Biologia 65/1: 23—27, 2010
Section Botany
DOI: 10.2478/s11756-009-0215-3
Karyotype analysis and new chromosome number reports
in Silene species (sect. Auriculatae, Caryophyllaceae)
Abbaas Gholipour2 & Masoud Sheidai1*
1
2
Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran; e-mail: [email protected]
Payeme Noor University (PNU) Iran
Abstract: Karyotype study was performed in 13 populations of 11 Silene species (sect. Auriculatae L., Caryophyllaceae)
growing in Iran. All the species studied showed 2n = 2x = 24 chromosome number supporting the earlier report on S.
meyeri, while the chromosome number of S. palinotricha, S. sojakii, S. gertraudiae, S. elymaitica, S. pseudonurensis, S.
dschuparensis, S. eriocalycina, S. araratica, S. prilipkoana and S. commelinifolia are new to science. The chromosomes
were mainly metacentric or sub-metacentric and their size varied from 1.10 µm in S. pseudonurensis to 7.11 µm in S.
dschuparensis. The species studied differed significantly in the total size of the chromosomes, the size of the short arms
and the long arms, indicating the role of quantitative genomic changes in the Silene species diversification. They also differ
in their karyotype formulae indicating the occurrence of structural changes in their chromosomes. The Silene species were
placed in 1A, 2A, 1B and 2B classes of Stebbins karyotype symmetry showing symmetrical karyotypes. Clustering of the
species based on karyotype features grouped the species of S. palinotricha, S. prilipkoana, S. commelinifolia, S. eriocalycina,
S. meyeri, S. araratica and S. Sojakii together while the species of S. gertraudiae and S. elymaitica showed more similarity
and were placed close to each other.
Key words: Silene; karyotype; Iran
Introduction
The genus Silene L. (Caryophyllaceae) is a very large
genus of world-wide distribution, containing about 700
species which are mostly hermaphrodite, although a
few species are diocious or gynodioceious (Bari 1973,
Greuter 1995). Silene species are mostly distributed
throughout the northern hemisphere, Europe, Asia and
northern Africa. They are annual, biennial, or perennial
herbs with the basic chromosome number x = (10) 12.
The available literature from the other parts of the
world dealing with cytogenetics of Silene indicates the
importance of such studies (Heaslip 1951; Bari 1973;
Melzheimer 1978; Markova et al. 2006), while such data
is totally lacking for the species growing in Iran. Most
of the Silene species are diploid having 2n = 2x = 24,
or 2n = 2x = 20 (Bari 1973), some others are tetraploid
(2n = 4x = 48) and hexaploid (2n = 6x = 72) and a
few species show higher polyploidy level for e.g. 2n = c.
96, 120 and 192 (Bari 1973). In addition 2n = 3x = 30
is reported for S. fortunei (Heaslip 1951).
About 110 Silene species grow in Iran out of which
about 35 species are endemic with very limited geographical distribution (Melzheimer 1988). Chowdhuri
(1957) placed the Silene in 44 sections but recent molecular studies do not support such sectional classifications
particularly for the endemic North American taxa (Ox-
elman et al. 1997, 2000; Burleigh & Holtsford 2003).
The section Auriculatae (Boiss.) Schischkin is the
largest section of the genus containing about 35 species,
out of which 21 species are endemic to Iran (Melzheimer
1980). The members of this section are caespitose
mountainous plants with large flowers placed at the end
of short stems.
There has not been any biosystematic study in Iranian Silene species up to now and the present work reports the karyotype features of 13 populations of 11
Silene species (sect. Auriculatae L.) growing in Iran for
the first time. Morphometric analysis of these taxa is in
hand and when it is done the results may be compared.
Material and methods
Plant material
Karyotype study was performed in 13 populations of 11
Silene species (sect. Auriculatae L.) growing in Iran. The
species studied are: 1 – Silene palinotricha Fenzl ex Boiss.
(two populations), 2 – S. sojakii Melzh., 3 – S. gertraudiae
Melzh., 4 – S. elymaitica Bornm., 5 – S. meyeri Fenzl ex
Boiss., 6 – S. pseudonurensis Melzh., 7 – S. dschuparensis
Bornm., 8 – S. eriocalycina Boiss. (two populations), 9 – S.
araratica Schisck., 10 – S. prilipkoana Schisck, and 11 – S.
commelinifolia Boiss. The voucher specimens are deposited
in Herbarium of Shahid Beheshti University (HSBU).
* Corresponding author
c
2009
Institute of Botany, Slovak Academy of Sciences
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24
Cytological studies
For karyotype study, freshly grown root tips were collected
from the germinated seed of at least ten randomly selected plants in each species, pretreated with 2 mmol 8hydroxyquinolin (2–2.5 hrs) and fixed in ethanol : acetic acid
(3:1) for 24 hrs. The fixed tips were then washed thoroughly
in distilled water and macerated in 60 ◦C 1N HCl for about 5
min. Squash technique was used for cytological studies with
2% aqueous aceo-orcein as the stain. The somatic chromosome number and karyotype details were studied in at least
5 well-prepared metaphase plates. The chromosomes were
photographed by digital camera and measured by Image
Tools3 software (Sheidai & Rashid 2007).
The chromosomes were identified according to Levan et
al. (1964), karyotype symmetry was determined according
to Stebbins (1971), while other karyotype parameters like
haploid total chromosome length (Total sum of the size of
the chromosomes by using only one chromosome from each
pair), mean chromosome length (Total haploid chromosome
A. Gholipour & M. Sheidai
length/number of chromosome pairs), total form percentage
(TF % = Sum of short arms of the chromosomes/Total chromosome length), coefficient of variation (CV) of the chromosome size as well as A1 and A2 indices of Romero-Zarco
(1986) were determined (Sheidai & Jalilian 2008).
Statistical analyses
In order to reveal significant difference the analysis of variance (ANOVA) followed by the least significant difference
test (LSD) were performed on the size of chromosomes, size
of the long arms and size of the short arms as well as arms
ratio among the species and populations studied (Sheidai
& Jalilian 2008). Moreover, principal components analysis
(PCA) was performed to identify the most variable karyotype characters. SPSS ver. 9 (1998) was used for ANOVA
and PCA analysis.
In order to group the species studied based on similarity in their karyotype features, UPGMA (Unweighted Paired
Group with Arithmetic Average) and Neighbor Joining (NJ)
Fig. 1. Representative somatic metaphase cells in Silene species studied. A – Metaphase cell showing 2n = 24 inS. Sojakii, B –
Metaphase cell showing 2n = 24 in S. pseudonurensis, C – Metaphase cell showing 2n = 24 in Ahvan population of S. palinotricha,
D – Metaphase cell showing 2n = 24 in S. meyeri, E – Metaphase cell showing 2n = 24 in S. commalinifolia, F – Metaphase cell
showing 2n = 24 in S. araratica, G – M etaphase cell showing 2n = 24 in S. gertraudiae, H – Metaphase cell showing 2n = 24 in S.
dschuparensis, I – Metaphase cell showing 2n = 24 in Sabzkooh population of S. eriocalycina, J – Metaphase cell showing 2n = 24 in
S. prilipkoana, K – Metaphase cell showing 2n = 24 in Shahrood population of S. palinotricha, L – Metaphase cell showing 2n = 24
in Palangan population of S. eriocalycina, M – Metaphase cell showing 2n = 24 in S. elymaitica. Scale bar = 10 µm.
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Karyotype analysis in Silene
25
Table 1. Karyotype features of the Silene species studied.
Species
Locality
2n Ploidy level TL (µm) L (µm) S (µm) L/S X (µm) ST A1
A2 CV TF%
Silene palinotricha
S. palinotricha
S. gojakii
S. gertraudiae
S. elymaitica
S. meyeri
S. pseudonurensis
S. dschuparensis
S. eriocalycina
S. eriocalycina
S. araratica
S. prilipkoana
S. commelinifolia
Ahvan
Shahrood
Semnan
Sorkheh
Sabzkooh
Zanjan
Kerman
Kerman
Palangan
Sabzkooh
Ilam
Paveh
Touchal
24
24
24
24
24
24
24
24
24
24
24
24
24
0.20
0.16
0.13
0.16
0.18
0.14
0.20
0.22
0.15
0.15
0.10
0.19
0.18
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
85.58
79.00
92.80
107.49
99.36
75.82
120.44
140.44
81.55
88.91
87.61
77.00
79.78
3.82
3.37
3.99
4.91
4.32
3.11
5.62
7.11
3.87
3.54
3.52
3.44
3.43
1.86
1.98
2.51
2.70
2.46
1.93
2.90
3.34
2.11
2.34
2.48
1.79
1.69
1.15
0.94
0.92
1.06
1.07
0.88
1.09
1.29
0.90
0.94
0.85
1.14
1.20
7.13
6.58
7.73
8.96
8.28
6.32
10.00
11.70
6.80
7.41
7.30
6.42
6.65
2B
1A
1A
1A
1A
1A
2A
1B
1A
1A
1A
1A
1B
0.30
0.32
0.30
0.31
0.37
0.24
0.39
0.31
0.24
0.26
0.26
0.28
0.29
30
32
30
31
37
24
39
31
24
26
26
28
29
40.00
43.00
42.70
41.00
39.00
43.00
38.00
41.00
43.00
39.50
45.70
42.00
41.00
Karyotype
formulae
9 m + 3 sm
12 m
12 m
11 m + 1 sm
8 m + 4 sm
11 m + 1 sm
7 m + 5 sm
9 m + 3 sm
12 m
11m + 1sm
12 m
11 m + 1 sm
12 m
L – Total diploid chromosome length, L – size of the longest chromosome, S – size of the shortest chromosome, L/S – ratio of the
longest to shortest chromosome, X – mean chromosome length, ST – Stebbins’ class, A1 & A2 – symmetry indices of Romero-Zarco,
CV – coefficient of variation, TF% – total form percentage
clustering methods as well as ordination based on principal coordinate analysis (PCO) were performed. NTSYS Ver.
2.02 (1998) was used for clustering and PCO analyses. Standardized karyotype data (mean = 0, variance = 1) were used
to determine taxonomic distance among the species, which
were used in clustering (Sheidai & Jalilian 2007). Cophenetic correlation was estimated to determine the goodness
of fit of the clusters to the original data (Sheidai & Jalilian
2008).
Results and discussion
Details of karyotype analyses in the Silene species studied are presented in Table 1, Fig. 1. All the species studied showed 2n = 2x = 24 chromosome number. The results obtained support the earlier report on S. meyeri
(Nersesian & Goukasian 1995), while the chromosome
number of S. palinotricha, S. sojakii, S. gertraudiae,
S. elymaitica, S. pseudonurensis, S. dschuparensis, S.
eriocalycina, S. araratica, S. prilipkoana and S. commelinifolia are new to science.
The size of the longest chromosome varied from
3.11 µm in S. meyeri to 7.11 µm in Kerman population of S. dschuparensis, while the size of the shortest
chromosomes varied from 1.69 µm in S. commelinifolia
to 2.70 µm in S. gertraudiae. The chromosomes were
mainly metacentric (m) and sub-metacentric (sm) (Table 1).
The highest total diploid chromosome length and
the highest mean chromosome length occurred in S.
dschuparensis (140.44 & 11.70 µm respectively), while
the lowest value of the same parameters occurred in S.
meyeri (75.82 µm & 6.32, Table 1). The highest value
of the ratio of longest to shortest chromosome also occurred in S. dschuparensis (1.29) while the lowest value
of the same occurred in S. araratica (0.85).
The highest value of coefficient of variation (CV)
(37.00) for the size of chromosomes occurred in S. elymaitica, indicating the highest degree of size variation
among its chromosomes, while the lowest CV value
(24.00) occurred in Palangan population of S. eriocalycina and S. meyeri.
The ANOVA and LSD tests revealed a significant
difference (p < 0.02) for the total size of the chromosomes and the size of the short arms and the long arms
among the species and populations studied, indicating
the role of quantitative genomic changes in the Silene
species diversification.
Pearson correlation determined among the karyotype features showed a positive significant correlation
between the mean chromosome length and the size of
the short and long arms of the chromosomes (r > 0.80,
p < 0.05). Therefore, the significant quantitative change
in the chromatin material has occurred in the size of
both chromosome arms during the species diversification.
The Silene species studied also differ in their karyotype formulae indicating the occurrence of structural
changes in their chromosomes (Table 1). The total form
percentage (TF%) varied from 38 in S. pseudonurensis to 45 in S. araratica (Table 1), a higher value of
TF% indicates the presence of relatively more symmetrical karyotype. The Silene species were placed in 1A,
2A, 1B and 2B classes of Stebbins karyotype symmetry,
which are considered relatively primitive in this system.
Therefore, it seems that the Silene species studied are
having symmetrical karyotypes.
Ahvan population of S. palinotricha shows the
highest asymmetric karyotype among the species studied as it stands in 2B class of Stebbins’ classification.
Shahrood population of this species stands in 1A class
showing relatively more symmetrical karyotype, possibly due to the occurrence of chromosomal structural
changes.
Among the species placed in 1A class, S. pseudonurensis shows a higher value of A1 index (0.39) of
Romero-Zarco and therefore has relatively more asymmetrical karyotype. All these results indicate the role
of both quantitative and qualitative changes in the
genome during the Silene species diversification.
Pearson correlation showed a negative significant
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26
A. Gholipour & M. Sheidai
Fig. 2. UPGMA clustering of Silene species based on karyotype data. Species code: pal1 & 2 – Ahvan and Shahrood populations of S.
palinotricha respectively; commel – S. commelinifolia; prilip – S. prilipkoana; erio1 & 2 – Palangan and Sabzkooh populations of S.
eriocalycina respectively; meyeri – S. meyeri; sojaki – S. sojakii; ararat = S. araratica; great – S. gertraudiae; elymai – S. elymaitica;
pseudo – S. pseudonurensis; dschup – S. dschuparensis.
correlation between TF% and the ratio of the longest
to shortest chromosome, and also between TF% and the
ratio of the long arm to short arm of the chromosome
pair number 2, 9, 10 and 11 (r = −0.563, p < 0.05).
Therefore it seems that the symmetric karyotype of the
Silene species occurs partly due to reduction in the ratio of the longest to shortest chromosome of the complement and also reduction in the arm ratio of the chromosome pair numbers 2, 9, 10 and 11.
PCA analysis (data not given) shows that the first
2 components comprise about 80% of the total variation. In the first component with about 71% of total
variance, the mean chromosome length, size of the short
arms and long arms as well as total length of the chromosomes are the most variable characters (r > 0.90),
supporting the results of ANOVA stated earlier.
In the second component with about 8% of total
variance, the ratio of the long arm to the short arm
of the chromosomes pair number 11, 12 and TF% are
the most variable characters (r > 0.70), supporting our
earlier suggestion about the role of qualitative changes
in the Silene species diversification.
Different clustering methods and PCO ordination
of the Silene species based on karyotype data produced
similar results and UPGMA clustering produced higher
cophenetic correlation (r > 0.80), therefore UPGMA
dendrogram is discussed here (Fig. 2). In general, 3 major clusters are formed; the species of S. palinotricha,
S. prilipkoana, S. commelinifolia, S. eriocalycina, S.
meyeri, S. araratica and S. sojakii comprise the first
major cluster, out of which Shahrood population of S.
palinotricha shows more similarity to S. commelinifolia, while S. sojakii shows more similarity to S. araratica. Two species of S. gertraudiae and S. elymaitica
form the second major cluster while two species of S.
pseudonurensis and S. dschuparensis comprise the third
cluster. PCO ordination supports the clustering result,
revealing karyotype distinctness of S. pseudonurensis
and S. dschuparensis, as well as S. gertraudiae and S.
elymaitica, from the other Silene species studied.
At present, the morphometric studies of the species
reported here are going on and when the results are
ready, we shall discuss the species relationships in both
morphological and karyotype grounds. Also, no data
is available on the genome size of the studied species;
therefore no more detailed comparison or discussion
could be made on karyotype data available.
Acknowledgements
This project was partly supported by Iran Scientific Foundation (INSF), with project No. 8611850.
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Received June 3, 2008
Accepted May 27, 2009
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