151
Key words: Caryophyllaceae, Iran, 
ecology, phenotypic plasticity, 
Canonical Correspondence Analysis. 
Ključne besede: Caryophyllaceae, 
Iran, ekologija, fenotipska 
plastičnost, kanonična korelacijska 
analiza.
Corresponding author:
Iraj Mehregan
E-mail: iraj@daad-alumni.de
Received: 12. 6. 2022
Accepted: 26. 6. 2023
Genetic diversity and morphological 
variability of Iranian Silene aucheriana 
populations inferred from nrDNA ITS 
sequences and morphological analysis
Abstract
We conducted a comprehensive study on the diversity of Silene aucheriana 
Boiss. populations by analyzing both morphological data and nrDNA ITS 
sequences. Maximum parsimony and Bayesian methods on representative material 
from 15 populations throughout Iran demonstrated that they did not form 
a monophyletic group. Strong positive correlation occurred between epipetalous 
filaments length to claw (EFLC), basal leaf length (BLL), basal leaf width (BLW), 
and plant height (PLH) with elevation. While negative correlation achieved 
between anthophore length (AnL) with the average air temperature and rainfall. 
Canonical Correspondence Analysis (CCA) showed that most morphological 
traits were more correlated to elevation than to average rainfall and temperature. 
Multivariate analysis of morphological traits with ITS analysis displayed a slight 
divergence between two types of regions based on their elevation.
Izvleček
Izvedli smo celovito študijo o raznolikosti populacij vrste Silene aucheriana Boiss. 
z analizo morfoloških meritev in zaporedij ITS nrDNA. Največja parsimonija 
in statistične metode po Bayesu na reprezentativnem materialu iz 15 populacij 
po vsem Iranu so pokazale, da ne tvorijo monofiletične skupine. Ugotovili smo 
močno pozitivno korelacijo med dolžino epipetalnih filamentov in žebice (EFLC), 
dolžino spodnjih listov (BLL), širino spodnjih listov (BLW) in višino rastline 
(PLH) z nadmorsko višino. Negativno korelacijo smo ugotovili med dolžino 
antofore (AnL) in povprečno temperaturo zraka ter količino padavin. Kanonična 
korespondenčna analiza (CCA) je pokazala, da je večina morfoloških lastnosti bolj 
povezana z nadmorsko višino, kot pa s povprečno količino padavin in temperaturo. 
Multivariatna analiza morfoloških lastnosti z analizo ITS je pokazala rahlo 
odstopanje med dvema skupinama regij glede na njihovo nadmorsko višino.
Masoumeh Safaeishakib1, Mostafa Assadi2, Shahina A. Ghazanfar3 &  
Iraj Mehregan1
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 
2 Department of Botany, Research Institute of Forests and Rangelands, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
3 Royal Botanic Gardens Kew, Richmond, Surrey, UK.
DOI: 10.2478/hacq-2023-000123/1 • 2024, 151–163
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Evaluation of genetic and morphological diversity in Iranian Silene aucheriana 
populations
Introduction
Silene aucheriana Boiss. is in the Caryophyllaceae subge-
nus Silene, section Auriculatae (Boiss.) Schischk. (Mel-
zheimer, 1988; Oxelman & Lidén, 1995). This species 
is found in Anatolia, Iraq, Turcomania and it is wide-
spread in Iran (Melzheimer, 1988). Genetic diversity is 
traditionally explored via analysis of morphological traits 
(Rautenberg et al., 2012). This approach includes some 
restrictions like the effect of environmental factors, giv-
ing outputs that present just a fraction of total genetic 
variability (Živković et al., 2012). Applying an approach 
integrating morphological and genetic data is generally 
necessary to unravel species delimitation (Duminil & 
Di Michele, 2009; Heinrichs et al., 2009). Regarding 
genus Silene, Oxelman (1996) used morphological and 
molecular markers including Internal Transcribed Spacer 
DNA (ITS) to study the species relationships in the sect. 
Sedoides. Populations of S. aucheriana are widely distrib-
uted in different regions of Iran with different ecologi-
cal conditions and therefore, it is expected to show some 
morphological variation (Safaeishakib et al., 2023). The 
genetic structure based on ISSR data of these studied 
populations was previously reported by Safaeishakib et 
al. (2023), which revealed that genetic divergence among 
populations most likely reflects their climatic conditions. 
The current morphometric data and internal transcribed 
spacer (ITS) sequences were performed from two differ-
ent types of geographical regions. With these data, the 
aims of the present study are a) to examine the morpho-
logical variation in different populations of S. aucheriana, 
b) to investigate the correlation between quantitative 
morphological traits and some meteorological data, and 
c) to evaluate which morphological features are more sig-
nificantly impacted by environmental factors.
Materials and methods
Plant material and Morphological 
studies
Five samples (for morphological analysis) from each of 
the fifteen S. aucheriana populations were collected from 
May to early July 2017 from two types of regions with dif-
ferent average temperatures, elevations, and rainfall. For 
better comprehension, 15 populations were divided into 
two groups according to their elevation (Table 1, Figure 
1). Populations with an elevation cut-off limit higher than 
2800 m (mountainous area and dry cold) in one group 
and those with an elevation cut-off limit lower than 
1800 m (semi-arid, and sub-humid) placed in another 
group. These cut-off limits were based on the mean eleva-
tions of localities (Safaeishakib et al., 2023). The assess-
ment of these climate differences and classification was 
based on the Köppen-Geiger (KG) climate classification 
scheme (Hijmans et al., 2005; Peel et al., 2007; Fick and 
Hijmans, 2017), www.WorldClim.org, and www.weath-
erunderground.com definitions. Meteorological data on 
localities including average temperature and rainfall were 
obtained from www.en.climate-data.org (O’Donnell & 
Ignizio, 2012; Safaeishakib et al. 2023). Voucher speci-
mens are deposited at the Islamic Azad University Her-
barium (IAUH) (Table 1, Figure 1). Seventeen morpho-
logical characteristic were measured for each individual 
material (see Table 2 for characters used). These charac-
ters have been used by various authorities in identifying 
 Silene species (Chowdhuri, 1957; Coode & Cullen, 1967; 
Ghazanfar, 1984; Firat & Yıldız, 2016; Townsend et al., 
2016). All measurements were done by using a Stereo 
 Microscope (NTB-3A). 
Statistical analysis
Statistical analysis was made using software package SPSS 
18.0 for Windows (SPSS Inc. Chicago, USA). Morpho-
logical results are shown as mean ± standard error obser-
vations (mean ± SE). Duncan’s test followed by a Bonfer-
roni correction (ɑ'=ɑ/k and ɑ=0.05) was used to detect 
possible differences in the studied populations (Rice, 
1989). Differences among mean values obtained were 
determined at p<0.05 by Duncan’s test. The relationship 
between populations and environmental variation was 
assessed using canonical correspondence analysis (CCA) 
(Ter Braak, 1986). Clustering was performed according 
to the method of Ward with PAST software version 4.06b 
based on 1000 bootstrap replicates to explore relation-
ships among samples with high variability where data and 
grouping are unclear (Hammer et al., 2001). Principal 
Component Analysis (PCA) was constructed using Vari-
max rotation to obtain variation in morphological traits 
(Safaei et al., 2016). Pearson’s correlation coefficient was 
analyzed by using GraphPad Prism version 6.04 for Win-
dows (GraphPad Software, La Jolla California USA), to 
identify the relationships between the seventeen morpho-
logical traits and the environmental variables.
Genomic DNA extraction and 
amplification
Total genomic DNA was extracted from silica gel dried 
leaves following the protocol Nucleo Spin™ Plants Kits 
(Machery-Nagel, Germany) (Doyle & Doyle, 1987). The 
quality of the extracted DNA was evaluated on a 1% aga-
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populations
rose gel. The concentration of DNA was also estimated 
through Nano-Drop™ 2020 (Thermo Fisher Scientific, 
USA) at 260 nm. Amplification of ITS region was carried 
out by using forward primer AB101 (5’-ACG AAT TCA 
TGG TCC GGT GAA GTG TTC G-3’) and reverse 
primer AB102 (5’-TAG AAT TCC CCG GTT CGC 
TCG CCG TTAC-3’) (Douzery et al., 1999). The ampli-
fication was gained by 35 cycles of 1 min, denaturation 
(94 °C), 1 min annealing (54 °C), and 2 min, 30-second 
elongation (72 °C), (Oxelman & Lidén, 1995). Finally, 
PCR products were evaluated qualitatively by electro-
phoresis on 1% agarose gel. The concentration of DNA 
was also estimated through Nano-Drop™ 2020 (Thermo 
Fisher Scientific, USA) at 260 nm. Sequencing of am-
plicons was performed on an ABI 3730 DNA Analyzer 
(Hitachi-Applied Biosystems, Waltham, Massachusetts, 
USA). Sequences of the ITS regions for 15 populations 
were submitted to GenBank (http://www.ncbi.nim.nih.
gov/genbank/). In addition to these newly generated se-
quences, ITS sequences of 37 accessions of other Silene 
species from Safaeishakib et al. (2020) were added to 
analysis for further comparison (see Table1).
Sequence editing and phylogenetic 
analysis
Sequences were visually checked and edited with the 
software tool Sequencher* version 5.4.6. (Gene Codes 
Corporation, Ann Arbor, Michigan, USA). New data-
set was aligned using the software tool MacClade 4.08 
Figure 1: Geographic distribution of S. aucheriana populations used in this study. Blue-colored circles reflect elevation cut-off limit higher than 
2800 m, lower average temperature, and less rainfall (8.66 °C, 566.34 mm), whereas brown-colored circles show different climate conditions with 
an elevation cut-off limit lower than 1800 m, higher average temperature, more average rainfall, (15.57 °C, 600 mm). Local names are presented in 
Table 1. (Map designed using Arc GIS 10.2).
Slika 1: Geografska razširjenost populacij vrste S. aucheriana uporabljenih v študiji. Modra barva odraža nadmorsko višinsko mejo nad 2800 m, 
nižjo povprečno temperaturo in manj padavin (8,66 °C, 566,34 mm), rjava barva pa drugačne podnebne razmere nadmorsko višinsko mejo nižjo 
od 1800 m, višje povprečje temperatura, več povprečne količine padavin, (15,57 °C, 600 mm). Krajevna imena so predstavljena v tabeli 1. (Zem-
ljevid je bil izdelan z Arc GIS 10.2).
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(Maddison & Maddison, 2000). Phylogenetic analyses 
were carried out with PAUP v. 4.0b10 (Swofford, 2003). 
The consistency index (CI) and retention index (RI) were 
estimated to assess the amount of homoplasy present in 
the data. Maximum parsimony (MP) analyses was per-
formed using the heuristic search option with 1000 ran-
dom taxon additions and tree bisection and reconnection 
(TBR) as the branch swapping algorithm (Felsenstein, 
1985). The model of molecular evolution for each data-
set was selected using the Akaike Information Criterion 
(AIC) in MrModeltest v. 2.3 (Posada & Crandall, 1998). 
A Markov Chain Monte Carlo (MCMC) sampling was 
performed for 4,000,000 generations with sampling oc-
curring at every 100 generations. The posterior probabil-
ity (PP) values were calculated using MrBayes version. 
3.1.2 (Ronquist & Huelsenbeck, 2003). Twenty-five per-
cent of the initial trees was discarded, and the remaining 
sampled trees were combined into a 50% majority rule 
consensus tree. 
Results
Nuclear ribosomal DNA ITS
The ITS region varied in length from 650 to 840 bp 
and parsimony-informative characters gained 52. The 
shortest trees found by PAUP* had 231 steps resulted 
in CI=0.77, RI=0.65. The (GTR+I+G) was selected 
as the best-fit model by MrModeltest. The frequencies 
of nucleotides were estimated as following: A=0.2182, 
C=0.2955, G=0.2883 and T=0.1980). The majority-rule 
consensus tree generated via Bayesian inference analysis 
and is highly consistent with the strict consensus tree 
inferred from MP analysis. Therefore, only the Bayesian 
tree is shown. Posterior Probabilities (PP) are indicated 
by numbers above each branch. Bootstrap supports for 
this branch retrieved in the Maximum Parsimony (MP) 
assessment were displayed by the numbers below each 
branch. The phylogenetic analysis revealed that Tarom 
(Pop 13) and Silvana (Pop 7) populations are well sup-
ported (PP=0.95); furthermore, Dona (Pop 11), Qoshchi 
(Pop 5) and Heydareh (Pop 3) populations showed close 
affinity to each other (populations belonging to elevation 
cut-off limit lower than 1800 m). On the other hand, 
four populations such as Emamzadeh Hashem (Pop 1), 
Gaduk (Pop 8), HezarMasjed (Pop 15), and Rineh (Pop 
10), belong to elevation cut-off limit higher than 2800 m, 
and were separately placed close to each other. Other val-
ues of posterior probability (PP) and maximum parsi-
mony bootstrap (MP) were achieved from other species 
of sect. Auriculatae like S. bornmuelleri and S. sisianica 
(PP=1, PB=90%) (Figure 2).
Figure 2: Bayesian majority-rule consensus tree derived from nuclear 
ribosomal DNA internal transcribed spacer sequences (ITS). Posterior 
probability (≥ 0.90) and MP bootstrap support (≥70%) values are 
presented on above and below branches. Blue color reflects elevation 
cut-off limit higher than 2800 m, lower average temperature, and less 
rainfall (8.66 °C, 566.34 mm), whereas brown color shows different 
climate conditions with an elevation cut-off limit lower than 1800 m, 
higher average temperature, more average rainfall (15.57 °C, 600 mm). 
Local names are presented in Table 1.
Slika 2: Konsenzno drevo Bayesovega večinskega pravila, izpeljano iz 
regije ITS jedrne ribosomalne DNK. Vrednosti posteriorne verjetnosti 
(≥0,90) in podpora s samovzorčenjem analize MP (≥70%) so predstav-
ljene na zgornjih in spodnjih vejah. Modra barva odraža nadmorsko 
višinsko mejo nad 2800 m, nižjo povprečno temperaturo in manj 
padavin (8,66 °C, 566,34 mm), rjava barva pa drugačne podnebne 
razmere z nadmorsko višinsko mejo nižjo od 1800 m, višje povprečje 
temperature, večjo povprečno količine padavin (15,57 °C, 600 mm). 
Krajevna imena so predstavljena v tabeli 1.
0.951
90
0.95
0.87
0.82
82
S. bornmuelleri
S. sisianica
S. aucheriana (Pop 8)
S. aucheriana (Pop 1)
S. aucheriana (Pop 15)
S. aucheriana (Pop 10)
S. aucheriana (Pop 13)
S. aucheriana (Pop 7)
S. aucheriana (Pop 4)
S. aucheriana (Pop 14)
S. aucheriana (Pop 6)
S. aucheriana (Pop 2)
S. aucheriana (Pop 9)
S. aucheriana (Pop 12)
S. aucheriana (Pop 11)
S. aucheriana (Pop 3)
S. aucheriana (Pop 5)
S. mishudaghensis
S. albescens
S. tragacantha
S. geriraudiae
S. breviauriculata
S. pravitziana
S. eriocalycina
S. pseudomurensis
S. gynodioica
S. hirticalyx
S. indeprensa
S. oligophylla
S. gynodioica
S. ecrispans
S. alymaitica
S. erysimifolia
S. daenensis
S. palinotricha
S. oxelmanii
S. microphylla
S. persica
S. meyeri
S. nurensis
S. commelinifolia
S. araratica
S. perepolitana
S. dschuparensis
S. cephalantha
S. prilipkoana
S. brahuica
S. spergulifolia
S. virgata
S. muradica
S. montbretiana
S. goniucaula 0,0 100.0
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Statistical analysis
Correlation coefficients demonstrated that correlations 
were mostly either non-significant or null. However, sig-
nificant correlations in some relations were observed, for 
example, a strong positive correlation occurred between 
epipetalous filaments length to claw (EFLC), basal leaf 
length (BLL), basal leaf width (BLW), and plant height 
(PLH) with elevation. Similarly, positive correlation be-
tween seed width (SEW) and average air temperature 
was also obtained. The correlations between anthophore 
length (AnL) with the average air temperature and rain-
fall were mainly negative and non-significant at p<0.05 
level respectively (Figure 3). Although no significant dif-
ferences in a single trait were observed in all populations, 
basal leaf width (BLW) in Silvana (Pop 7) displayed a 
noteworthy variation compared to the other populations. 
Moreover, basal leaf length (BLL) trait in Qoshchi (Pop 
5), petal limb length (PLL) in HezarMasjed and Klishom 
(Pop 15, Pop 9), petal limb division length (PLDL) in 
Klishom (Pop 9), epipetalous filaments length to claw 
(EFLC) in Tochal (Pop 12), and capsule length (CAPL) 
in Golestan Kooh (Pop 4) revealed a significant differ-
ence among the studied populations. Noticeably, mete-
orological data exhibited significant differences with all 
quantitative traits (Table 3). 
Cluster analysis and principal 
component analysis of 
morphological traits
Morphological variation in 17 studied traits (using PCA) 
showed populations from Zangoleh (Pop 6), Emamzadeh 
Hashem (Pop 1), Rineh (Pop 10), Gaduk (Pop 8) were 
clustered at the first component while the second compo-
nent clustered populations from Tarom (Pop 13), Silvana 
(Pop 7), Qoshchi (Pop 5), Heydareh (Pop 3), Tochal (Pop 
12), Dona (Pop 11), Golestan Kooh (Pop 4), Gorsfid 
(Pop 2). The populations of Dizin (Pop 14),  HezarMasjed 
(Pop 15), and Klishom (Pop 9) were positioned in the 
third dimension (Figure 4).
Cluster analysis based on Ward’s method showed 
that populations of Rineh (Pop10), Zangoleh (Pop 6), 
Emamzadeh Hashem (Pop 1), were placed together with 
similar environmental conditions while populations from 
Silvana (Pop 7), Qoshchi (Pop 5), Heydareh (Pop 3), 
Dona (Pop 11), and Tochal (Pop 12) were located at close 
distance (Figure 5). These results were in agreement with 
the PCA plot result. In order to estimate the relative im-
portance of each eco-geographical trait populations dis-
tribution, CCA was used, and showed that most of the 
evaluated traits are influenced by elevation (Figure 6).
Figure 3: Correlation analysis between eco-geographical variables and morphological traits achieved from populations of S. aucheriana. 
The abbreviations of traits are presented in Table 2. Note: Ave t: average temperature; RF: rainfall; Alt: Altitude.
Slika 3: Korelacijska analiza med ekološko-geografskimi spremenljivkami in morfološkimi znaki, pridobljenimi iz populacij vrste S. aucheriana. 
Okrajšave znakov so predstavljene v tabeli 2. Opomba: Ave t: povprečna temperatura; RF: padavine; Alt: nadmorska višina.
Alt 
Ave t
RF
PLH
BLL
PLW
SLL
SLW
APL
CL
CTL
PCL
PLL
PLDL
ESA
EFLC
CAPL
AnL
SEL
SEW
1
0.8
0.6
0.4
0.2
0
–0.2
–0.4
–0.6
–0.8
–1.0
Al
t 
Av
e 
t
R
F
PL
H
BL
L
PL
W
SL
L
SL
W
AP
L
C
L
C
TL
PC
L
PL
L
PL
D
L
ES
A
EF
LC
C
AP
L
An
L
SE
L
SE
W
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Discussion 
Cluster analysis and PCA based on morphological traits 
indicated Klishom’s population (Pop 9) is close to Dizin 
(Pop 14), and HezarMasjed (Pop 15) populations be-
longing to elevation cut-off limit higher than 2800 m. 
While the genetic structure study among 15 populations 
of S. aucheriana (Safaeishakib et al., 2023) showed slight 
gene flow has occurred in Klishom’s population (Pop 9) 
and revealed that this population is grouped with po-
pulations at elevation cut-off limit lower than 1800 m, 
more average rainfall, and higher average temperature 
(600 mm, 15.57 °C). From the morphological point 
of view and based on the results of the sequence, we 
found that the population of Tochal (Pop 12) (belong-
ing to the elevation cut-off limit higher than 2800 m) is 
placed close to the populations from the elevation cut-off 
limit lower than 1800 m. However, this result was not 
obtained based on ISSR sequencing (Safaeishakib et al., 
2023). In this study, the molecular data achieved from 
S. aucheriana populations exhibited an almost consistent 
pattern that nearly aligned with the results obtained from 
the morphological analysis. Taxonomically, S. aucheriana 
Figure 4: Principal component analysis (PCA) of the morphometric 
data. Blue-colored circles reflect elevation cut-off limit higher than 
2800 m, lower average temperature, and less rainfall (8.66 °C, 
566.34 mm), whereas brown-colored circles show different climate 
conditions with an elevation cut-off limit lower than 1800 m, higher 
average temperature, more average rainfall, (15.57 °C, 600 mm). 
Slika 4: Analiza glavnih komponent (PCA) morfometričnih podatkov. 
Modro obarvani krogi odražajo mejno nadmorsko višino nad 2800 m, 
nižjo povprečno temperaturo in manj padavin (8,66 °C, 566,34 mm), 
medtem ko rjavi krogi prikazujejo različne podnebne razmere z mejno 
nadmorsko višino, nižjo od 1800 m, višjo povprečno temperaturo, 
večjo povprečno količino padavin (15,57 °C, 600 mm).
Figure 5:  Canonical correspondence analysis (CCA) was carried out 
with seventeen morphological data and three geographical traits of 
fifteen accessions of S. aucheriana collected from different locations. 
The abbreviation of traits is presented in Table 2. Note: AVE t: average 
temperature; RF: rainfall; ALT: Altitude
Slika 5:  V kanonično korespondenčno analizo smo vključili sedem-
najst morfoloških in tri geografske znake petnajstih nabirkov vrste 
S. aucheriana, nabranih na različnih nahajališčih. Okrajšave znakov 
so predstavljene v tabeli 2. Opomba: AVE t: povprečna temperatura; 
RF: količina padavin; ALT: nadmorska višina.
is distinct from S. montbretiana, while they have been 
treated synonymous in Flora of Turkey and Flora of Iraq 
(Coode & Cullen 1967; Townsend et al., 2016). Mel-








































 





	








 


 










 








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and alar pedicel length traits affected by average tempera-
ture. Additionally, stem leaf width, stem leaf length and 
basal leaf width appear to be strongly affected by rainfall. 
While other traits such as epipetalous filaments length to 
claw, seed length, anthophore length, seed width, calyx 
tooth length, calyx length, plant height, petal limb di-
vision length, capsule length, and epipetalous stamens 
are affected mainly by elevation. Additionally, based on 
the morphological observations in the collected samples 
of S. aucheriana populations, we noticed two traits of 
basal leaf width and calyx length are more variable than 
other traits. Some studies have shown that genetic diver-
sity in Silene species is more representative of geography 
and climate than taxonomy; for example, in S. latifolia, 
divergence of populations showed that two kinds of cli-
mate regions were involved that resulted in variation of 
morphological characters (Brothers et al., 2016; Durović 
et al., 2017; Del Valle et al., 2019). Similarly, the study 
conducted by Karrenberg et al. (2018) utilized environ-
mental data consisting of 19 bioclimatic variables to ana-
lyze populations of S. dioica and S. latifolia. The findings 
suggest that speciation has been driven by ecological di-
vergence.
Conclusions
The present study intended to examine the genetic 
and morphological diversity of two different groups of 
S. aucheriana populations, with a focus on elevation 
cut-off limits above 2800 m and below 1800 m. Cluster 
analysis of molecular data was shown to be similar to 
morphological traits results. Accordingly, Emamzadeh 
Hashem (Pop 1), Gaduk (Pop 8), Zangoleh (Pop  6), 
and Rineh (Pop 10) populations belong to elevation 
cut-off limit higher than 2800 m, and were separately 
placed close to each other. Subsequently Silvana (Pop 7), 
Qoshchi (Pop 5), Heydareh (Pop 3), and Dona (Pop 11) 
populations were located in another group (elevation 
cut-off limit lower than 1800 m) at close distance. The 
phylogenetic results of nrDNA ITS revealed that the 
two populations of Tarom (Pop 13), and Silvana (Pop 7) 
formed a well-supported branch as a monophyletic group 
(PP=0.95). Similarly, in terms of morphological analyses, 
apart from the Tochal (Pop 12) and Klishom (Pop 9), 
other populations were placed together based on their 
elevation grouping. Having such a relatively clear-cut 
grouping from both methods could suggest the existence 
of a genetic background supporting morphological di-
versity. Some morphological variables such as basal leaf 
length, and alar pedicel length showed a positive corre-
lation with average air temperature while negative cor-
Figure 6: Cluster analysis of S. aucheriana populations using Ward’s 
linkage based on morphological data. Blue color reflects elevation 
cut-off limit higher than 2800 m, lower average temperature, and 
less rainfall (8.66 °C, 566.34 mm), whereas brown color shows dif-
ferent climate conditions with an elevation cut-off limit lower than 
1800 m, higher average temperature, more average rainfall, (15.57 °C, 
600 mm). 
Slika 6: Klastrska analiza populacij vrste S. aucheriana z Wardovo 
metodo povezovanja na osnovi morfoloških podatkov. Modra barva 
odraža nadmorsko višinsko mejo nad 2800 m, nižjo povprečno 
temperaturo in manj padavin (8,66 °C, 566,34 mm), rjava barva 
pa drugačne podnebne razmere z nadmorsko višinsko mejo, nižjo 
od 1800 m, višjo povprečno temperaturo, večjo povprečno količino 
padavin (15,57 °C, 600 mm).
zheimer (1988) has separated Iranian S. aucheriana from 
the Turkish S. montbretiana on the basis of leaf characters 
(Ghazanfar & Edmondson, 2013). 
Differences in habitat can be reflected in morphologi-
cal features, so different environmental conditions cannot 
be ignored in population diversity studies (Thorpe, 1987; 
Peppe et al., 2011). Elevation is one of the important 
environmental factors that affect population differentia-
tion and genetic diversity. High elevation, especially in 
alpine regions, has significant effect on plants. Recently, 
plant-based ecology studies have raised many fundamen-
tal questions of morphological variation as populations 
have obvious adaptation strategies by changing their mor-
phological and physiological characteristics (Royer et al., 
2005; Verheijen et al., 2013). The ordination techniques 
are useful in the elucidation of cluster pattern as well as 
in the detection of the eco-geographical variation (Mu-
cino, 1982). In this regard, CCA showed basal leaf length 
Qoshchi (Pop 5)
Heydareh (Pop 3)
Silvana (Pop 7)
Dona (Pop 11)
Dizin (Pop 14)
Hezar Mas (Pop 15)
Klishom (Pop 9)
Tochal (Pop 12)
Gorsfid (Pop 2)
Gol-kooh (Pop 4)
Tarom (Pop13)
Gaduk (Pop 8)
Em-Hash (Pop 1)
Zangoleh (Pop 6)
Rineh (Pop 10)
200 150
100Distance
50100
23/1 • 2024, 151–163
158
Safaeishakib et al.
Evaluation of genetic and morphological diversity in Iranian Silene aucheriana 
populations
relation was seen between capsule length, and petal limb 
division length with elevation. The evaluation conducted 
by CCA demonstrated that elevation had a stronger cor-
relation with most of the morphological traits as com-
pared to average rainfall and temperature. Among all in-
vestigated traits, only basal leaf length (BLL), basal leaf 
width (BLW), petal limb length (PLL) and epipetalous 
filaments length to claw (EFLC), and capsule length 
(CAPL) displayed a significant difference among the 
studied populations. Two quantitative traits, basal leaf 
width and calyx length, were variable compared to sev-
enteen other quantitative traits.
Acknowledgments
The authors would like to thank Dr. Abbas Gholipour 
for his help in confirmation of our species identification.
Funding
Present work is supported by the Science and Research 
Branch, Islamic Azad University, Tehran, Iran
Masoumeh Safaeishakib  https://orcid.org/0000-0002-3557-0287
Mostafa Assadi  https://orcid.org/0000-0001-9014-585X
Shahina A. Ghazanfar  https://orcid.org/0000-0002-0482-2023
Iraj Mehregan  https://orcid.org/0000-0002-5108-2558
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23/1 • 2024, 151–163
161
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Evaluation of genetic and morphological diversity in Iranian Silene aucheriana 
populations
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68
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162
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Evaluation of genetic and morphological diversity in Iranian Silene aucheriana 
populations
Table 3: The Duncan’s means of the traits, assessed from fifteen populations of S. aucheriana. Note: The data are reported 
as Mean ± SD (Standard deviation). Mean values with the same letters are not significantly different at (p < 0.05) according 
to Duncan’s test followed by Bonferroni’s correction (ɑ'=ɑ/k, ɑ=0.05). The abbreviation of traits is presented in Table 2. 
AvgT: average temperature; RF: rainfall; Sig: significance level. Highlighted cells show significant differences.
Tabela 3: Duncanove povprečne vrednosti znakov, ocenjenih iz petnajstih populacij vrste S. aucheriana. Opomba: podatki so 
navedeni kot povprečje ± SD (standardni odklon). Srednje vrednosti z istimi črkami se statistično značilno ne razlikujejo (p < 0,05) 
glede na Duncanov test, ki mu sledi Bonferronijeva korekcija (ɑ'=ɑ/k, ɑ=0,05). Okrajšave znakov so predstavljene v tabeli 2. 
AvgT: povprečna temperatura; RF: padavine; Sig: stopnja statistične značilnosti. Osenčene celice kažejo statistično značilne razlike.
Table 2: The morphological characters used for morphometric analyses among populations of S. aucheriana (description of each 
character is given separately in the Results).
Tabela 2: Morfološki znaki, uporabljeni za morfometrične analize med populacijami vrste S. aucheriana (opis vsakega od znakov je 
podan posebej v Rezultatih).
No Quantitative morphological characters
1 Plant height (PLH; cm)
2 Basal leaf length (BLL; mm)
3 Basal leaf width (BLW; mm)
4 Stem leaf length (SLL; mm)
5 Stem leaf width (SLW; mm)
6 Alar pedicel length (APL; mm)
7 Calyx length (CL; mm)
8 Calyx tooth length (CTL; mm)
9 Petal claw length (PCL; mm)
No Quantitative morphological characters
10 Petal limb length (PLL; mm)
11 Petal limb division length (PLDL; mm)
12 Epipetalous stamens to alternate (ESA; mm)
13 Epipetalous filaments length to claw (EFLC; mm)
14 Capsule length (CAPL; mm)
15 Anthophore length (AnL; mm)
16 Seed length (SEL; mm)
17 Seed width (SEW; mm)
Pop SLL Sig. SLW Sig. APL Sig. CL Sig. CTL Sig. PCL Sig.
1 22.8±8.90d,f,g 0.57 0.725±0.43g,f 0.34 9.97±6.11b,c,d 0.81 10.55±1.75g 0.37 0.42±0.29d 0.35 6±2.00f 0.34
2 14.2±7.70e,f,g, 0.44 1.37±0.89d,e,f 1 7.25±5.53c,d,e,f 0.86 12.75±2.33d,e,f 0.66 1.05±0.58b,c 0.80 6.17±1.11e,f 0.37
3 18.45±5.83e,f 0.62 2.13±1.03d 0.54 5.96±5.36d 0.74 9.8±3.03g 0.27 1.11±0.76b,c 0.87 6.42±1.94e,f 0.47
4 15.2±7.50e,f,g 0.47 2.96±1.31c 0.45 7.27±5.30c,d,e 0.93 15.12±0.47a,b,c 0.59 0.85±2.31b,c 0.93 8±2.31b,c,e 0.77
5 36.35±1.65a 0.25 3.95±1.01a 0.32 17.35±1.59a,c,e,f 0.45 11.6±3.45c,e,f,g 0.61 1.1±0.57b,c 0.87 8.57±2.93a,b,c 0.72
6 32.4±1.37a,b 0.83 3.37±1.27a,b,c 0.34 13.05±6.41a,b 0.55 16.55±3.10a 0.28 0.89±0.64b,c 0.93 9.92±1.64a 0.42
7 34.8±8.10a 0.37 3.45±1.07a,b,c 0.33 10.65±5.41a,c,d,e,f 0.78 11.65±2.65e,f,g 0.21 1±0.75b,c 0.87 6.92±1.11d,e,f 0.54
8 10.95±4.46f,g 0.77 1.15±0.92e,g,f 1.04 5.87±3.35c,d,e 0.73 13.5±2.62c,d,e 0.66 1.72±0.82a 0.12 9.35±1.51a,b 0.55
9 27.6±1.23b,c 0.53 3.8±2.39a,b 1 11.97±8.43b,c 1.35 15.9±0.52a,b 0.43 0.89±0.52b,c 0.93 9.47±2.45a 0.54
10 26.2±1.09b,c,d,e 0.52 1.79±0.82d,e 0.45 4.6±4.64e 0.65 16.55±3.03a 0.28 1.17±0.65b,c 0.88 7.97±1.55b,c,d 0.77
11 19.42±1.08d,e 0.65 1.75±1.02d,e 0.45 7.15±4.42c,d,e 0.12 14.55±2.53b,c,d 0.61 1.2±0.50b, 0.90 8.7±2.27a,b,c 0.69
12 26.5±9.80a,b,c 0.53 1.95±1.11d,e 0.48 12.65±1.16b,d,c 0.59 13.77±2.63d 0.34 1.18±0.55b,c 0.89 8.97±1.80a,b 0.66
13 30±1.14a,b,c 0.47 3.05±1.30b,c 0.43 6±4.82d,c,e,f 0.75 11.4±2.98f,g 0.59 0.82±0.42b,c 0.93 9.35±1.70a,b 0.55
14 14.15±8.27e,f,g,e 0.44 1.57±1.23d,e 0.49 4.2±4.49d,c,e,f 0.63 13.37±3.25c,d,e,f 0.65 1.12±0.49b,c 0.87 7.42±1.70c,d,e 0.69
15 12±3.20f,g, 0.45 0.48±0.28g 0.23 2.63±2.16d,c,e,f 0.54 13.15±1.92c,d,e,f 0.64 0.76±0.41d 0.93 7.36±1.87c,d,e 0.67
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Evaluation of genetic and morphological diversity in Iranian Silene aucheriana 
populations
Pop Elevation Sig. AveT Sig. RF Sig. PLH Sig. BLL Sig. BLW Sig.
1 2.715±00 .000 11.69±00i .000 12.91±00m .000 180.5±40d,e,f 0.75 22.45±7.86c,d,e,f 0.78 0.56±0.33d 0.85
2 1.31±00 .000 17.4±00b .000 10.75±00o .000 226±40.70c,d,e 0.86 15.05±5.18g,h 0.52 0.75±0.46c,d 0.93
3 3.3±00 .000 11.32±00j .000 32±00e .000 283±0.58a,b,c 0.66 24.75±8.15b,d,e 0.63 1.96±1.37b,c,d 0.93
4 2.46±00 .000 10.52±00k .000 16.66±00j .000 247.5±52.8a,b,c,d 0.85 19.1±7.83e,f,g 0.67 2.47±1.33a,b,c,d 0.93
5 2.1±00g .000 51.8±00a .000 31±00f .000 268.5±53.24a,b,c 0.75 37.25±8.96a 0.03 3.47±1.16b 1.00
6 2.32±00 .000 15.2±00f .000 90.41±00a .000 191±40.89d,e,c 1.64 26.5±1.22b,d 0.52 2.04±1.39b,c,d 0.93
7 2.3±00k .000 9.49±00m .000 39.83±00d .000 283.5±62.68a,b 0.65 28.3±6.38b,c 0.42 7.85±1.25a 0.00
8 2.22±00 .000 12.81±00g .000 11.31±00n .000 199±55.71c,d,e 0.86 11.55±5.79h 0.38 0.72±0.45c,d 0.93
9 1.94±00 .000 10.4±00l .000 25±00g .000 158.5±37.17e,f 1.33 29.1±1.28b 0.42 3.25±2.08b,c 0.93
10 2.01±00 .000 11.82±00h .000 13.66±00l .000 195±53.90c,d,e 0.85 18.9±7.69e,f,g 1.32 1.32±1.05b,c,d 0.93
11 2.51±00 .000 15.73±00d .000 90.08±00b .000 313.75±6.51a 0.47 16.62±0.36f,g,h 0.56 1.36±0.77b,c,d 0.93
12 1.91±00 .000 16.38±00c .000 18.33±00i .000 231±57.93d,e,c 0.93 27.15±9.72b,d 0.46 1.36±1.19b,d 0.93
13 2.1±00 .000 15.45±00e .000 56.41±00c .000 227.5±51.79d,e 0.93 22±8.98d,e,f 0.85 1.7±0.86b,c,d 0.93
14 2.72±00 .000 7.33±00n .000 16.25±00k .000 168±44.79e,f 0.67 15.2±1.12g,h 0.53 1.79±1.48b,c,d 0.93
15 2.91±00 .000 11.8±00i .000 21.25±00h .000 110.5±22.35f 0.40 18.25±8.18f,g 0.63 0.53±0.27d 0.84
Pop PLL Sig. PLDL Sig. ESA Sig. EFLC Sig.
1 4.47±1.22e,f 0.73 1.25±0.61f,g 0.82 4.47±0.73d 0.57 1.25±0.61b,c 0.93
2 4.77±2.01d,e 0.61 1.91±1.20f 0.68 4.7±0.86d 0.58 0.53±30b,c 0.93
3 4.87±1.38d,e 0.63 1.95±0.63f 0.68 3.7±0.42e 0.75 0.5±0.30b,c 0.93
4 5.78±1.28b,c,d 0.70 2.46±1.01d,e 0.69 6.31±1.37b 1.88 0.84±0.50b,c 0.93
5 3.75±0.85f 0.47 2.65±1.08c,d,e 0.68 5±1.71c,d 0.56 0.81±0.32b,c 0.93
6 6.27±1.95b,c 0.37 2.65±1.08c,d,e 0.68 7.52±3.04a 0.56 0.76±0.41b,c 0.93
7 4.75±0.76d,e 0.61 2.52±0.96d,e 0.68 5.4±0.78b,c,d 0.51 1.05±0.75b 0.93
8 5.32±2.02d,e 0.73 2.92±1.57c,d 0.72 7.9±2.01a 0.2 0.94±0.41b,c 0.93
9 7.82±1.87a 0.04 5.21±0.88a 0.03 7.44±2.22a 0.29 0.68±0.30b,c 0.93
10 6.5±1.51b,c 0.77 4.02±1.62a 0.16 5.9±1.35b,c 0.48 0.32±0.18c 0.93
11 4.92±1.26d,e 0.65 2.46±0.69d,e 0.69 5.05±0.87c,d 0.54 0.55±0.27b,c 0.93
12 6.29±1.26b,c 0.06 3.5±1.04a,c 0.49 7.87±1.57a 0.21 2.33±3.26a 0.002
13 4.59±1.14e,f 0.6 2.26±0.81d,e 0.68 3.34±1.33e 0.15 0.52±0.45b,c 0.93
14 4.97±1.37d,e 0.66 2.08±0.81d,e,f 0.67 5.9±1.72b,c 0.48 1.07±0.76b 0.93
15 2.24±0.86g 0.008 0.65±0.28g 0.10 3.6±1.16e 0.32 1.02±0.64b,c 0.93