Karyological and flow cytometric studies of

Tulipa (Liliaceae) species from Iran

Rahele Abedi, Alireza Babaei & Ghasem

Karimzadeh

Plant Systematics and Evolution

ISSN 0378-2697
Volume 301
Number 5

Plant Syst Evol (2015) 301:1473-1484

DOI 10.1007/s00606-014-1164-z

1 23
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 Author's personal copy
Plant Syst Evol (2015) 301:14731484
DOI 10.1007/s00606-014-1164-z
ORIGINAL ARTICLE
Karyological and flow cytometric studies of Tulipa (Liliaceae)
species from Iran
Rahele Abedi Alireza Babaei Ghasem Karimzadeh
Received: 7 January 2014 / Accepted: 13 October 2014 / Published online: 9 November 2014
Springer-Verlag Wien 2014
Abstract Chromosomal and karyotype parameters and
genome size were examined in 22 Iranian populations of
nine different Tulipa species, using squash technique and
1 % (w/v) aceto-orcein stain. Flow cytometric studies were
performed using PI staining method and Vicia faba cv.
Inovec (2C DNA = 26.9 pg) as a reference standard. Most
species were diploid (2n = 2x = 24), having three chro-
mosome types (m, sm, st). The chromosome length currently the most important bulbous geophyte in the world
(TL) varied from 6.4 lm (S2) to 12.90 lm (S3). Total
chromosome volume (TCV) ranged from 32.9 lm3 (S2) to
66.0 lm3 (S8). Statistical analyses confirmed more intra-
specific chromosomal and genome size variations than
interspecific. S1E2, S2E1 and S5E2, with the highest in-
trachromosomal (MCA) and relatively high interchromo-
somal asymmetry (CVCL), indicated higher asymmetry in
the karyotype. The genome size in Tulipa species of Iran is
being reported for the fist time. The mean 2C DNA was
54.52 pg, and ranged from 77.40 in S6E2 to 31.51 pg in
S9E2. Significant positive correlations were found between
2C DNA and either TL or TCV.
Handling editor: Frank H. Hellwig.
R. Abedi  A. Babaei (&)
Department of Horticultural Sciences, Faculty of Agriculture,
Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran
e-mail: arbabaei@modares.ac.ir
R. Abedi
e-mail: ra_abedy@yahoo.com
G. Karimzadeh
Department of Plant Breeding and Biotechnology,
Faculty of Agriculture, Tarbiat Modares University,
P.O. Box 14115-336, Tehran, Iran
e-mail: karimzadeh_g@modares.ac.ir
Keywords Tulipa  Karyotype  Chromosome 
Flow cytometry  DNA 2C-value
Introduction
The genus Tulipa L., originated from Central Asia, is
and contains around 35 species in the subg. Tulipa with
glabrous stamens, and 20 species in the subg. Eriostemones
with bossed, usually hairy stamens (Booy and Van Ra-
amsdonk 1998; Van Tuyl and Van Creij 2006; Christen-
husz et al. 2013). Of these, 18 species including T.
ulophylla Wendelbo, T. montana Lindl., T. humilis Herbert,
T. clusiana DC., T. stylosa Stapf, T. wilsoniana Hoog, T.
kuschkensis B.Fedtsch., T. micheliana Hoog., T. lehman-
niana Merckl, T. harazensis Rech.f., T. urumiensis Stapf,
T. biflora Pall., T. linifolia Regl, T. sogdiana Bunge, T.
schrenkii Regl, T. hoogiana B.Fedtsch., T. florenskyi Wo-
ron. and T. aucheriana Baker occur naturally in different
regions of Iran (Mozaffarian 1996). Thus, high genetic
diversity of Tulipa is expected due to the wide range of
geographical conditions and diverse environments. Tulips
are widely grown for bulb production, cut flowers, flow-
ering potted plants, and landscaping (Van Tuyl and Van
Creij 2006). In plant taxonomy, genetic studies and
breeding information about karyotypes can be useful in
species identification and analysis of hybrid populations
(Anjali and Srivastava 2012). Besides, newly developed
analytical tools such as flow cytometry (FCM) has been
performed for various aspects, e.g., for estimating ploidy
level and genome size for a wide range of plant community
and for physiological, ecological, taxonomical, cell and
molecular biology, plant breeding and genome evolution
studies (Bennett 1998; Mahdavi and Karimzadeh 2010;
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1474
Karimzadeh et al. 2010, 2011; Naghavi et al. 2013).
Monoploid genome size refers to the amount of DNA of
one chromosome set, 1-Cx-value, with chromosome base
number x as described by Greilhuber et al. (2005), Mahdavi
and Karimzadeh (2010) and Karimzadeh et al. (2011).
Leitch et al. (2007) reported 2C DNA = 30.25 pg for Tu-
lipa montana (2n = 2x = 24). Afterwards, the genome
size of a wide range of Tulipa species was studied in the
Netherlands, showing a range of genome size from diploid
30 pg to tetraploid 123 pg (Zonneveld 2009). A karyotype
study was previously performed on some Iranian species of
Tulipa (Sheidai et al. 2002). Yet, little is known about
genome size and ploidy levels of tulip species of Iran,
precluding a clear understanding of evolutionary processes.
Therefore, the main objective of this study was to inves-
tigate the chromosomal parameters and DNA 2C-value
variations between tulip populations of Iran.
Materials and methods
Plant materials
The bulbs of 22 populations of nine different Tulipa spe-
cies belonging to the two subgenera of Tulipa and Erios-
temones were collected from over 15,000 km exploratory
trips in Iran in spring 2011 and used in this study (Table 1).
The population codes and geographical descriptions (lati-
tude, longitude and altitude) are shown in Table 1.
Chromosome analysis
At first, healthy bulbs were placed in a 1:3 (v/v) mixture of
peat moss:perlite in a growth chamber under dark conditions
at 810 C. The growth medium was kept moist with distilled
water until shoot emergence. For the cytological prepara-
tions, actively around 1 cm long growing roots were removed
and placed in 0.05 % colchicine at room temperature (RT) for
4 h in the dark to induce metaphase arrest. Then, the samples
were incubated in 0.1 N (0.56 w/v) KCl for 60 min at RT. The
root samples were fixed in freshly 3:1 (v/v) absolute etha-
nol:glacial acetic acid at least for 24 h at 4 C and followed
by keeping in 70 % (v/v) aqueous absolute ethanol at 4 C
until required (Jafari et al. 2014). The fixed roots were then
washed in dsH2O, hydrolyzed in 1 M HC1 for 10 min at RT
followed by staining in 1 % (w/v) aceto-orcein for 3 h at RT.
The five well-spread monolayer metaphase plates from dif-
ferent individuals were analyzed per tulip populations. High-
resolution microscopic digital photographs were taken using
an Olympus BX50 (Olympus Optical Co., Ltd., Tokyo,
Japan) microscope equipped with an Olympus DP12 digital
camera. Nine chromosomal parameters were either measured
as long arm (L) and short arm (S) lengths, or calculated as total
123
R. Abedi et al.
chromosome length (TL) and volume (TCV), arm ratio (AR),
relative length of chromosome (RL) and centromeric index
(CI). Their formulas are shown as follows:
L
TL L S Arm ratio AR TCV
S
2 S
pr  TL CI
TL
The determination of positions of centromeres as well as
the measurements of S, L and TCV was performed, using
MicroMeasure 3.3 (available from the MicroMeasure Web
site). Moreover, for analysis of karyotype asymmetry, the
following parameters were estimated: (1) coefficient of
variation of chromosome length (CVCL); (2) mean cen-
tromeric asymmetry (MCA); and (3) Stebbins asymmetry
categories. A review of each method and their formulas are
given in Stebbins (1971), Paszko (2006), Peruzzi et al.
(2009), Zuo and Yuan (2011) and Peruzzi and Eroglu
(2013). For both CVCL and MCA, the larger the value, the
greater is the asymmetry in the karyotype.
Flow cytometric analyses
The 2C-value of each tulip population was estimated using
FCM. Nuclear suspensions were obtained from 1 cm2 pieces
of young and well-developed leaves. FCM studies were
performed using PI (Propidium Iodide) staining method.
Vicia faba cv. Inovec leaves with a 2C DNA value of 26.9 pg
were used as an internal reference standard. The nuclei sus-
pension was analyzed by the BD FACSCanto ll flow
cytometer (BD Biosciences, Bedford, MA, USA), using BD
FACSDivaTM Software. Data were then transferred to
Flowing Software version 2.5.0 (Cell Imaging Core, Turku
Centre for Biotechnology) to be editable in Partec FloMax
ver. 2.4e. Gating region range was performed on FCM his-
tograms using the latter software. Healthy fresh young leaves
of the standard reference and of the tulip sample were
chopped with a sharp razor blade in ice-cold nucleic extrac-
tion buffer of Partec CyStain PI absolute P, Code No.
05-5022, Germany. The chopped leaves were filtered through
a 30 lm Partec green nylon mesh. One ml of staining buffer,
12 ll of PI solution and 6 ll of RNase stock solution were
added to each sample. The measurements of relative fluo-
rescence intensity of stained nuclei were performed on a
linear scale. At least 5,000 nuclei were typically analyzed for
each sample. The absolute DNA amount of a sample was
calculated based on the values of the G1 peak means (Dolezel
et al. 2003, 2007; Dolezel and Bartos 2005) as follows:
Sample 2C DNA (pg) = (Sample G1 peak mean/Stan-
dard G1 peak mean) 9 Standard 2C DNA (pg). Monoploid
genome size in the form of base pair was calculated based
on a converting formulas proposed by Dolezel et al. (2003),
when 1 pg of DNA represents 978 mega base pairs (Mbp).
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Cytogenetic and flow cytometry analyses of Tulipa 1475

Table 1 Tulip populations with their species and geographical description

Family and Subgenus Population Species Local collection locations Latitude Altitude
genus name name codes longitude (m)

LiliaceaeTulipa Tulipa S1E1 T. montana Boghati, Hamedaan, Iran 35320 N 1,750

0
4844 E
S1E2 T. montana Boghati, Hamedan, Iran 35320 N 2,235
48430 E
S1E3 T. montana Khoshyeylagh, Golestan, Iran 36500 N 979
55220 E
S1E4 T. montana Padegan Haraz, Tehran, Iran 35470 N 1,697
51570 E
S2E1 T. montana var. chrysanta Ahovan, Semnan, Iran 318450 N 1,430
53370 E
S3E1 T. systola Golestan kooh, Esfahan, Iran 3390 N 2,756
0
5024 E
S3E2 T. systola Chalruqani, Charmahalbakhtiari, Iran 3280 N 2,633
50220 E
S4E1 T. micheliana Shahrud, Semnan, Iran 36150 N 1,279
5500 E
S4E2 T. micheliana Jandaq, Semnan, Iran 35210 N 1,799
0
5427 E
S4E3 T. micheliana Dare Alme, Golestan, Iran 37210 N 1,366
56120 E
S5E1 T. hoogiana Park Meli, Golestan, Iran 36500 N 979
55220 E
S5E2 T. hoogiana Khoshyeylagh, Golestan, Iran 36590 N 1,363
55180 E
S6E1 T. schrenkii Salehabad, Khorasan, Iran 35430 N 913
6130 E
S6E2 T. schrenkii Miane, Azarbayjanesharqi, Iran 37410 N 1,921
0
4730 E
Eriostemones S7E1 T. humilis Dashtelale, Lorestan, Iran 33230 N 1,701
49410 E
S7E2 T. humilis Miane, Azarbayjanesharqi, Iran 37410 N 1,914
47300 E
S8E1 T. biflora Sorkhehesar, Azarbayjanesharqi, Iran 37420 N 1,616
0
4741 E
S8E2 T. biflora Kohenova, Kermanshah, Iran 34190 N 2,000
4670 E
S8E3 T. biflora Tange Sayad, Charmahalbakhtiari, Iran 32130 N 2,323
5130 E
S9E1 T. biebersteiniana Boghati, Hamedan, Iran 35320 N 1,750
48440 E
S9E2 T. biebersteiniana Sefidkhani, Markazi, Iran 33580 N 2,472
49330 E
S9E3 T. biebersteiniana Khoshyeylagh, Golestan, Iran 36500 N 979
0
5522 E

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1476 R. Abedi et al.

S1E3

S1E1
S1E2

S3E2

S1E4 S2E1 S3E1

S5E2

S4E1 S4E2 S5E1

S6E1 S6E2 S7E1 S7E2

S8E1

S7E3 S8E2 S8E3

S9E1

S9E2 S9E3

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Cytogenetic and flow cytometry analyses of Tulipa
b Fig. 1 Somatic chromosomes of 22 tulip populations of nine Tulipa
species: S1 (T. montana), S2 (T. montana var. chrysanta), S3 (T.
systola), S4 (T. micheliana), S5 (T. hoogiana), S6 (T. schrenkii), S7
(T. humilis), S8 (T. biflora), S9 (T. biebersteiniana). Bars = 5 lm.
The asterisks and arrows show the satellite and the B chromosomes,
respectively
Statistical analyses
After initially testing the normal distribution of data, the
cytological and flow cytometric data were subjected to
analysis of variance (ANOVA), using the GLM procedure
of SAS (SAS Institute Inc 2002) for a nested design with
five replications. The appropriate error term for species and
population F tests was population (species) and replication
(population), respectively. Within each species, to deter-
mine population effect on 2Cx DNA content, flow cyto-
metric data were subjected to a completely randomized
design (CRD) ANOVA. Differences between means were
determined by the least significant difference (LSD).
Multivariate statistical analysis was carried out in Minitab
software package (Minitab ver. 16.1.0, Minitab Ltd.). A
cluster analysis on chromosomal parameters was per-
formed, using the UPGMA (Unweighted Pair Group
Method Arithmetic Mean) and the Euclidean distance to
evaluate variations and similarities among the populations.
The raw data matrices were standardized to a common
scale by subtracting the means and dividing by the standard
deviation prior to perform the cluster analysis. Simple
scatter plot between MCA and CVCL was produced to fur-
ther explore relations among the tulip populations. Peruzzi
and Eroglu (2013) showed definitively what and how to
measure to correctly infer karyotype asymmetry, by pro-
posing to couple CVCL with MCA in a new way by means
of a bidimensional scatter plot, where the two asymmetry
estimators are put in the x and y axes and points represent
each sample.
Results
Among 22 tulip populations examined, 21 were diploid
(2n = 2x = 24). Interestingly, the chromosome number
2n = 2x = 26 was evidenced in S6E2 (Fig. 1).
Karyotypes of somatic complement and the idiograms of
haploid complement of studied tulip populations are shown in
Figs. 1 and 2, respectively. There were significant differ-
ences among populations within species for S, L, TL, arm
ratio (AR), TCV and CI (Table 2). The mean value of TL was
9.30 lm and varied from 6.27 lm (S1E2) to 13.08 lm
(S3E1). The mean TCV was 54.31 lm3 and ranged from
31.94 lm3 (S5E2) to 80.73 lm3 (S7E2). The mean CI of the
complement was 25 and varied from 21 (S1E2) to 29 (S6E2).
1477
ANOVA showed that species had significant differences in
terms of short (S) and long (L) arms of the chromosomes, total
chromosome length (TL) and total chromosome volume
(TCV). The lowest values for such parameters were obtained
in S2, while the highest values were found in either S3 or S7
(Table 3). In addition, according to karyotype symmetry
parameters examined, studied Tulipa species showed differ-
ent symmetrical groups (Table 3). As Table 4 shows, the
highest value of CVCL was identified in either S8E2 or S9E1
(29 %) and the lowest value was in S8E3 (16 %). The highest
and the lowest values of MCA were evidenced in S1E2 (59 %)
and S3E1 (45 %), respectively. On the other hand, karyotypes
of the most populations (20) were classified within A4 cate-
gory of qualitative Stebbins classification (Stebbins 1971;
Table 4) which corresponds to relatively symmetrical kary-
otypes. For more detailed studies of asymmetry, mean MCA
values were plotted against the mean values for CVCL. This
scatter plot presents five groups of populations (Fig. 3).
S1E2, S2E1 and S5E2, with the highest intrachromosomal
(MCA) and relatively high interchromosomal asymmetry
(CVCL), indicated higher asymmetry in the karyotype.
Among species, S2 and S5 showed the most asymmetrical
karyotypes (Table 3). Moreover, there was a pair of satellites
(ranging 2.012.72 lm) on long arms of the first chromo-
some in S1E1 and on the eighth chromosome in S9E3
(Table 4; Fig. 2).
In the present report, supernumerary chromosomes (B
chromosomes) were identified in two populations of S3E1
and S6E2 (Fig. 1), both from subg. Tulipa.
Cluster analysis of chromosome parameters indicated
the presence of five major population groups (Fig. 4). The
first group included six populations (S1E1-2, S2E1, S5E2
and S9E1-2); the second group, seven populations (S1E3-4,
S7E1-2, S8E1-2, S9E3); the third group, six populations
(S4E1-3, S5E1, S6E1, S8E3); the fourth group, two pop-
ulations (S3E1-2); and the fifth group, only S6E2.
Figure 5 shows the flow cytometric histograms of nuclear
DNA amounts and their resultant data for 18 tulip populations
belonging to the eight examined species are presented in
Table 3. Note that bulbs of four populations (S9E3, S4E1-3)
did not produce enough leaves to measure their genome size.
There were highly significant differences (P \ 0.001) between
tulip populations for nuclear DNA amount. The coefficients of
variation of this amount for all populations varied between 2
and 5 %. The mean 2C DNA value of studied Iranian 16
diploid tulip populations was (2n = 24 = 50.25 pg) and
ranged from 31.58 pg in S6E1 to 73.86 pg in S8E2 (Table 5).
This value for a diploid S3E1 was (2n = 24 ? 2B chromo-
some = 73.40 pg). On the other hand, the 2C DNA value of a
diploid S6E2 was (2n = 26 ? 2B chromosome =
77.40 pg). Comparing populations within species, in S1
species (4 populations), ranged from 31.67 pg (S1E2) to
51.36 pg (S1E3; 38.3 % increase; P \ 0.01). In S3 species (2
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1478 R. Abedi et al.

Fig. 2 Haploid chromosome S1E1 S1E2 S1E3

12 12 12

idiograms of 22 tulip 9 9 9

populations of nine Tulipa 6 6 6

3 3 3
species: S1 (T. montana), S2 (T. 0 0 0
montana var. chrysanta), S3 (T. -3
1 2 3 4 5 6 7 8 9 10 11 12
-3
1 2 3 4 5 6 7 8 9 10 11 12
-3
1 2 3 4 5 6 7 8 9 10 11 12

systola), S4 (T. micheliana), S5 -6 -6 -6

-9 -9
(T. hoogiana), S6 (T. schrenkii), -9

-12 -12 -12

S7 (T. humilis), S8 (T. biflora), -15 -15 -15

S9 (T. biebersteiniana) 12 S1E4 12 S2E1 S3E1

9 9

6 6

3 3

0 0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
-3 -3

-6 -6

-9 -9

-12 -12

-15 -15

S3E2 S4E1 S4E2

Chromosome length (m)

S4E3 S5E1 S5E2

S6E1 S6E2 12 S7E1

9

0
1 2 3 4 5 6 7 8 9 10 11 12
-3

-6

-9

-12

-15

S7E2 S8E1
12 12 S8E2
9 9

6 6

3 3

0 0
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
-3 -3

-6 -6

-9 -9

-12 -12

-15 -15

S8E3 S9E1 S9E2

Chromosome length (m)

S9E3

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Cytogenetic and flow cytometry analyses of Tulipa 1479

Table 3 Mean (standard deviation) chromosomal and karyotype parameters of nine Tulipa species: S1 (T. montana), S2 (T. montana var. chrysanta), S3 (T. systola), S4 (T. micheliana), S5
Table 2 Chromosomal parameters of 22 tulip populations of nine

Species of range
Max
Tulipa species: S1 (T. montana), S2 (T. montana var. chrysanta), S3

S6
S3

S2
S3

S7
S3
S2
S5
(T. systola), S4 (T. micheliana), S5 (T. hoogiana), S6 (T. schrenkii),
S7 (T. humilis), S8 (T. biflora), S9 (T. biebersteiniana)
Population S L TL AR TCV CI

Min

S2
S2

S3
S2

S2
S2
S4
S3
S1

Mean

2.27
6.97

3.28
9.01

0.25
54.17

23.58
50.49
E1 1.60b 5.79b 7.39b 3.75b 35.2b 0.23b
c b c a b
E2 1.25 5.02 6.27 4.11 38.4 0.21c
a a a c b
E3 2.35 6.84 9.19 2.97 40.3 0.26a

1.89 0.6
6.05 1.2

3.39 0.4
7.94 1.8

0.24 0.0
24.82 1.4
51.91 3.7
48.45 10
a a a c a
E4 2.53 7.26 9.80 2.90 51.8 0.26a
S2
E1 1.31 5.08 6.39 3.94 32.9 0.22

S9
S3

2.52 0.4
7.27 0.4

3.01 0.2
9.79 0.8

0.26 0.0
22.13 3.3
48.51 3.5
3.50a 9.58a 13.08a 2.77a 68.1a 0.27a

65.98 15
E1
a a a a b
E2 3.31 9.41 12.71 2.87 62.1 0.26a
S4

S8
E1 2.38a 7.67a 10.04a 3.32a 41.7b 0.24a
b b b a a
E2 1.91 6.35 8.26 3.50 63.5 0.23a

2.49 0.1
7.17 0.2

2.99 0.0
9.66 0.2

78.79 2.7
0.26 0.0
25.86 0.1
47.80 0.1
a a a a ab
E3 2.49 8.00 10.49 3.25 54.3 0.24a
S5

S7
E1 2.16a 6.62a 8.78a 3.16b 44.7a 0.25a
E2 1.54b 6.21a 7.74b 4.12a 31.9b 0.21b

2.83 0.9
8.03 1.0

2.98 0.6
10.86 2.0

64.07 6.5
0.26 0.0
24.77 3.8
48.48 7.4
S6
E1 2.17b 7.33b 9.50b 3.44a 47.8b 0.23b
a a a b a
E2 3.44 8.68 12.12 2.55 79.1 0.29a
S6

S7
2.44a 7.05a 9.48a 3.01a 76.9a 0.26a
1.85 0.4
6.41 0.3

3.64 0.7
8.26 0.7

38.33 9.0
0.23 0.0
22.44 4.1
54.46 5.5
E1
E2 2.54a 7.29a 9.83a 3.00a 80.7a 0.26a
(T. hoogiana), S6 (T. schrenkii), S7 (T. humilis), S8 (T. biflora), S9 (T. biebersteiniana)

S8
S5

E1 2.83a 7.66a 10.50a 2.85b 69.1a 0.27a

a a a b a
E2 2.63 7.36 10.00 2.90 79.4 0.26a
2.26 0.3
7.34 0.8

3.36 0.1
9.60 1.1

0.24 0.0
21.21 1.3
52.67 0.6
53.17 11

b a a a a
E3 2.10 6.79 8.89 3.29 49.4 0.24b
S9
1.65b 5.36b 7.01b 3.42ab 47.4ab 0.24ab
S4

E1
E2 1.45b 5.36b 6.81b 3.77a 38.5b 0.22b
3.4 0.1
9.49 0.1
12.90 0.2

65.08 4.2

47.00 0.0
2.82 0.0

0.27 0.0
23.64 1.0

E3 2.58a 7.43a 10.02a 2.99b 59.4a 0.26a

For a given species, means within a column followed by the same
letter are not significantly different according to the LSD at the 0.05
S3

probability level
1.31 0.3
5.08 1.6
6.39 1.7

32.89 6.0

51.31 8.2
3.94 1.3

0.22 0.0
26.55 4.1

populations), S3E1 showed 26.5 % more increase

S2

(P \ 0.001) in DNA amount than S3E2 (Table 5). In S6

species (2 populations) was ranging from 31.58 pg (S6E1) to
1.93 0.6

77.40 pg (S6E2; 59.47 % increase; P \ 0.001). In S8 spe-

6.23 1.0
8.16 1.6

41.42 7.2

52.26 5.1
3.43 0.6

0.24 0.0
24.06 3.8

cies (3 populations), DNA content varied from 54.61 pg

Species

(S8E1) to 73.86 pg (S8E2; 27.4 % increase; P \ 0.001). In

S1

S9 species (2 populations), DNA content was ranging from

31.51 pg (S9E2) to 56.30 pg (S9E1; 43.8 % increase;
Parameters

TCV(lm3)

CVCL (%)

P \ 0.01). Furthermore, among geographical, cytological

MCA (%)
TL (lm)
L(lm)
S(lm)

and karyotype parameters, TL, TCV and MCA had significant

AR

CI

correlation with 2C DNA (Fig. 6).

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1480 R. Abedi et al.

Table 4 Some karyotypic Population Stebbins type Karyotype formula CVCL % MCA % SAT
symmetry formula of 22 tulip
populations of nine Tulipa S1E1 3A 2sm ? 2 m ? 20st 27.49 53.96 1L
species: S1 (T. montana), S2 (T.
S1E2 4A 22st ? 2sm 25.76 58.72
montana var. chrysanta), S3 (T.
systola), S4 (T. micheliana), S5 S1E3 4A 8st ? 16sm 23.54 48.53
(T. hoogiana), S6 (T. schrenkii), S1E4 4A 8st ? 16sm 19.45 47.82
S7 (T. humilis), S8 (T. biflora), S2E1 4A 20st ? 4sm 26.55 57.11
S9 (T. biebersteiniana)
S3E1 4A 4st ? 20sm 22.24 45.5
S3E2 4A 4st ? 20sm 25.05 47
S4E1 4A 16st ? 8sm 17.81 52.38
S4E2 4A 18st ? 6sm 22.14 53.35
S4E3 4A 18st ? 6sm 23 52.28
S5E1 4A 10st ? 14sm 19.5 50.55
S5E2 4A 20st ? 4sm 25.39 58.36
S6E1 4A 18st ? 6sm 21.98 53.71
S6E2 3A 2st ? 22sm ? 2m 27.57 43.25
S7E1 4A 8st ? 16sm 22.15 47.7
S7E2 4A 10st ? 14sm 22.97 47.9
S8E1 4A 4st ? 20sm 21.08 45.9
S8E2 4A 6st ? 18sm 29 47.19
S8E3 4A 16st ?8sm 16.06 52.45
S9E1 4A 16st ? 8sm 29 51.83
S9E2 4A 18st ? 6sm 23.45 55.62
S9E3 4A 10st ? 14sm 21.65 48.27 8L

30.0
S8E2 S9E1
7.21
S6E2 S1E1
27.5 S2E1
S1E2
S3E2 S5E2
25.0
Distance

4.81
S1E3 S9E2
CVCL

S7E2 S4E3
S3E1
22.5 S7E1 S4E2
S6E1
S9E3
S8E1

20.0 S1E4 S5E1 2.40

S4E1
17.5
S8E3
0.00
15.0
S1E1
S9E2
S9E1
S1E2
S2E1
S5E2
S1E3
S1E4
S9E3
S7E1
S7E2
S8E2
S8E1
S4E1
S6E1
S4E3
S5E1
S8E3
S4E2
S3E1
S3E2
45 50 55 60 S6E2
MCA Populations

Fig. 3 Scatter plot of interchromosomal and intrachromosomal
asymmetries (CVCL and MCA, respectively) of tulip populations of
nine Tulipa species: S1 (T. montana), S2 (T. montana var. chrysanta),
S3 (T. systola), S4 (T. micheliana), S5 (T. hoogiana), S6 (T.
schrenkii), S7 (T. humilis), S8 (T. biflora), S9 (T. biebersteiniana).
The oval or circle around the centroid indicates the 95 % confidence
interval for the class
Discussion
We studied 22 tulip populations from Iran in this work.
The chromosome number for 21 tulip populations exam-
ined was 2n = 24. These results are in agreement with
Fig. 4 Dendrogram showing the phenetic relationships among the
studied populations of tulip of nine Tulipa species: S1 (T. montana),
S2 (T. montana var. chrysanta), S3 (T. systola), S4 (T. micheliana), S5
(T. hoogiana), S6 (T. schrenkii), S7 (T. humilis), S8 (T. biflora), S9 (T.
biebersteiniana)
those reported by Ramanna et al. (2012). In addition, the
chromosome number 2n = 26 was recorded for the sec-
ond time. To our knowledge, only the findings of Newton
(1926) has indicated such a chromosome number
2n = 2x = 26 for a tulip. Probably, a hyper-aneuploidy or
tetrasomy may be involved, but chromosome banding is
necessary to prove this hypothesis. When there is an

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Cytogenetic and flow cytometry analyses of Tulipa 1481

S1E1 S1E2 S1E3 S1E4

S2E1 S3E1 S3E2 S5E1

S5E2 S6E1 S6E2 S7E1

Number of nuclei

S7E2 S8E1 S8E2 S8E3

S9E1 S9E2

Relative nuclear DNA content

Fig. 5 Flow cytometric histograms of 2Cx DNA content of 18 tulip S7 (T. humilis), S8 (T. biflora), S9 (T. biebersteiniana). The left peaks
populations of eight Tulipa species: S1 (T. montana), S2 (T. montana refer to G1 of tulip samples and the right peaks to G1 of the Vicia
var. chrysanta), S3 (T. systola), S5 (T. hoogiana), S6 (T. schrenkii), faba var. Inovec (2C DNA = 26.9 pg) reference standard

increase in the number of chromosomes compared to the
chromosome number of an organ, then the condition is
called as hyper-aneuploidy. In this study, the hyper-
aneuploidy may be two chromosome pairs in triplicate
which is also classified as double trisomy. The variability
in chromosome number can be due to the events of
polyploidy and aneuploidy (Felix et al. 2011a). In the
present study, Tulipa species were less variable and
polyploidy and aneuploidy events may be related to their
karyotype evolution (Felix et al. 2011b; Urdampilleta
et al. 2013).
In the present study, scatter plot presents five groups of
populations. As a whole, based on the two parameters, MCA
and CVCL, Tulipa species are characterized by symmetrical
karyotypes. In general, a symmetric karyotype does not
necessarily imply primitivity. Indeed, an independent
source of information is always needed to assess the
direction in karyotype changes (Peruzzi et al. 2009; Peruzzi

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1482 R. Abedi et al.

Table 5 Information obtained Populations 2n Ploidy level B chromosome Mean 2Cx DNA (pg) 1Cx monoploid
from flow cytometric evaluation Se genome size (Mbp)
of Iranian tulip populations of
nine Tulipa species: S1 (T. S1
montana), S2 (T. montana var.
E1 24 2x 33.03 0.01b 16151.67
chrysanta), S3 (T. systola), S4
(T. micheliana), S5 (T. E2 24 2x 31.67 0.16c 15486.63
a
hoogiana), S6 (T. schrenkii), S7 E3 24 2x 51.36 0.17 25115.04
(T. humilis), S8 (T. biflora), S9 E4 24 2x 32.87 0.05b 16073.43
(T. biebersteiniana)
S1 mean 37.23 18206.69
S2
E1 24 2x 55.35 0.30 27.66.15
S3
E1 24 2x 2 73.40 0.23a 35892.60
E2 24 2x 52.62 0.12b 25.761.18
S3 mean 63.01 30811.89
S5
E1 24 2x 57.13 3.26a 27961.02
E2 24 2x 59.85 0.01a 29266.62
S5 mean 58.51 28611.39
S6
E1 24 2x 31.58 0.05b 15442.62
a
E2 26 2x 2 77.40 0.09 37848.60
S6 mean 54.49 26645.61
S7
E1 24 2x 58.65 2.13a 28679.85
E2 24 2x 62.09 1.15a 30362.01
S7 mean 60.37 29520.93
S8
E1 24 2x 54.61 0.39c 26704.29
E2 24 2x 73.86 018a 36117.54
E3 24 2x 61.42 0.19b 30034.38
S8 mean 63.29 30948.81
S9
For a given species, means
within a column followed by the E1 24 2x 56.30 0.36a 25731.18
same letter are not significantly E2 24 2x 31.51 0.20b 27530.70
different according to the LSD S9 mean 43.9 21467.10
at the 0.05 probability level

and Eroglu 2013). Seijo and Fernandez (2003) pointed out
that the utilization of asymmetry indices for the estab-
lishment of the evolutionary relationships may not be
straightforward. In relation to the number and position of
satellites in tulip populations of Iran, there were differences
between the findings of this study and those of Sheidai
et al. (2002), probably due to chromosome polymorphism
(Anjali and Srivastava 2012). Generally, some differences
in karyotype formula and asymmetry indices found among
species suggest that structural changes may have contrib-
uted to the diversification of the genus (Seijo and Fernan-
dez 2003). As aforementioned, S2 and S5 have a greater
degree of asymmetry, perhaps because of the presence of
st chromosomes in more complements. Within each
species, populations from higher altitude had more st
chromosomes than those from lower elevations (Tables 1
and 4). Similar results were also observed by Zhang et al.
(2010) when the karyotype formula was arranged alongside
altitude biotopes in Campanumoea (Campanulaceae
family).
To the best of our knowledge, there has been no cyto-
logical report of the presence of B chromosomes in Tulipa
species of Iran; hence, this study is the first report in Iranian
tulip. B chromosomes may play either a positive role as an
adaptive function or a negative role as a parasitic genome
(Felix et al. 2011b; Jones 2012). In the present study, the B
chromosome seems to have some positive adaptive advan-
tages in S3 and S6 due to improvements in some morpho-
logical traits such as plant height, tepal and leaf area and the
size of reproductive organs (data not shown).

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Cytogenetic and flow cytometry analyses of Tulipa 1483

90 2x = 56.70 pg (T. biebersteiniana), 2x = 48.80 pg (T.

2Cx DNA =17.43 + 3.87 * (1.57) TL
80
2Cx DNA amount (pg)

70
hoogiana) and 2x = 61.50 pg (T. schrenkii) (Zonneveld
60 2009; Bennett and Leitch 2012). Furthermore, significant
50 correlation between TL and TCV and 2C DNA may show
40
that there is homogeneity of the karyotypes between pop-
30
20 ulations within each of the species (Sheidai et al. 2002). In
10 agreement with our data, such a relationship between
0
6 7 8 9 10 11 12 13 14 nuclear DNA content and chromosome parameters has been
TL reported in Vicia (Naranjo et al. 1998) and in Thymus spe-
90
2Cx DNA =24.10 + 0.54 ** (0.17) TCV
cies (Mahdavi and Karimzadeh 2010). Overall, an analysis
80
2Cx DNA amount (pg)

70
of the relationship between 2C DNA and MCA showed
60 these two parameters were negatively correlated (Fig. 6).
50 This result agree broadly with that previously reported by
40
30
Peruzzi et al. (2009) based on an analysis at genus level,
20 namely that an increase in genome size in Amana, Ery-
10 thronium and Tulipa through the addition of equal amounts
0
25 35 45 55 65 75 85 of DNA to each chromosome regardless of length, led to a
TCV decrease in both their inter- and intrachromosomal asym-
90
metries. In the present report, there was no significant
2Cx DNA =143.90 - 1.79 * (0.67) MCA relationship between 2C DNA and altitude (r = 0.16,
2Cx DNA amount (pg)

80
70
60
50
40
30
20
40 45 50
M CA
Fig. 6 Linear relationship between 2Cx DNA amount and TL
(r = 0.526*) and TCV (r = 0.616**) and MCA (r = 0.554*) of tulip
populations
As Figs. 3 and 4 show, the populations belonging to
each individual species are scattered across the entire
diversity space or across the clusters in the dendrogram.
Most of this difference can be attributed to geographical
differences and weather conditions, as these species and
populations were distributed to very wide areas in Iran as
shown in Table 1. Zhang et al. (2010) stated that in the
experiments for karyotype comparisons in related species,
it is more useful to choose population from similar altitudes
to reduce the degree of error.
The highest correlations (0.99) occurred for L vs. TL
and for CI vs. AR. This indicates that changes in chro-
mosome length related to the changes of L. In relation to
the genome size variation, one of the reasons for smaller
genome sizes could have resulted from the transfer of DNA
sequences from the nuclear into mitochondria and chloro-
plasts (Karimzadeh et al. 2010). In the present study, the
nuclear 2C DNA amount is in agreement with the previous
study which indicated the approximate 2C DNA values of
2x = 32 pg (T. montana), 2x = 65 pg (T. humilis),
2x = 43 pg (T. biflora), 2x = 56.10 pg (T. systola),
P [ 0.05). This result is contrary to the findings of Ray-
burn and Auger (1990) who observed a close relationship
between altitude and genome size of Zea mays subsp.
mays. As mentioned above, S3 and S6 had the highest
values for some morphological traits, the size of repro-
ductive organs (unpublished data), chromosome and
55 60
karyotype parameters and 2C DNA. These observations are
in agreement with a considerable amount of data that show
that the size of reproductive organs may be related to the
genome size, and both increases and decreases of genome
size may have participated in the evolution and diversifi-
cation of the genus, even within a related group of species
(Seijo and Fernandez 2003).
These findings may provide useful information for Tu-
lipa evolutionary, genetic and breeding studies. Further
research using C-banding and molecular cytogenetic tech-
niques such as FISH (Fluorescence in situ hybridization)
may help these studies in more detail.
Acknowledgments The authors gratefully acknowledge the support
provided for this survey by the Tarbiat Modares University, Iran. We
also thank Ms M. Mohseni at the Department of Immunology, Faculty
of Medical Sciences, Tarbiat Modares University (TMU) for her
technical assistance in flow cytometric analysis.
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