Karyological Studies of Fritillaria (Liliaceae) Species from Iran
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© 2016 The Japan Mendel Society Cytologia 81(2): 133–141 Karyological Studies of Fritillaria (Liliaceae) Species from Iran Marzieh Ahmadi-Roshan1, Ghasem Karimzadeh1*, Alireza Babaei2 and Hadi Jafari2 1 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran P. O. Box 14115–336, Iran 2 Department of Horticultural Sciences, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Received September 26, 2015; accepted March 14, 2016 Summary Five species (13 ecotypes) belonging to three subgenera of ornamental-medicinal Iranian Fritillaria were karyotypically studied, using a standard squash technique. All species were diploid (2n=2x=24) having mean chromosome lengths of 15.8 µm (15.2–16.7 µm). Their satellites varied in number (1–3 pairs) and in size (1.2–2.6 µm), mostly being located on long arms. Four chromosome types (“m”, “sm”, “st”, “T”) formed 10 dif- ferent karyotype formulas: “T” type chromosome is reported for the first time in most species (with the exception of S4, Fritillaria. reuteri Boissi). ANOVA confirmed significant intra- and inter-specific chromosomal variation across the Iranian Fritillaria species. Twelve different methods were used to assess the degree of karyotype asymmetry. Among those, one qualitative parameter (Stebbins classification) and eight quantitative (CVTL, DI, A1 & A2, AI, A, AsK%, MCA, CVCI) parameters verified that S2 (F. gibbosa Boiss.) and S5 (F. zagrica Stapf.) species represented the most asymmetrical and symmetrical karyotypes, respectively. Key words Fritillaria, Cytogenetics, New chromosome type, Karyotype, Iran. The name Fritillaria is likely based on the word “fri- Fritillaria subgenus is morphologically classified into six tullus” which means a cup in Latin (Ulug et al. 2010). complexes: the F. crassifolia Boiss. & Noe, F. kotschyana Fritillaria was one of the earliest horticultural products ssp. Herbert, F. graeca Boiss. & Spruner, F. meleagris in Europe, and was described by Linnaeus (Linnaeus L., F. cirrhosa D-Don and F. cuncasica Adams. groups. 1753). Iran supports a great range of unique plants and Jafari et al. (2014) reported chromosomal and karyotypi- habitats, and it is one of the main diversity centers of cal parameters and their association with either the evolu- Fritillaria in the world (Rix 1977). There are 14 species tion or intra/inter-specific variation through five species of local importance (De Hertogh and Le Nard 1993). An (F. persica, F. kotschiana, F. imperialis Lutea Maxima, additional 18 species of Fritillaria and Rhinopetalum F. crassifolia, F. straussii) belonging to three subgenera have been recorded, seven of which (F. zagrica L., F. (Theresia, Fritillaria, Petillium) of Iranian Fritillaria. In ariana Losinsk. & Vved., F. kotschyana Herb., F. straus- this study, twelve different methods were used to assess sii Bommuller., F. olivieri Baker, and F. raddeana Regel.) the degree of karyotype asymmetry. Among those, one are endemic to Iran (Mozaffarian 1992, Khaniki 2005). qualitative (Stebbins classification) and seven quantitative Overall, Fritillaria, a genus of about 100 species of bul- (TF%, CVTL, DI, AsK%, A2, Rec, CG%) parameters veri- bous plants within the family Liliaceae, comprises peren- fied that F. kotschiana and F. straussii species are recog- nial temperate herbs that grow on mountain slopes and in nized as having the most asymmetrical and symmetrical sub-alpine meadows, typically located on open, stony and karyotypes, respectively. moist hillsides of the Northern Hemisphere (Maharjan et The identification of extensive variation provides the al. 2012). The last reconsideration of the genus Fritillaria, genus Fritillaria with agronomic and economic impor- according to Rix (2001), divided this genus into eight tance (Metin et al. 2013). Members of this genus in par- subgenera: Davidii Rix, Japonica Rix, Liliorhiza Benth. ticular are the source of components having medicinal & Hook, Fritillaria L., Petilium L. Baker, Rhinopetalum properties, and these are finding increased medical appli- Fisch. ex Alexander, the monotypic Theresia Koch and cations (Rønsted et al. 2005, Maharjan et al. 2012). Com- Korolkowia Regel (Rix 2001, Rønsted et al. 2005, Leitch parative karyotype analysis of related species has been et al. 2007, Ambrožová et al. 2011), two sections, and used to describe patterns and directions of chromosomal 165 taxa (Rix 2001). All Iranian species belong to four evolution through a group, and to estimate the evolution- subgenera: Fritillaria, Theresia., Petilium and Rhinopeta- ary role that such karyotype changes may have played lum. The Petilium subgenus comprises two well-known (Peruzzi et al. 2009). The chromosomes of Liliaceae have species, F. imperialis L. and F. raddeana Regel. The long attracted attention, given their diversity in number, size, and structure (Peruzzi et al. 2009). Chromosome * Corresponding author, e-mail: [email protected] numbers have been reported for more than 50 species of DOI: 10.1508/cytologia.81.133 Fritillaria. Most species have a basic chromosome num- 134 M. Ahmadi-Roshan et al. Cytologia 81(2) ber x=12, although x=9 (3 species), x=11 (2 species), and treatment was applied to break the dormancy of bulbs: x=13 (2 species) also have been reported, but no special the bulbs were cultured in a medium containing peat and pattern is obvious (Darlington 1937, Noda 1964, Fedorov perlite (1 : 3). The cold treatment found to be most effec- 1969, Li and Shang 1989). There is a great uniformity in tive involved a temperature of 8–10°C for 45–60 d, and chromosome morphology (Khaniki 2002a, b, c, Khaniki this was found to encourage growth of primary roots. For 2003, Khaniki 2005). In Iran, wild ecotypes of important the analysis of somatic chromosomes, 1–1.2 cm long fresh species, such as F. imperialis L. and F. persica L., are in root tips were excised from the cultivated bulbs. Different danger of elimination and extinction due to a lack of pro- protocols for pretreatment were tested, and the best result tecting rules, the irregular grazing of Fritillaria stands, was obtained using 0.05 M of colchicine in darkness, at and the alteration of pastures to dry farmlands and pest room temperature (RT) for 6 h. Sample roots were then overflow (Ebrahimie et al. 2006). washed three times with distilled water (each 5 min) at The main aim of this research was to study chromo- RT. In order to achieve best separation of the chromo- somal parameters and their association with the evolu- somes at metaphase, roots were placed in KCl (0.1 M, tion and intra/inter-specific variations through five spe- 0.56% (w/w)) for 60 min at RT. They were subsequently cies of Iranian Fritillaria. fixed in Carnoy’s fixative (glacial acetic acid : ethanol; 1 : 3 v/v) overnight at 4°C (Karimzadeh et al. 2010, 2011, Materials and methods Ebadi-Almas et al. 2012, Shariat et al. 2013, Jafari et al. 2014, Abedi et al. 2015). After thorough washing with The first step of this program involved sampling 13 distilled water, the roots were transferred to 70% (v/v) ecotypes of related species including: Fritillaria imperial- aqueous ethanol, and stored in a refrigerator until used. lis, F. zagrica (an endangered species), F. gibbosa Boiss., Hydrolysis was carried out with 1 M HCl for 15 min at F. rhaddeana, and F. reuteri Boissier. This involved over RT. Thereafter, root tips were stained in 2% (w/v) aceto- 15000 km of exploratory trips in Iran in Spring, 2011 orceine for 5 h at 20°C, according to Karimzadeh et al. (Table 1 and Fig. 1). The exploratory trips were arranged (2011) and Jafari et al. (2014). The stained root tips were according to the logical climatic pattern of different afterwards squashed in a driblet of 45% (v/v) acetic acid. zones of Iran, from the warmest climates to the coolest. The cover slips were then removed after quick freezing The sampled species belong to three subgenera of Petil- the slides on dry ice (Rechinger 1990). At least five well- lium, Rhinopetalum and Fritillaria. After collection, cold spread metaphase plates from different individuals were Table 1. Locations of the collected exotic and endemic Fritillaria spp. Mean Mean Family & Ecotypes Exotic/ Latitude (N) Altitude Subgenera Species name Local collection locations temp rainfall genera names codes Endemic Longitude (E) (m) (°C) (mm) Liliaceae̶ Petillium F. imperialis S1-E1* Aligudarz, Lorestan, Iran Exotic 33°08′ 2734 11.0 409 Fritillaria 49°25′ F. imperialis S1-E2 Aligudarz, Lorestan, Iran Exotic 33°08′ 2497 11.0 409 49°27′ F. imperialis S1-E3 Marivan, Kordestan, Iran Exotic 35°18′ 1978 14.7 831 46°11′ F. imperialis S1-E4 Sarmil, Kermanshah, Iran Exotic 34°19′ 1663 15.4 484 46°07′ F. imperialis S1-E5 Khonsar, Isfahan, Iran Exotic 33°09′ 2743 19.5 264 50°24′ F. imperialis S1-E6 Malayer, Hamedan, Iran Exotic 34°17′ 1778 11.3 290 48°51′ F. imperialis S1-E7 Khoshyeylaq, Golestan, Iran Exotic 36°50′ 1573 17.8 515 55°22′ Rhinopetalum F. gibbosa S2-E1 Maimai, Semnan, Iran Exotic 36°22′ 1300 17.4 152 55°47′ Petillium F. raddeana S3-E1 Aladagh, Golestan, Iran Exotic 37°21′ 2410 17.8 515 57°07′ F. raddeana S3-E2 Cheshmeh Khan, Golestan, Iran Endemic 37°20′ 1307 17.8 515 56°02′ Fritillaria F. reuteri S4-E1 Sabze Kooh, Shahrekord, Iran Exotic 31°45′ 2698 19.4 328 50°59′ F. zagrica S5-E1 Sanandaj, Kordestan, Iran Endemic 35°16′ 1818 14.7 389 47°07′ F. zagrica S5-E2 Khonsar, Isfahan, Iran Endemic 33°09′ 2818 19.5 264 50°23′ * S: species, E: ecotype 2016 Karyological Studies of Fritillaria (Liliaceae) 135 analyzed per ecotype. The best metaphase spreads were Twelve different methods were used to assess the de- recorded photographically, using a DP12 digital camera gree of karyotype asymmetry, comprising the dispersion (Olympus Optical Co., Ltd., Tokyo, Japan) mounted on the index (DI; Lavania and Srivastava 1999), the total form BX50 Olympus microscope (Olympus Optical Co., Ltd., percentage (TF%; Huziwara 1962), the total chromatin Tokyo, Japan).