Cytomorphological Study of Trigonella Disperma (Fabaceae) in Iran

Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran

Massoud Ranjbar*, Roya Karamian and Zahra Hajmoradi

Department of Biology, Herbarium Division, Bu-Ali Sina University, P.O. Box 65175/4161, Hamedan, Iran

Received May 5, 2010; accepted April 28, 2011

Summary Chromosome number, meiotic behaviour and morphological characters related to habit and pollen grains were studied in 8 populations of Trigonella disperma of Trigonella sect. Ellipticae native to Iran. All populations are diploid and possess a chromosome number of 2n=2x=16, which is consistent with the proposed base number of x=8. This taxon displayed regular bivalent pairing and chromosome segregation at meiosis. However, some meiotic abnormalities observed included varied degrees of sticky chromosomes with laggards and bridges in metaphase I or II, asynchronous nuclei in metaphase II, desynapsis in metaphase I, and cytomixis. This paper reports the ﬁrst known study of the meiotic chromosome number and behaviour of T. disperma. We evaluated and determined the population limits within T. disperma, employing multivariate statistics. Results from meiotic behaviour supports the phenetic grouping. We found a striking association between morphological patterns of the pollen grains, meiotic behaviour and habit morphology. Our results showed that Iranian populations of T. disperma represent the same chromosome, suggesting that the pollen size in different populations can be served to obtain useful information. The environment of origin seems to have an effect on the chromosomes within different populations.

Key words Fabaceae, Meiosis, Morphology, Pollen, Trigonella disperma.

The tribe Trifolieae in the family Fabaceae consists of 6 genera: Medicago L., Melilotus Mill., Ononis L., Parcochetus Buch.-Ham. ex D. Don, Trifolium L. and Trigonella L. (Heyn 1981, Lock and Simpson 1991, Mabberley 1997). This tribe as proposed by Berchtold and Presl (1820) is characterised by having trifoliate leaves and stipules that are adnate to the petioles. In the early 20th century, Schulz (1901) used seed characters to form a tribe called Trifolieae, uniting the genera, Parochetus, Ononis and Trifolium. Hutchinson (1964) separated the genus Ononis into the monogeneric tribe Ononideae and he expanded the tribe Trifolieae to include the genera Factorovskya, Medicago, Melilotus, Parochetus, Trifolium and Trigonella. This treat- ment of the genera has been adopted by subsequent authors (Davis 1970, Townsend and Guest 1974, Meikle 1977). Small et al. (1981, 1990) separated the genus Ononis and subdivided the other 6 genera into 2 subtribes: subtribe Trifoliinae consisting only of the genus Trifolium, and subtribe Trigonellinae including the rest of the genera. Progression of modern tools in taxonomy led to a ﬂux of taxonomic opinion of these genera and tribes. Comprising of ca. 135 species, the genus Trigonella is one of the largest genera in the tribe Trifolieae and widely distributed in the dryer regions around the eastern Mediterranean, western Asia, southern Europe, northern and southern Africa, with only 1 species found in southern Australia (Townsend and Guest 1974, Polhill 1981, Kawashty 1998). Trigonella consists of perennial or annual herbs with pinnately trifoliate leaves, often exhaling an odour and are, like other grain legumes, important for food and medicine (Chopra et al. 1956,

* Corresponding author, e-mail: [email protected] 280 M. Ranjbar et al. Cytologia 76(3)

Girardon et al. 1989, Balodi and Rao 1991, Bhatti et al. 1996, Dangi et al. 2004). In Flora Iranica (Rechinger 1984) the genus is represented by 58 annual and perennial species in 12 sections. Little is known about the nature of genetic variability in diploid species of Trigonella and the taxonomic relationships between different taxa (Singh and Roy 1970). An important part of perennial members (nearly 7) of the genus endemic to Iran belong to T. sect. Ellipticae. This section is characterised by having glabrous leaves, stems and pods, and yellow or occasionally violet flowers. The taxonomy of the T. sect. Ellipticae continues to be subject of much confusion, mainly because of different approaches to species delimitation, resulting in various numbers of recognised species (Boissier 1872, Hedge 1970). Hence, investigations in different aspects such as morphology, chromosome number, meiotic behaviour and pollen morphology may be useful to solve taxonomic problems within this section. Multivariate statistical methods provide an objective analysis of patterns of morphological variation within a taxon and are a suitable tool for systematists in deciding on taxon delimitation. Some recent studies employing this method are e.g. Baum and Bailey (1992), Tyteca and Dufrene (1994), Matos (1995), Eakes and Lammers (1996), Eriksen (1997), Aldasoro et al. (1998), Sepp and Paal (1998), Shaw (1998), Verboom and Linder (1998), Brunell and Whitkus (1999), Eddie and Ingrouille (1999), Wilkin (1999), and Cupido (2003). Cytogenetic investigations were conducted on Trigonella by Singh and Roy (1970), Singh and Singh (1976), Agarwal and Gupta (1983), Ahmad et al. (1999) and Dundas et al. (2006). The mitotic chromosome number of an accession from Nisyros (Greece) of Trigonella balansae Boiss. & Reuter, an annual pasture legume of Eurasian origin, was first reported by Kamari and Papatsou (1973). Studies on the impact of karyotypic data on the interspecific and phylogenetic relationships and also on meiotic behaviour in the genus are still limited. Study on the pollen grains of legumi- nous plants (Clarke and Kupicha 1976, Ferguson and Skvarla 1981, Ferguson 1990, Ferguson and Stirton 1993, Diez and Ferguson 1994, Hughes 1997, among others), dealt mainly with the descrip- tion of the pollen grains of certain genera or tribes. This article follows previous studies conducted on fodder legumes in Iran (Ranjbar et al. 2004. 2006, 2009a, 2009b, 2010a, 2011a, 2011b, 2011c) and aims at increasing the knowledge about patterns of morphological variation, chromosome numbers, meiotic behaviour and pollen morphology in different populations of Trigonella disperma Bornm. (1910: 326) of section Ellipticae in Iran. It is an endemic and also a widespread perennial species of the genus in the western Iran. As a mem- ber of Leguminosae, it improves the nitrogen content of the soil by fixing the atmospheric nitrogen. The species is an aromatic plant; often emitting an odour consists of hydrocarbons and oxygenated sesquiterpenes as the most abundant components (Ranjbar et al. 2009b). In our previous study, ana- tomical characters related to epidermis cells of the leaves and peduncles were used to assess intraspecific relationships within T. disperma and their taxonomic significance was discussed (Ranjbar et al. 2010a). The present study establishes relationships between chromosomal criteria, pollen morphology and taxonomic delimitation. Further investigations conducted on native populations of T. disperma coming from other parts of Iran would help us to verify the emitted hypothesis for better understanding of the mechanism of evolution in these populations.

Materials and methods

Morphology Plants were collected from different regions within the natural geographical distribution of T. disperma during several excursions in Iran (Table 1). The collected materials were in the vegetative or fruiting phase and deposited at BASU, Hamedan, Iran. Several herbarium sheets have been examined for each population from the following herbaria: FUMH, PR, TARI, W, WU, Herbarium of Isfahan University, Herbarium Research Centre of Natural Resource and Animal Affairs of 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 281

Table 1. Collection data of 8 populations of T. disperma

Taxa Locality Altitude (m) Voucher specimen Abbreviation

T. disperma Kurdestan: Serishabad 1750 m 14482 (BASU) DIS 82 T. disperma Kurdestan: The ﬁrst village after Serishabad 1750 m 14483 (BASU) DIS 83 T. disperma Hamedan: Mahnian to Avaj 2062 m 14484 (BASU) DIS 84 T. disperma Kurdestan: Hamekasi 2035 m 14485 (BASU) DIS 85 T. disperma Hamedan: Avaj to Abgarm, 15 km to Abgarm 1732 m 14486 (BASU) DIS 86 T. disperma Hamedan: Kaboudrahang, 5 km after Kantapeh village 2200 m 14479 (BASU) DIS 79 T. disperma Zanjan: Abhar to Gheydar, before Kinevers village 1700 m 14480 (BASU) DIS 80 T. disperma Hamedan: 16 km before Avaj 1900 m 14481 (BASU) DIS 81

Table 2. Mean values of morphological characters and character state matrixes of 8 populations of T. disperma (abbreviations are as listed in Table 1)

DIS DIS DIS DIS DIS DIS 7 DIS DIS Morphological characters 82 83 84 85 86 9 80 81

Stem length (cm) 34 31.5 36 24.5 34.5 30 27 30 Internodes length (cm) 1.75 2.6 2 2 1.75 3.25 2.5 2.4 Leaf length (mm) 13.5 9 12 8 8 9 13 11.5 Leaflet length (mm) 6.5 8.5 7.5 6.5 7 8.5 9.5 8.5 Leaflet width (mm) 4.5 5.5 6 5 5 5.75 6 15 Petiole length (mm) 8.5 6.5 10 6.5 5.5 7 10.5 10 Stipule length (mm) 3.5 4 4 3.5 2.5 5 4.5 4 Stipule width (mm) 0.65 0.75 0.75 0.75 1.25 0.75 0.75 0.75 Peduncle length (cm) 3.25 4 2.1 3.25 1.5 2.9 2.75 2.85 Calyx tube length (mm) 3 3.5 3 3 2.5 3 3 3 Calyx tooth length (mm) 1 1.5 2 1 1.5 1 1 2 Calyx length (mm) 4 5 5 4 4 4 4 5 Standard length (mm) 7 9 10 9 8 10 8 10 Standard width (mm) 6 7 7 8 6 9 6 7 Wing length (mm) 8 9 10 9 8 10 8 9 Wing width (mm) 2.25 3 3 2.5 2 2.75 2 2.5 Wing claw length (mm) 3 3 3 3 3 2.5 3 3 Keel length (mm) 7 8 9 8 7 9 8 9 Keel width (mm) 3 3 3 3 3 3.5 3 3 Number of flowers 10 9 10 9 9 8 6 8 Pedicel length (mm) 2.5 3 4 2.5 3.5 3 2.5 2.5 Ovary stipa 1 1.5 1 0.75 1 1.25 1 1 Hair density on leaflet 1 1 1 1 1 0 1 1 (loose=1, sparse=0) Hair density on calyx 0 1 0 1 1 0 1 1 (sparse-loose=1, sparse-glabrous=0) Hair density on stipule 0 1 1 1 1 0 1 1 (loose=1, sparse=0) Hair density on bract 2 2 0 1 1 2 2 2 (loose=1, glabrous=0, sparse-glabre=0) Standard colour 1 1 1 0 1 2 0 1 (yellow=0, mild yellow=1, dark yellow=2) Standard shape 0 0 0 0 0 1 0 2 (orbit fan-shaped=0, obovatum=1, emarginate=2)

Tabriz, Mashhad, Isfahan, Shiraz, Kerman and Zahedan. In total, 28 quantitative/qualitative morphological characters related to vegetative and reproductive organs were investigated. A list of morphological characters studied is presented in Table 2. 282 M. Ranjbar et al. Cytologia 76(3)

Cytogenetics Chromosome number and meiotic behaviour were analysed in different populations of T. disperma. Fifteen flower buds from at least 5 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% ethanol at 4°C until used. Anthers were squashed and stained with 2% acetocarmine. All slides were made permanent by Venetian turpentine. Photographs of chromosomes were taken with an Olympus BX-41 photomicroscope at initial magnification of ×1000. Chromosome counts were made from well-spread metaphases in intact cells, by direct observation and from photo- micrographs. Voucher specimens are kept at BASU, Hamedan, Iran (Table 1). All data obtained from morphology and cytogenetics were analysed by MVSP software and the relationships between different populations were discussed.

Pollen morphology Pollen samples were obtained from herbarium specimens and prepared using the standard method described by Erdtman (1960). They were then mounted on unstained glycerine jelly and observations were made with a Nikon Type-2 microscope. The measurements were based on 25 readings from each specimen. Polar axis (P) and equatorial diameter (E), colpus length (L), colpus width (G in granule site and NG in none granule site), and the shape index (P/E), were measured. Data was analysed by MVSP version 3.1 and SPSS software and the relationship between different populations was discussed. The terminology used here follows Faegri (1956).

Phenetics Eight populations of T. disperma in Iran were used as operational taxonomic units (OTUs). A numerical taxonomic analysis of the different individuals from these populations was carried out based on 28 quantitative/qualitative characters related to vegetative and reproductive organs. Data was entered into a Microsoft Excel version 7 spreadsheet. This spreadsheet was later converted into a ﬁle format suitable for phenetic analysis by MVSP software version 3.2 (Kovach 1985–2002). Principal coordinate analysis (PCO) was carried out using MVSP, with a matrix of standardised data. The data was standardised to eliminate distorting effects in the output results caused by different measurement scales. Standardisation was performed by subtracting the character mean and dividing by the standard deviation. For PCO, an average-distance-matrix of standardised data was obtained. The average distance was used because the data set contained both metric and binary (mixed) data. The distance matrix was double centred and the eigenvectors were calculated and plotted. The PCO gives the distances between OTUs rather than the correlation between the characters. This method is therefore suitable for mixed character data, as it will not be distorted by binary characters. This added the advantage of being able to handle missing data well.

Results and discussion

Morphology Morphological characters show intraspecific variation within T. disperma. Results from PCO analysis on the matrix of correlations are presented in Fig. 1. It is possible to distinguish 4 main groups when plotted on the first 2 eigenvectors. Group 1 with DIS 85, DIS 80; Group 2 with DIS 81, DIS 82, DIS 83, DIS 84; Group 3 with DIS 79; and Group 4 with DIS 86. DIS 79 is placed sep- arately for the shape and colour of standard and DIS 86 for the small size of corolla, peduncle and leaflets (Table 2). A distribution map showing the location of different populations and the shape and colour of the standard petal are presented in Fig. 2. 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 283

Fig. 1. Relationships between 8 populations of T. disperma illustrated by the ﬁrst and second egenvectors of PCO analysis based on morphological characters (abbreviations are as listed in Table 1).

Fig. 2. Distribution maps of 8 populations of T. disperma in western Iran and different shape and colour of their standards. 284 M. Ranjbar et al. Cytologia 76(3)

Table 3. Number of pollen mother cells (PMCs) analyzed and percentage of PMCs meiotic behaviour in 8 populations of T. disperma

Meiotic characters DIS 82 DIS 83 DIS 84 DIS 85 DIS 86 DIS 79 DIS 80 DIS 81

Cell number 1110 1266 1260 714 810 535 560 330 D/MI 219 396 415 224 236 206 98 80 % D/MI 19.7 31.27 32.93 31.28 29.13 38.5 17.5 24.24 % Cytomixis 5.47 0 1.92 0.44 0.84 8.25 0 11.25 % Cytoplasmic channel 0 1.76 5.3 0.86 3.81 18.93 0 7.5 % Univalent 0 0 0 0.44 0 0 0 0 % B-Chromosome 0 0 0.24 1.33 0 0.97 0 0 % Fragmented chromosome 6.84 6.81 3.85 7.14 5.08 10.67 18.36 17.5 AI/TI 330 372 277 106 210 89 136 47 % AI/TI 29.7 29.38 21.98 14.8 25.92 16.63 24.28 14.24 % Laggard chromosome 0.6 1.61 1.44 0 0.47 3.37 0 4.25 % Bridge 0.9 2.15 3.24 0 0.95 4.49 0.73 0 % Micronucleus 0 0 0 1.88 3.33 0 0 0 % Asynchronous nucleus 0 0.53 0 0 0 1.12 0 0 % Cytoplasmic channel 0 0 2.88 0 2.85 8.98 2.94 2.12 % Precocious segregation 0 0 0 0 0 1.12 0 0 % Cytomixis 0 0 2.52 0 0 0 2.2 0 MII 101 154 177 80 118 37 196 61 % MII 9.09 12.16 14.04 11.17 14.56 6.91 35 18.48 % Fragmented chromosome 1.98 0.64 0 5 3.38 5.4 1.02 3.27 % Cytoplasmic channel 0.99 0.64 0 0 3.38 0 2.04 0 % Asynchronous nucleus 14.85 18.18 16.38 6.25 28.81 24.32 25.51 14.75 % Laggard chromosome 0 0.64 0 2.5 0 0 0 0 % Cytomixis 0 0 0 1.25 1.96 8.1 2.04 0 % Micronucleus 0 0 0 0 0 0 0 0 AII/TII 460 344 391 306 246 203 130 142 % AII/TII 41.44 27.17 31.03 42.73 30.37 37.94 23.21 43.03 % Cytoplasmic channel 0.21 0 0 0 0 0.49 1.53 0 % Hexapolar cell 0.21 0 0 0 0 0 0 0 % Asynchronous nucleus 0.21 0 0 0 0 0 0.76 0 % Pentapolar cell 0 0 0 0.32 0 0.49 0 0 % Micronucleus 0 0.58 0.25 1.96 0.4 0 2.3 0 % Cytomixis 0 0.29 3.83 0 0 7.38 0.76 0 % Bridge 0 0 1.79 0 0.4 0.49 0 1.4 % Laggard chromosome 0 0 1.02 0 0 0 0 0 % Tripolar cell 0 0 0 0 13 0 0 0 % Ring 1.8 5.7 4.7 5.6 2.9 6.3 5.4 3.5 % Rod 6.2 2.2 3.2 2.3 5.1 1.3 2.5 2.3 % Nucleolar bivalent 2.3 2.8 1.7 2.7 1.2 3.8 2.8 2.6

Abbrevaitions: D/MI=Diakinesis/Metaphase I; AI/TI=Anaphase I/Telophase I; MII=Metaphase II; AII/TII=Anaphase II/Telophase II.

Cytogenetics Data with regard to meiotic chromosome number, meiotic stages, as well as abnormalities observed in each stage are presented in Table 3. A total of 1874 diakinesis/metaphases I (D/MI), 1567 anaphase I/telophase I (AI/TI), 924 metaphase II (MII), and 2222 anaphase II/telophase II (AII/MII) cells were analysed. The meiotic irregularities observed in the different studied populations of T. disperma included chromosomes stickiness, B-chromosomes, precocious division of centromeres, chromosome bridges resulting from stickiness, the occurrence of laggard chromosomes, formation of micronuclei in tetrad cells, formation of multipolar and tripolar cells, and cytomixis, which are discussed bellow. 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 285

Figs. 3–14. M eiosis in DIS 82 population. 3=Diplotene with nucleolar bivalent. 4=Diakinesis with 8 bivalents (arrows show ring bivalents). 5=Metaphase I with fragmented chromosomes. 6=Bridge. 7=Laggard chromosomes. 8=Hexapolar cell with micronocleus. Scale bar=3 μm. Figs. 9–11. Meiosis in DIS 83 population. 9=Diakinesis with 8 bivalents (7 ring and 1 rod bivalents). 10=Laggard chromosomes. 11=Micronucleus. Scale bar=3 μm. Figs. 12–14. Meiosis in DIS 84 population. 12=Fragmented chromosomes. 13=Cytomixis. 14=Bridge. Scale bar=3 μm.

Laggards, fragmented and sticky chromosomes Fragmented chromosomes, for being unable to orient at the metaphase plate, were observed during metaphase I or metaphase II (Figs. 5, 12). The highest frequency of fragmented chromosomes of metaphase I cells was observed in DIS 80 (Table 3). 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 terminalisation (Souza et al. 2006). These laggards might have degenerated or may have resulted in the formation of poly- ads, particularly at the resting phase (Basi et al. 2006). Most of the populations that were analysed in this study showed laggards in anaphase I (Figs. 10 and 22) and the DIS 84 population was the only species that forms laggards in anaphase II.

B-Chromosomes 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 286 M. Ranjbar et al. Cytologia 76(3)

Figs. 15–23. Meiosis in DIS 85 population. 15=B-chromosome. 16=Univalent. 17=Precocious segregation. Scale bar=3 μm. Figs. 18–21. Meiosis in DIS 86 population. 18=Micronuclei. 19=Asynchronous nuclei. 20=Tripolar cells. Scale bar=3 μm. Figs. 21–23. Meiosis in DIS 79 population. 21=Precocious segregation. 22=Laggard chromosomes. 23=Bridge. Scale bar=3 μm. association with them. B-Chromosomes when present in high numbers negatively affect the growth and vigour of the plants, while in low numbers they may be beneﬁcial to the plant (Jones and Houben 2003). B-Chromosomes were observed only in 3 populations with the highest percentage in DIS 85 population (Fig. 15).

Univalent Desynapsis, which occurred in the bivalent, can produce univalents. During metaphase I the number of cells with univalents representing precocious migration to the poles in the DIS 85 population was high (Fig. 16), while the other populations did not show this abnormality. Because univalent usually do not suffer regular segregation in the ﬁrst division, the frequency of univalents in diakinesis/metaphase I has been used as a standard measure of meiotic disturbances in other species (Scoles and Kaltsikes 1974). In general, univalents migrate precociously to the poles or behave as laggards in anaphase, but in both cases they can produce micronuclei in telophase I, which normally remain until the tetrad stage (Koduru and Rao 1981).

Cytomixis The phenomenon of cytomixis consists of the migration of chromosome between meiocytes through cytoplasmic connection. Since cytomixis creates variation in the chromosome number of the gametes, it could be considered as a mechanism of evolution (Ghaffari 2006). This phenomenon occurred in all populations at different stages and the DIS 81 population showed the highest percentage in D/M I stage (Table 3).

Chromosome bridges Chromosome bridges resulting from stickiness were observed in all populations at anaphase I cells, except the DIS 81 and DIS 85 populations, while only 4 populations show this abnormality in 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 287 anaphase II (Table 3). The number of chromosomes involved in their formation varied among different meiocytes. Genetic as well as environmental factors have been considered as the reason for chromosome stickiness in different plant species (Nirmala and Rao 1996).

Micronucleus Chromosomes that produced micronuclei during meiosis were eliminated from microspores as microcytes. The micronucleus reached the microspore wall and formed a kind of bud, separated from the microspore. The eliminated microcytes gave origin to small and sterile pollen grains (Baptista-Giacomelli et al. 2000). Micronuclei were seen in some populations (Table 3), with the highest percentage in the DIS 86 population (Fig. 18).

Tripolar cells Failure of chromosome movement occurred in one of the poles of anaphase cells, leading to the formation of tripolar cells. Such cells normally produce reduced and unreduced daughter cells. This phenomenon was found only in the DIS 86 population (Fig. 20). Such unreduced meiocytes may lead to the formation of 2n pollen grains (Sheidai et al. 2007).

Multipolar cells The spindle apparatus is normally bipolar and acts as a single unit, playing a crucial role in chromosome alignment during the metaphase. Any distortion or breakage in the spindle may result in random sub-grouping of the chromosome (Nirmala and Rao 1996). Hexapolar cells were observed in the DIS 82 population (Fig. 8), while pentapolar cells were only found in the DIS 85 and DIS 79 populations (Table 3). Such cells may lead to the formation of abnormal tetrads and infertile pollen grains.

Ring and rod chromosomes Bivalents are found in 2 forms: open ring chromosomes and closed rod chromosomes. Ring bivalents usually are created by metacentric and sub-metacentric chromosomes. In this bivalent, chiasma exists in each arm of homologous chromosomes (Fig. 4), but in rod bivalents, chiasma is formed in acrocentric or telocentric chromosomes. In this case, chiasma exists in the long arm of homologous chromosomes (Sheidai 2002). The average numbers of ring and rod chromosomes are given in Table 3.

Nucleolar bivalent The mean value of nucleolar bivalents for every population is shown in Table 3 and illustrated in Fig. 3. All data obtained from meiotic behaviour analysed by MVSP version 3.1. They showed an intraspeciﬁc variation similar to the variation resulted from the analyses of morphology and pollen morphology. Group 1 included the DIS 80, DIS 82, DIS 83 and DIS 85 populations and group 2 included the DIS 81 and DIS 84 populations. The DIS 79 population individually formed group 3. The reason for separating the DIS 79 population is that it has a high degree of meiotic abnormality. The DIS 86 population also has a high abnormality especially in the formation of micronucleus (3.33%). It is also the only population that shows tripolar cells (13%). Results from PCO analysis on the matrix of correlations are given in Fig. 24.

Pollen morphology Pollen grains in this species are small (rarely medium) sized ranging from: P=24 (25.2) 27; E=15 (19.5) 21 μm to P=28 (28.9) 30; E=21 (22.2) 23 μm. The smallest are the pollen grains of DIS 84, while the largest ones belong to DIS 81 (Table 4). They are prolate-spheroidal, subprolate or prolate, occasionally perprolate, colporate or colpate. The colpi are long-extending to the poles 288 M. Ranjbar et al. Cytologia 76(3)

Fig. 24. Relationships between 8 populations of T. disperma illustrated by the ﬁrst and second eigenvectors of PCO analysis based on meiotic variation (abbreviations are as listed in Table 1).

Table 4. Pollen characters of 8 populations of T. disperma

Pollen characters Population E P G NG l P/E

T. disperma82 15(19.5)21 25(27.2)30 13(15.2)17 8(9)10 20(20.4)24 1.39 T. disperma83 18(20.5)24 26(26.7)30 14(15.4)19 9(9.9)14 20(21.0)25 1.30 T. disperma84 16(18.3)18 24(25.2)27 11(12.7)15 8(8.6)10 17(19.9)22 1.37 T. disperma85 20(22.7)25 25(27.7)29 16(18.7)21 10(11.3)13 20(21.0)23 1.21 T. disperma86 21(22.2)23 24(25.5)27 18(19.1)20 12(13.3)14 20(20.7)23 1.14 T. disperma79 17(19.5)21 24(26.3)28 12(15.2)19 8.0(9.5)11 17(20.5)24 1.34 T. disperma80 17(19.5)22 24(26.7)29 9(14.8)17 7.0(9.9)14 19(21.4)24 1.36 T. disperma81 17(19.3)24 28(28.9)30 6(9.6)15 5.0(8.5)13 20(23.2)25 1.50

Abbrevaitions: E=Equatorial diameter; P=Polar axis; L=Colpus length; G=Colpus width in granule site; NG=Colpus width in none granule site; P/E=Shape index. with tapering ends, with coarsely granulated membranes and either smooth or ornamented margins. The apocolpi are usually ornamented, rarely smooth, the endoaperture is well-developed (if pres- ents), oval in shape and protruding at the equator. The exine is reticulate, foveolate granulate or rarely tectate, beset with ﬁne granules (Figs. 25–39). The mean values and ranges of 5 quantitative characters are given in Table 4. Three characters of polar axis length (P), equatorial diameter (E) and polar axis length/equatorial diameter ratio (P/E) were occasionally useful in separating closely related taxa. However, analysis of pollen morphological data (Fig. 40) indicates that the ranges of most characters overlap among populations of T. disperma and separation of the populations ap- pears rather weak as they show overlapping ranges. Except for a slight overlap, the range of colpus width in granule site (Fig. 40D) shows a separation in DIS 81 population. Also, the equatorial axis length (Fig. 40A) and colpus width in non granule site (Fig. 40C) separate DIS 85 and DIS 86 populations from other populations. PCO analysis using average distance showed phenetic relationships between different populations of T. disperma (Fig. 41). In this analysis, 3 main groups were distinguished. Group 1 included 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 289

Figs. 25–39. Light micrographs from polar and equatorial views of the pollen grains in 8 populations of T. disperma (abbreviations are as listed in Table 1). 25–26=DIS 82 population. 27–28=DIS 83 population. 29=DIS 84 population. 30–31=DIS 85 population. 32–33=DIS 86 population. 34–35=DIS 79 population. 36–37=DIS 80 population. 38–39=DIS 81 population. Scale bar=6 μm.

DIS 85 and DIS 86 populations; group 2 included DIS 79, DIS 80, DIS 82 and DIS 83 populations. The ﬁrst group placed relatively far from the other populations, because of its spheroid pollen grains. DIS 81 population along with DIS 84 populations formed a single group because of their small colpi width in the granule sites (G). DIS 84 population was separated from others, because it has the smallest pollen grains. The mean values of the pollen characters and their ranges are presented in Fig. 40.

Discussion

The meiotic data showed an intraspecific variation like morphological and pollen morphological characters. We determined chromosome numbers of 8 populations of T. disperma in Iran as 2n=2x=16 for the first time. Comparison of our results with previous studies on chromosome number in Trigonella, do not show any variation and confirm that 2n=16 is the most common chromosome number in this genus (Tutin and Heywood 1964). However, there are not enough 290 M. Ranjbar et al. Cytologia 76(3)

Fig. 40. Box and whisker plots depicting the pollen characters in 8 populations of T. disperma. A=Equatorial diameter. B=Polar axis. C=Colpus width in none granule site. D=Colpus width in granule site. E=Colpus length. F=Shape index (P/E) (abbreviations are as listed in Table 1).

Fig. 41. Relationships between 8 populations of T. disperma illustrated by the first and second eigenvectors of PCO analysis based on pollen characters (abbreviations are as listed in Table 1). 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 291 literature data on chromosome number of Trigonella species. Previous reports show that all investigated Trigonella species are diploid with 2n=14 and 16 (Biddak and Amin 1996, Das et al. 2000) chromosome numbers. Aykut et al. (2009) showed that the basic haploid chromosome numbers of Trigonella may be n=7, 8 and 9. Differences in chromosome numbers and their total lengths might be result of addition or deletion of DNA (Narayan and Durrant 1983). Hence, comprehensive study involving different sections and species of Trigonella in Iran is necessary for understanding Trigonella taxonomy effectively. All populations of T. disperma were diploid and polyploidy was not observed in this species. Polyplooidy is probably an infrequent phenomenon in perennial Trigonella species based on cytological evidences. Polyploids of the genus are encountered mainly in north-west Asia, especially in Anatolia, Iran, Turkey and Turkmenistan, occuring in annual Trigonella, which are probably associated with chromosome duplication, intrachromosomal variability and reduction of mean chromosome length. However, in perennial species of Trigonella, cytomixis creates variation in the chromosome number of the gametes. It could be considered as a mechanism of evolutionary significance (Ranjbar et al. 2007, 2010b). The presence of tetraploidy and hexaploidy at the same time has been reported for T. monantha subsp. noeana and monantha belonging to T. sect. Bucerates has annual species (Martin et al. 2008). B-Chromosomes were observed in 3 populations of T. disperma with the highest percentage in the DIS 85 population. This finding was earlier reported only in T. sect. Bucerates (Martin et al. 2008). In this study, different meiotic abnormalities were observed in varied degrees in different populations. Cytomixis occurred in all populations at different stages. Chromosome bridges resulting from stickiness were observed in all populations at anaphase I cells except for the DIS 81 and DIS 85 populations. Most of the populations analysed here showed laggards in anaphase I, but the DIS 84 population was the only species that showed laggards in anaphase II. Micronucleus was seen in some populations, with the highest percent in the DIS 86 population. Tripolar cells were found only in the DIS 86 population. Such unreduced meiocytes may lead to the formation of 2n pollen grains (Sheidai et al. 2007). Hexapolar cells were observed in the DIS 82 population, while pentapolar cells were found in the DIS 85 and DIS 79 populations. Such cells may lead to the formation of abnormal tetrads and infertile pollen grains. All the above meiotic abnormalities observed in T. disperma are reported for the first time for the genus Trigonella. The factors endangering living creatures, such as destruction of natural habitats, field clear- ances, tourism, dams and irrigation systems, may affect the genus Trigonella as well. Thus it is an obligation to protect biological treasures especially endangered endemic species as ex-situ or in-situ. The Trigonella species are generally localized in the Mediterranean regions, western Anatolia and southwest Asia, especially in Iran, but are also partially seen in western Europe and eastern Anatolia.

Acknowledgments

This research has received ﬁnancial support from the Bu-Ali Sina University. The great help of Dr. E. Vitek, Dr. B. Wallnofer and Dr. W. Till during our visit to W, WU in Vienna is much ap- preciated. We thank the Director of the Herbarium of Ferdowsi University of Mashhad (FUMH), and the Herbarium Research Centres of Natural Resources and Animal Affairs of Isfahan, Kashan, Kerman, Mashhad, Semnan, Shiraz, Tabriz and Zahedan for making the herbarium facilities avail- able for our study.

References

Agarwal, K. and Gupta, P. K. 1983. Cytological studies in the genus Trigonella Linn. Cytologia 48: 771–779. 292 M. Ranjbar et al. Cytologia 76(3)

Ahmad, F., Acharya, S. N., Mir, Z. and Mir. P. S. 1999. Localization and activity of rRNA genes on fenugreek (Trigonella foenum-graecum L.) chromosomes by fluorescent in situ hybridization and silver staining. Theor. Appl. Genet. 98: 179–185. Aldasoro, J. J., Aedo, C., Navarro C. and Garmendia F. M. 1998. The genus Sorbus (Maloideae, Rosaceae) in Europe and in North Africa: Morphological analysis and sys tematics. Syst. Bot. 23: 189–212. Aykut, Y., Martin, E., Unal, F. and Akan, H. 2009. Karyological study on six Trigonella L. species (Leguminosae) in Turkey. Caryologia 6(2): 89–94. Balodi, B. and Rao, R. R. 1991. The genus Trigonella L. (Fabaceae) in the Northwest Himalaya. J. Econ. Tax. Bot. 5: 11–16. Baptista-Giacomelli, F. R., Pagliarini, M. S., Almeida, J. L. 2000. Elimination of micronuclei from microspores in a Brazilian oat (Avena sativa L.) variety. Genet. Mol. Biol. 23(3): 681–684. Basi, S., Subedi, L. P. and Adhikari, N. R. 2006. Cytogenetic effects of gamma rays on indica rice radha-4. J. Inst. Agri. Anim. Sci. 27: 25–36. Baum, B. R. and Bailey, L. G. 1992. Morphometric study of three closely related South American species of Hordeum section Stenostachys (Poaceae). Can. J. Bot. 70: 496–502. Berchtold, F. G. and Presl, J. S. 1820. Tribe Trifolieae. O. Prirozenosti Rostlin, Prague. Bhatti, M., Khan M., Ahmad, B., Jamshaid, M. and Ahmad, W. 1996. Antibacterial activity of Trigonella foenum-graecum seeds. Fritoletapia 67: 372–374. Biddak, L. and Amin, A. W. 1996. Inter- and intraspecific chromosomal variations in four species of Trigonella L. Journal of Union of Arab Biologists (Cairo) 3: 203–215. Boissier, E. 1872. Trigonella. Flora Orientalis 2. Georg, H. Basel, pp. 526–553. Bornmüller, J. 1910. Collectiones Straussianae novae, weitere Beiträge zur Kenntnis der Flora West-Persiens. Beih. Bot. Centralbl. 27(2): 288–347. Brunell, M. S. and Whitkus, R. 1999. Assessment of morphological variation in Eriastrum densifolium (Polemoniaceae): implications for subspecies delimitation and conservation. System. Bot. 23: 351–368. Chopra, R. N., Nayyarand, S. L. and Chorpa, I. E. 1956. Glossary of Indian Medicinal Plants. 1st ed., CSIR, New Delhi, p. 248. Clarke, G. C. S. and Kupicha, F. L. S. 1976. The relationships of the genus Cicer L. (Leguminosae): the evidence from pollen morphology. Bot. J. Linn. Soc. 72: 35–44. Cupido, C. N. 2003. Systematic studies in the genus Merciera (Campanulaceae): A re-assessment of species bound- aries. Adansonia 25: 33–44. Dangi, R. S., Lagu, M. D., Choudhary, L. B., Ranjekar, P. K. and Gupta, V. S. 2004. Assessment of genetic diversity in Trigonella foenum-graecum and Trigonella caerulea using ISSR and RAPD markers. BMC Plant Biol. 4: 13–23. Das, A. B., Mohanty, S., Thangaraj, T. and Das P. 2000. Variation of 4C DNA Content and Karyotype in Nine Cultivars of Fenugreek (Trigonella foenum-graecum L.). J Herbs Spices Med Plants 7(1): 25–32. Davis, P. H. 1970. Flora of Turkey. Edinburgh University Press, London. Diez, M. I. and Ferguson, L. K. 1994. The pollen morphology of the tribes Loteae and Coronilleae, Lotus L. and related genera (Papilionoideae: Leguminosae). Rev. Palaeobot. Palynol. 81: 233–255. Dundas, I. S, Nair, R. M. and Verlin, D. C. 2006. First report of meiotic chromosome number and karyotype analysis of an accession of Trigonella balansae (Leguminosae). New Zeal. J. Agri. Res. 49: 55–58. Eakes, M. J. and Lammers, T. G. 1996. Phenetic analy sis of the Lobelia laxiflora complex (Campanulaceae: Lobelioideae) reveals a previously undescribed species in Mexico (abstract). Am. J. Bot. 83: 154. Eddie, W. M. M. and Ingrouille, M. J. 1999. Polymorphism in the Aegean “five-loculed” species of the genus Campanula, section Quinqueloculares (Campanulaceae). Nord. J. Bot. 19: 153–169. Eriksen, B. 1997. Morphometric analysis of Alaskan members of the genus Potentilla sect. Niveae (Rosaceae). Nord. J. Bot. 17: 621–630. Erdtman, G. 1960. The acetolysis method. Svensk Bot. Tidskr. 54: 561–564. Faegri, K. 1956. Recent trends in palynology. Bot. Rev. 22: 639–664. Ferguson, I. K. 1990. The significance of some pollen morphology characters of the tribe Amorpheae and the genus Mucuna (tribe Phaseoleae). Rev. Palaeobot. Palynol. 64: 129–136. ̶ and Skvarla, J. I. 1981. The pollen morphology of the subfamily Papilionoidea e (Leguminosae). In: Polhill, R. M. and Raven, P. H. (Eds.). Advances in legume systematics. Royal Botanic Gardens, Kew. pp. 859–896. ̶ and Stirton, C. H. 1993. Pollen morphology of the genera Leguminosae: (Papilionoideae: Sophoreae). Panurea and Bowdichia. Grana 2: 44–48. Ghaffari, S. M. 2006. Occurrence of diploid and polyploidy microspores in Sorgum bicolor (Poaceae) is the result of cytomixis. Afr. J. Biotechnol. 5(16): 1450–1453. Girardon, P., Sauvaire, Y., Baccou, U. C. and Bessiere, J. M. 1989. Identification de la 3-hydroxy-4,5-dimethyl-2 (SH)- furanone dans L’arome des grains de fenugrec (Trigonella foenum-graecum L.). Lebensm. Wiss. Technol. 19: 2011 Cytomorphological Study of Trigonella disperma (Fabaceae) in Iran 293

44–46. Hedge, I. C. 1970: Trigonella. In: Davis, P. H. (Ed.), Flora of Turkey and the Aegean Islands 3. Edinburgh University Press, London. pp. 452–482. Heyn, C. C. 1981. Trifolieae. In: Polhill, R. M. and Raven, P. H. (Eds.), Advances in legume systematics; part 1. Royal Botanic Gardens, Kew. pp. 383–385. Hughes, C. E. 1997. Variation in anther and pollen morphology in Leucaena Benth. (Leguminosae: Mimosoideae). Bot. J. Linn. Soc. 123: 177–196. Hutchinson, J. 1964. The Genera of Flowering Plants, vol. 1. Clarendon Press, Oxford. Jones, N. and Houben, A. 2003. B chromosomes in plants: Escapees from the A chromosome genome? Trends Plant Sci. 8: 417–423. Kamari, G. and Papatsou, S. 1973. Chromosome studies in some Mediterranean angiosperms. Nord. J. Bot. 126: 266–268. Kawashty, S. A., Abdalla, M. F., Gamal El Din, N. E. M. and Saleh, N. A. M. 1998. The chemosystematics of Egyptian Trigonella species. Biochem. Syst. Ecol. 26: 851–856. Koduru, P. R. K. and Rao, M. K. 1981. Cytogenetics of synaptic mutants in higher plants. Theor. Appl. Genet. 59: 197–214. Kovach, W. 1985–2002. Institute of Earth Studies, University College of Wales, ABERYSTWYTH, (Shareware), MVSP Version 3.2, 1985–2002 Kovach Computing Services, http://www.kovcomp.com/MVPs/downl2.html Lock, J. M. and Simpson, K. 1991. Legumes of west Asia. Royal Botanic Gardens, Kew. Mabberley, D. J. 1997. The plant book, a portable dictionary of the vascular plants. 2nd ed. University Press, Cambridge. Martin, E., Akan, H., Ekici, M. and Aytaç, Z. 2008. Karyomorphological studies on section Bucerates Boiss. of Trigonella L. (Leguminosae) from Turkey. Caryologia 61(3): 225–236. Matos, J. A. 1995. Pinus hartwegii and P. rudis: a critical assessment. Syst. Bot. 20: 6–21. Meikle, R. D. 1977. Flora of Cyprus. Royal Botanical Gardens, Kew. Narayan, R. K. J. and Durrant, A. 1983. DNA distribution in chromosomes of Lathyrus species. Genetica 61: 47–53. Nicklas, R. B. and Ward, S. C. 1994. Elements of error correction in mitosis: microtubule capture, release and tension. J. Cell Biol. 126: 1241–1253. Nirmala, A. and Rao, P. N. 1996. Genetics of chromosome numerical mosaism in higher plants. The Nucleus 39: 151–175. Pagliarini, M. S. 1990. Meiotic behavior and pollen fertility in Aptenia cordifolia (Aizoaceae). Caryologia 43: 157–162. Polhill, R. M. 1981. Galegeae. In: Polhill, R. M. and Raven, P. H. (Eds.), Advances in legume systematics. Royal Botanic Gardens, Kew, pp. 367–370. Ranjba r, M., Amirabadizadeh, H., Karamian, R. and Ghahremani, M. A. 2004. Notes on Onobrychis sect. Heliobrychis (Fabaceae) in Iran. Willdenowia 34: 187–190. ̶, Hajmoradi, Z. and Karamian, R. 2010a. The taxonomic importance of leaf epidermis morphology and anatomy in Trigonella disperma. Taxonomy and Biosystematics 1(2): 15–26. ̶, ̶ and ̶. 2011a. Trigonella biflora Griseb. and Trigonella tenuis Fisch. (Fabaceae), two new records of Trigonella L. from Iran. Iran. J. Bot. (in press). ̶, ̶ and ̶. 2011b. Taxonomic notes on Trigonella sect. ellipticae (Fabaceae, Trifolieae) in Iran. Ann. Bot. Fenn. (in press). ̶, Karamian, R. and Hadadi, A. 2009a. Biosystematic study of Onobrychis viciifolia Scop. and Onobrychis altissima Grossh. (Fabaceae) in Iran. Iran. J. Bot. 15(1): 85–95. ̶, ̶ and Hajmoradi, Z. 2009b. Comosition of the essential oil of Trigonella disperma from Iran. Chem. Nat. Compd. 45(1): 116–117. ̶, ̶ and ̶. 2011c. A revision of Trigonella sect. Ellipticae (Fabaceae) in I ran. Nord. J. Bot. (in press). ̶, ̶ and Johartchi, M. R. 2006. Notes on the taxonomy of Hedysarum (Fabaceae) in Iran. Ann. Bot. Fenn. 43: 152–155. ̶, ̶ and Nouri, S. 2010b. Diploid-tetraploid mixoploidy in an interesting new species of Astragalus sect. Incani DC. (Fabaceae) from Iran. Ann. Bot. Fenn. (in press). ̶, ̶, Tolui, Z. and Amirabadizadeh, H. 2007. Onobrychis assadii (Fabaceae), a new species from Iran. Ann. Bot. Fenn. 44: 481–484. Rechinger, K. H. 1984. Trigonella. In: Rechinger, K. H. (Ed.), Flora Iranica 157. Akademische Druck- und Verlagsanstalt, Graz. pp. 207–253. Scoles, G. J and Kaltsikes, P. J. 1974. The cytology and cytogenetics of Triticale. Z. Pflanzenzucht. 73: 13–43. Schulz, O. E. 1901. Monographie der gattung Melilotus. Bot. J. Syst. Pflanzengeschich. und Pflanzengeogr. 29: 660–735. Sepp, S. and Paal, J. 1998. Taxonomic continuum of Alchemilla (Rosaceae) in Estonia. Nord. J. Bot. 18: 519–535. Shaw, P. J. A. 1998. Morphometric analyses of mixed Dactylorhiza colonies (Orchidaceae) on industrial waste sites in England. Bot. J. Linn. Soc. 128: 385–401. Sheidai, M., Sottodeh, M. and Akbarei. B. 2007. Cytogenetic variability in several oil seed rape cultivars. Pak. J. Biol. Sci. 10(4): 553–560. ̶. 2002. Cytogenetic. Adena publication, Tehran. pp. 85–89. Singh, A. and Roy, R. P. 1970. Karyological studies in Trigonella, Indigofera and Phaseolus. The Nucleus 13: 41–54. 294 M. Ranjbar et al. Cytologia 76(3)

̶ and Singh, D. 1976. Karyotype studies in Trigonella. Nucleus 19: 13–16. Small, E., Brooker, B. and Lassen, P. 1990. Circumscription of the genus Medicago (Leguminosae) by seed characters. Can. J. Agric. Res. 39: 631–641. ̶, Crompton, C. W. and Brooker, B. 1981. The taxonomic value of ﬂoral characters in tribe Trigoneleae (Leguminosae) with special reference to Medicago. Can. J. Bot. 59: 1578–1598. Souza, M. M., Martins, E. R., Pereira, T. N. S. and Olivera, L. O. 2006. Reproductive studies on Ipecac (Cephalis ipecacua- nha (Brot.) A. Rich, Rubiaceae): Meiotic behavior and pollen viability. Braz J Biol 66: 151–159. Townsend, C. C. 1974. Tribe Trifolieae. In: Townsend C. C. and Guest E. (Eds.), Flora of Iraq 3. Ministry of Agriculture and Agrarian Reform, Baghdad. Tutin, T. G. and Heywood, V. H. 1964. Flora Europaea I and II. Cambridge University Press, Cambridge. Tyteca, D. and Dufrene, M. 1994. Biostatistical studies of Western European allogamous populations of the Epipactis hel- leborine (L.) Crantz species group (Orchidaceae). System. Bot. 19: 424–442. Verboom, G. A. and Linder, H. P. 1998. A re-evaluation of species limits in Chaetobromus (Danthonieae: Poaceae). Nord. J. Bot. 18: 57–77. Wilkin, P. 1999. A morphometric study of Dioscorea quartiniana A.Rich. (Dioscoreaceae). Kew Bull. 54: 1–18.