Karyological studies of six taxa of the genus Allium L. (Amaryllidaceae) from Turkey

Abstract

Karyotype structures in representative taxa of the genus Allium L. are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of Allium arlgirdense, A. anacoleum and A. microspathum (in subg. Allium), and A. rhetoreanum and A. shirnakense (in subg. Melanocrommyum) are described for the first time. The karyological analyses indicate the relationships among the species with respect to asymmetry indices. All Allium taxa studied are diploid with 2n = 2x = 16 chromosomes. One B chromosome is detected in the chromosomes of A. microspathum. All chromosome sets of the studied taxa consist of seven median chromosomes and one submedian chromosomes and all submetacentric pairs bear a satellite on the short arms. The lengths of the chromosomes show remarkable differences particularly at the subgenus level. The three subgenus of genus Allium show a clear tendency to have karyotypes distinct on asymmetry grounds. Karyotype asymmetry relationships are discussed according to the bidimensional scatter plots of A₁–A₂ and CV_CL–M_CA.

Keywords:

• Chromosome number
• karyotype asymmetry
• Allium
• Amaryllidaceae
• Turkey

Introduction

According to Koyuncu (2012), the genus Allium L. is represented in Turkey by 196 taxa, many of which endemic (c.39%) to this territory. Chromosome numbers have been reported for approximately 75% of these taxa. Most of these taxa are diploid. However polyploidy has been reported some previous counts for Allium (Goldblatt and Johnson 1979–; Özhatay et al. 2000). A basic chromosome number of x = 8 dominates in most subgenera whereas nearly all taxa of subg. Amerallium are characterized by x = 7 chromosomes (Fritsch and Astanova 1998).

This study focuses on six taxa including Allium arlgirdense, A. anacoleum, A. microspathum (in subg. Allium), A. rhetoreanum, A. shirnakense (in subg. Melanocrommyum) and A. oreophilum (in subg. Poryphrason) (Friesen et al. 2006). These taxa are rare for Flora of Turkey owing to populations with a very restricted area of occupancy, but A. oreophilum is more widely distributed than the others (Kollmann 1984).

Material and methods

The plant materials used in this study are listed in Table 1. Voucher specimens have been deposited in the Herbarium of University of Yüzüncü Yıl (VANF), Van, Turkey.

Table 1. Localities and voucher specimens of Allium taxa examined in the present study.

Taxon	Locality	Voucher
A. arlgirdense Blakelock	Hakkari, Yüksekova, Sat Mountains, South of Sat Lake, 3200 m, 11 August 2013 M. Fırat s.n.	VANF 30417
A. anacoleum Hand.-Mazz.	Hakkari, Yüksekova, Sat Mountains, 2700 m, 11 August 2013, M. Fırat s.n.	VANF 30454
aA. microspathum Ekberg	Van, Gürpınar, Hoşap, Güzeldere pass, 2700 m, 18 August 2014. M. Fırat s.n.	VANF 30670
aA. rhetoreanum Nábelek	Hakkari, Şemdinli, Alan River, 1481 m, 29 May 2014, M. Fırat s.n.	VANF 31040
aA. shirnakense L. Behçet & Rüstemoğlu	Şırnak, Beytüşşebap, near Üçağaç village, 1300 m, 27 May 2014, M. Fırat s.n.	VANF 30740
A. oreophilum C. A. Mey	Van, Başkale, İspiriz Mountain, 3300 m, 7 June 2014, M. Fırat s.n.	VANF 31011

^a Endemic taxon.

For karyological observations, four to eight individuals were used for each taxon in this study. Mitotic metaphase cells of root tips were obtained from planted bulbs which were collected in natural habitats from Turkey. Root tips were pretreated in 0.002 M 8-hydroxyquinoline at 4°C overnight, washed with distilled water and fixed in Carnoy’s solution (3:1 absolute ethanol: glacial acetic acid) for a minimum of 1 h. The root tips were hydrolysed for 10 minutes in 1N HCl at 60°C, stained using the standard Feulgen technique and squashes were prepared.

Microphotographs of good quality metaphase plates were taken using an Olympus BX53 (Tokyo, Japan) microscope equipped with a high-resolution digital camera. The somatic chromosome number and karyotype details were studied in a minimum of five spread metaphase plates from different individuals; mean values were used for the analysis. Metaphase observations and chromosome measures were made using the image analysis systems KAMERAM (ARGENİT Microsystems, İstanbul, Turkey). The karyotype was determined using the above-mentioned software for the recognition of homologues, couple ordering, chromosome classification and karyotype formula based on centromere position (Levan et al. 1964). Karyotype symmetry was determined calculating both CV_CI and CV_CL parameters (Paszko 2006), together with the recent M_CA index (Peruzzi and Eroğlu 2013). Karyotype asymmetry was determined calculating both A₁ and A₂ parameters (Romero Zarco 1986).

Results

All Allium taxa studied were diploid with 2n = 2x = 16 chromosomes, but A. microspathum has a B chromosome. It is fixed for all cells and individuals analysed in this study (Figure 1). The ideograms and parameters obtained are based on the mean values presented in Figure 2. All chromosome sets of the studied taxa consist of seven median chromosomes and one submedian chromosome and all submetacentric pairs bear a satellite on the short arms. Although one of the metacentric pair bears a satellite on the short arms of A. rhetoreanum (Figure 1d, 2).

Figure 1 Somatic chromosomes in the studied taxa. (a) Allium arlgirdense; (b) A. anacoleum; (c) A. microspathum; (d) A. rhetoreanum; (e) A. shirnakense; (f) A. oreophilum. Scale bars 10 μm.

Figure 2 Haploid ideograms in the studied taxa. (a) Allium arlgirdense; (b) A. anacoleum; (c) A. microspathum; (d) A. rhetoreanum; (e) A. shirnakense; (f) A. oreophilum.

Chromosome measures, karyotype formulae and symmetry indices are given in Tables 2 and 3 for all investigated taxa.

Table 2. Karyotype formula according to Levan et al. (1964) and characteristics of the investigated taxa.

Species	Range	HCL (μm)	p (μm)	q (μm)	Karyotype formula
	SC–LC (μm)	mean (± SD)	mean (± SD)	mean (± SD)
A. arlgirdense	6.11–9.25	61.61 (± 1.02)	4.31 (±0.70)	3.39 (±0.67)	14m + 2sm^sat
A. anacoleum	7.43–10.97	72.89 (± 1.13)	5.09 (±0.84)	4.02 (±0.54)	14m + 2sm^sat
A. microspathum	7.60–10.75	72.83 (± 1.01)	5.01 (±0.61)	4.09 (±0.63)	14m + 2sm^sat + 1stc
A. rhetoreanum	9.17–12.84	85.23 (± 1.09)	6.17 (±0.48)	4.48 (±0.75)	12m + 2m^sat + 2sm^sat
A. shirnakense	8.16–11.56	81.55 (± 1.04)	5.97 (±0.43)	4.22 (±0.71)	14m + 2sm^sat
A. oreophilum	11.59–16.26	109.73 (± 1.52)	7.98 (±0.66)	5.74 (±1.21)	14m + 2sm^sat

Abbreviations: SC: shortest chromosome length; LC: longest chromosome length; HCL: haploid chromosome length; p: mean long arm length; q: mean short arm length; SD: standard deviation.

Table 3. Karyo-morphometric parameters and symmetry indices for the investigated taxa.

Taxon	A1	A2	CVCL	CVCI	MCA
A. arlgirdense	0.19 ± 0.03	0.13 ± 0.02	13.04	13.91	12.04
A. anacoleum	0.19 ± 0.04	0.12 ± 0.03	12.03	10.40	11.39
A. microspathum	0.17 ± 0.01	0.11 ± 0.03	11.11	9.43	10.24
A. rhetoreanum	0.27 ± 0.03	0.10 ± 0.01	10.20	8.97	16.28
A. shirnakense	0.29 ± 0.02	0.10 ± 0.03	10.18	9.31	17.66
A. oreophilum	0.28 ± 0.04	0.11 ± 0.02	11.09	13.18	16.97

Differences were not observed in the karyotype formulae of the studied taxa. However remarkable differences were identified in the lengths of the chromosomes, particularly at the subgenus level.

The ratio of shortest and longest chromosome ranged from 6.11 μm in A. arlgirdense (subg. Allium) to 16.26 μm in A. oreophilum (subg. Poryphrason). The ratio of shortest and longest haploid chromosome length ranged from 61.61 μm in A. arlgirdense (subg. Allium) to 109.73 μm in A. oreophilum (subg. Poryphrason). The smallest arm ratio was observed in A. arlgirdense (3.39) and the largest was observed in A. oreophilum (7.98) (Table 2).

According to the intrachromosomal (A₁) asymmetry indices, A. microspathum (0.17 ± 0.01) had the most symmetrical karyotype and A. oreophilum (0.29 ± 0.02) had the most asymmetrical karyotype among the Allium species analysed. According to values of A₁, subg. Allium had the most symmetrical karyotype, then subg. Melanocrommyum and subg. Poryphrason (Table 3 and Figure 3).

Figure 3 Scatter plot for the investigated taxa reported in Table 3: (a) against A₂ (x axis) and A₁ (y axis); (b) against CV_CL (x axis) and CV_CI (y axis); (c) against M_CA (x axis) and CV_CL (y axis); (d) against M_CA (x axis) and CV_CI (y axis).

Discussion

The current paper presents the somatic chromosome numbers and karyotypes of six rare taxa in three subgenera of the genus Allium distributed in the east Anatolian part of Turkey.

The chromosome number of A. oreophilum was reported as 2n = 16 in previous studies (Badr and Elkington 1977; Pogosian 1983; Hanelt et al. 1989; Özhatay 1993). Additionally for this species, Pogosian (1983) reported two different chromosome numbers of 2n = 14, 16, whereas we found 2n = 2x = 16 as in other reports. The chromosome number, the karyotype formula and the ideogram of other five taxa were reported for the first time in this study.

The best way to represent karyotype asymmetry relationships among organisms is by means of bidimensional scatter plots (Romero Zarco 1986; Peruzzi et al. 2009). These scatter plots were generated with A₁ and A₂ (Romero Zarco 1986) and/or CV_CI and CV_CL (Paszko 2006; Peruzzi et al. 2009) parameters. A new parameter was added by Peruzzi and Eroğlu (2013) which is called M_CA (mean centromeric asymmetry).

According to the results of present investigation CV_CL results positively correlated with M_CA; CV_CI did not correlate with CV_CL and M_CA. This evidence has endorsed the results of Peruzzi and Eroğlu (2013).

As can be seen in Figure 3c, the three subgenus of genus Allium show a clear tendency to have karyotypes distinct on asymmetry grounds: subg. Allium, with clearly low intrachromosomal (M_CA) and relatively high interchromosomal asymmetry (CV_CL), subg. Melanocrommyum with higher intrachromosomal asymmetry and subg. Poryphrason with higher interchromosomal asymmetry. The results of a previous paper by the first author about the karyology of subg. Melanocrommyum showed values of A₁ ranging from 0.23 to 0.39 (Genç et al. 2013), which endorses the results of the present investigation.

Subg. Poryphrason is close to subg. Melanocrommyum (Friesen et al. 2006). Therefore these subgenera are not distinct from each other.

In conclusion, according the results of this study, A₁ and M_CA parameters have separated species at the subgenus level for Allium.

Disclosure statement

No potential conflict of interest was reported by the authors.