Contribution to the cytotaxonomic knowledge of 10 species of Klasea Cass. (Asteraceae) from Turkey

Abstract

In the present study, karyotype analyses of 10 taxa belongs to the genus Klasea Cass. (Asteraceae) were conducted. Somatic chromosome numbers are not a distinctive character of classification of Klasea taxa. All of the taxa were counted as 2n = 30 chromosomes, except for Klasea radiata subsp. biebersteiniana (2n = 60). Although all the taxa have a chromosome number of 2n = 30, they could be differentiated by their karyotype formula and quantitative parameters of the karyotype. Karyotype analysis was performed by means of an Image Analysis System and karyotype asymmetry indices were measured. According to these parameters, karyological relationships among studied taxa were inferred.

Keywords:

• Compositae
• PCoA
• karyotype
• karyological relationships
• Klasea

Introduction

Asteraceae, with approximately 1620 genera and more than 23,600 species, is the largest family of flowering plants (Stevens 2001). This family is widely distributed across diverse regions ranging from southwest USA, Mexico, southern Brazil, South Africa, Middle and Southwest Asia as well as Australia. South America is accepted to be phylogenetically the geographic origin of the family (Bremer and Anderberg 1994). There are 1209 species of Asteraceae in the Flora of Turkey, of which 447 are endemic, with an endemism ratio of 37%. With a total number of 134 genera this family constitutes the second largest family of Turkish flora (Davis et al. 1988; Özhatay and Kültür 2006).

Klasea Cass. is an Old World genus of perennial herbs (Martins 2006; Ranjbar et al. 2012). This genus was treated as a section in Serratula L. for a long time, and finally was accepted at the generic level. Klasea is divided into 10 sections by Martins (2006): Grandifoliae L. Martins, Melanolepis L. Martins, Lasiocephalae L. Martins, Leuzeopsis (Boriss.) L. Martins, Nikitinia (Iljin) L. Martins, Quinquefoliae L. Martins, Coriaceae L. Martins, Schumeria (Iljin) L. Martins, Klasea, and Demetria (Boriss.) L. Martins. In Turkey, the Eastern Mediterranean and Irano-Turanian phytogeographical region Klasea is represented by 15 species. Five of them are endemic and endemism rate is 31.25% (Doğan et al. 2012). In this study, chromosome numbers are reported for 11 Klasea taxa belonging to six sections. Karyomorphological observations were made on 10 of these taxa because the chromosomes of Klasea kurdica were not possible to measure.

Material and methods

According to the classification of Martins (2006), we analyzed 10 Klasea taxa in six sections: Klasea, Quinquefoliae, Lasiocephalae, Melanolepis, Demetria, and Schumeria (for species see Table 1). The plant samples were collected from different localities in Turkey (Table 1). For karyotypes, root tips were obtained from germinating cypselae; this process took about 10 days on wet filter paper in Petri dishes. Root tips were pretreated with α-monobromonaphthalene for 16 h at 4°C, and then fixed in Carnoy’s solution absolute ethanol and glacial acetic acid (3:1) for 24 h and stored at 4°C until use. Root tips were washed in distilled water to remove the fixative, hydrolyzed in 1N HCl for 13 min at room temperature and stained in 2% aceto-orcein for 2 h. Permanent slides were made with the standard liquid nitrogen method; slides were dried for 24 h at room temperature and mounted in DEPEX. Karyotype analyses were made by using Bs200Pro Image Analysis Software (http://www.bab.com.tr/). Chromosomes were classified using the nomenclature of Levan et al. (1964) as median (m), submedian (sm), subterminal (st) and terminal position (T). Idiograms for each taxon were built according to decreasing chromosome length.

Table 1. Localities and dates of collection for the Klasea material used in the present study.

Taxa	Locality	Voucher no
Sect. Quinquefoliae
Klasea quinquefolia	Turkey, Artvin, Ardanuç, Boyalı village, 1210 m, 41°06.967′ N, 042°07.283′ E, 11 August 2009	B. Doğan 2139 & A. Duran (KNYA)
Sect. Klasea
Klasea radiata subsp. biebersteiniana	Turkey, Kars, Kağızman, Akçay to Cumaçay, 1830 m, 20 July 2009	B. Doğan 2124 & A. Duran (KNYA)
Klasea erucifolia	Turkey, Erzurum, Köprüköy, Eğirmez village, 1635 m, 39°57.056′ N, 041°51.530′ E, 9 August 2009	B. Doğan 2137 & A. Duran (KNYA)
Klasea haussknechtii	Turkey, Muş, Malazgirt, Karıncalı village, 1840 m, 39°21.219′ N, 042°20.010′ E, 18 July 2009	B. Doğan 2113 (KNYA)
Sect. Melanolepis
Klasea kotschyi	Turkey, Bitlis, Tatvan, Sapur village, 1965 m, 38°26.154′ N, 042°24.413′ E, 6 August 2009	B. Doğan 2131 & A. Duran (KNYA)
Klasea hakkiarica	Turkey, Hakkari, Cilo mountain, Kırıkdağ, near dez stream, 2210 m, 37°32.974′ N, 043°57.615′ E, 7 August 2009	B. Doğan 2132 & A. Duran (KNYA)
Sect. Demetria
Klasea oligocephala	Turkey, Kahramanmaraş, Ahir mountain, 995 m, 37°36.475′ N, 036°52.947′ E, 16 July 2009	B. Doğan 2108 & A. Duran (KNYA)
Sect. Schumeria
Klasea cerinthifolia	Turkey, Kahramanmaraş, Ahir mountain, 990 m, 37°36.470′ N, 036°52.917′ E, 16 July 2009	B. Doğan 2107 & A. Duran (KNYA)
Klasea kurdica	Turkey, Osmaniye, Yarpuz, 1465 m, 37°00.774′ N, 036°26.683′ E, 15 July 2009	B. Doğan 2106 & A. Duran (KNYA)
Sect. Lasiocephalae
Klasea bornmuelleri	Turkey, Malatya, Darende, near Akçatoprak, 1010 m, 38°30.064′ N, 037°33.907′ E, 17 July 2009	B. Doğan 2110 & A. Duran (KNYA)
Klasea lasiocephala	Turkey, Antalya, Gazipaşa, Çayıryaka mountain pasture, 1730 m, 36°30.027′ N, 032°32.181′ E, 30 June 2009	B. Doğan 2105 & A. Duran (KNYA)

Karyological characters and data analysis

According to Peruzzi and Eroğlu (2013) and Peruzzi and Altınordu (2014), we used chromosome number (2n), basic chromosome number (x), total haploid length of chromosome set (THL), together with CV_CI (coefficient of variation of centromeric index) and CV_CL (coefficient of variation of chromosome length) (Paszko 2006) and the karyotype asymmetry M_CA (mean centromeric asymmetry) (Peruzzi and Eroğlu 2013). These data were subjected to Principal Coordinate Analysis (PCoA) analysis using Gower’s general coefficient of similarity. To determine karyological relationships among studied taxa of Klasea, we used the software Past 3.03 (Hammer et al. 2001; Hammer 2013).

Results

Section Melanolepis

Klasea kotschyi (Boiss.) Greuter & Wagenitz

The chromosome number of Klasea kotschyi is 2n = 30 (Figure 1a). The chromosome size ranges from 1.13 μm to 2.39 μm, and haploid chromosome length is 12.33 μm. The karyotype formula of this taxon consists of 10m+5sm pairs. The idiogram is given in Figure 1b.

Figure 1. Klasea kotschyi. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Klasea hakkiarica (P. H. Davis) Greuter & Wagenitz

The chromosome number of Klasea hakkiarica is 2n = 30 (Figure 2a). The chromosome size ranges from 1.18 μm to 2.15 μm, and haploid chromosome length is 12.48 μm. The karyotype formula of this taxon consists of 13m+2sm pairs. The idiogram is given in Figure 2b.

Figure 2. Klasea hakkiarica. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Section Quinquefoliae

Klasea quinquefolia (Willd.) Greuter & Wagenitz

The chromosome number of Klasea quinquefolia is 2n = 30 (Figure 3a). The chromosome size ranges from 1.18 μm to 2.93 μm, and haploid chromosome length is 13.36 μm. The karyotype formula of this taxon consists of 9m+6sm pairs. The idiogram is given in Figure 3b.

Figure 3. Klasea quinquefolia. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Section Klasea

Klasea erucifolia (L.) Greuter & Wagenitz

The chromosome number of Klasea erucifolia is 2n = 30 (Figure 4a). The chromosome size ranges from 1.23 μm to 2.75 μm, and haploid chromosome length is 13.84 μm. The karyotype formula of this taxon consists of 8m+7sm pairs. The idiogram is given in Figure 4b.

Figure 4. Klasea erucifolia. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Klasea haussknechtii (Boiss.) Holub

The chromosome number of Klasea haussknechtii is 2n = 30 (Figure 5a). The chromosome size ranges from 1.48 μm to 2.61 μm, and haploid chromosome length is 14.79 μm. The karyotype formula of this taxon consists of 14m+1sm pairs. The idiogram is given in Figure 5b.

Figure 5. Klasea haussknechtii. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Klasea radiata subsp. biebersteiniana (Grossh.) Greuter

The chromosome number of Klasea radiata subsp. biebersteiniana is 2n = 60 (Figure 6a). The chromosome size ranges from 1.12 μm to 2.52 μm, and haploid chromosome length is 13.11 μm. The karyotype formula of this taxon consists of 18m+12sm pairs. The idiogram is given in Figure 6b.

Figure 6. Klasea radiata subsp. biebersteiniana. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Section Lasiocephalae

Klasea lasiocephala (Bornm.) Greuter & Wagenitz

The chromosome number of Klasea lasiocephala is 2n = 30 (Figure 7a). The chromosome size ranges from 1.71 μm to 3.14 μm, and haploid chromosome length is 16.09 μm. The karyotype formula of this taxon consists of 7m+8sm pairs. The idiogram is given in Figure 7b.

Figure 7. Klasea lasiocephala. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Klasea bornmuelleri (Azn.) Greuter & Wagenitz

The chromosome number of Klasea bornmuelleri is new to science and 2n = 30 (Figure 8a). The chromosome size ranges from 1.26 μm to 2.01 μm, and haploid chromosome length is 12.21 μm. The karyotype formula of this taxon consists of 6m+9sm pairs. The idiogram is given in Figure 8b.

Figure 8. Klasea bornmuelleri. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Section Demetria

Klasea oligocephala (DC.) Greuter & Wagenitz

The chromosome number of Klasea oligocephala is 2n = 30 (Figure 9a). The chromosome size ranges from 1.43 μm to 2.81 μm, and haploid chromosome length is 15.18 μm. The karyotype formula of this taxon consists of 12m+3sm pairs. The idiogram is given in Figure 9b.

Figure 9. Klasea oligocephala. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Section Schumeria

Klasea cerinthifolia (Sm.) Greuter & Wagenitz

The chromosome number of Klasea cerinthifolia is 2n = 30 (Figure 10a). The chromosome size ranges from 1.10 μm to 1.97 μm, and haploid chromosome length is 11.10 μm. Chromosome arm ratios are measured as 1.23–3.78. The karyotype formula of this taxon consists of 9m+4sm+2st pairs. The idiogram is given in Figure 10b.

Figure 10. Klasea cerinthifolia. (a) Metaphase chromosomes (scale bar 10 μm); (b) idiogram.

Klasea kurdica (Post) Greuter & Wagenitz

The chromosome number of Klasea kurdica is 2n = 30 (Figure 11). Since the chromosomes were not possible to measure, the karyotype of this taxon could not be analyzed.

Figure 11. Metaphase chromosomes of Klasea kurdica (scale bar 10 μm).

Consequently, chromosome morphology and karyotype asymmetry of Klasea kotschyi, K. quinquefolia, K. erucifolia, K. hakkiarica, K. lasiocephala, K. oligocephala, K. haussknechtii, K. cerinthifolia, K. bornmuelleri and K. radiata subsp. biebersteiniana were identified. Chromosome numbers and karyomorphological features are given (Table 2). Also detailed karyotype asymmetry indices and variability of asymmetry indices among species are given (Table 3). PCoA analysis was used to show karyological relationships among Klasea taxa and PCoA cumulative variance explained by the first two axes: 62.5%. In this case, the taxa belonging to the same section tend to cluster together (Figure 12).

Table 2. Karyomorphological features of Klasea taxa.

Taxon	2n	Chromosome length (μm)		Haploid chromosome length (μm)	Arm ratio		Relative length		Karyotype formula
		Min	Max		Min	Max	Min	Max
K. kotschyi	30	1.13	2.39	12.32	1.18	2.68	4.56	9.69	10m+5sm
K. quinquefolia	30	1.18	2.93	14.40	1.17	2.19	4.09	10.17	9m+6sm
K. erucifolia	30	1.23	2.75	13.84	1.25	2.64	4.44	9.92	8m+7sm
K. haussknechtii	30	1.48	2.61	14.79	1.31	1.82	4.99	8.83	14m+1sm
K. hakkiarica	30	1.18	2.15	12.48	1.24	1.84	4.73	8.60	13m+2sm
K. bornmuelleri	30	1.26	2.01	12.21	1.34	2.74	5.17	8.23	6m+9sm
K. cerinthifolia	30	1.10	1.97	11.10	1.23	3.78	4.93	8.85	9m+4sm+2st
K. lasiocephala	30	1.71	3.14	16.09	1.12	2.43	5.33	9.76	7m+8sm
K. oligocephala	30	1.43	2.81	15.18	1.11	2.04	4.69	9.24	12m+3sm
K. radiata subsp. biebersteiniana	60	1.12	2.52	13.11	1.06	2.63	2.14	4.81	18m+12sm

Table 3. Karyological characters including karyotype symmetry indices for studied taxa.

Species	THL	2n	x	CVCI	CVCL	MCA
K. erucifolia	13.84	30	15	12.32	20.35	24.8
K. hauksknectii	14.79	30	15	5.18	16.46	20.33
K. hakkarica	12.48	30	15	7.34	16.59	19.54
K. cerinthifolia	11.10	30	15	19.8	16.64	26.37
K. bornmuelleri	12.21	30	15	13.2	13.29	26.93
K. kotschyi	12.32	30	15	15.48	18.63	23.87
K. lasiocephala	16.09	30	15	12.91	17.97	24.07
K. oligocephala	15.18	30	15	10.55	19.21	16.94
K. quinquefolia	14.40	30	15	11.41	26.73	20.13
K. radiata subsp. biebersteiniana	13.11	60	15	15.93	22.35	22.31

Figure 12. PCoA for Klasea taxa based on karyological quantitative data.

Discussion

All taxa were counted as 2n = 30 except for Klasea radiata subsp. biebersteiniana. The karyomorphologic and asymmetry characters are useful to determine the karyological relationship of studied taxa.

The lowest CV_CI value, indicating a chromosome complement with a more homogeneous centromere position, was observed in K. hauksnectii. The most heterogeneous centromere positions occur in K. bornmuelleri. CV_CL values range between 13.29 and 26.73 and K. quinquefolia shows the highest interchromosomal asymmetry with a value of 26.73. K. bornmuelleri has the highest intrachromosomal asymmetry (M_CA) value, 26.93, and K. oligocephala possess lowest M_CA of 16.94.

Section Quinquefoliae is sister to sect. Demetria (Martins 2006). Klasea quinquefolia and K. oligocephala showed similar karyological features. Also within sect. Klasea, K. hauksnectii, K. radiata subsp. biebersteiniana and K. erucifolia clustered together and were karyologically similar to sect. Quinquefoliae and sect. Demetria. Klasea bornmuelleri and K. lasiocephala, in Lasiocephalae sect. (Martins 2006), were karyologically similar. In Melanolepis sect., Klasea hakkiarica and Klasea kotschyi were considered and our results were congruent with Martins (2006).

According to Hidalgo et al. (2007), the basic chromosome number of Klasea was reported as x = 15. Chromosome numbers of Klasea cerinthifolia (Sm.) Greuter & Wagenitz and K. grandifolia (P. H. Davis) Greuter & Wagenitz, were reported as 2n = 30 (Garcia-Jacas et al. 1998; Hidalgo et al. 2007).

Cueto Romero and Blanca Lopez (1987) reported the chromosome number of Klasea nudicaulis (L.) Fourr. as 2n = 30. Cantó (1982) reported a diploid chromosome number of 2n = 30 + (0–4B) for this taxon. Cantó (1982) also studied Klasea taxa, and obtained the following chromosome numbers: Klasea baetica subsp. alcalae Cantó & R. Mart. as 2n = 30; K. baetica subsp. lusitanica (Cantó) Cantó & R. Mart. as 2n = 30, 60; K. legionensis (Lacaita) J. Holub as 2n = 30; K. integrifolia (Vahl) Greuter as 2n = 30, 90; K. flavescens subsp. leucantha (Cav.) Cantó & R. Mart. as 2n = 30; K. flavescens subsp. mucronata (Desf.) Cantó & R. Mart. as 2n = 60; K. pinnatifida (Cav.) Talavera as 2n = 60 and 90. Also Fernandez and Queiros (1971) expressed the chromosome number for Klasea pinnatifida as 2n = 90.

Chromosome numbers of Klasea lycopifolia (Vill.) Á. Löve & D. Löve, K. radiata subsp. biebersteiniana and K. radiata (Waldst. & Kit.) Á. Löve & D. Löve were reported as 2n = 30 and 60 (Pogan et al. 1989). Conversely, 2n = 28 and 2n = 26 chromosome numbers were determined for K. serratuloides (DC.) Greuter & Wagenitz and K. coriacea Holub, respectively, by Tonian (1980).

In Central and Northwest Europe, some taxa of Klasea were studied and the diploid chromosome number of 2n = 30 has been reported (Löve and Löve 1961).

But according to the literature, no karyotype information is available for this genus. In this study, we performed karyotype analyses of 10 Klasea taxa.

As well as having the same chromosome number, karyotype formulae were found to be different. The karyotypes are mostly formed of metacentric and submetacentric chromosome pairs. Only Klasea cerinthifolia has a subtelocentric chromosome type.

The chromosome numbers and karyotype details of Klasea taxa were identified and this has contributed to the systematic knowledge of the genus.

Acknowledgments

We express our thanks to TUBITAK [project no. TBAG-109-T-243] and the Scientific Investigation Projects Coordinate Office of Selçuk University [project no. 11401048] for financial support. I would like to thank Turan Arabacı, Meryem Öztürk and Özlem Çetin for their valuable contributions.

Disclosure statement

No potential conflict of interest was reported by the authors.