendrograms. Only the data for Euclidean coefcient are shown here (Table 4). 4. Discussion During the last decade, the number of Astragalus species described as new from Iran has been steadily increasing. This fact resulted in strong doubts in delimitation of the species in this genus, particularly because the morphological

Fig. 2. Dendrogram obtained from Euclidean distance and UPGMA based on isoenzyme data. Solid lines on the right side of dendrogram indicate the populations distributed on Zagros mountain range, and patterned lines indicate the populations distributed on Alborz mountain range.

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Table 4 Euclidean distances: pair-wise comparisons of A. persicus and A. gossypinus populations Populations Astragalus persicus ara1 ara1 nesa emam tuch zarr ara2 pal1 pal2 west 0.000 1.633 1.913 2.075 2.107 2.783 2.645 2.700 2.629 nesa 0.000 1.266 1.607 1.794 2.784 2.437 2.454 2.522 emam tuch zarr Astragalus gossypinus ara2 pal1 pal2 west

0.000 2.136 2.307 3.152 2.761 2.764 2.851

0.000 2.034 2.886 2.969 2.992 3.018

0.000 2.682 2.644 2.547 2.753

0.000 1.639 1.278 1.716

0.000 1.251 1.372

0.000 1.378

0.000

differences used for separation of the new species from their closest relatives are rather negligible. A. gossypinus and A. persicus are used in the present study as the core species from which either several new species have been separated (Rechinger et al., 1958; Maassoumi, 1994; Podlech and Maassoumi, 2001), or several synonyms have been considered under few polymorphic species (Chamberlain and Mathews, 1970; Zarre and Podlech, 1996). Based on the Euclidean distance, the interspecic distance between A. gossypinus and A. persicus is rather high (3.152), which was expected based on major morphological differences between these species. The intraspecic distance among different populations of both species is also relatively high and varies between 1.251 and 1.716 in A. gossypinus, while for A. persicus these values range between 1.266 and 2.307. In a pervious study conducted on Astragalus using other isoenzyme systems (Zarre et al., 2004) the intraspecic Euclidean distance was measured between 0.265 and 1.133, which is signicantly lower than the results obtained for A. gossypinus and A. persicus. Interestingly, the morphological variability in the species studied here is much higher than in Astragalus submitis. This high variation resulted in publishing several valid taxonomic names corresponding to A. gossypinus (six names) and A. persicus (14 names), compared with A. submitis (four names). Most probably, high morphological variation exhibited by some Astragalus species is accompanied with high genetic diversity based on isoenzyme patterns. Among different populations of A. persicus complex analysed here, the population zarr ts the type of A. persicus (according to Podlech and Maassoumi, 2001) and shows the highest distance (2.307 with the population emam) with the rest. However, the morphological differences between A. persicus and other putative taxa related to it are not signicant. Sparse short hairs on the calyx, and stipules longer than 12 mm in A. persicus are the only characteristic features of this species, which can in turns overlap with the features of other related putative taxa. According to the diagnostic key performed by Podlech and Maassoumi (2001) among the populations of A. persicus complex analysed here, ara1 represents A. bounophilus, emam represents A. capax, nesa represents A. naftabensis, and tuch represents A. rubriorus. As the distances between each pair of these populations and the next pair change continuously, no sharp limits can be determined between these pairs. Therefore, considering any of these populations as a separated taxon does not seem reasonable (Table 4). In the case of A. gossypinus, the differences in Euclidean distance vary from a minimum of 1.251, between two different populations collected from different elevations of Palang Darreh (pal1 and pal2), south west of Qom (Central Zagros range), and a maximum of 1.716, between the populations of west and ara2. According to the pub irjaev, 1939; Chamberlain and Mathews, 1970), pal1 and pal2 t the type of A. gossypinus var. lished references (S nervulosus, west represents A. xanthogossypinus, and ara2 represents A. gossypinus. It is likely that due to relatively low morphological polymorphism exhibited by this species, the Euclidean distances obtained based on isoenzyme banding pattern among different populations of A. gossypinus selected here are also rather low. Similar to the case of A. persicus, due to the continuous range in a pair-wise comparison of distances, division of A. gossypinus into separated taxa nds no support from this study either. A. persicus shows signicantly higher diversity (H0 ) than A. gossypinus based on seven putative isoenzymes assessed here. This conclusion is in agreement with higher inter-populational distances measured for the former species (see below). For A. persicus complex, among all the populations analysed here, the population of nesa, representing A. naftabensis, shows the highest H0 (0.936). By contrast the population emam, representing A. capax, shows the lowest H0 (0.406), indicating the high value of the former population for plant protective purposes (Baskauf and

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Snapp, 1998). In A. gossypinus complex, the populations of pal1 and pal2 (both representing A. gossypinus var. nervulosus Boiss.) show the lowest and highest H0 , respectively. Interestingly, the population pal2 was collected at about 200 m higher elevation than pal1, indicating the effect of higher altitudes on increasing the intraspecic diversity based on isoenzyme banding patterns in Astragalus. Although the number of populations analysed here is not high, the dendrogram presented in Fig. 2 clearly shows that there is a signicant distance between the populations distributed on Alborz (indicated by patterned lines) and those distributed on Zagros mountain ranges. This result indicates a clear genetic distance between geographically separated populations, even when they belong to the same species. It should be noted that based on the isoenzyme patterns obtained here, geographical factors affect the isoenzyme patterns more signicantly than ecological factors such as the elevation at which the plants grow. For this reason, two populations of A. gossypinus collected in Palang Darreh area at different elevations cluster in the same group. However, together with another population from Zagros (west), they make a separated cluster from the populations collected from Alborz (ara2). In conclusion, based on the examples of spiny Astragalus selected here, depicting a narrow specic concept in this group is not justied. A comparative analysis of isoenzymes would be useful in nding more support in delimitation of taxa at specic level in Astragalus, especially when morphological data do not provide any clear evidence in separating the related taxa. In most cases, a eld study based on morphological data on different populations of certain species of Astragalus will probably reveal signicant number of transitional forms between previously considered species, and this, together with isoenzyme variation analysis will result in synonymy of several taxonomical names. Acknowledgements We are grateful to Dr. A.A. Maassoumi (Tehran) and Prof. Dr. Podlech (Munich) for their useful suggestions and help in determination of the species and morphs. We thank H. Nouroozi and S.M. Rajaiy for their technical assistance. We are also grateful to Dr. M. Pedram (Tehran University of Medical Sciences) for his useful comments and careful editing of the manuscript. Research Council of the University of Tehran supported this study through a grant to the rst author. References
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