Internal transcribed spacer (ITS) polymorphism in the wild Primula (Primulaceae) taxa of Turkey

Abstract

Twenty-three populations belonging to 10 wild Primula L. (Primulaceae) taxa of Turkey, some of which are morphologically quite similar, were investigated based on nrDNA ITS regions. The plant materials were collected from different geographical areas of Anatolia in vegetation periods of 2005 and 2006. Total genomic DNAs were isolated from the healthy leaves of each population. The entire ITS regions of the populations were amplified by universal primers with the aid of a polymerase chain reaction (PCR), and then the PCR products were sequenced. Neighbour-Joining (NJ) and Maximum Parsimony (MP) trees were constructed in order to identify the relationships among Primula taxa. According to ITS data, 724 characters were determined among the aligned sequences of the populations and the divergence values were found to be between 0.0% and 20.9%. ITS sequences from 23 specimens provided a number of variable and sufficient characters to explore the relationships. As a result, it was determined that the dendrograms obtained by NJ and MP analysis are concordant with the traditional taxonomical order at subgeneric level.

Full text

Internal transcribed spacer (ITS) polymorphism in the wild
Primula (Primulaceae) taxa of Turkey
Mutlu GÜLTEPE1, Uğur UZUNER2, Kamil COŞKUNÇELEBİ1,
Ali Osman BELDÜZ1, Salih TERZİOĞLU3
1Karadeniz Technical University, Faculty of Arts & Sciences, Department of Biology, 61080 Trabzon - TURKEY
2Texas A&M University, Department of Plant Pathology & Microbiology, College Station, TX 77843 - USA.
3Karadeniz Technical University, Faculty of Forestry, Department of Forest Botany, 61080 Trabzon - TURKEY
Received: 27.05.2009
Accepted: 16.02.2010
Abstract: Twenty-three populations belonging to 10 wild Primula L. (Primulaceae) taxa of Turkey, some of which are
morphologically quite similar, were investigated based on nrDNA ITS regions. e plant materials were collected from
dierent geographical areas of Anatolia in vegetation periods of 2005 and 2006. Total genomic DNAs were isolated from
the healthy leaves of each population. e entire ITS regions of the populations were amplied by universal primers with
the aid of a polymerase chain reaction (PCR), and then the PCR products were sequenced. Neighbour-Joining (NJ) and
Maximum Parsimony (MP) trees were constructed in order to identify the relationships among Primula taxa. According
to ITS data, 724 characters were determined among the aligned sequences of the populations and the divergence values
were found to be between 0.0% and 20.9%. ITS sequences from 23 specimens provided a number of variable and sucient
characters to explore the relationships. As a result, it was determined that the dendrograms obtained by NJ and MP
analysis are concordant with the traditional taxonomical order at subgeneric level.
Key words: Turkey, ITS PCR, nrDNA, phylogeny, Primula
Türkiye’de doğal olarak yayılış gösteren Primula (Primulaceae)
taksonlarının ITS polimorzmi
Özet: Bu çalışmada, morfolojik olarak birbirine oldukça benzeyen 10 doğal Primula L. (Primulaceae) taksonuna ait 23
populasyon nrDNA ITS bölgeleri bakımından incelenmiştir. Bitki örnekleri 2005-2006 vejetasyon döneminde
Anadolu’nun farklı coğrak alanlarından toplanmıştır. Her populasyonun genomik DNA’ları sağlıklı yapraklardan elde
edilmiştir. ITS bölgeleri evrensel oligonükleotidler kullanılarak PCR yardımıyla elde edilmiş ve baz dizin analizleri
yapılmıştır. Primula taksonları arasındaki logenetik ilişkiyi belirlemek için Neighbour-Joining (NJ) ve Maksimum
Parsimoni (MP) ağaçları çizilmiştir. Populasyonlara ait baz dizin verilerinin hizalanmasıyla 724 karakterlik bir veri
matriksi elde edilmiş ve divergens değerlerinin de % 0,0-20,9 arasında değiştiği bulunmuştur. ITS baz dizinleri 23 örnekte
akrabalık ilişkilerini açıklamada yeterli veri sağlamaktadır. Sonuç olarak, NJ ve MP analizlerinden elde edilen
dendrogramların altcins seviyesinde geleneksel taksonomik verilerle uyumlu olduğu bulunmuştur.
Anahtar sözcükler: Türkiye, logeni, ITS PCR, nrDNA, Primula
147
Research Article
Turk J Bot
34 (2010) 147-157
© TÜBİTAK
doi:10.3906/bot-0905-23
* E-mail: mutlugultepe61@gmail.com

148
Internal transcribed spacer (ITS) polymorphism in the wild Primula (Primulaceae) taxa of Turkey
Introduction
The genus Primula L. (Primulaceae) includes
about 400 species belonging to 6 subgenera and 37
sections (Richards, 1993). It is widely distributed
outside of the Asian highlands and in the high
altitudes of North America, Europe, and the eastern
Sino-Himalayan region, considered the primary
centre of diversity for this genus (Hu & Kelso, 1996).
Many Primula species are also widely cultivated
throughout the world as ornamental plants (Mizuhiro
et al., 2001). Plant collectors have supplied enough
materials for horticultural and scientific purposes and
have played an important role in recognising the
genus (Hu & Kelso, 1996; Zhang & Kadereit, 2004)
and this genus has been recently studied by many
taxonomists, ecologists, geneticists, and gardeners
(Mast et al., 2001), but it is still not well known in the
wild. Heterostyly, recognised as a complex
reproductive syndrome with significant ecological
and evolutionary implications, is a well-known
process for the genus Primula (Barrett et al., 2000).
Heterostyly has played an important role in systematic
treatments of the genus Primula at the generic and
infrageneric levels (Richards, 1993). The suggested
base chromosome number is x = 11, but it varies from
x = 8 to x = 12 (Wendelbo, 1961; Mast et al., 2001;
Abou-el-Enain, 2005). Systematic investigations of
this genus have also shown that the chromosome
numbers are an important character in its
classification (Wendelbo, 1961; Richards, 1993).
A few characters used in the traditional systematic
studies for the genus Primula, besides heterostyly and
homostyly, are the chromosome base number, leaf
vernation, and pollen exine morphology (Mast et al.,
2001). Despite their putative importance in
elucidating relationships among Primula taxa, the
characters listed here do not align consistently with
one another (Mast et al., 2001), and therefore,
molecular sampling might provide an independent
systematic value (Conti et al., 2000). The internal
transcribed spacer (ITS) region and many other
molecular markers have been used in numerous plant
systematic studies at the family, generic, and specific
levels (Baldwin et al., 1995; Ogundipe & Chase, 2008).
This region is often used in closely related species for
phylogenetic analyses in several plant genera
(Anderberg & Stahl, 1995) and also in the genus
Primula (Conti et al., 2000; Kovtonyuk & Goncharov,
2009). Conti et al. (2000) used nuclear DNA
sequences to reconstruct the infrageneric phylogeny
of Primula, while Kovtonyuk and Goncharov (2009)
analysed the sequences of nuclear DNA and
confirmed Richards’ (2003) sectional rank proposal.
Mast et al. (2001) examined the specific chloroplast
DNA regions (the atpB, ndhF, and rblC genes) and
stressed that subg. Aleuritia needs a revision of its
delimitation. Analysis of the ITS sequences provided
new insights into the phylogeny of Primula (Conti et
al., 2000; Martins et al., 2003). According to Wendelbo
(1961) and Richards (1993), involute leaf vernation is
an ancestral trait, based on phenetic analysis, but
Conti et al. (2000) stress that the revolute traits are
ancestral, based on ITS data. Therefore, we thought
that performing molecular analyses based on nuclear
rDNA could provide additional useful information
about the Primula taxa of Turkey.
According to Lamond (1978), the genus Primula
is represented by only 8 species in Turkey. Of these, 2
members, P. d av i s ii W. W. S m . a n d P. l o ng i pe s Freyn &
Sint, are endemic to Turkey, and they are assessed as
vulnerable (VU) and endangered (EN), respectively
(Ekim et al., 2000). Turkish representatives of Primula
have been the subject of morphological, anatomical
(Beyazoğlu, 1989), cytotaxonomical (Hayırlıoğlu-
Ayaz & İnceer, 2003), and palynological (Pınar et al.,
2005) studies that have improved our understanding
of the systematics of the genus, but the Primula taxa of
Turkey have not been evaluated by analysis of ITS
region, except for P. davisii, which was used as an
outgroup to investigate the phylogeny and
biogeography of Dionysia Fenzl (Trift et al., 2004).
Thus, the main purpose of the current research was
to determine the ITS polymorphism among the taxa
of Primula distributed naturally in Turkey and to
elucidate their relationships.
Materials and methods
Plant Material
All plant materials used in this research were
collected from various regions of Anatolia during field
work in 2005 and 2006. For each population,
information related to the collection region and the
subgenera are shown in Table 1. The samples were

Table 1. Locality information.
Pop. Subg. Taxa Locality
no.
1P. vu lg ar is Huds. subsp. vulgaris A7 Trabzon: Beşikdüzü, Yeni Camii village, 01.v.2005, 970 m, Uzuner P11, KTUB
2P. vu lg ar is Huds. subsp. vulgaris A7 Trabzon: Beşikdüzü, Yeni Camii village, 01.v.2005, 970 m, Uzune r P12, KTUB
3P. vu lg ar is Huds. subsp. sibthor pii A7 Trabzon: Beşikdüzü, Yeni Camii village, 01.v.2005, 970 m, Uz uner P13, KTUB
(Hoffmanns.) W.W.Sm. & Forrest
4P. vu lg ar is Huds. subsp. vulgaris A4 Kastamonu: Bozkurt, 11.v.2005, 1200 m, Uz une r P15, KTUB
5P. vu lg ar is Huds. subsp. vulgaris A4 Kastamonu: Bozkurt, 11.v.2005, 1200 m, Uzuner P16, KTUB
6P. vu lg ar is Huds. subsp. sibthorpii A4 Kastamonu: Bozkurt, 11.v.2005, 1200 m, Uz une r P17, KTUB
(Hoffmanns.) W.W.Sm. & Forrest
7P. megaseifolia Boiss. & Bal. A7 Trabzon: Beşikdüzü, Şahmelik village, 10.iv.2005, 450 m, Uz uner P6, KTUB
8P. megaseifolia Boiss. & Bal. A8 Trabzon: Araklı, Sularbaşı village, 14.iv.2005, 1120 m, Uzune r P7, KTUB
9P. el at io r (L.) Hill subsp. meyerii (Rupr.) A8 Rize: İkizdere, Ovit Mountain, 24.vii.2005, 2850 m, Uzuner P26, KTUB
Valentine & Lamond
10 P. el at io r (L.) Hill subsp. meyerii (Rupr.) A7 Trabzon: Çaykara, Demirkapı village, 28.v.2005, 2920 m, Uzuner P29, KTUB
Valentine & Lamond
11 P. ve ri s L. subsp. columnae (Ten.) Lüdi A7 Trabzon: Çaykara, Ablaryas Plateau, 01.vi.2005, 2052 m, Uzuner P18, KTUB
12 P. ve ri s L. subsp. columnae (Ten.) Lüdi A7 Trabzon: Maçka Sümela Monastery, 24.v.2006, Coşkunçelebi 700, KTUB
13 P. v e r i s L. subsp. macrocalyx (Bunge) Lüdi A9 Artvin: Karagöl, 08.vi.2005, 1950 m, Uzuner P22, KTUB
14 P. lo ng ip e s Freyn & Sint. A8 Rize: İkizdere, Ovit Mountain, 24.vii.2005, 2850 m, Uzuner P25, KTUB
15 P. lo ng ip e s Freyn & Sint. A7 Trabzon: Çaykara, Demirkapı village, 28.v.2005, 3067 m, Uzuner P27, KTUB
16 P. auriculata Lam. A7 Gümüşhane: Kadırga village, 13.vi.2005, 2150 m, Uzuner P20, KTUB
17 P. auriculata Lam. A4 Kastamonu: Bozkurt, 1200 m 11.vi.2005, Coşkunçelebi 701, KTUB
18 P. auriculata Lam. A8 Erzurum: İspir, Moryayla, 21.x.2005, 2450 m, Uz une r P30, KTUB
19 P. auriculata Lam. A8 Kop Dağı, 2290 m, 18.v.2006, Coşkunçelebi 699, KTUB
20 P. algida Adams A9 Ardahan: Posof, Gönülaçan, 09.vi.2005, 2130 m, Uzuner P23, KTUB
21 P. algida Adams A8 Rize: İkizdere, Ovit Mountain, 24.vii.2005, 2850 m, Uzuner P24, KTUB
22 P. algida Adams A7 Trabzon: Çaykara, Demirkapı village, 28.v.2005, 2920 m, Uzuner P28, KTUB
23 P. davisii W.W.Sm C10 Hakkari: Çukurca, Stream path fork, 7.3 km, limy rock splits, 1200 m,
10.vi.2006, MF 10124, KTUB
Primula L.Aleuritia (Duby)
Wendelbo
Sphondylia
(Duby) Rupr.
149
M. GÜLTEPE, U. UZUNER, K. COŞKUNÇELEBİ, A. O. BELDÜZ, S. TERZİOĞLU
then identified according to traditional methods,
mainly by using Flora of Turkey (Lamond, 1978) and
Flora Europaea (Valentine & Kress, 1972), and stored
as herbarium specimens in the herbarium of the
Department of Biology, Faculty of Arts and Sciences,
Karadeniz Technical University.
DNA extraction
Total genomic DNAs were extracted from silica-
dried leaves or herbarium materials following the
modified CTAB extraction procedure of Doyle and
Doyle (1987). The gDNAs were resuspended in TE
(tris HCl-EDTA) and stored at +4 °C. The isolated
genomic DNAs were checked in a 1% agarose-TAE
(tris, acetate, and EDTA) gel containing 0.5 µg/L of
ethidium bromide and examined under UV light.
PCR amplification
The entire ITS regions (ITS1, 5.8S, and ITS2) were
amplified using a Biometra Personal Thermal Cycler.

The PCR reactions were performed using universal
ITS4 (5´- TCCTCCGCTTATTGATATGC- 3´) and
ITS5 (5´- GGAAGTAAAAGTCGTAACAAGG -3´)
primers, designed by White et al. (1990). The
amplification process was performed in 50 µL of PCR
reaction volume, containing 10 mM of Taq
polymerase reaction buffer, 2 mM of magnesium
chloride (MgCl2), 200 mM of dNTP, 1 M (ITS4 and
ITS5) each of the primer, 1-2 units of Taq DNA
polymerase, 2-6 ng (1 L of 2-6 ng/L) of total
template DNA, and 14 L of ddH2O. Reaction
mixtures were sealed with 1 or 2 drops of mineral oil
to prevent evaporation during thermal cycling.
Thermal cycling amplification was performed with an
initial denaturation step of 94 °C for 4 min, followed
by 35 cycles of strand denaturation at 94 °C for 1 min,
annealing at 50 °C for 45 s, and primer extension at
72 °C for 1 min, and a final elongation at 72 °C for 7
min.
Sequence analysis
PCR product purification and DNA sequence
analysis were performed by Macrogen Inc. (Seoul,
Korea). The sequencing process was conducted with
BigDyeTM terminator cycling protocols (Applied
Biosystems Inc., Foster City, CA, USA). PCR products
were purified using ethanol precipitation and run on
an Automatic Sequencer (ABI 3730x1) by a contract
laboratory. Sequencing of the 5’ end of the ITS region
was carried out using the primer ITS4. Sequences
with ambiguous sites were resequenced from the 3’
end with the primer ITS5. The sequence data were
submitted to GenBank under the accession number
of EU643642-EU643664 (Table 1).
Data analysis
The nucleotide sequences were automatically
aligned using BioEdit v.7.0 software (Hall, 1999).
Neighbour-Joining (NJ) and Maximum Parsimony
(MP) trees were built using the Molecular
Evolutionary Genetics Analysis (MEGA v.3.1)
program (Kumar et al., 2004). DNA sequences were
analysed based on Kimura’s 2-parameter model
(K2P). All characters were unordered and equally
weighted, and gaps were treated as missing data. The
topology of the consensus tree was constructed and
evaluated with 1000 bootstrap replications
(Felsenstein, 1985) for both the MP and NJ (Saitou &
Nei, 1987) analysis. For the phylogenetic analyses of
the ITS regions, Lysimachia nemorum L. (GenBank:
AY855153) and Anagallis serpyllifolia Dumort.
(GenBank: AY855154) were selected as outgroups.
Results
The total lengths of the ITS (ITS1, 5.8S, and ITS2)
regions of the examined populations ranged from 695
to 714 bp. The shortest ITS length among the
examined taxa was identified in P. algida (Pop. No.
21), collected from A8 Rize, İkizdere, Ovit Mountain.
P. v er i s subsp. macrocalyx, collected from A9 Artvin,
was identified as the population with the longest ITS
length, at 714 bp (Table 1). Alignments of entire ITS
sequences resulted in 724 characters, except for the
outgroup. The entire ITS region contained 153
(21.1%) parsimony informative sites (Table 2), 252
(34.8%) variable sites, and 466 (64.3%) conserved
sites.
The NJ tree obtained from the analysis of the ITS
regions provided many useful data (Figure 1). All of
the investigated taxa settled explicitly into 3 clusters
corresponding to subgenera of Primula, namely subg.
Sphondylia (Clade-I), Aleuritia (Clade-II), and
Primula (Clade-III). Clade-I included only one taxon,
P. davisii, which is the single species of subg.
Sphondylia in Turkey. Clade-II, with a bootstrap value
of 63%, consisted of the populations of P. algida, P.
auriculata, and P. l o n g i p e s , which belong to subg.
Aleuritia. Among the representatives of subg.
Aleuritia, P. auriculata and P. algida were linked to
each other and formed a sister group with P. l on gi p es
(Figure 1). Clade-III contained the taxa P. v ul g ar is , P.
veris, P. megaseifolia, and P. e l a t i o r of subg. Primula,
with a bootstrap value of 98% (Figure 1), and this
clade also divided into 2 distinct subgroups. The first
subgroup was composed of P. veris and the second,
which was split again into 2 small clusters, was
composed of the rest of the members of subg.
Primula. While P. megaseifolia and P. el a t i o r were
linked to a low bootstrap value of 51%, the taxa of P.
vulgaris were linked to each other with bootstrap
values of 92% in the second subgroup.
Explanatory information related to the parsimony
informative sites inferred by MEGA software is given
in Table 2. The analysis of the entire ITS sequence of
P. ve ri s exhibited notable base alterations from the rest
Internal transcribed spacer (ITS) polymorphism in the wild Primula (Primulaceae) taxa of Turkey
150

M. GÜLTEPE, U. UZUNER, K. COŞKUNÇELEBİ, A. O. BELDÜZ, S. TERZİOĞLU
151
of the taxa of subg. Primula in the parsimonic
nucleotide sites at the positions of 143, 159, 300, 453,
567, 577, 620, 631, and 672. The examined subspecies
of P. v e r i s are distinguished from each other by
position 659 of their ITS sequences (see Table 2). In
addition, the 2 populations of P. megaseifolia included
different nucleotides at 91, 228, 456, 474, 512, 553,
and 600 when compared to the rest of the taxa of subg.
Primula, and they were also distinguished from each
other at the nucleotide positions of 60, 78, 94, and 108.
P. e l a t i o r , however, included fewer parsimony
informative sites and was separated from the rest of
the members of subg. Primula at the positions of 54,
457, 598, and 634. Furthermore, the distinct
population of P. e l a t i o r had only one different base
alteration, at the position of 624.
The members of subg. Aleuritia (Pop. No. 13-22)
displayed the highest parsimony informative sites
within all the taxa of the study. In this subgenus, the
population of P. au ri cu la t a displayed a number of base
alterations differing from the rest of the taxa of subg.
Aleuritia, at the positions of 54, 76, 82, 111, 230, 232,
304, 454, 456, 484, 497, 555, 567, 599, 601, 606, and
621 (see Table 2). The populations of P. a ur ic ul at a also
exhibited some differences at the positions of 259,
465, 478, 512, 552, 565, 572, 580, 600, 648, and 659.
The populations of P. algida demonstrated some
differences from other populations of subg. Aleuritia
at the positions of 82, 86, 94, 152, 190, 437, 454, 517,
540, 555, 563, 568, 575, 597, 620, 630, and 672. We
also observed some base variations at the positions of
37, 60, 61, 78, 102, 103, 105, 111, 219, 452, 475, and
563 among the populations of P. algida. P. l o n g i p e s ,
with the highest parsimony informative sites, differed
from the rest of the species of subg. Aleuritia at the
positions of 6, 61, 74, 75, 82, 89, 154, 166, 168, 184,
190, 217, 218, 219, 225, 231, 232, 234, 248, 259, 432,
442, 443, 445, 446, 447, 459, 481, 514, 537, 543, 556,
561, 562, 566, 567, 581, 583, 584, 603, 604, 618, 620,
624, 625, 630, 649, 650, 659, 674, 676, 677, 681, and
682. The 2 populations of P. l o n g i p e s also contained
Primula v ulg aris subsp. vulgaris (1)
Primula v ulg aris subsp. vulgaris (2)
Primula v ulg aris subsp. sibthorpii (3)
Primula v ulg aris subsp. v ulg ar is (5)
Primula vulgaris subsp. vulgaris (4)
Primula vulg aris subsp. sibthorpii (6)
Primu la meg aseifolia (7)
Primula megaseifolia (8)
Primula elatior subsp. mey erii (9)
Primula eletior subsp. mey e ri i (10)
Primula veris subsp. columnae (11)
Primula veris subsp. co lumnae (12)
Primu la veris subsp. macrocalyx (13)
CLA DE I II
Primula longipes (14)
Primu la lo ng ipe s (15)
Primula algida (21)
Primula algida (22)
Primula algida (20)
Primula auricu lata (19)
Primula auricu lata (17)
Primula auriculata (16)
Primula auriculata (18)
CLA DE II
CLAD E I
Primula davisii (23)
Lys ima ch ia n emo ru m
Anagallis serpyllifolia
OUTGROUP
99
99
91
99
72
67
99
59
63
86
99
96
89
98
51
62
70
79
92
0.1
Figure 1. The dendrogram showing the genetic relationships of the Turkish Primula. Primula species recovered from ITS sequences,
evaluated by the neighbour-joining method, with Lysimachia nemorum and Anagallis serpyllifolia as outgroups. Values above
the branch indicate bootstrap values supporting the respective cluster. Values higher than 50% are displayed.

Table 2. Parsimony informative sites’ aligned sequences of Primula taxa. For the pop. no. explanation, see Table 1.
1
TAAGCGCATGGACAACGACTGAAGATGGTAGGTCTGTTAAGAATGCCTATATTTAAGGCTCTTGTAACCTATGGCAG
2
.............................................................................
3
.............................................................................
4
...............................................................A.........A...
5
...............................................................A.........A...
6
...............................................................A.........A...
7
.....C....T.....A.T...T.C....................T.................T......G...A..
8
................A.......C....................T.................T......G...A..
9
....A...................C-.....................................T.......C.....
10
....A...................C-.....................................T.......C.....
11
..........................A...A..........................A.....T....G........
12
..........................A...A..........................A.....T....G........
13
..........................A...A..........................A.....T....G........
14
CG.A..ATA..TT.T..T.A.G..CG..CG.TATAAGATCA.GCA..ATGGG.ATG...A.CGCAC.....CT...A
15
CG.A..ATA..TT.T..T.A.G..CG..CG.TATAAGATCA.GCA..ATGGG.ATG...A.CGCAC.....CT...A
16
.G..A.T..A.TG.GG.T.A.GGACG...G....AA.A...G.C..A.G.GGG..GA.T-...T..G..AGCA....
17
.G..A.T..A.TG.GG.T.A.GGACG...G....AA.A...G.C..A.G.GGG..GA.T-...T..G..AGCA...A
18
.G..A.T..A.TG.GG.T.A.GGACG...G....AA.A...G.C..A.G.GGG.GGA.T-...T..G..AGCA....
19
.G..A.T..A.TG.GG.T.A.GGACG...G....AA.A...G.C..A.G.GGG..GA.T-...T..G..AGCAA...
20
.TCA.CG...TTATGG.TTGCCG.CG.A.G....AAC....G.CA.....GGG..GA..-T..T..G..G.CA.-..
21
.G.A..T....TATGG.TTC.GGTCG.A.G....AAC...TG.CA.....GGG..GA..-T..T..GG.G.CA.-C.
22
.G.A..T....TATGG.TTCAGG.CG.A.G....AAC...TG.CA.....GGG..GA..-T..T..GG-G.CA.-C.
23
.GC.TAT....TA.GG.T.CAGGACG...G....AA.A.....C....TCGGG..G...-.C.T..G....CA.-..
6
37
52
53
54
60
61
74
75
76
78
79
82
86
89
90
91
93
94
102
103
105
108
111
116
126
143
152
154
156
159
166
168
184
187
189
190
215
217
218
219
224
225
226
227
228
230
231
232
234
236
239
246
248
259
271
291
300
304
432
437
442
443
445
446
447
449
452
453
454
456
457
459
465
474
475
478
Pop. no.
Dashes (-) indicate alignment gaps within the sequence
152
Internal transcribed spacer (ITS) polymorphism in the wild Primula (Primulaceae) taxa of Turkey
different nucleotides at the positions of 623 and 631.
P. dav i si i , the single population of subg. Sphondylia in
Turkey, differed from subg. Primula and subg.
Aleuritia at the positions of 54, 60, 234, 566, 612, and
674.
The pair-wise distances obtained by Kimura’s 2-
parameter model for the examined populations varied
from 0.0% to 20.9% (see Table 3). The divergence
values within the subg. Primula based on ITS
sequence variations ranged from 0.0% to 5.4%; for
subg. Aleuritia, it was determined to range from 0.0%
to 19.7%. The pair-wise distance matrix of genetic
divergence values among all of the examined species
is given in Table 4 and includes various outputs
ranging from 0.0% to 18.9%. The base variations of
investigated species of subg. Primula ranged from
0.0% to 6.0% and varied from 0.0% to 16.1% for the
subg. Aleuritia. The values of the pair-wise distances
of subg. Sphondylia,represented byP. d av is ii , to subg.
Primula and subg. Aleuritia are 15.7% and 12.6%,
respectively.
Discussion
Internal transcribed spacer (ITS) sequences have
been widely used in plant molecular phylogenetics
and evolutionary studies (Zheng et al., 2008).
Alignment of the nrDNA ITS sequences for many
plant taxa has supplied useful data for solving
taxonomic problems, especially below the generic
levels (Baldwin et al., 1995; Gravendeel et al., 2001).
As an explicit result of the present study, all of the
examined taxa were found to be clustered within the
3 main clades of the phylogenetic tree obtained by NJ
analysis. The clades, which represent 3 different
subgenera, included the closely related taxa congruent
with the conventional taxonomic order of Turkey’s
Primula (Table 1). The present data confirmed the
tree topologies of Mast et al. (2001), in which subg.
Primula and subg. Aleuritia taxa are clustered in a
different clade. The molecular results of these 2
studies supported the view of Lamond (1978) at the
subgenera level.

1
CCAAGGGAGCTCCGATTAACCCCCCCGTGGAAGGACAACGCGAGATGAGAGCCAAGTGACGCATTCAGAAGGAATA
2
............................................................................
3
............................................................................
4
.....A......................................................................
5
...............................G............................................
6
.....A......................................................................
7
.....AA..........C........................G......................T...G......
8
.....AA..........C........................G................-.....T...G......
9
.....A..................................A.............C....T.....T...G......
10
.....A..................................A..................T.....T...G......
11
.....A...................T......A..................T......T......T...GA.....
12
.....A...................T......A..................T......T......T...GA.....
13
.....A...................T......A..................T......T......T.A.GA.....
14
ATGC...T.TC.TAGCA..ATG..TT--AAG.AATGCGT..A..GATCC.AG..GACCG.A-GAGG.A.G.TTGAG
15
ATGC...T.TC.TAGCA..ATG..TT--AAG.AATGCGT..A..GATCC.AG.GGACC..A-GAGG.A.G.TTGAG
16
.TTCT.A..TG...GCA.C..A.T.A-GAAG-A.CT.GT..T.A..ACCC..T...C...AAG..GGTTG......
17
.TTCT.A..TG...GCG.C..A.T.A-GAAG-A.CT.GT..T.A..ACCC..T...C...AAG..GGATG......
18
.TTCT.A..TG...GCA.C..A.T.A-GAAG-A.CT.GT..TGA..ACCC..T...C...AAG..GGTTG......
19
.TTCT....TG...GCA.C..A...A-GA.G-AACT.GT..T.A..ACCC..T...C...AAT..GGTTG......
20
.TGC....ATGTT..CG.T..AG...AGAAGGA.CT...A.A....TCCC.T.G..CA..AAG..GGTTGA.....
21
.TGC....ATGTT..CG.T..AG...AGAAGGA.CT...A.A....TCCC.T.G..CA..AAG..GGTTGA.....
22
.TGC....ATGTT..CG.T..AA...AGAAGGA.CT...A.A....TCCC.T.G..CA..AAG..GGTTGA.....
23
.T.CT....T..T.GCG.T..G..GAAGAAGGA.CT.G...-.....TCCA..G..C.....G..GGATGAC....
481
483
484
490
497
508
512
514
517
533
537
540
541
543
545
550
552
553
555
556
561
562
563
565
566
567
568
569
570
572
573
575
577
580
581
583
584
586
595
597
598
599
600
601
603
604
606
612
613
617
618
620
621
623
624
625
629
630
631
634
636
641
648
649
650
653
654
659
660
667
672
674
676
677
681
682
Pop. no.
Table 2. Continued.
Dashes (-) indicate alignment gaps within the sequence
153
M. GÜLTEPE, U. UZUNER, K. COŞKUNÇELEBİ, A. O. BELDÜZ, S. TERZİOĞLU
According to Lamond (1978), the species of P.
vulgaris, which does not have well- developed scape
structures, morphologically differs from P.
megaseifolia, P. e l at i o r ,and P. v e r i s . In the present
study, it was found that all examined populations of
P. v u l g a r i s formed a distinct cluster from the rest of
the taxa of subg. Primula, having well-developed
scape structures. As seen in Figures 1 and 2, the
examined populations of P. v u l g a r i s clustered into 2
lineages with high bootstrap values of 92% and 78%,
respectively. These genetic dissimilarities can also be
inferred from the pair-wise distance values given in
Table 3. The first 3 populations of P. v ul ga ri s (Pop. no.
1, 2, and 3), which have diverse corolla colours as the
single apparent morphological differentiation, were
collected from the same location (A7 Trabzon) and
clustered into one subgroup in relation to their
ecological closeness. The second 3 populations of
Primula (Pop. no. 4, 5, and 6; A4 Kastamonu) were
also accumulated together in the subgroup due to
similar conditions. Although both population
members of P. v ul g a ri s were collected from 2 distinct
regions, they exhibited high similarity (only 2 base
diversities; see Table 2) with respect to their ITS
sequence data. We also identified several parallel
alignments that demonstrate congruency with
geographical dispersion within the other examined
populations. Many studies have shown that there is a
clear affinity between populations and the
environments in which they exist (Aston & Bradshaw,
1966). The current and previously introduced data
showed that closer geographical distances yield more
homology in ITS genotypes (Wardill et al., 2005). On
the other hand, the genetic variations within the
subspecies could be affected by regional climates
(Noyes, 2006).
Both P. e l a t i o r and P. v e r i s have leaves that are
rugose, efarinose, and glabrous to densely villous
below, and so they carry relatively similar
morphological characteristics (Lamond, 1978). Yet, as
seen in Figure 1, P. e l a t i o r is closely linked to P.

Table 3. Pair-wise distance matrix of genetic divergence values of 23 populations, according to Kimura’s 2-parameter model. For the pop. no. explanation, see Table 1.
Pop. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
no.
1 0.000
2 0.000
3 0.000 0.000
4 0.004 0.004 0.004
5 0.003 0.003 0.003 0.001
6 0.004 0.004 0.004 0.000 0.001
7 0.047 0.047 0.047 0.047 0.049 0.047
8 0.016 0.016 0.016 0.016 0.018 0.016 0.03
9 0.013 0.013 0.013 0.013 0.015 0.013 0.046 0.015
10 0.013 0.013 0.013 0.013 0.015 0.013 0.044 0.015 0.003
11 0.018 0.018 0.018 0.018 0.020 0.018 0.054 0.023 0.020 0.020
12 0.018 0.018 0.018 0.018 0.020 0.018 0.054 0.023 0.020 0.020 0.000
13 0.040 0.040 0.040 0.040 0.041 0.040 0.076 0.044 0.041 0.041 0.021 0.021
14 0.160 0.160 0.160 0.164 0.162 0.164 0.207 0.170 0.160 0.162 0.164 0.164 0.186
15 0.162 0.162 0.162 0.166 0.164 0.166 0.209 0.172 0.162 0.164 0.168 0.168 0.190 0.009
16 0.112 0.112 0.112 0.116 0.114 0.116 0.145 0.112 0.109 0.109 0.119 0.119 0.143 0.138 0.140
17 0.116 0.116 0.116 0.119 0.117 0.119 0.149 0.116 0.112 0.112 0.123 0.123 0.144 0.138 0.140 0.006
18 0.119 0.119 0.119 0.123 0.121 0.123 0.149 0.116 0.116 0.116 0.126 0.126 0.150 0.143 0.145 0.006 0.012
19 0.126 0.126 0.126 0.126 0.124 0.126 0.163 0.130 0.122 0.122 0.133 0.133 0.157 0.154 0.156 0.023 0.029 0.029
20 0.163 0.163 0.163 0.167 0.165 0.167 0.190 0.171 0.163 0.163 0.165 0.165 0.190 0.197 0.195 0.117 0.120 0.124 0.136
21 0.117 0.117 0.117 0.121 0.119 0.121 0.155 0.125 0.117 0.117 0.119 0.119 0.143 0.143 0.141 0.067 0.070 0.074 0.085 0.059
22 0.119 0.119 0.119 0.123 0.121 0.123 0.157 0.127 0.119 0.119 0.121 0.121 0.145 0.145 0.143 0.070 0.073 0.077 0.088 0.060 0.006
23 0.150 0.150 0.150 0.154 0.152 0.154 0.191 0.158 0.148 0.148 0.154 0.154 0.177 0.173 0.171 0.099 0.097 0.106 0.118 0.154 0.109 0.109 0.000
Internal transcribed spacer (ITS) polymorphism in the wild Primula (Primulaceae) taxa of Turkey
154

M. GÜLTEPE, U. UZUNER, K. COŞKUNÇELEBİ, A. O. BELDÜZ, S. TERZİOĞLU
155
magaseifolia rather than P. v e r i s , by virtue of sharing
the same character (C) at site 116 (Table 2). This
evidence indicates that molecular data may
sometimes present findings that are in contrast to
morphological data and could not always resolve the
relationships in Primula (Martins et al., 2003).
Although several hybrids naturally occur between
P. v u l ga ri s and P. v e r i s (Kalman et al., 2004), P. v e r i s is
a distinct species among the subg. Primula based on
the NJ analysis (Figure 1). In addition, P. v u l g a r i s
(Hayırlıoğlu-Ayaz & İnceer, 2003) and P. v er is (Abou-
El-Enain, 2005) have the same (2n = 22) chromosome
Table 4. Pair-wise distance matrix of genetic divergence values of 8 taxa, according to Kimura’s 2-parameter model.
Subg. Species P. vulgaris P. megaseifolia P. elatior P. veris P. longipes P. auriculata P. algida P. davisii
P. vulgaris
Primula P. megaseifolia 0.032
P. elatior 0.014 0.030
P. v er i s 0.040 0.060 0.041
P. l on gi pe s 0.162 0.189 0.162 0.188
Aleuritia P. auriculata 0.119 0.135 0.115 0.148 0.144
P. algida 0.135 0.154 0.133 0.159 0.161 0.092
Sphondylia P. davisii 0.152 0.174 0.148 0.177 0.172 0.105 0.124
Primula vulgaris s ubsp. vulgaris (1)
Primula vulgaris s ubsp. vulgaris (2)
Primula vulgaris s ubsp. sibthorpii (3)
Primula vulgaris s ubsp. vulgaris (5)
Primula vulgaris subsp. vulgaris (4)
Primula vulgaris subsp. sibthorpii (6)
Primula megaseifolia (7)
Primula megaseifolia (8)
Primula elatior subsp. mey e rii (9)
Primula eletior subsp. meye rii (10)
Primula veris subsp. columnae (12)
Primula veris subsp.columnae (11)
Primula veris subsp. macroc alyx (13)
Primula longipes (14)
Primula longipes (15)
Primula auriculata (19)
Primula auriculata (17)
Primula auriculata (16)
Primula auriculata (18)
Primula algida (20)
Primula algida (21)
Primula algida (22)
Primula davisii (23)
Lys ima ch ia n emo ru m
Anagallis serpyllifolia
97
100
60
100
100
99
92
63
53
56
78
100
100
100
50
Figure 2. One of 16 parsimony trees for the Primula nuclear ITS region. Bootstrap values were obtained by 1000 replicates; numbers
above the branches are bootstrap values > 50, tree length 669, consistency index = 0.8714, retention index = 0.9198, rescaled
consistency index = 0.8016.

number. This is probably due to the few base
variations that emerged as a result of geographical and
ecological effects.
P. e l a t i o r and P. megaseifolia are linked to each other
with a low bootstrap value (51%) (Figure 1) and they
revealed a few more base dissimilarities than the rest
of the subg. Primula taxa (Table 2). The different
corolla colours and chromosome numbers of these
species also support their differences. Furthermore, the
earlier recorded different chromosome numbers for P.
megaseifolia (2n = 18) and P. v u l g a r i s (2n = 22)
(Hayırlıoğlu-Ayaz & İnceer, 2003) can be presented as
additional evidence for genetic dissimilarity. Although
P. megaseifolia morphologically resembles P. v u l g a r i s
(Lamond, 1978), the results based on phylogenetic
analyses showed a distinctive relationship between P.
elatior and P. megaseifolia (Table 4 and Figure 1),
despite the differences in their chromosome numbers
(2n = 16 and 2n = 18, respectively) (Hayırlıoğlu-Ayaz
& İnceer, 2003). The current data also indicate that P.
veris and P. megaseifolia are the most distant species in
the subg. Primula because of the high base variation
(Table 2), which may be caused by ecological effects
(Scheiber et al., 2000).
The subg. Aleuritia is represented by 3 species in
the present study. Among these species, P. l o n g i p e s is
the most distinct, based on inflorescence shape and
length of scape (Lamond, 1978). The results of the
sequence analyses of the entire ITS region also
confirmed this taxonomical differentiation (Figure 1).
According to the genetic divergence values of the pair-
wise distance matrix (Table 4), P. a u r i c u l a t a and P.
algida are the most closely related species, while P.
algida and P. l on gi pe s are the most discrete taxa of the
subg. Aleuritia. P. l o ng i p e s was obviously separated
from the other taxa of subg. Aleuritia since it
contained many different base substitutions (Table 2)
compared to P. auriculata and P. algida, which are the
closer taxa in Clade-II (Figure 1). Several earlier
studies related to ITS regions have shown that this
region supplies sufficient genetic information to
explore the relationships at both specific and generic
levels (Bain & Jansen, 1995).
Leaf vernation is a useful taxonomic character for
Primulaceae (Wendelbo, 1961); however, both
revolute and involute leaf vernation can occur in the
genus Primula (Richards, 1993). According to
Wendelbo (1961), the involute vernation is a primitive
morphological trait which is one of the distinguishing
characters of the subg. Sphondylia. The current
findings based on the NJ tree and other related data
also support the positioning of P. d a v i s i i as a primitive
taxon consisting of traits that are present in the
common ancestor (Figure 1).
It is well known that molecular sampling might
provide an independent phylogenetic hypothesis
(Mast et al., 2001) for traditional taxonomic studies.
Thus, our current study can be illustrated as
preliminary molecular and phylogenetic research
carried out on the wild Primula taxa of Anatolia. All
the results showed that the ITS region supplies useful
molecular information for exploring the relationships
among the Turkish Primula taxa. However, to
reconstruct an accurate phylogenetic relationship, the
taxa should be examined with different molecular
markers and intensive sampling.
Acknowledgements
The authors would like to express their thanks to
Dr. Sabriye Dülger and Dr. Yusuf Bektaş for kindly
helping with the laboratory studies, Mehmet Fırat for
providing the sample of P. davisii, and TÜBİTAK
(TBAG-HD/356-107T918) and the Research
Foundation of Karadeniz Technical University (KTU-
BAP-2007.111.004.07) for the financial support.
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Bain JF & Jansen RK (1995). A phylogenetic analysis of the aureoid
Senecio (Asteraceae) complex based on ITS sequence data. Plant
Syst Evol 195: 209–219.
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