Introduction

Astragalus L. (Gavan in Persian) is probably the largest genus of flowering plants, containing up to 3000 species (Polhill, 1981; Lock and Simpson, 1991; Yakovlev et al., 1996; Ranjbar and Karamian, 2002, 2003; Bagheri et al., 2011). Iran is one of the largest centers of diversity for this genus, with approximately 700 species and an endemic rate of 57% (Ghahreman et al., 2002; Podlech, 2001). Astragalus sect. Onobrychoidei DC. with more than 80 species is a rather large section within genus. The section was revised for the flora of the former Soviet Union (Gontscharov, 1946) and for certain areas, e.g. the flora of Iran (Rechinger et al., 1958), the flora of Turkey (Chamberlain and Matthews, 1970), and the flora of Iraq (Townsend and Guest, 1974). In the course of the work on A.sect. Onobrychoidei inIran have been investigated (Rechinger et al., 1958; Ranjbar and Maassoumi, 1998; Podlech and Sytin, 2002).

Most of the cytological studies in the genus have concentrated on the chromosome count (Aryavand, 1983; Maassoumi, 1987, 1989; Sheidai et al., 1996, 2000; Ghaffari, 2006; Akhavan and Saeidi, 2010; Faramarzi and Saeidi, 2011; Jalilian and Rahiminejad, 2011; Ranjbar and Mahmoudian, 2013). The basic chromosome number (x = 8) and four ploidy levels (2n = 2x = 16, 2n = 4x = 32, 2n = 6x = 48, 2n = 8x = 64 and 2n = 12x = 96) are present in the genus. The present study reports that there are chromosome abnormalities in A. sect. Onobrychoidei. It also shows the existence of polyploidy in A. vegetus99.

Materials and Methods

For mitosis, materials of 10 populations belonging to 5 species of Astragalus sect. Onobrychoidei were collected from different localities in Iran (Figure 1), in 2000 through 2008 and pods were separated from healthy plants. Voucher specimens were deposited at the Herbarium of the Bu-Ali Sina University (BASU), Hamedan, Iran (Table 1). Then, pods were left to dry at room temperature, and seeds obtained from dry pods and kept at 4 °C until used. Young root tips were obtained from seeds germinated in Petri dishes pretreated with 0.05% colchicines for 3 h and fixed in 3 : 1 ethanol: glacial acetic acid for 24 h. Root tips were hydrolyzed for 6 minuts in 1 M HCl at 60 °C, washed briefly in dd H₂O and stained in Feulgen's solution for 1-2 h. All permanent slides were made using Venetian turpentine (Wilson, 1945). The slides were examined under an Olympus BX-41 photomicroscope.

Also chromosome number and meiotic behavior were analyzed in three species of A. vegetus and A. brevidens and A. aduncus. 15 flower buds from at least 5 plants at an appropriate stage of development were fixed in Piennr's fluid containing ethanol (96%), chloroform and propionic acid, 6 : 3: 2 (v/v/v), for 24 h at room temperature and then stored in 70% alcohol at 4 °C until used. Anthers were squashed and stained with 2% acetocarmine. All permanent slides were made using Venetian turpentine (Wilson, 1945).

Figure 1. Distribution map of Astragalus cancellatus (1), A. aduncus (2), A. arguricus (3), A. vegetus (4), A. lilacinus (5), and A. brevidence (6)

Table 1. Taxa studied and acronyms

Results and discussion

Mitosis

1. Astragalus cancellatus Bunge, Mém. Acad. Imp. Sci. Saint Pétersbourg 11(16): 102 in clave [et l.c. 15(1): 178. 1869].

Iran. West Azerbaijan: Misho Dagh, 1830 m, Ranjbar 4933 (BASU). East Azerbaijan: Kaleybar, 1215 m, Ranjbar 15751 (BASU).

The basic chromosome number of 2n = 2x = 16 is reported for A. cancellatus. According to our literature review, this is the first chromosome count for this taxon (Figures 1-2).

2. Astragalus aduncus Willd. 1802, Sp. Pl. 3: 1269.

Iran. West Azerbaijan: Urmieh, 1300 m, Ranjbar 4953 (BASU); Oshnaviyeh, 2150 m, Ranjbar 4949 (BASU). East Azerbaijan: Kandavan, 2010 m, Ranjbar 15822 (BASU); 25 km to Marand, 1300 m, Ranjbar 5498 (BASU). Hamedan: Tuyserkan, 2000-2200 m, Ranjbar 7821 (BASU).

In this study, chromosome count for this taxon reported with 2n = 2x = 16 (Figures 1-2).

3. Astragalus arguricus Bunge, Mém. Acad. Imp. Sci. Saint Pétersbourg 11(16): 103 in clave [et l.c. 15(1): 181. 1869].

Iran. West Azerbaijan: Misho Dagh, 1830 m, Ranjbar 4947 (BASU).

Morphologically, it is a variable species, normally found in open fields and along roadsides, and flourishes in spring. Chromosome count of 2n = 2x = 16(Figures 1-2).

4. Astragalus vegetus Bunge, Mém. Acad. Imp. Sci. Saint Pétersbourg 11(16): 102 in clave [et l.c. 15(1): 181. 1869].

Iran. East Azerbaijan: Varzaqan, 1415 m, Ranjbar 15798 (BASU).

For this species, chromosome count of 2n = 2x = 16 (Figures 1-2) is the first report.

5. Astragalus lilacinus Boiss., Diagn. Pl. Orient., ser. 1, 9: 42. 1849.

Iran. East Azerbaijan: Bostan Abad to Sarab, 1660 m, Ranjbar 15451 (BASU).

For this species, chromosome count of 2n = 2x=16 is the first report (Figures 1-2).

Figure 2. Mitosis in Astragalus cancellatus (15751) species studied: a. Prophase. Mitosis in A. cancellatus (4933) species studied: b. Prophase. Mitosis in A. aduncus (5498) species studied: c. Prophase. Mitosis in
A. aduncus (15822) species studied: d and e. Prophase. Mitosis in A. aduncus (4953) species studied:
f. Prophase. Mitosis in A. aduncus (7821) species studied: g. Prophase. Mitosis in A. arguricus (4947) species studied: h. Prophase. Mitosis in A. vegetus (15798) species studied: i. Prophase

Meiotic

The cytological data for the examined taxa is summarized in Table 2 and Figures 3-4. Chromosome numbers based on the basic number of x = 8 are found in the majority of the studied taxa, a diploid number of 2n = 2x = 16 is recorded in the majority taxa, whereas a tetraploid number (2n = 4x = 32) is recorded in only one sample of A. vegetus99. Ledingham (1960) found that the Astragalus species from the old world have a basicchromosome number of x = 8, while those from the new world have x = 11, 12 and 13. This reportwas substantiated by Ledingham and Rever(1963). The chromosome number of Astragalus, based on x = 8 have been reported in the vast majority of the old world in Astragalus genus while counts based on base numbers x = 7 or x = 6 have been encountered in only a few species (Maassoumi, 1987; Badr et al., 1996; Malallah et al., 2001; Badr and Sharawy, 2007). The preponderance of Astragalus species with a basic number of x = 8 led Badr et al. (1996) to conclude that it is the primary basic number of Astragalus species. They claimed that the x = 7 and x = 6 numbers have been derived from x = 8 by aneuploid loss of chromosomes. Polyploidy in this species have been reported by Badr and Sharawy (2007). They determined the chromosome number in a karyological study on 24 species of Egyptian Astragalus and found different ploidy levels such as diploid (2n = 16, 14 and 12), triploid (2n = 24), tetraploid (2n = 32, 30 and 28), pentaploid (2n = 30), hexaploid (2n = 48) and octaploid (2n = 64) and Sheidai et al. (2009) have reported 2n = 16, 32 and 48, in some species of Iranian Astragalus, indicating the role of polyploidy in the evolution of this genus.Only Astragalus exhibits aneuploidy, and it does so consistently only in the New World (though sporadically elsewhere). Some 95% of all Eurasian Astragalus species have euploid numbers based on n = 8. Recent molecular systematic studies (Liston, 1992a, 1992b; Sanderson and Doyle, 1993; Wojciechowski et al., 1993; Wojciechowski et al., 1999), indicating the use of cytological data in studying the phylogenetic relationship of Astragalus species (Hu et al., 2008). Chromosome migration may also occur through cell wall dissolution among the neighboring meiocytes and forming syncyte (Falistocco et al.,1995). The distribution of several meiotic abnormalities observed include a various degree of fragmented chromosomes; cytomixis; asynchronous nucleus; laggard chromosome; B chromosome and unequal distribution chromosome, which is likely to result in many structural changes and rearrangements at meiosis, and which could then lead to speciation. In this study, a total of 754 prophase (55.2%), 135 diakinensis/metaphases I (D/MI) (9.89%), 199 anaphase I/telophase I (AI/TI) (14.57%), 45 metaphase II (MII) (3.29%), 232 anaphase II/telophase II (AII/TII) (16.1%) cells in A. brevidence of Ghochan (Figures 3: a-c). A total of 697 prophase (48.63%), 185 diakinensis/metaphases I (D/MI) (12.9%), 425 anaphase I/telophase I (AI/TI) (29.6%), 22 metaphase II (MII) (1.53%), 104 telophase II (TII) (7.25%) cells in A. aduncus21of Abgarm of Qazvin (Figures 3: d-h). A total of 372 prophase (33.60%), 133 diakinensis/metaphases I (D/MI) (12.01%), 208 anaphase I/telophase I (AI/TI) (18.7%), 98 metaphase II (MII) (8.85%), 296 anaphase II/telophase II (AII/MII) (26.73%) cells in A. vegetus99of Toyserkan of Hamedan(Figure 3: i-l).

Table 2. Number of pollen mother cells (PMCs) analyzed and percentage of PMCs meiotic behavior

Figure 3. Different stages of meiosis in Astragalus brevidense with 2n = 2x = 16: a. Porophase; b. Telophase; I and c. Telophase II. Different stages of meiosis in A. aduncus21 with 2n = 2x = 16: d. Diakinesis;
e. Metaphase; I and f. Anaphase; I and g. Telophase; I and h. Anaphase II. Different stages of meiosis in
A. vegetus99 with 2n = 4x = 32: i. Diakinesis; J. Metaphase II; k. Telophase II; l. Tetrad in telophase II

Figure 4. Meiotic behavior in Astragalus brevidence with 2n = 2x=16: m, n and o. Cytomixis in prophase I. Meiotic behavior in A. aduncus21 with 2n = 2x = 16: p. Cytomixis in prophase I; q. B chromosome in methaphase I; r and s. Unequal distribution chromosome in telophase I; t. Fragmented chromosome in methaphase I; u. Unequal distribution chromosome in telophase I. Meiotic behavior in A. vegetus99 with 2n = 4x = 32: v. Cytomixis in prophase I; w. laggard chromosome in anaphase I; x. Pentapolar.

The meiotic irregularities included the occurrence of various degree of binuclear in prophase; anucleus in prophase; desynapsis in diakinesis/metaphase I; fragmented chromosomes in metaphase I; sticky chromosome in anaphase I; cytomixis indiakinesis/metaphase I, anaphase I/telophase I, anaphase II/telophase II; cytoplasmic channel in prophase, diakinesis/metaphase I, anaphase I/telophase I, metaphase II, anaphase II/telophase II; B Chromosome in diakinesis/metaphase I; micronucleus in anaphase I/telophase I, anaphase II/telophase II; laggard chromosome in anaphase I/telophase I, anaphase II/telophase II; forword chromosome in anaphase I/telophase I, anaphase II/telophase II; bridge in anaphase I/telophase I, anaphase II/telophase II; tripolar and polypolar in anaphase II/telophase II; asynchronous nucleus intelophase I; some abnormalities in meiotic behavior of the taxa are described (Table 2, Figure 4: m-x). The phenomenon of cytomixis consists of the migration of chromosome between meiocytes through cytoplasmic channel. Since cytomixis creates variation in the chromosome number of the gametes, it could be considered as a mechanism of evolutionary significance (Ghaffari, 2006). Cytomixis is not considered to be of great evolutionary importance, but it may lead to production of aneuploid plants, or results in the production of unreduced gametes, as reported in several grass species (Falistocco et al.,1995). Unreduced gamete formation is of evolutionary importance as it can lead to the production of plants with higher ploidy levels. It was found in 2.38% P in A. brevidence of Ghochan, 0.03% P in A. aduncus21, 0.05% AI/TI in A. vegetus99of Toyserkan(Table 2, Figure 4). Migration of chromatin material among the adjacent meiocytes occurs through cytoplasmic channels originated from the pre-existing system of plasmodesmata formed within the tissues of the anther. It was found in 0.13% AI/TI in in A. vegetus99of Toyserkan.

According to Nicklas and Ward (1994), non-oriented bivalents may be related to impaired attachment of kinetochores to the spindle fibers. Pagliarini (1990) reported that laggards may result from late chiasma terminalization. Ascending chromosomes are the result of precocious migration and, according to Utsunomiya et al. (2002), generally consist of univalent chromosomes formed during late prophase stages by precocious chiasma terminalization in early metaphase I or may even result from low chiasma frequency or from the presence of asynaptic or desynaptic genes (Pagliarini, 2000). Laggards and non-oriented chromosomes may produce micronuclei, if they fail to reach the poles in time to be included in the main telophase nucleus (Koduru and Rao, 1981; Utsunomiya et al.,2002), leading to the formation of micro-pollen and probably to gametes with an unbalanced chromosome numbers (Mansuelli et al.,1995), such as aneuploids (Defani-Scoarize et al.,1995). The highest score for laggard chromosomes expressed in 0.5% D/MI in A. brevidence of Ghochan, 0.06% D/MI, 1.01% AI/TI in A. aduncus21 and0.41% D/MI, 0.02% AI/TI in A. vegetus99ofToyserkan.

B chromosomes or accessory chromosomes that occur in addition to the standard or A chromosomes in some of the plants are smaller than other chromosomes and do not form any association with them. B chromosomes, when present in high numbers affect negatively the growth and vigor of the plants, while in low numbers may benefit the plant possessing them (Jones and Houben, 2003). The highest percent of B chromosomes were observed in 1.71% D/MI and 0.03% AI/TI, in A. brevidence of Ghochan, 1.23 % D/MI in A. aduncus21of Abgarm, 1.06% D/MI in A. vegetus 99of Toyserkan.

Fragmented chromosomes, because of being unable to orient at the metaphase plate were observed during diakinesis and metaphase I and II. The highest percent of fragmented chromosomes were observed in 0.03% D/MI in A. aduncus21of Abgarm.

A considerable number of cells showed the unequal distribution of chromosomes that might be attributed to abnormalities in spindle formation causing unequal distribution of chromosomes. The highest percent of unequal distribution chromosome 1.2% AI/TI in A. aduncus21of Abgarm.

In conclusion, original mitotic chromosome counts were presented for 10 populations belonging to 6 species of Astragalus sect. Onobrychoidei: A. cancellatus, A. aduncus, A. arguricus, A. vegetus and A. lilacinus. All taxa were diploid and possessed 2n = 2x = 16 chromosome number, consistent with the proposed base number of x = 8. In addition, meiotic chromosome number of 2n = 2x = 16 for A. aduncus21and A. brevidens 2n = 4x = 32 for A. vegetus99. All studied taxa displayed regular bivalent pairing and chromosome segregation at meiosis. However, some abnormalities which were observed in the taxa are discussed. 3905 cells were examined in this study. Although chromosomes of the investigated species showed regular behavior during meiosis, some abnormalities were observed as laggard chromosomes in diakinesis/methaphase I; anaphase I/telophase I; B chromosomes in diakinesis/methaphase I; anaphase I/telophase I; fragmented chromosomes in diakinesis/methaphase I; unequal distribution chromosome in telophase I; multipolar cells in telophase II; channels in the cytoplasmic anaphase I/telophase I; cytomixis phenomenon in prophase I and anaphase I/telophase I.

Acknowledgements

The fieldwork in Iran was supported by grants from the Bu-Ali Sina and Shahid Beheshti Universities.