مطالعه سیتوتاکسونومی چهار جمعیت Astragalus anserinifolius Boiss. بخش Malacothrix Bunge از ایران

نویسندگان

گروه زیست‌شناسی، دانشکده علوم، دانشگاه بوعلی سینا، همدان، ایران

چکیده

رفتار و تعداد کروموزوم‌های میوزی چهار جمعیت ایرانی گونه Astragalus anserinifolius Boiss. از جنس گون، بخش Malacothrix مورد مطالعه قرار گرفت. تمامی این جمعیت‌های خودرو دیپلوئید بوده، عدد کروموزومی 16=x2=n2 را نشان دادند که مطابق با عدد پایه پیشنهادی 8=x از ICPN است. گرچه در تمامی تاکسون‌ها جفت شدن کروموزوم‌ها و جدا شدن آنها در مرحله میوز منظم بود، لیکن بی‌نظمی‌های میوز شامل درجات متفاوتی از کروموزوم‌های جدا افتاده و چسبندگی کروموزوم‌ها در متافاز I، چند هسته‌ای و شمار متفاوتی از کروموزوم‌های تأخیری، پیشرو و پل در آنافاز I/تلوفاز I، ناهمزمانی هستک‌ها و مهاجرت زود هنگام کروموزوم‌ها در متافاز II و تأخیر و تشکیل پل‌ها و سیتومیکسی در آنافاز II/تلوفاز II مشاهده شد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Cytotaxonomy study of four populations of Astragalus anserinifolius Boiss. of section Malacothrix Bunge from Iran

نویسندگان [English]

  • Massoud Ranjbar
  • Bahareh Mahmoudian
Department of Biology, Faculty of Sciences, Bu-Ali Sina University, Hamedan, Iran
چکیده [English]

In this research, meiotic chromosome number and the behavior of four populations of Astragalus anserinifolius Boiss. of Astragalus sect. Malacothrix were studied. All wild populations were diploid and showed 2n=2x=16 chromosome number, consistent with the proposed base number of x=8 from IPCN. Although all taxa displayed regular bivalent pairing and chromosome segregation at meiosis, some meiotic abnormalities included varied degrees of fragmented and sticky chromosomes in metaphase I, polynucleate and a variable number of laggards, forwarded chromosomes and bridges in anaphase I/telophase I, asynchronous nucleus and precocious chromosome migration in metaphase II and laggards, bridges and cytomixis in anaphase II/telophase II were observed.

کلیدواژه‌ها [English]

  • Astragalus sect. Malacothrix
  • A. anserinifolius
  • Chromosome number
  • meiotic abnormalities

Introduction

Astragalus L. (Fabaceae) is the most diverse genus in the southwest Asia (ca. 1000 spp). With more than 840 species, it is the largest genus in the flora of Iran and the most problematic group in the legume systematic (Lock and Simpson 1991; Yakovlev et al., 1996; Ranjbar and Maassoumi, 1998; Ranjbar and Karamian, 2002, 2003; Ranjbar et al., 2005, 2010a, 2010b, 2010c, 2010d, 2011). Astragalus sect. Malacothrix, with about 90 species in Iran, is one of the largest sections within the genus (Podlech et al., 2010). Bunge (1868) placed the section together with seven other sections into subgenus Hypoglottis. All members of the subgenus share similar morphological characters such as dense capitates or spike like inflorescences. In recent Podlech's typification system (1982), this subgenus is no longer upheld, because of nearly continuous transitions to other groups of the genus. Most of the cytological studies in the genus have concentrated on the chromosome count (Aryavand, 1983; Maassoumi, 1987; Bader and Sherif, 2007; Sheidai et al.,1996). The basic chromosome number (x=8) and the two ploidy levels (2n=2x =16, 2n=4x=32) are present in the genus of the old world.

 

Hence, investigations in different aspects can be useful to resolve taxonomic problems of this problematic group. This work follows previous studies conducted on leguminous fodder species in Iran (Ranjbar and Karamian, 2004; Ranjbar, 2007a, 2007b; Ranjbar et al.,2009) and aims to: increase the knowledge about patterns of morphological variation, chromosome numbers and meiotic behaviour in the four populations of A. anserinifolius belonging to A. sect. Malacothrix. Such findings can be helpful in understanding the relationships between the chromosomal criteria and taxonomic delimitations.

 

Materials and methods

Morphology

This study is mainly based on field observations during several excursions in Iran and on herbarium materials. The field studies were carried out in different parts of Iran, primarily in central and southeastern Iran (Figure 1). All vouchers have been preserved in BASU, Hamedan, Iran. Also, several sheets were examined for each taxon from the following herbaria: W, P, TARI, BASU, Herbarium of Isfahan University and Herbarium of Research Centers of Natural Resources and Animal Affairs of Yazd and Hormozgan. The specimens studied morphologically are listed in Table 1 and used as operational taxonomic units (OTUs). A total of 14 quantitative characters related to vegetative and reproductive organs were studied in the 4 populations of A. anserinifolius (Table 2). Data were entered onto a computerized spreadsheet program, Microsoft Excel version 7. The spreadsheet was later transformed into a file format suitable for phenetic analysis. Principal component analysis (PCA) was performed using MVSP version 3.2 (Kovach, 1985-2004) and used to determine inherent or natural groupings.

 

 

Figure 1. Distribution map of A. anserinifolius in Iran

 

Cytogenetics

Chromosome number and meiotic behavior were studied in 4 populations of
A. anserinifolius. For each population, 15 flower buds from at least 2 plants at an appropriate stage of development were fixed in 96% ethanol, chloroform and propionic acid (6 : 3 : 2) 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 slides were made permanent by the Venetian turpentine. Photographs of chromosomes were taken on an Olympus BX-41 photomicroscope at an initial magnification of 1000 X. Chromosome counts was made from well-spread metaphases in intact cells, by direct observation and from photomicrographs. Voucher specimens were kept at BASU, Hamedan, Iran (Table 1).

 

Table 1. Localities of the species used in this study

Species

Voucher
specimens

Locality

Altitude
(m)

Collector
name

Date

Abbreviation

A. anserinifolius

BASU 22653

Yazd: Tang-e Chenar toward Mehriz, 15 km to Mehriz

2018

Ranjbar & Mahmoudian

10.4.2010

ANS53

A. anserinifolius

BASU 22649

Kerman: Anar toward Shahr-e Babak, 30 km after Anar, 60 km before Shahr-e Babak

1982

Ranjbar & Mahmoudian

11.4.2010

ANS49

A. anserinifolius

BASU 22264

Kerman: Sarcheshmeh toward Pariz, 5 km before Pariz

2295

Ranjbar & Mahmoudian

11.4.2010

ANS64

A. anserinifolius

BASU 22528

Kerman: Baft, Azad University of Baft

2174

Ranjbar & Mahmoudian

13.4.2010

ANS28

               

 

Table 2. Morphological characters of four populations of A. anserinifolius compared with its type specimen

Morphological characters

ANS64

ANS53

ANS49

ANS28

ANS (Type)

Plant height (cm)

19

35

19

18

17.5

Leaf length (cm)

2.5

2.5

2.5

2.25

3

Petiole length (cm)

0.75

1

0.85

0.75

0.75

Leaflet number

9.5

6.5

10

11

11

Leaflet length (mm)

5.5

6.5

6

4.5

6

Leaflet width (mm)

2

2.5

2.5

1

4

Stipule length (mm)

4

4

4

4

2.5

Peduncle length (cm)

4

5.5

3.75

4.5

5

Bract length (mm)

3.5

5

3.5

2.5

4

Calyx length (mm)

13

14.5

16.5

12.5

13

Calyx teeth length (mm)

8

8

9.5

6.5

7

Standard length (mm)

12

12.5

13

11.5

15

Standard width (mm)

5

5

5.5

5

5

Keel length (mm)

9

9

10

8.5

13

Wing length (mm)

11.5

11

12.5

10

15

 

Results and discussion

Morphology

Results from morphological study showed an inter population variation within
A. anserinifolius, so that its different populations were divided into two groups. The populations ANS-type, ANS28, ANS64 and ANS49 were placed in group 1 and the population ANS53 in group 2 (Figure 2). Group 2 with a single population (ANS53) was separated from the other populations by its higher plant height and lower number of leaflets (Table 2).

 

 

Figure 2. PCA analysis of different populations of A. anserinifolius based on morphological characters (abbreviations as listed in Table 1).

 

A. anserinifolius Boiss. 1843, Diagn. pl. orient., ser. 1, 2: 76 - Malacothrix - Holotype: [Iran] ad sinum Persicum, P.M.R. Aucher-Eloy 4410(G-BOIS!; iso: BM, FI: foto MSB, G!, K!, LE, OXF, P!, W!) [1 sheet in W!: foto MSB Aucher 4410, 'erronneously ad Ispahan'!] (Figure 3).

 

Plants 11-24 cm tall, with developed stems. Stipules ca. 2-3 mm long, pilose, free from one another and the petiole. Leaves ca. 3 cm long, petiole 0.5-1 cm, both petiole and rachis covered with appressed or subpatent hairs. Leaflets 9-13 pairs, contiguous, complicate, obovate, ca. 6 mm long and 4 mm broad, both sides covered with appressed hairs. Inflorescence axillary, spherical or elliptic in flowering, elongated in fruit, flowers numerous, peduncle ca. 5 cm long, covered with short appressed hairs. Bracts linear, ca. 4 mm long, pilose. Pedicels ca. 1 mm long, pilose. Calyx campanulate or shortly tubular, at the base gibbous, ca. 10 mm long, covered with white hairs, rarely with some scattered black hairs, the teeth subulate, ca. 7 mm long. Corolla yellow or pale violet. Standard ca. 15 mm long, the limb elliptic, emarginated at apex. Wing-petals ca. 15 mm long, the limb narrowly elliptic, dilated toward the apex, round-tipped, at the base auriculate, equaling the claw. Keel ca. 10 mm long, the limb oblique-elliptic, equaling the claw. Ovary pilose, sessile. Pods ca. 9 mm long and 4 mm broad, covered with spreading long soft white hairs, bilocular.

 

Specimens seen

Kerman: Sarcheshmeh toward Pariz, 5 km before Pariz, 2295 m, 2010.4.11, Ranjbar & Mahmoudian 22264 (BASU); Sarcheshmeh toward Sirjan, After Pariz, Pasujan village, 2206 m, 2010.4.12, Ranjbar & Mahmoudian 22609 (BASU); Baft, Azad University of Baft, 2174 m, 2010.4.13, Ranjbar & Mahmoudian 22528 (BASU); Anar toward Shahr-e Babak, 30 km after Anar, 60 km before Shahr-e Babak, 1982 m, 2010.4.11, Ranjbar & Mahmoudian 22649 (BASU); 66 km to Sirjan, 35 km after Shahr-e Babak, 1816 m, Ranjbar & Mahmoudian 22601 (BASU).

 

Isfahan: 10 km SW Mourchekhort to Natanz, 1600 m, 2002.5.11, Rahiminejad, Sahebi & Ghaemmaghami 13224 (Isfahan University Herbarium); Isfahan University Campus, 4574 (Isfahan University Herbarium); Isfahan University Campus, Ahmad Zarre-dehabadi 6792 (Isfahan University Herbarium).

Yazd: Tang-e Chenar toward Mehriz, 15 km to Mehriz, 2018 m, 2010.4.10, Ranjbar & Mahmoudian 22653 (BASU); Bafgh, Bahabad, 1994.7.3, Jafarynejad & Javadian 404 (Animal & Natural Resources Research Center of Yazd).

 

Figure 3. Isotype of A. anserinifolius Boiss. (Aucher-Eloy 4410 P)

 

Cytogenetics

All the wild populations of A. anserinifolius studied here possessed 2n=2x=16 chromosome number and showed regular bivalent pairing and chromosome segregation at meiosis. They were similar in life history, breeding system, ecology, and geographical distribution in Iran (Figure 1). However, some meiotic abnormalities were observed  in different populations included the occurrence of varied degrees of sticky, fragmented and forward chromosomes in anaphase I, laggards and bridges in anaphase I to telophase II, asynchronism, precocious chromosome migration and cytomixis (Table 3; Figures 4-7).

 

Table 3. Number of pollen mother cells (PMCs) analyzed and percentage of PMCs meiotic behavior in different populations of A. anserinifolius

Meiotic characters

ANS53

ANS49

ANS64

ANS28

Number of counted cells

1573

660

1161

1424

% D/MI

17.16

21.81

15.15

18.53

% Fragmented chromosome

19.25

25.6

7.59

1.13

% Bridge

0.3

0

0

0

% Chromosome stickiness

18.14

39.58

17.61

0

% Precocious migration

10.74

7.63

4.54

0.37

% AI/TI

29.43

17.42

13.52

13.55

% Laggard chromosome

2.59

2.6

0.63

0

% Bridge

1.29

2.6

1.91

0

% Forward chromosome

0.64

1.73

0.63

0

% Micronucleus

0

0

0

0.37

% Polynucleate

0.43

0

3.18

5.69

% Asynchronous nucleus

0.64

0

0

0

% Cytomixis

0

0.86

0

0

% MII

8.77

13.78

5.25

8.14

% Asynchronous nucleus

48.55

15.38

21.31

43.1

% Fragmented chromosome

1.44

2.19

0

3.44

% Bridge

0.72

0

0

0

% Precocious migration

7.24

10.98

14.75

4.31

% AII/TII

44.62

46.96

66.06

100

% Laggard chromosome

0

0.32

1.17

0

% Forward chromosome

0

0

0

0

% Bridge

0.56

0

0.13

0

% Precocious migration

0

0

0

0

% Multipolar cell

0.14

0

0

0

% Cytomixis

0

0

0.13

0

 

Laggard, forward, sticky and fragmented chromosomes

Fragmented chromosomes, being unable to orient at the metaphase plate, were observed during metaphase I or metaphase II (Figures 4-I, J, R, 5-I, 6-H, 7-H). The highest frequencies of fragmented chromosomes of metaphase I and metaphase II cells were observed in populations ANS49 and ANS28, respectively. Laggard chromosomes were observed during anaphase I in populations ANS53, ANS49 and ANS64 (Figures 4-M; 5-K, L; 6-K); and during anaphase II in populations ANS49 and ANS64 (Figures 5-R; 6-P). According to Niclas 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 (Souza et al., 2006). These laggards might have degenerated or may have resulted in the formation of polyads particularly at the resting phase (Basi et al., 2006). Forward chromosomes were seen during anaphase I in the populations ANS53, ANS49 and ANS64, among which ANS49 showed the highest frequency (Table 3). Sticky chromosomes were observed at diakinesis and metaphase I in the populations ANS53, ANS49 and ANS64 (Figures 4-J; 5-H, I; 6-I), among which ANS49 had the highest frequency. Chromosome stickiness might have been caused by genetic and environmental factors. However, several agents have been reported to cause chromosome stickiness (Pagliarini, 2000).

 

Chromosome bridges

Chromosome bridges resulting from stickiness were observed at anaphase I and anaphase II stages in the populations ANS53, ANS49 and ANS64 (Figures 4-N, S, U; 5-M; 6-L, Q). The population ANS53 also showed chromosome bridges at metaphase II. The thickness of bridges observed and the number of the chromosomes involved in their formation varied among different meiocytes in different species.

 

 

Figure 4. A-L) Representative meiotic cells in the population ANS53. A) Diakinesis; B) Metaphase I;
C) Anaphase I; D) Telophase I; E) Metaphase II; F) Anaphase II; G) Telophase II; H) Sticky chromosomes in diakinesis; I) Fragmented chromosomes in metaphase I; J) Sticky and fragmented chromosomes in metaphase I; K-L) Precocious migration of chromosomes to the poles in metaphase I; M-U) Representative meiotic cells in the population ANS53. M) Laggards in anaphase I; N) Bridges in anaphase I; O) Trinucleate in telophase I; P-Q) Asynchronism in meiosis; R) Fragmented chromosome in metaphase II; S) Bridge in metaphase II plates; T) Precocious chromosome migrating to the poles; U) Bridge in late anaphase II. Scale bar: 3 μm.

 

Cytomixis

The phenomenon of cytomixis consists in the migration of chromosome between meiocytes through cytoplasmic connection. cytomixis, which is principally a type of meiotic abnormality resulting in changes in gametic chromosome number through migration of chromosomes between adjacent PMCs, could be considered as a process of evolutionary significance in plant populations (Ghanima and Tallat, 2003; Ghaffari, 2006). The factors responsible for cytomixis are rather ambiguous. Some possible causes attributed to cytomixis are the effect of fixation (Gottschalk, 1970), mechanical injury (Sarvella, 1958), pathological conditions (Boback and Herich, 1978), temperature anomalies (Basavaiah and Murthy, 1987), polyploid level (Verma et al., 1984), hybrid condition (Yen et al., 1993), cell response as a consequence of pesticides and antibiotic dosages (Kumar and Sinha, 1991), abnormal genetic behavior due to treatment with a chemical mutagen (Kumar and Srivastava, 2001; Kumar and Sharma, 2002), crop culture condition (Pierozzi and Benatti, 1998), failure of cell wall formation during premeiotic mitosis (Kamra, 1960), and genetically controlled behavior (De Mantu and Sharma, 1983). This phenomenon was observed in the population ANS49 at telophase I (Figure 5-O) and in the population ANS64 at telophase II (Figure 6-R, Table 3).

 

Asynchronous nucleus

Asynchrony in nucleus was observed in all populations at metaphase II (Figures 4-P, Q; 5-P; 6-M, N; 7-K), among which the population ANS53 showed the highest frequency. Asynchronism was also seen in the population ANS53 at anaphase I (Table 3).

 

Precocious migration

Precocious migration of chromosomes to the poles wasobserved in all populations at metaphase I and II stages (Figures 4-K, L, T; 5-J, Q; 6-J, O; 7-L). The highest frequencies of such chromosome migrations at metaphase I and II were observed in the populations ANS53 and ANS64, respectively (Table 3).

 

Micronucleus

As a consequence of precocious migration of univalent, non-oriented bivalents and laggard chromosomes, some micronuclei were observed in telophase I only in the population ANS 28 (Figure 7-J).

Results obtained from PCA analysis based on cytogenetic data showed an inter population variation as well as morphological characters and resulted in dividing taxa into two groups but with different members in comparison to morphology. So that the populations ANS28, ANS64, ANS53 were divided in group 1 and the population ANS49 in group 2 (Figure 8). The population ANS49 is separated from other populations by high score in the formation of fragmented and sticky chromosomes at metaphase I (25.6% and 39.58%, respectively).

Results from PCA analysis of morphological characters represented variation only between different populations of A. anserinifolius. On the other hand, the responsible reasons were not strong enough to justify variation at interspecific level leading to a new species or even at intraspecific level for separating a new subspecies or a new variety. According to some previous works on the genera Astragalus and Onobrychis of the family Fabaceae (Ranjbar et al., 2009, 2010b, 2011), the agreement between groupings resulted from morphological and meiotic analyses, occurred when the taxa demonstrate well inter/intraspecific variations in phenetic analysis. Quantitative/qualitative morphological characters responsible for such variations affected meiotic behavior of the taxa, and thus, results from meiotic analysis support morphological groups well.

 

 

Figure 5. A-L) Representative meiotic cells in the population ANS49. A) Diakinesis; B) Metaphase I;
C) Anaphase I; D) Telophase I; E) Metaphase II; F) Anaphase II; G) Telophase II; H) Sticky chromosomes in diakinesis; I) Fragmented and sticky chromosomes in metaphase I; J) Precocious chromosome migration in metaphase I; K-L) Laggard in anaphase I. M-R) Representative meiotic cells in the population ANS49. M) Bridges in Anaphase I; N) Forward chromosome in anaphase I; O) Cytomixis in telophase I; P) Asynchronous nucleus; Q) Precocious chromosome migration to the pole in metaphase II; R) Laggard in anaphase II. Scale bar: 3 μm.

 

 

 

 

 

Figure 6. A-L) Representative meiotic cells in the population ANS64. A) Diakinesis; B) Metaphase I;
C) Anaphase I; D) Telophase I; E) Metaphase II; F) Anaphase II; G) Telophase II; H) Fragmented chromosomes in metaphase I; I) Sticky chromosomes in metaphase I; J) Precocious chromosome migration  to the poles in metaphase I; K) Laggard in anaphase I; L) Bridge in anaphase I. M-R) Representative meiotic cells in the population ANS64. M-N) Asynchronous nucleus; O) Precocious migration of chromosomes to the poles in metaphase II; P) Laggards in anaphase II; Q) Bridge in anaphase II; R) Cytomixis in telophase II. Scale bar: 3 μm.

 

 

 

 

Figure 7. A-L. Representative meiotic cells in the population ANS28. A) Diakinesis; B) Metaphase I;
C) Anaphase I; D) Telophase I; E) Metaphase II; F) Anaphase II; G) Telophase II; H) Fragmented chromosome in metaphase I; I) Precocious migration of chromosomes to the pole in metaphase I; J) Trinucleate in telophase I and one micronucleus; K) Asynchronous nucleus; L) Precocious migration of chromosomes in metaphase II. Scale bar: 3 μm.

 

Figure 8. PCA analysis of different populations of A. anserinifolius based on cytogenetic data (abbreviations are as listed in Table 1).

References

Aryavand, A. (1983) IOPB chromosome number reports LXXX. Taxon 32: 504-511.

Bader, A. and Sherif, M. S. (2007) Karyotype analysis and systematic relationships in the Egyptian Astragalus L. (Fabaceae). International Journal of Botany 3: 147-159.

Basavaiah, D. and Murthy, T. C. S. (1987) Cytomixis in pollen mother cells of Urochloa panicoides P. Beauv. (Poaceae). Cytologia 52: 69-74.

Basi, S., Subedi, L. P. and Adhikari, N. R. (2006) Cytogenetic effects of gamma rays on indica rice radha-4. Institute of Agriculture and Animal Sciences 27: 25-36.

Boback, M. and Herich, R. (1978) Cytomixis as a manifestation of pathological changes after the application of trifluraline. The Nucleus 20: 22-28.

De Mantu, D. E. and Sharma, A. K. (1983). Cytomixis in pollen mother cells of an apomictic ornamental Ervatamia divericata (Linn.) Alston. Cytologia 48: 201-207.

Ghaffari, S. M. (2006) Occurrence of diploid and polyploidy microspores in Sorgum bicolor (Poaceae) is the result of cytomixis. African Journal of Biotechnology 5: 1450-1453.

Ghanima, A. M. and Talaat, A. A. (2003) Cytomixis and its possible evolutionary role in a Kuwaiti population of Diplotaxis harra (Brassicaceae). Botanical Journal of the Linnean Society 143: 169-175

Gottschalk, W. (1970) Chromosome and nuclear migration during microsporogenesis of Pisum sativum. The Nucleus 13: 1-9.

Kamra, O. P. (1960) Chromatin extrusion and cytomixis in pollen mother cells of Hordeum. Hereditas 46: 592-600.

Kovach, W. L. (1985-2004) MVSP- A multivariate statistical package for windows (version 3.1). Pentraeth Wales, UK: Kovach Computing Services.

Kumar, G. and Sharma, V. (2002) Induced cytomixis in chickpea (Cicer arietinum L.). The Nucleus 45: 24-26.

Kumar, G. and Sinha, S. S. N. (1991) Genotoxic effects of two pesticides (Roger and Bavistin) and antibiotic (Streptomycin) in meiotic cells of grasspea (Lathyrus sativus). Cytologia 56: 209-214.

Kumar, G. and Srivastava, U. (2001) Cytomictic variation in isabgol (Plantago ovata Forsk.). The Nucleus 44: 180-182.

Lock, J. M. and Simpson, K. (1991) Legumes of West Asia, a check-list. Royal Botanic Gardens, Kew.

Maassoumi, A. A. (1987) Notes on the genus Astragalus in Iran I, cytotaxonomic studies on some species. Iranian Journal of Botany 3: 117-128.

Maassoumi, A. A. (1993) Revision of Astragalus L. sect. Malacothrix Bunge (Leguminosae) in Iran. Sendtnera 1: 157-240.

Podlech, D., Zarre, Sh., Maassoumi, A. A., Ekici, M. and Sytin, A. (2010) Papilionaceae VI, Astragalus IV. Astragalus sect. Malacothrix Bunge. In: Flora Iranica (ed. Rechinger K. H.) 178: 58-146. Verlag des Naturhistorischen Museums,Wien.

Niclas, R. B. and Ward, S. C. (1994) Elements of error correction in mitosis: microtubule capture, release and tension. Cell Biology 126: 1241-1253.

Pagliarini, M. S. (1990) Meiotic behavior and pollen fertility in Aptenia cordifolia (Aizoaceae). Caryologia 43: 157-162.

Pagliarini, M. S. (2000) Meiotic behavior of economically important plant species: the relationship between fertility and male sterility. Genetics and Molecular Biology 23: 997-1002.

Pierozzi, N. I. and Benatti, J. R. (1998) Cytological analysis in the microsporogenesis of ramie, Boehmeria nivea Gaud. (Urticaceae) and the effect of colchicines on the chiasma frequency. Cytologia 63: 213-221.

Podlech, D. (1982) Neue Aspekte zur Evolution und Gliederung der Gattung Astragalus L. Mitteilungen der Botanischen Staatssammlung München. 18: 359-378.

Ranjbar, M. (2007a) Astragalus sect. Dissitiflori (Fabaceae) in Iran. Nordic Journal of Botany 24: 523-531.

Ranjbar, M. (2007b) Note on Astragalus sect. Incani (Fabaceae) in Iran. Novon 17: 390-392.

Ranjbar, M. and Karamian, R. (2002) Astragalus sect. Astragalus (Fabaceae) in Iran, complementary notes with a key to the species. Nordic Journal of Botany 22: 177-181.

Ranjbar, M. and Karamian, R. (2003) Caraganeae, a new tribe with note on the genus Chesneya Lindl. ex Endel. (Fabaceae) from Flora of Iran. Thaiszia Journal of Botany 13: 67-75.

Ranjbar, M. and Karamian, R. (2004) Taxonomic study of Astragalus sect. Erioceras (Fabaceae) in Iran with additional notes and key to the species. Nordic Journal of Botany 22: 713-717.

Ranjbar, M. and Maassoumi, A. A. (1998) New species and new records of the genus Astragalus L. (Leguminosae) from Iran. Iranian Journal of Botany 7: 235-238.

Ranjbar, M., Assadi, A. and Karamian, R. (2011) Systematic study of Astragalus chrysostachys Boiss. (Fabaceae) in Iran, with the description of a new species. Annalen des Naturhistorischen Museums in Wien B 112: 221-237.

Ranjbar, M., Karamian, R. and Enayati Akmal, A. (2010a) Meiotic behavior and chromosome number of seven populations on the Astragalus sect. Megalocystis Bunge (Fabaceae) in Iran. Cytologia 75: 49-58.

Ranjbar, M., Karamian, R. and Hadadi, A. (2009) Biosystematic study of Onobrychis vicifolia Scop. and Onobrychis altissima Grossh. (Fabaceae) in Iran. Iranian Journal of Botany 15: 85-95.

Ranjbar, M., Karamian, R. and Hadadi, A. (2010b) Cytotaxonomics of three Onobrychis species (Fabaceae) in Iran. Caryologia 63: 237-249.

Ranjbar, M., Karamian, R. and Hajmoradi, F. (2010c) Chromosome number and meiotic behavior of two populations of Onobrychis chorassanica Bunge (O. sect. Hymenobrychis) in Iran. Journal of Cell and Molecular Research 2: 49-51.

Ranjbar, M., Nouri, S. and Karamian, R. (2010d) Systematic study of simple-leaved group of Astragalus sect. Incani DC. in Iran. Journal of Taxonomy and Biosystematics 3:  33-46.

Ranjbar, M., Rahiminejad, M. R. and Assadi, M. (2005) New findings on the genus Astragalus sect. Incani DC. in Iran. Willdenowia 35: 117-124.

Sarvella, P. (1958) Cytomixis and the loss of chromosome in meiotic and somatic cells of Gosspium. Cytologia 23: 14-24.

Sheidai, M., Maassoumi, A. A. and Pakravan, M. (1996) Karyotypes of some Astragalus taxa (sect. Xiphidium BGE.) from Iran. Nucleus 39: 111-113.

Souza, M. M., Martins, E. R., Pereira, T. N. and Oliveira, L. O. (2006) Reproductive studies on Ipecac (Cephalis ipecacuanha (BROT.) A. RICH; Rubiaceae): Meiotic behavior and pollen viability. Brazilian Journal of Biology 66: 151-159.

Verma, R. C., Sarkar, A. and Das, B. C. (1984) Cytomixis in mulberry. Current Science 53: 1258-1260.

Yakovlev, G. P., Sytin, A. K. and Roskov, Yu. R. (1996) Legumes of Northern Eurasia, a check-list. Royal Botanic Gardens, Kew.

Yen, C., Yang, J. L. and Sun, G. L. (1993) Intermeiocyte connections and cytomixis in intergeneric hybrid of Roegneria ciliaris (Trin.) Nevski with Psathyrostachys huashanica Keng. Cytologia 58: 187-193.