Karyological studies of some species of the genus Vicia L. (Leguminosae) in Turkey

Abstract

In this study, 11 Vicia taxa naturally distributed in Turkey were karyologically studied. Chromosome numbers, karyotypes and idiograms of five taxa that belong to section Cracca of the genus Vicia (Vicia cracca subsp. gerardii, V. cracca subsp. atroviolacea, V. cracca subsp. stenophylla, V. canescens subsp. canescens, V. palaestina) and six taxa that belong to section Vicia (Vicia michauxii var. stenophylla, V. pannonica var. pannonica, V. hybrida, Vicia sativa subsp. nigra var. nigra, Vicia sativa subsp. nigra var. segetalis, Vicia sativa subsp. incisa var. cordata) were done. The chromosome numbers of these taxa were found as 2n = 10, 12, 14, 24. The chromosome numbers and karyotype analysis of five of these were reported for the first time and chromosome morphology of two taxa was identified for the first time. Also chromosome numbers of six taxa were verified. These results will contribute to the taxonomy of the genus Vicia which has a high diversity in Turkey.

Keywords:

• chromosome number
• karyotype
• Leguminosae
• Turkey
• Vicia

Introduction

Leguminosae (Fabaceae), with 18,000 flowering plants, is highly varied and one of the world's three largest families. It has approximately 650 genera. There are 69 genera and 1128 taxa of the Leguminosae family in Turkey. The number of endemic species in Turkey is 375; the rate of endemism is 39.1% (Davis 1970). Genus Vicia L. is represented by 166 species in the world, and is distributed mainly from Europe, Asia and North America to Tropical Africa and the temperate regions of South America (Maxted 1993). There are 87 species of genus Vicia L. in Turkey, and five of them are endemic. The rate of endemism is 8.5% (Davis et al. 1988). Turkey, in particular some parts of eastern and southeastern Anatolia, is one of the three most genetically diverse regions in terms of the number of Vicia taxa. This region is also the gene center for some cereal crops (Vavilov 1950).

Some cytotaxonomic, genetic, cytogenetic, phenetic, genotoxic and biochemical studies were realized on the genus Vicia (Maxted et al. 1991; Pandey and Upadhyay 2007; Raina et al. 2010). The chromosome numbers of half of the 166 Vicia species are known (Maxted 1993). The basic chromosome number in Vicia species was assigned as x = 5, 6 or 7 at most (Darlington and Janaki-Ammal 1945; Maxted et al. 1991); only six of them are polyploid (Cremonini et al. 1992). In the karyological study of Yamamoto (1973) on various Vicia taxa, satellites were observed in two species in the genus Vicia studied. In addition, karyotypes of 27 species (2n = 10, 12 and 14) belonging to the subgenus Vicia have been identified through the training set for linear discriminant analysis (a statistical procedure that uses the variables of known groups or species to classify/identify new unknown groups or species using the same variables) by Venora et al. (2008).

The aim of this study was to determine the chromosome numbers and karyotype morphologies of some species of Vicia. This will contribute to the taxonomy of Vicia.

Materials and methods

Seeds of Vicia taxa that make up the study materials were collected from natural populations in Turkey. The first two characters (e.g. C4, B5) in the line related to the locality of the taxa, referring to the square adopted by Davis (1970), in Flora of Turkey. Chromosome counts were made at somatic metaphase using the squash technique. Root meristems from germinating seeds that were collected in the wild were used. Root-tips were pre-treated with α-monobromo naphthalene solution at 4°C for 16 h, washed and fixed in 3:1 absolute ethanol/glacial acetic acid overnight. Samples were hydrolyzed with 1 N HCl at 60°C, washed for 12 min and stained in aceto-orcein solution for 1–2 h. Stained tips were squashed with a drop of 1% aceto-orcein and mounted in 45% acetic acid (Gray 1964). Cells with a good spread of chromosomes were photographed using an Olympus photomicroscope. Chromosomes were counted from well-spread metaphase in intact cells, by direct observation and from photomicrographs. The chromosome counts of the species studied were confirmed in at least five cells. As Levan et al. (1964) described, long and short arm measurements of the chromosomes were made. Idiograms were drawn for each species.

Results and discussion

As a result of cytogenetic investigations, mitotic metaphase chromosome numbers, karyotype analysis and idiograms were determined for 11 taxa of the genus Vicia, belonging to the Leguminosae family, that grow naturally in Turkey. Chromosome numbers and karyotype analysis of five Vicia taxa, and the chromosome morphology of two taxa are reported here for the first time. The chromosome numbers of six taxa are confirmed. Detailed karyological features of mitotic metaphase chromosomes obtained are given below. Chromosome numbers, ploidy levels, basic numbers, long-arm length (L), short-arm length (S) total chromosome length (C = S + L), arm ratio (r = L/S), relative length (RL = total length (C)/total haploid length × 100) and chromosomal formula of all taxa studied are presented in Table 1.

Table 1. Karyological data of the Vicia species studied.

Taxa	2n	x	Ploidy level	Total chromosome length (C)	Long arm length (L)	Short arm length (S)	Arm ratio (r = L/S)	Relative length (%) (RL)	Karyotype formula
				(μm)	(μm)	(μm)	(μm)
V. cracca subsp. gerardii	12	6	2x	3.35–4.51	2.48–3.43	0.83–1.53	1.70–3.20	14.50–19.48	3st+3sm
V. cracca subsp. atroviolacea	24	6	4x	3.35–6.78	1.74–3.75	1.07–3.03	1.08–3.00	6.28–12.73	5m+6sm+1st
V.cracca subsp. stenophylla	24	6	4x	2.13–3.72	1.22–1.95	0.61–1.77	1.03–2.67	6.90–12.02	5m+7sm
V. canescens subsp. canescens	10	5	2x	3.66–4.76	2.04–3.24	1.26–1.85	1.1–2.14	18.06–23.45	1m+4sm
V. palaestina	12	6	2x	3.63–6.98	3.01–6.02	0.62–1.50	2.18–6.28	11.25–21.65	2sm+4st
V. michauxii var. stenophylla	12	6	2x	5.76–7.92	4.38–5.28	1.38–2.70	1.80–3.17	14.06–19.34	3sm+3st
V. pannonica var. pannonica	12	6	2x	4.08–5.73	3.05–4.27	1.04–1.83	1.67–3.22	14.07–19–75	3sm+3st
V. hybrida	14	7	2x	3.99–5.88	2.82–4.47	0–2.2	0–3.86	12.13–17.88	1m+4sm+1a+1st
V. sativa subsp. nigra var. nigra	12	6	2x	1.96–2.82	1.23–2.82	0–1.12	0–1.68	13.13–18.90	1m+1sm+4a
V. sativa subsp. nigra var. nigra	14	7	2x	4.78–7.43	4.18–5.76	0–2.59	0–9.24	12.04–18.73	1sm+1st+5a
V. sativa subsp. nigra var. segetalis	12	6	2x	1.96–4.62	1.96–4.31	0–1.57	0–2.95	8.89–20.97	1m+1sm+4a
V. sativa subsp. incisa var. cordata	10	5	2x	3.28–3.85	2.26–3.85	0–1.27	0–2.78	18.64–21.89	2sm+3a

Sect. Cracca S.F. Gray

Vicia cracca subsp. gerardii Gaudin

C4 Turkey, Antalya: Alanya turnout, 0–10 m, 23.v.2005, YB 3242 TU, 2n = 12 (Figure 1).

Figure 1 Karyotypes and metaphase plates of the Vicia species studied. Scale bar = 10 μm.

The chromosome number and morphology of V. cracca subsp. gerardii taxon were determined in this study for the first time. In the karyotype analysis of the taxon, the chromosome number was determined as 2n = 2x = 12 and the karyotype formula as 3 subtelocentric (st) +3 submetacentric (sm) (Figure 2).

Figure 2 Idiograms of the Vicia species studied.

Vicia cracca L. subsp. atroviolacea Bornm.

C4 Turkey, Konya: Beyşehir-Konya road, 1170 m, 11.vii.2005, KE 3534 OT, 2n = 24 (Figure 1).

Both the chromosome number and chromosome morphology of the V. cracca subsp. atroviolacea taxon were determined in this study for the first time. In the karyotype analysis, tetraploid chromosome number of this taxon was determined as 2n = 4x = 24. The karyotype formula of the taxon is as 5 metacentric (m) + 6sm + 1st (Figure 2).

Vicia cracca L. subsp. stenophylla Vel.

B5 Turkey, Yozgat: Cayiralani, Pinus nigra forest skirts, 1680 m, 22.vii.2005, YB 3429 HD, 2n = 24 (Figure 1).

Diploid chromosome numbers of the V. cracca subsp. stenophylla were previously reported by several authors as 2n = 14 and the karyotype formula as 2sm + 5st (Şahin and Babaç 1995; Bağcı and Şahin 2000). In the karyotype analysis of this taxon in this study, tetraploid chromosome number was determined as 2n = 4x = 24 and the karyotype formula as 5m + 7sm (Figure 2).

In contrast to the basic chromosome number (x = 7) of the V. cracca subsp. stenophylla taxon determined by the researchers mentioned above, in this study we determined it as x = 6 and also discovered the presence of metacentric chromosomes instead of subtelocentric chromosomes. Both in V. cracca subsp. stenophylla and V. cracca subsp. atroviolacea the chromosome numbers were found to be tetraploid. Raina and Rees (1983) reported tetraploidy for V. cracca subsp. cracca.

Chromosome number in subsp. cracca, which is another subspecies of V. cracca, was determined as 2n = 14 (Şahin and Babaç 1990; Beyazoğlu and Hayırlıoğlu 1991; Akpınar and Bilaloğlu 1997; Inceer et al. 2002; Inceer and Hayırlıoğlu-Ayaz 2005).

In contrast to the researchers reporting that subspecies of V. cracca were formed as submetacentric and subtelocentric chromosomes (Bağcı and Şahin 2000), some other researchers reported the presence of metacentric chromosomes as we did in this study (Inceer and Hayırlıoğlu-Ayaz 2005). The chromosome numbers of V. cracca and its subspecies were previously reported as 2n = 12, 14, 28 by many researchers (Rousi 1961; Şahin and Babaç 1990, 1995; Beyazoğlu and Hayırlıoğlu 1991; Maxted et al. 1991; Akpınar and Bilaloğlu 1997; Bağcı and Şahin 2000; Inceer et al. 2002; Inceer and Hayırlıoğlu-Ayaz 2005). However, in the V. cracca subsp. stenophylla and V. cracca subsp. atroviolacea taxa, chromosome numbers of 2n = 24 were reported for the first time here.

Vicia canescens Lab. subsp. canescens

B5 Turkey, Aksaray : Hasan Dağı, sandy rocky areas, 2066 m, 22.vii.2005, YB 3443 HD, 2n = 10 (Figure 1).

The chromosome number of V. canescens species was previously studied by Akpınar and Bilaloğlu (1997) and the diploid chromosome number was found as 2n = 10, but no chromosome morphology was encountered. The chromosome morphology of V. canescens subsp. canescens was determined in this study for the first time. The chromosome number of the taxon was noted as 2n = 2x = 10 and the karyotype formula as 1m + 4sm (Figure 2).

Vicia palaestina Boiss.

C6 Turkey, Gaziantep: 5 km between Fevzi Pasa-Osmaniye, 1000 m, 23.v.2005, KE 3547 OT 2n = 12 (Figure 1).

Both the chromosome number and the chromosome morphology of V. palaestina was determined for the first time in this study. In the karyotype analysis the chromosome number was found as 2n = 2x = 12 and the karyotype formula as 2sm + 4st (Figure 2).

Sect. Vicia L.

Vicia michauxii Sprengel var. stenophylla Boiss

C4 Turkey, Antalya: Kepezustu, 281 m, 23.v.2005, YB 3249 TU, 2n = 12 (Figure 1).

The diploid chromosome number and morphology of V. michauxii var. stenophylla were previously reported by several researchers as 2n = 14 (Yamamoto 1973; Şahin and Babaç 1990; Bağcı and Şahin 2000). Unlike these researchers, we found the chromosome number of V. michauxii var. stenophylla to be 2n = 12. In contrast to reports in the studies either that all chromosome formulas were subtelocentric (Yamamoto 1973) or 6 st + 1 acrocentric (a), one pair of them with satellite (Bağcı and Şahin 2000), we found the karyotype formula to be 3sm + 3st without satellite (Figure 2).

Vicia pannonica Crantz. var. pannonica

B3 Turkey, Afyon: between Afyon-Eskisehir, 1230 m, 13.vii.2005, OT 3644 TU, 2n = 12 (Figure 1).

The diploid chromosome number of V. pannonica was previously reported by several researchers as 2n = 12 (Cincura 1962; Hanelt and Mettin 1970; Yamamoto 1973; Návratilová et al. 2003; Neumann et al. 2006; Venora et al. 2008). The chromosome morphology of this species was reported as four pairs submetacentric and two pairs subtelocentric (Navratilova et al. 2003). But in this study the chromosome morphology of V. pannonica var. pannonica was determined for the first time. In accordance with the previous studies the chromosome number is 2n = 2x = 12, which shows similarity with our karyotype formula as 3sm + 3st in terms of containing sm and st chromosomes (Figure 2).

Vicia hybrida L.

C6 Turkey, Osmaniye: Osmaniye Highway turnout, under Eucalyptus trees, 50 m, 21.v.2005, KE 3449 OT, 2n = 14 (Figure 1).

Diploid chromosome numbers and morphology of V. hybridia species have been examined many times. Some studies reported the chromosome number of this species as 2n = 12 (Yamamoto 1973; Roti-Michelozzi and Caffaro 1984; Şahin and Babaç 1990; Beyazoğlu and Hayırlıoğlu 1991; Meriç and Olgun 1994; Akpınar and Bilaloğlu 1997; Hayıroğlu-Ayaz et al. 1999; Bağcı and Şahin 2000; Venora et al. 2008). The chromosome number determined by us as 2n = 14, verifies the results of Tabur et al. (2002). We determined the karyotype formula of this species as 1m + 4sm + 1a + 1st (Figure 2).

Previous studies have reported the chromosome morphology of this species as follows: Yamamoto (1973) 6st; Roti-Michelozzi and Caffaro (1984) 3sm + 3st; Hayıroğlu-Ayaz et al. (1999) 5sm + 1st; Bağcı and Şahin (2000) 1m^sat + 5st. Our results comply with these studies since they include metacentric, submetacentric and subtelocentric chromosome types but they contradict these studies due to the acrocentric chromosome.

Vicia sativa L. subsp. nigra (L.) Ehrh. var. nigra (L.) Ehrh.

C6 Turkey, Hatay: between Antakya-Yayladag, Cabala Village, 630 m, 22.v.2005, KE 3528 OT, 2n = 12 (Figure 1).

C6 Gaziantep: between Fevzipasa-Osmaniye, 5 km, 1000 m, 23.v.2005, KE 3546 OT, 2n = 14 (Figure 1).

The chromosome number of the V. sativa subsp. nigra var nigra was previously reported as 2n = 12 (Davis 1970, Şahin and Babaç 1990; Bağcı and Şahin 2000). In this study, in the karyotype analysis of this taxon, the diploid chromosome number was found both as 2n = 12 and 2n = 14. The karyotype formula of this taxon was determined as 1m + 1sm + 4a for 2n = 12 (Figure 2) and as 1sm + 1st + 5a for 2n = 14. However, the diploid chromosome number of V. sativa subsp. nigra was reported as 2n = 12 (Akpınar and Bilaloğlu 1997), and has also been reported as 2n = 14 (Beyazoğlu and Hayırlıoğlu 1991; Akpınar and Bilaloğlu 1997). The chromosome numbers that we found as 2n = 12 and 2n = 14 belonging to the V. sativa subsp. nigra var nigra support these other studies. However, in contrast to the karyotype morphology of Bağcı and Şahin (2000) who found that all chromosomes had subtelocentric centromeres, in this study we determined one metacentric, one submetacentric and many acrocentric chromosomes. Although some studies detected satellites in this taxon (Bağcı and Şahin 2000), we did not observe any satellites.

The taxon with a chromosome number of 2n = 12 could have been formed by centric fusion of two separate acrocentric chromosomes in the taxon with a chromosome number of 2n = 14; vice versa, 2n = 14 may have been caused by centric fusion that occurred in the bi-armed chromosome in 2n = 12. The reason for the different number of bi-armed chromosomes in two separate populations may be caused by pericentric inversion and Robertson translocations that occur in the chromosome.

Vicia sativa L. subsp. nigra (L.) Ehrh. var. segetalis (Thuill) Arc.

C6 Turkey, Gaziantep, between Fevzipasa-Islahiye, 2 km, 516 m, 23.v.2005, KE 3540 OT, 2n = 12 (Figure 1).

The chromosome number and morphology of V. sativa subsp. nigra var. segetalis was previously determined by Tabur et al. (2002). In this study the diploid chromosome number was verified as 2n = 12. As a result of the karyotype analysis the karyotype formula was determined as 1m + 1sm + 4a (Figure 2).

Vicia sativa L. subsp. incisa (Bieb.) Arc. var. cordata (Wulfen ex Hoppe) Arc.

C4 Turkey, Antalya: Kepezustu, 281 m, 23.v.2005, YB 3250 TU, 2n = 10 (Figue 1).

The chromosome number and morphology of V. sativa subsp. incisa var. cordata were determined as the diploid chromosome number 2n = 10 (Davis 1970; Kamari et al. 1994). This chromosome number was verified in this study.

However, the karyotype formula is given as 2sm + 3a here for the first time (Figure 2). In contrast with our results in V. sativa subsp. incisa var. incisa, the other taxon, of the same subspecies, Meriç and Dane (1999) reported the chromosome number as 2n=14, and determined the karyotype formula as 1 sm +6 st, three of which had satellite. This subspecies showed differences in variety level in terms of both chromosome number and karyotype morphology. The chromosome numbers and karyotype formula of accessions collected from different localities might be different. These cytogenetic diversions may be a result of infrageneric and infraspecific variations (Martin et al. 2008). The karyotype of a species is generally subject to little variation and Venora et al. (2008) suggested that two similar species can be different for a number of chromosome rearrangements correlated with phyletic distance between them (Stebbins 1971).

Conclusion

A pair of satellites was observed in all species belonging to section Cracca by Şahin and Babaç (1995), in contrast with our results. The chromosome number reported was similar to our results, as 2n = 10 for the V. anatolica and V. mollis, and as 2n = 12 for the V. seriocarpa and V. noeana in the section Vicia. In accordance with the basic chromosome numbers x = 5, 6, 7 that we obtained for this section, Yamamota (1973) reported the diploid chromosome number of many species in the Vicia section as 2n = 10, 12, 14. He reported that the chromosomes of species that he studied were submetacentric and subtelocentric and most of them had satellite and secondary constriction. However, our results showed that Vicia section members also contain acrocentric chromosomes.

Although there is no sectional distinction in terms of chromosome numbers between the taxa studied, the basic chromosome numbers of the members of Cracca section were determined as x = 6 in all except one taxon (V. canescens subsp. canescens, 2n = 10). Yamamoto (1973) reported that among the taxa belonging to Cracca section, the species (V. atropurpurea, V. villosa, V. eriocarpa, V. dasycarpa, V. varia, V. cappadocica) except V. neglecta (2n = 12), had the chromosome number 2n = 14 and the chromosome morphology of them consisted of submetacentric and subtelocentric chromosomes, which complies with our study.

In case of polyploidy, pairs of chromosomes with the same size can be different in terms of the position of the centromer (Yousefzadeh et al. 2010). This difference is usually brought about by unequal translocation; pericentric inversion was the reason for the chromosomal rearrangement and arm differences (Stebbins 1971). But, some irregularities in the mitotic stages such as delayed chromosomes in anaphase or telophase and centric fusion and fission responsible for infrequently a variable chromosome number in the intra-section and between populations (Ranjbar et al. 2010; Yousefzadeh et al. 2010).

The differences in karyotype formula and asymmetric indices found among the species suggest that structural changes of chromosomes may contribute to the diversification of the genus. These genomic differences could be used for breeding purposes (Hesamzadeh Hejazi and Nasab 2010) Also taxonomic criteria such as chromosome number and chromosome morphology have been developed and these results have been used mostly in plant taxonomy and phylogenetic consideration of the genus (Yılmaz et al. 2009; Hesamzadeh Hejazi and Nasab 2010)

In conclusion, our results confirmed that the basic chromosome numbers of the taxa belonging to the genus Vicia were x = 5, 6, 7, as has been reported so far.

Acknowledgements

We owe great thanks to The Scientific Investigation Projects Coordinate Office of the Selcuk University (Project no: 07201051) for financial support. We would like to thank Dr. Atilla Arslan for the arrangement of figures, Elif Gülbahçe for her invaluable help in laboratory studies, and Dr. Ahmet Tamkoç, Dr. Osman Tugay, Dr. Yavuz Bağcı, Dr. Tuna Uysal and Dr. Hakkı Demirelma for providing plant specimens.