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 different 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 chromosomal 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). The morphologies of the chromosomes length (X) and the coefficient of variation of total chro- was described using the nomenclature proposed by (Le- mosome length (TL; Paszko 2006), the mean centromeric van et al. 1964). asymmetry (MCA; Peruzzi and Eroglu 2013), the coef- Nine chromosomal parameters were either measured ficient of variation of centromeric index (CVCI; Paszko or calculated, including long (L) and short (S) arms, total 2006), the degree of karyotype asymmetry (A; Watanabe chromosome length (TL) and volume (TCV), arm ratio et al. 1999), the Asymmetry index (AI; Paszko 2006), (AR), r-value, relative length of chromosome (RL), chro- the index of karyotype symmetry (Syi) and the index mosome form percentage (F%) and centromeric index of chromosome size resemblance (Rec; Greilhuber and (CI). Their formulas are as follows: Speta 1978), the centromeric gradient (CG%; Lavania LSand Srivastava 1999), the percentage karyotype asym- TLLS=+ Arm ratio (AR )= r -value = SLmetry index (AsK%; (Arano 1963)), the Romero-Zarco ’ TL S (1986) method, and Stebbins classification (1971). Their RL%=× 100F % =× 100 formulas are as follows: TL TL S CI = TL

S XCI×× CV TL CG%% CV  DI = = TF%= × 100 X=×2  TL 100 100  TL n  Li− Si Sd Li+ Si CV× CV CV%= CV % = × 100 =×AA 100 =i =1 ×100 AI = TL CI TL Xn2 100 n  CLi LC X Rec =×i =1 100 Syi =S nXL L SXCI  S CVCI = ×100 AsK%= ASI % = × 100CG % == 100 XXCI  TL L n S   x =1 L Sd AA12= 1 − = n X

In order to examine differences between species Results (interspecific variation) and differences between ecotypes within the same species (intraspecific variation), All five species (13 ecotypes) were diploid a nested design was performed with 5 replications of (2n=2x=24). The karyotypes of the somatic comple- metaphase cells. The resultant chromosomal data were ment and the idiograms of haploid complement of the first tested for normality, followed by the Bartletts and studied Fritillaria species are given in Figs. 2 and 3, re- Levene’s tests for the homogeneity of variances, us- spectively. The ANOVA of chromosomal parameters was ing Minitab 16 statistical software and then they were performed to assess inter- and intraspecific chromosomal analyzed. The LSD test was carried out for mean com- variations. Hence, in term of interspecific variation, parisons of ecotypes within species, using Minitab 16 ANOVA indicated significant differences for four chro- statistical software. Principal component analysis (PCA) mosomal parameters, including AR, r-value (p<0.05), was carried out to differentiate the studied species based F% and CI (p<0.01), but in term of intraspecific varia- on chromosomal parameters (Jolliffe 1986). tion, only TCV was highly significant (p<0.001; Table 2). 136 M. Ahmadi-Roshan et al. Cytologia 81(2)

Fig. 1. Geographic distribution of sampled Fritillaria on the map of Iran using ArcGIS.

At the level of subgenera, ANOVA verified significant symmetric; Table 3), whereas the lowest was in S2 (19.4; differences for the all chromosomal parameters (data not the most asymmetric). The highest and the lowest values shown). CV% were identified in S4 (23.8%; the most asymmet- Comparing species with each other, the mean total ric) and S5 (19.3%; the most symmetric), respectively. chromosome length (TL) was determined as 15.8 µm, Similar to the result of CV%, the highest value of DI% with a range varying from 15.2 µm (S3) to 16.7 µm (S5); was detected in S4 (5.0%; the most asymmetric) while these differences were not significant. The mean TCV S5 showed the lowest (4.0%; the most symmetric). AI was 109.8 µm3, ranging from 88.4 µm3 (S3) to 132.3 µm3 varied from 0.6 (S1) to 0.64 (S2). The highest value of

(S5). The mean CI of the complement varied from 18 MCA was identified in S2 (63.9%) while S1 demonstrated (S2, S3) to 21 (S1). Using Levan et al. (1964) chromo- the lowest value (56.1%). The mean CVCI was deter- some nomenclature, four chromosome types, types “m” mined as 44.7 µm, varying from 38.5 µm (S3) to 55.2 µm (centromere at the median region), “sm” (centromere at (S2). The highest and the lowest values of CG% were the submedian region), “st” (centromere at the subtermi- distinguished in S1 (23.2%) and S2 (19.4%), respec- nal region), and “T” (centromere at the terminal point), tively. The highest value of A was identified in S2 (0.64) formed ten distinctive karyotypes (Table 3). The “T” while S1 gave the lowest value (0.56). The highest and chromosome type is being reported for the first time the lowest values of Syi were found in S1 (29.6) and S2 in most species (excluding S4). Comparing ecotypes (24.1), respectively. The highest value of Rec was recog- within species, in the S1 species (seven ecotypes), the nized in S5 (73.0%) while S2 showed the lowest (66.3%). mean TCV was 100.4 µm3, ranging from 82.9 µm3 (S1- The highest value of AsK% was identified in S2 (80.6%) E3) to 104.7 µm3 (S1-E2; 26.3% increase; p<0.001). while S1 demonstrated the lowest value (77.2%). To In the S3 species (two ecotypes), the mean TCV was determine the total variation in species and parameters, 128.2 µm3, varying from 99.1 µm3 (S3-E1) to 157.3 µm3 PCA was performed, showing that the first three princi- (S3-E2; 58.7% increase; p<0.001). In the S5 species (two pal components account for the 99% of the cumulative ecotypes), the mean TCV was 117.7 µm3, ranging from variation. The first two components were projected in a 78.3 µm3 (S5-E2) to 148.1 µm3 (S5-E1; 89.1% increase; two-dimensional graphic format (Fig. 4). p<0.001). The satellites varied in number (1–3 pairs) and Moreover, cluster analysis was performed (using the in size (1.2–2.6 µm), typically being located on the long matrix of karyotype similarities, UPGMA, Cophenetic arms. The karyotypes of all five species (13 ecotypes) correlation r=0.92, data not shown), confirming the re- were classified as the 3B type of Stebbins (1971) clas- sults obtained from the PCA’s three main groups (S1 & sification. According to various indices of karyotype S2, S3 & S4, S5; Fig. 4). symmetry, the studied Fritillaria species showed different behavior within these groups. For example, the highest value of TF% was detected in S1 (23.2; the most 2016 Karyological Studies of Fritillaria (Liliaceae) 137

Fig. 2. Somatic chromosomes of ﬁve species (13 ecotypes) Iranian Fritillaria (2n=2x=24). Scale bars=5 µm.

Discussion species that were studied. Different chromosome types have been reported in Fritillaria. For example, Peruzzi All five Fritillaria species (13 ecotypes) of Iran were et al. (2009) reported four chromosome types in 27 dif- diploid (2n=2x=24). This is in agreement with previous ferent species of Fritillaria, including “m”, “sm”, “st” and studies (Fedorov 1969, Marchant and Macfarlane 1980, “t”, with the “st” and “sm” types appearing at the highest Khaniki 2002a). According to previous reports (Zhang and the lowest frequencies, respectively. In the present et al. 1992, Wang et al. 2006, Gao et al. 2009), the genus study, four chromosome types (“m”, “sm”, “st” and “T”) Fritillaria has more obvious secondary constrictions were identified in five different species of Fritillaria, (dips) in comparison with those reported for other spe- with the “st” and “T” types at the highest and the lowest cies within the Liliaceae. These dips were positioned frequencies, respectively; the “T” chromosome type is on the long arms of Fritillaria chromosomes, and were being reported for the first time in most Iranian Fritil- stable through the population. On the chromosomes laria species (except for ecotypes of the S4 species). of some of the species, 1–3 pairs of satellites were de- The existence of “T” chromosome type in these spe- tected, the majority of which were located on the long cies probably indicates their high degree of evolution arms. Furthermore, this finding is in agreement with in comparison to other species in the present study and that obtained by Khaniki (2002a). On the other hand, in to those reported by Peruzzi et al. (2009) and Jafari et the case of F. zagrica, up to four B chromosomes have al. (2014). In the Peruzzi et al. (2009) report, which in- been reported (Khaniki 2002b) whereas, in the current cluded 61 accessions and 27 distinct species, the average study, no B chromosomes were seen for the Fritillaria TL was 12.8 µm (ranging 8.6–18.8 µm), which was 3% 138 M. Ahmadi-Roshan et al. Cytologia 81(2)

Fig. 3. Haploid chromosomes idiograms of ﬁve species (13 ecotypes) Iranian Fritillaria (2n=2x=24).

Table 2. ANOVA for chromosomal parameters of Fritillaria spp. from Iran.

MS SOV Df S L TL AR r-value RL% F% TCV CI

Sp.1 4 6.96ns 57.76ns 4.71ns 9.79* 9.79* 0.03ns 9.74** 17.28ns 9.79** E2 in Sp. 8 2.31ns 26.25ns 3.20ns 1.98ns 1.98ns 0.04ns 1.91ns 14.89*** 1.98ns Ch3 in (E in Sp.) 143 3.17*** 21.58*** 4.31*** 3.07*** 3.07*** 5.19*** 3.22*** 2.04*** 3.07*** Error 624 0.44 2.58 0.18 0.45 0.45 0.05 0.42 0.47 0.45

ns non-significant difference (p>0.05) and the significances are indicated as *p<0.05, **p<0.01, ***p<0.001. 1Species, 2Ecotype, 3Chromo- some. less than that in the present study. In other words, the TCV within species can be attributed to environmental chromosomes of Iranian Fritillaria are 3% longer than changes (Swanson et al. 1981). those reported by Peruzzi et al. (2009). As a whole, in Karyotypic asymmetry was evaluated based on dif- the present study, remarkable interspecific variations ferences identified using either a qualitative classifica- were clearly identified for four chromosomal parameters tion method or quantitative indices. Thus, according (AR, r-value, F%, CI) and to a lesser extent, intraspe- to Stebbins (1971), karyotypes of different species of cific variation was observed for TCV. Such variation in Iranian Fritillaria studied in the present report were 2016 Karyological Studies of Fritillaria (Liliaceae) 139

Fig. 4. Diagram resulting from principal components analysis (PCA) of Fritillaria species.

Table 3. Mean chromosomal and karyotypic parameters of Fritillaria spp.

Species Species range Parameters Mean Range S1 S2 S3 S4 S5 Min Max

S (µm) 3.48 3.14 2.95 3.28 3.50 3.27 2.95–3.50 S3 S5 L (µm) 11.79 13.04 12.23 12.40 13.19 12.53 11.79–13.19 S1 S5 TL (µm) 15.28 16.19 15.19 15.69 16.69 15.80 15.19–16.69 S3 S5 AR 4.50 6.18 5.52 5.05 5.04 5.26 4.50–6.18 S1 S2 r-value 0.31 0.24 0.25 0.25 0.26 0.26 0.24–0.31 S2 S1 F% 1.89 1.62 1.62 1.75 1.75 1.72 1.62–1.89 S2, S3 S1 TCV (µm3) 105.55 104.71 88.44 118.18 132.27 109.83 88.44–132.27 S3 S5 CI 0.21 0.18 0.18 0.19 0.19 0.19 0.18–0.21 S2, S3 S1 TF% 23.23 19.44 19.45 20.95 20.97 20.80 19.44–23.23 S2 S1 X (µm) 356.90 388.56 364.61 376.64 400.74 377.49 356.90–400.4 S1 S5

CVTL 21.10 23.64 21.38 23.82 19.25 21.84 19.25–23.82 S5 S4 DI 4.90 4.59 4.16 4.99 4.03 4.54 4.03–4.99 S5 S4

A1 0.69 0.75 0.75 0.74 0.73 0.73 0.69–0.75 S1 S2, S3 A2 0.21 0.24 0.21 0.24 0.19 0.21 0.19–0.24 S5 S2, S4 AI 9.22 13.05 8.20 9.87 9.04 9.88 8.20–13.05 S3 S2 AsK% 77.22 80.56 80.50 79.04 79.00 79.26 77.22–80.56 S1 S2 Rec 68.93 66.31 69.32 66.66 73.03 68.85 66.31–73.03 S2 S5 Syi 29.57 24.13 24.24 26.51 26.58 26.21 24.13–29.57 S2 S1 A 0.56 0.64 0.62 0.61 0.61 0.61 0.56–0.64 S1 S2

MCA 56.06 63.92 62.48 61.28 60.84 60.95 56.06–63.92 S1 S2 CG% 23.24 19.44 19.46 20.95 20.97 20.81 19.44–23.24 S2 S1

CVCI 41.78 55.20 38.51 41.42 46.61 44.70 38.51–55.20 S3 S2 Species

S1 S2 S3 S4 S5

ST1 3B 3B 3B 3B 3B KF2 2(4m+4sm+16st), 2m+2sm+12st+8T (4sm+18st+2T), 2m+2sm+20st (2m+4sm+10st+8T), 2(2m+4sm+18st), (2m+2sm+14st+6T) (2m+2sm+20st) (4m+16st+4T), (2m+6sm+16st), (6m+16sm+2T)

1ST: Stebbins classification (1971), 2KF: Karyotype formula located in group 3B, which is classified as a relatively our results, those of F. raddeana and F. zagrica were. In asymmetric, or more developed, karyotype. In other the study of Gao et al. (2009) on two different species of reports (Khaniki 2002a, b, c), karyotypes of F. reuteri Fritillaria (F. cirrhosa, F. unibracteata), the symmetric and F. imperialis were not located in 3B but, similar to groups of Stebbins (3A, 2B and 3B) were reported, even 140 M. Ahmadi-Roshan et al. Cytologia 81(2) though Paszko (2006) describes Stebbins’ classification versity for financial support of this research project. We (1971) as a qualitative method, and therefore less power- acknowledge Professor David Galbraith, Department of ful and flexible in terms of the types of conclusions it Plant Sciences, The University of Arizona, U.S.A. for can provide. Thus, more quantitative indices need to be reviewing the manuscript and for his useful ideas. considered to archive greater measurement correctness. For example, in the current study, in case of TF%, all of References the species were asymmetric, having the mean value of 20.8%, ranging from 19.4% (S2) to 23.2% (S1), which Abedi, R., Babaei, A. and Karimzadeh, G. 2015. Karyological and is 0.2% more than the average (21%) reported by Peruzzi flow cytometric studies of Tulipa (Liliaceae) species from Iran. Plant Syst. Evol. 301: 1473–1484. et al. (2009). The gradual changes in the amounts of Ambrožová, K., Mandáková, T., Bureš, P., Neumann, P., Leitch, I. J., TF% are probably due to chromosomal abnormali- Koblížková, A., Macas, J. and Lysak, M. A. 2011. Diverse ret- ties. The morphological changes in the appearance of rotransposon families and an AT-rich satellite DNA revealed in chromosomes might be a consequence of chromosome giant genomes of Fritillaria lilies. Ann. Bot. 107: 255–268. duplication or translocation (Das et al. 1998). Peruzzi et Arano, H. 1963. Cytological studies in subfamily Carduoideae (Com- positae) of Japan. IX Bot Mag. 76: 32–39. al. (2009) reported the average of DI% as 4.6%, while Darlington, C. D. 1937. Recent Advances in Cytology. P. Blakiston’s this parameter in our study was more or less the same Son & Co., Inc, Philadelphia. (4.5%, ranging from 4% in S5 to 5% in S4). In our pres- Das, A. K., Cohen, P. T. and Barford, D. 1998. The structure of the ent study, the CV% across the species was 21.8%, rang- tetratricopeptide repeats of protein phosphatase 5: Implications ing from 19.3% (S5, the most symmetric) to 23.8% (S4, for TPR-mediated protein–protein interactions. EMBO J. 17: the most asymmetric). Peruzzi et al. (2009) reported the 1192–1199. De Hertogh, A. and Le Nard, M. 1993. Physiological and biochemi- CVCI as 62.6, while that in our study was 44.7 (ranging cal aspects of flower bulbs. In: De Hertogh, A. and Le Nard, M. from 38.5 in S3 to 55.2 in S2), about 29% less. In the (eds.). The Physiology of Flower Bulbs. Elsevier Science Publish- case of AI in the current study, it was 9.9, ranging from ers, Amsterdam, The Netherlands. pp. 53–69. 8.2 (S3) to 13.05 (S2) which is 4% less than that (13.8) Ebadi-Almas, D., Karimzadeh, G. and Mirzaghaderi, G. 2012. Karyo- achieved by (Peruzzi et al. 2009). Our results for the typic variation and karyomorphology in Iranian endemic ecotypes of Plantago ovata Forsk. Cytologia 77: 215–223. CVTL, A1, A2 and AsK% parameters are in agreement Ebrahimie, E., Mohammadi-Dehcheshmeh, M. and Sardari, M. with those reported by Peruzzi et al. (2009), whereas 2006. Fritillaria species are at risk of extinction in Iran: Study Gao et al. (2009) reported 10% fewer for the AsK% on effective factors and necessity of international attention. index. According to our understanding, there is no HortScience 41: 1002–1002. complete karyotype analysis on Iranian Fritillaria spe- Fedorov, A. 1969. Chromosome Numbers of Flowering Plants. Acad. Sci USSR, Leningrad (now St. Petersburg). cies within the literature. In the present report, the five Gao, Y., Zhou, S. and He, X. 2009. Karyotype of four genera in species (13 ecotypes) of Iranian Fritillaria were diploid Liliaceae (s. str.) from Hengduan Mountains of Southwest China. (2n=2x=24), having satellites mostly on their long arms. Acta Bot. Yunn. 31: 399–405. Four chromosome types (“m”, “sm”, “st”, “T”) were identi- Greilhuber, J. and Speta, F. 1978. Quantitative analyses of C-banded fied; the latter is being reported for the first time. Iranian karyotypes, and systematics in the cultivated species of the Scilla siberica group (Liliaceae). Plant Syst. Evol. 129: 63–109. Fritillaria species show low degree of karyotypic sym- Huziwara, Y. 1962. Karyotype analysis in some genera of Com- metry, probably indicating their high degree of evolu- positae. VIII. Further studies on the chromosomes of Aster. AJB. tion in comparison to other species. The present study 116–119. verifies more diversity between Fritillaria species rather Jafari, H., Babaei, A., Karimzadeh, G. and Ahmadi-Roshan, M. 2014. than within ecotypes of the same species. Cytogenetic study on some Fritillaria species of Iran. Plant Syst. As a conclusion, according to our knowledge, there Evol. 300: 1373–1384. Jolliffe, I. T. 1986. Principal Component Analysis, Vol. 487. Springer- is no complete karyotype analysis on Iranian Fritillaria Verlag, New York. species in the literature. In the present report, the stud- Karimzadeh, G., Danesh-Gilevaei, M. and Aghaalikhani, M. 2011. ied five species (13 ecotypes) of Iranian Fritillaria were Karyotypic and nuclear DNA variations in Lathyrus sativus diploid (2n=2x=24), having satellites mostly on their (Fabaceae). Caryologia 64: 42–54. long arms. Four chromosome types (“m”, “sm”, “st”, “T”) Karimzadeh, G., Mousavi, S. H., Jafarkhani-Kermani, M. and Jalali- Javaran, M. 2010. Karyological and nuclear DNA variation in were identified; the latter is being reported for the first Iranian endemic muskmelon (Cucumis melo var. inodorus). time. Hence, Iranian Fritillaria species show low degree Cytologia 75: 451–461. of karyotypic symmetry, probably indicating their deep- Khaniki, G. 2005. Giemsa C-banding studies on interphase nuclei of er evolution in comparison to other species. The present Iranian species of Fritillaria and Rhinopetalum (Liliaceae). Proc. study verifies more diversity between Fritillaria species Natl. Acad. Sci. India Sec. B. 75: 294. Khaniki, G. B. 2002a. Chromosome number of Fritillaria subgenera rather than ecotypes within species. Petilium and Theresia (Liliaceae). Nucleus 45: 6–11. Khaniki, G. B. 2002b. Chromosome number of all Iranian species of Acknowledgements Fritillaria caucasica group (Liliaceae). Nucleus 45: 103–108. Khaniki, G. B. 2002c. Chromosome number of Iranian species of The authors are grateful to Tarbiat Modares Uni- Fritillaria crassifolia group (Liliaceae). Nucleus 45: 109–113. 2016 Karyological Studies of Fritillaria (Liliaceae) 141

Khaniki, G. B. 2003. Fruit and seed morphology in Iranian species Peruzzi, L., Leitch, I. and Caparelli, K. 2009. Chromosome diversity of Fritillaria subgenus Fritillaria (Liliaceae). Pak. J. Bot. 35: and evolution in Liliaceae. Ann. Bot. 103: 459–475. 313–322. Rechinger, K. 1990. Fritillaria L. Flora Iranica 165: 61–76. Lavania, U. and Srivastava, S. 1999. Quantitative delineation of Rix, E. 1977. Fritillaria L. (Liliaceae) in Iran. Iranian J. Bot. 1: karyotype variation in Papaver as a measure of phylogenetic dif- 75–95. ferentiation and origin. Curr. Sci. 77: 429–435. Rix, E. 2001. Fritillaria. A Revised Classification. The Fritillaria Leitch, I., Beaulieu, J., Cheung, K., Hanson, L., Lysak, M. and Fay, Group of the Alpine Garden Society, UK. M. 2007. Punctuated genome size evolution in Liliaceae. J. Evol. Romero-Zarco, C. 1986. A new method for estimating karyotype Biol. 20: 2296–2308. asymmetry. Taxon 35: 526–530. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for Rønsted, N., Law, S., Thornton, H., Fay, M. F. and Chase, M. W. 2005. centromeric position on chromosomes. Hereditas 52: 201–220. Molecular phylogenetic evidence for the monophyly of Fritillaria Li, R. and Shang, Z. 1989. The chromosome observation on five spe- and Lilium (Liliaceae; Liliales) and the infrageneric classification cies of rare plants of China. J. Wuhan Bot. Res. 7: 217–220. of Fritillaria. Mol. Phylogenet. Evol. 35: 509–527. Linnaeus, C. V. 1753. Species Plantarum, 2 Vols. Laurentii Salvii, Shariat, A., Karimzadeh, G. and Assareh, M. H. 2013. Karyology of Holmiae. Iranian endemic Satureja (Lamiaceae) species. Cytologia 78: Maharjan, B. L., Devkota, H. K. and Baral, B. 2012. In vitro anti- 305–312. microbial activity and phytochemical screening of Fritillaria Stebbins, G. L. 1971. Chromosomal Evolution in Higher Plants. Ed- delavayi. Nepal J. Sci. Technol. 12: 85–90. ward Arnold Publisher, London. p. 216. Marchant, C. and Macfarlane, R. 1980. Chromosome polymorphism Ulug, B. V., Korkut, A. B., Sisman, E. and Muratozyavuz, E. in triploid populations of Fritillaria lanceolata Pursh (Liliaceae) 2010. Research on propagation methods of persian Lily bulbs in California. Bot. J. Linn. Soc. 81: 135–154. [Fritillaria Persica Linn.] with various vegetative techniques. Metin, Ö. K., Türktaş, M., Aslay, M. and Kaya, E. 2013. Evaluation of Pak. J. Bot. 42: 2785–2792. the genetic relationship between Fritillaria species from Turkey’s Wang, S., Gao, W., Chen, H. and Xiao, P. 2006. Studies on the mor- flora using fluorescent-based AFLP. Turk. J. Biol. 37: 273–279. phological, thermal and crystalline properties of starches sepa- Mozaffarian, V. 1992. New species and interesting plant records from rated from medicinal plants. J. Food Eng. 76: 420–426. Iran. Iranian J. Bot. 5: 83–90. Watanabe, K., Yahara, T., Denda, T. and Kosuge, K. 1999. Chromo- Noda, S. 1964. Cytology in the genus Fritillaria. I. variations in somal evolution in the genus Brachyscome (Asteraceae, As- karyotypes and B-chromosomes in F. amabilis. Bull. Osaka tereae): Statistical tests regarding correlation between changes in Gakuin University 2: 125–132. karyotype and habit using phylogenetic information. J. Plant Res. Paszko, B. 2006. A critical review and a new proposal of karyotype 112: 145–161. asymmetry indices. Plant Syst. Evol. 258: 39–48. Zhang, D., Sha, J. and Wang, X. 1992. Karyotypes of the genus Peruzzi, L. and Eroglu, H. 2013. Karyotype asymmetry: Again, how Frittilaria from south Anhui. Acta Phytotaxon. Sin. 30: 62–69. to measure and what to measure? Comp. Cytogen. 7: 1–9.