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Mitochondrial DNA Part A
DNA Mapping, Sequencing, and Analysis
ISSN: 2470-1394 (Print) 2470-1408 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn21
Phylogenetic relationships of Podarcis siculus
(Rafinesque-Schmaltz, 1810) and Podarcis tauricus
(Pallas, 1814) in Turkey, based on mitochondrial
DNA
Halime Koç, Ufuk Bülbül, Muammer Kurnaz, Ali İhsan Eroğlu & Bilal Kutrup
To cite this article: Halime Koç, Ufuk Bülbül, Muammer Kurnaz, Ali İhsan Eroğlu & Bilal Kutrup
(2018) Phylogenetic relationships of Podarcis�siculus (Rafinesque-Schmaltz, 1810) and Podarcis
tauricus (Pallas, 1814) in Turkey, based on mitochondrial DNA, Mitochondrial DNA Part A, 29:5,
664-673, DOI: 10.1080/24701394.2017.1342245
To link to this article: https://doi.org/10.1080/24701394.2017.1342245
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Published online: 04 Jul 2017.
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RESEARCH ARTICLE
Phylogenetic relationships of Podarcis siculus (Rafinesque-Schmaltz, 1810) and
Podarcis tauricus (Pallas, 1814) in Turkey, based on mitochondrial DNA
Halime Koc¸, Ufuk B€
ulb€
ul, Muammer Kurnaz, Ali
_
Ihsan Ero
glu and Bilal Kutrup
Department of Biology, Karadeniz Technical University, Trabzon, Turkey
ABSTRACT
The Italian wall lizard and the Balkan wall lizard have a series of taxonomic revisions. However, their
phylogenetic relationships still remain uncertain in Turkey. In the present study, we have assessed taxo-
nomic relationships, both of Podarcis siculus and Podarcis tauricus through estimation of phylogenetic
relationships among 43 and 42 specimens, respectively, using mtDNA (16 S rRNA and cytb) from great
main populations in Turkey. The genetic distances among the populations of P. siculus in Turkey were
very low and they were ranged from 0.2 to 1.6% in 16 S rRNA while they were ranged from 0.0% to
3.3% in cytb. On the other hand, the p-distances among the populations of P. tauricus were ranged
from 0.0 to 0.6% in 16S rRNA while they were 0.2% cytb in Turkey. Finally, most of the topologically
identical trees of phylogenetic analyses and p-distances showed that monophyly was found in extant
populations of P. siculus and P. tauricus. The nominate subspecies, P. s. siculus and P. t. tauricus are rep-
resentatives of these lizards in Turkey.
ARTICLE HISTORY
Received 6 April 2017
Accepted 11 June 2017
KEYWORDS
16S rRNA; P. s. siculus; cytb;
P. t. tauricus; monophyly
Introduction
Wall lizards of the genus Podarcis (Wagler 1830) comprise
currently 23 recognized species (Sindaco et al. 2013; Uetz and
Ho
sek 2016). The origin of the genus is western European
(Oliverio et al. 2000) and the genus is distributed in Europe,
North Africa, and North America. Most species of the genus
are restricted to the Mediterranean basin (Harris 1999; Harris
and Arnold 1999; Speybroeck et al. 2010; Silva-Rocha et al.
2012). Currently, the predominant reptile group in southern
Europe is distributed from Northwestern Africa through the
Iberian and the Italian peninsulas to the Balkans, northwest-
ern Asia Minor and the Crimean peninsula (Arnold 1973).
Taxonomy of Podarcis genus is complicated and continu-
ously needs revision, due to the substantial intra-specific vari-
ability (Arnold et al. 1978). The first phylogenetic studies on
the genus were conducted by Harris and Arnold (1999) and
Oliverio et al. (2000). The genus was separated into four geo-
graphic groups (the Western island group, the Balkan group,
the Italian group and the Southwestern group) but relation-
ships were mainly unresolved. It may be due to a large distri-
bution area of the genus.
The Italian wall lizard, Podarcis siculus, is one such species
that has a large distribution area in the central Mediterranean
region (Corti 2006). It is widespread in Italy (on many Adriatic
islands, the large islands Sicily, Sardinia and Corsica) and the
northern part of the east Adriatic coast. Apart from this distri-
bution, it is also distributed in the Mediterranean region (in
Portugal, Spain, France, Montenegro, Turkey, Libya and
Tunisia) and the USA (Behler and King 1979; Conant and
Collins 1991). Italy is thought to be the area of origin and the
expansion center of the species (Radovanovic 1956;
Schneider 1971; Gorman et al. 1975). In this large distribution
area, P. siculus has 23 subspecies.
In Turkey, the first specimens of P. siculus were recorded
from Anatolian part of the
_
Istanbul province by Berhold
(1942) and he described the specimens as the representatives
of P. s. hieroglyphicus based on their morphological charac-
ters. Other records were given from
_
Istanbul and islands of
Marmara (Bird 1936; Bodenheimer 1944; Mertens and
Wermuth 1960; Clark and Clark 1973; Bas¸o
glu and Baran
1977; Franzen 1990;C¸evik 1999; Jablonski and Stloukal 2012),
Bursa (U
gurtas¸ and Yıldırımhan 2000; Mollov 2009; Arslan
et al. 2013) and C¸anakkale (H€
ur et al. 2008; Tok and Cicek
2014; Tok et al. 2015) provinces in the Marmara Region and
Zonguldak (Ilgaz et al. 2013) and Samsun (Tok et al. 2015)
provinces in the Black Sea Region of Turkey. According to
current literature, the lizards belonging to Podarcis sicula cet-
tii,Podarcis sicula ragusae and Podarcis siculus hieroglyphicus
were approved as synonyms of P. s. siculus and the Turkish
specimens of P. siculus are considered as the representatives
of P. s. siculus (http://www.lacerta.de; Silva-Rocha et al. 2014).
However, there is no phylogenetic study on these specimens
belonging to Turkey populations.
The Balkan wall lizard, Podarcis tauricus is another species
of Podarcis genus that has a smaller distribution area than P.
siculus (ranging mainly in the southern Balkans and eastern
Europe). Currently, it is subdivided into three recognized sub-
species (Sindaco and Jerem
cenko 2008). The first one is P. t.
tauricus (Pallas 1811); the second one is P. t. ionicus (Lehrs
CONTACT Ufuk B€
ulb€
ul ufukb@ktu.edu.tr Faculty of Science, Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey
Supplemental data for this article can be accessed here.
ß2017 Informa UK Limited, trading as Taylor & Francis Group
MITOCHONDRIAL DNA PART A
2018, VOL. 29, NO. 5, 664–673
https://doi.org/10.1080/24701394.2017.1342245
1902); and the last one is P. t. thasopulae (Kattinger 1942).
The first two subspecies are geographically isolated by the
Pindos mountain while the third one has a small inhabiting
area on the islet of Thasopoula (north Aegean) (Psonis et al.
2017).
In Turkey, Podarcis tauricus is distributed in the provinces
of Thrace Region and
_
Istanbul (Schreiber 1912; Cyr
en 1924;
Bird 1936; Bodenheimer 1944; Mertens 1952; Clark and Clark
1973; Andren and Nilson 1976;C¸evik 1999), Kocaeli and
Sakarya (Baran 1977; Bas¸o
glu and Baran 1977; Nilson et al.
1988; Bergmann and Norstr€
om 1990, Franzen 1990; Teynie
1991; Baran et al. 1992; Mulder 1995; Sindaco et al. 2000) and
C¸anakkale (Tok and Cicek 2014) provinces in the Marmara
Region. Recently, B€
ulb€
ul et al. (2015) reported these lizards
from the western Black Sea Region of Turkey.
Mertens (1952) reported that all examined specimens in
the literature from the European and Anatolian parts of
Turkey belonged to P. t. tauricus. The current literature (based
on the morphological investigations) approves this view.
However, there is no phylogenetic study on the Turkish pop-
ulations of P. tauricus.
The phylogenetic relationships and phylogeography of the
P. tauricus subgroup found in Europe have previously been
investigated on the basis of mitochondrial DNA loci (Podnar
et al. 2004,2015; Poulakakis et al. 2005a,b; Psonis et al. 2017).
Although molecular studies were performed on the speci-
mens in European populations of P. siculus and P. tauricus,
the phylogenetic relationships of the Turkish populations of
these species were not investigated. For this reason, the pur-
pose of the present study is to appraise the phylogenetic
relationships of the P. siculus and P. tauricus specimens from
the great main distribution areas of Anatolia and to deter-
mine whether there is a potential new phylogenetic lineage
of these lizards in Turkey based on the results of mitochon-
drial DNA for the first time.
Material and methods
Collection of the samples
A total of 43 specimens of P. siculus and 42 ones of P. tauri-
cus were caught from different localities in Turkey (Figure 1)
(Tables 1 and 2). For each lizard, the longest finger of the
hind limb was clipped and preserved in 96% ethanol. After
registration and toe-clipping, all lizards were released back
into their natural habitats.
DNA extraction and PCR amplifications
The clipped toes obtained from lizards were stored in 96%
ethanol. Later, the toes were treated with 180 ll ATL, 20 ll
proteinase K and 4 ll RNAse in 2 ml eppendorf tubes over-
night at 56 C. Total genomic DNA of each specimen was
extracted using the NucleoSpin tissue isolation kit following
Manufacturer’s instructions.
For P. siculus, a 501 base-pair-fragment of the 16 S rRNA
gene (for 37 specimens) and a 469 base-pair-fragment of the
cytb gene (for 39 specimens) were amplified while a 501
base-pair-fragment of the 16 S rRNA gene (for 40 specimens)
and 425 base-pair-fragment of the cytb gene (for 40 speci-
mens) were amplified for P. tauricus, using 16SarL and
16SbrH (Palumbi et al. 1991); L14724 and H15175 (Palumbi
1996) primers, respectively. Each 16 S rRNA gene amplification
involved an initial incubation 3 min at 94 C; 35 cycles of 30 s
at 94 C; 30 s at the appropriate annealing temperature
(48–54 C); and 1 min at 72 C; followed by one cycle of 8 min
at 72 C. PCR amplifications for 16 S rRNA were conducted as
described by (Guo et al. 2011). Each cytb gene amplification
involved an initial incubation 5 min at 94 C; 35 cycles of 60 s
at 94 C; 60 s at the appropriate annealing temperature
(52–56 C); and 1 min at 72 C; followed by one cycle of 70 s
at 72 C. PCR amplifications for cytb were conducted as
described by (Poulakakis et al. 2003). Amplified DNA seg-
ments were purified and sequenced by Macrogen
Corporation in Netherlands.
Sequence alignment and phylogenetic analyses
The nucleotide sequences of each gene were aligned using
the Bioedit (Thompson et al. 1997) program. Haplotypes were
determined for each gene using TCS (Clement et al. 2000)
program. (GenBank accession numbers for each haplotype
sequence are given in (Tables 1 and 2). After confirming the
suitability for the combination of all the sequences of two
genes, we combined the data on these two genes for ML
and BI. For a comparison of our haplotypes with other
European P. siculus populations, we used six haplotypes from
Italy (AY770920.1 and AY770919.1, Podnar et al. 2005), Spain
(HM746963.1 and HM746964.1, Valdeon et al. 2010) and
Croatia (EU362073.6 and EU362074.1, Herrel et al. 2008) for
16 S rRNA and eight haplotypes from Italy (KF372034.1, Salvi
et al. 2013) and (AY770895.1, Podnar et al. 2005), Spain
(JX072939.1 and JX072941.1, Silva-Rocha et al. 2012), Croatia
(AY770882.1 and AY770892.1, Podnar et al. 2005), Portugal
(JX072953.1, Silva-Rocha et al. 2012) and Turkey (KP036398.1,
Silva-Rocha et al. 2014) for cytb gene. For similar aim, we
compared our P. tauricus haplotypes with European popula-
tions of the species and we used five haplotypes from Greece
(AY768728.1 and AY768727.1, Poulakakis et al. 2005a), Turkey
(KX658353.1 and KX658354.1, Psonis et al. 2017) and Albania
(KX658351.1, Psonis et al. 2017) for 16 S rRNA and three hap-
lotypes from Greece (KX658062.1, KX658057.1, Psonis et al.
2017) and Albania (KX658038.1, Psonis et al. 2017) for cytb
gene on Genbank.
Phylogenetic analyses based on the two genes (16 S rRNA
and cytb) separately and combined data. We conducted mul-
tiple complementary methods of data analysis, such as neigh-
bour-joining (NJ), maximum parsimony (MP), maximum
likelihood (ML) and Bayesian inference (BI) phylogenetic
approaches using MEGA 6.0 v (Tamura et al. 2013) for NJ and
MP and ML, and MrBayes 3.2.3 (Ronquist and Huelsenbeck
2003) for BI. Neighbour-joining (NJ), Maximum Parsimony
(MP) and maximum likelihood (ML) analyses were carried out
using a heuristic search method (10,000 random addition rep-
licates tree-bisection-reconnection, TBR, branch swapping)
and bootstrap analyses with 1000 replications for NJ, MP and
ML (Felsenstein 1985) were applied. Transitions and transver-
sions were equally weighted, and gaps were treated as
MITOCHONDRIAL DNA PART A 665
missing data. In the BI analysis, the following settings were
conducted: number of Markov Chain Monte Carlo (MCMC)
generations ¼six millions; sampling frequency ¼100; burn-in
¼25%. The burn-in size was determined by checking conver-
gence of –log likelihood (-ln L) using MrBayes 3.2.3 (Ronquist
and Huelsenbeck 2003). NJ, MP and ML trees were evaluated
using bootstrap analyses with 1000 replicates and statistical
support of the resultant BI trees was determined based on
Bayesian posterior probability (BPP). Best fit nucleotide substi-
tution models were determined for each gene region with
MEGA 6.0 v (Tamura et al. 2013) for NJ, MP, ML and BI analy-
ses based on Akaike’s information criteria (AIC). We a priori
regarded tree nodes with bootstrap values (BS) 70% or
greater as sufficiently resolved (Huelsenbeck and Hillis 1993),
and those between 50 and 70% as tendencies. In the BI ana-
lysis, we considered nodes with a BPP of 95% or greater as
significant (Leach
e and Reeder 2002). Uncorrected pairwise
sequence divergences for each gene were calculated using
MEGA 6.0 v (Tamura et al. 2013). Podarcis peloponnesiaca
(Gen-Bank accession number AY896179.1 (Poulakakis et al.
2005 b) and Eremias velox (Gen-Bank accession number
DQ658845.1 (Guo et al. 2011) for 16 S rRNA and Podarcis pelo-
ponnesiaca (Gen-Bank accession number AY896123.1
(Poulakakis et al. 2005 b) and Eremias velox (Gen-Bank acces-
sion number JQ690234.1 (Pouyani et al. 2012) for cytb were
selected as the outgroups for P. siculus.Podarcis hispanica
(Gen-Bank accession number HQ898057.1 (Kaliontzopoulou
et al. 2011) and Eremias velox (Gen-Bank accession number
Figure 1. Distribution ranges of the P. siculus (A) and P. tauricus (B) species in Turkey.
666 H. KOC¸ ET AL.
DQ658845.1 (Guo et al. 2011) for 16 S rRNA and Podarcis his-
panica (Gen-Bank accession number AY234154.1 (Busack
et al. 2005) and Eremias velox (Gen-Bank accession number
JQ690234.1 (Pouyani et al. 2012) for cytb were selected as
the outgroups for P. tauricus.
Results
Podarcis siculus
Phylogenetic analyses: sequence variation
A total of 501 homologous base pairs of the 16 S rRNA
sequences and 469 homologous base pairs of the cytb
sequences for 39 individuals were obtained, respectively.
There was 2 bp-deletion in 16 S rRNA gene, while there was
4 bp-deletion in cytb gene. In total, 15 mitochondrial haplo-
types for 16 S rRNA gene were identified and 19 haplotypes
for cytb gene were recognized. When we combined both the
16 S rRNA and cytb sequences, we identified 23 haplotypes.
In the NJ, ML and MP the best fit model selected by MEGA
6.0 v (Tamura et al. 2013), HKY þGþI (Kishino and Hasegawa
1989) for 16 S rRNA and HKY þGþI (Kishino and Hasegawa
1989) for 1st, 2nd and 3rd codon positions of cytb. Because
of the best fit model similarity, the sequences of 16 S rRNA
and cytb were combined and GTR þGþI (Tavar
e1986; Nei
and Kumar 2000) model was selected for the combined
sequences. In BI, the likelihood settings for the best-fit model
were selected as HKY (Kishino and Hasegawa 1989) in for
16 S rRNA and cytb genes. Because of the best fit model simi-
larity, the sequences of 16 S rRNA and cytb were combined.
GTR þGþI (Tavar
e1986; Nei and Kumar 2000) model was
selected for the combined data.
Phylogenetic relationships: genetic distances
NJ, MP, ML and BI phylogenetic analyses of each of the
studied genes and the combined dataset gave very similar
results and they showed only minor differences, mainly con-
cerning relationships between these groups and their support
values. The phylogenetic tree of the BI analysis of the 16 S
rRNA and cytb is shown Figures 2 and 3. Because the com-
bining tree and the tree belonging to 16 S rRNA is almost
similar, the combining tee is not shown.
The phylogenetic analyses of 16 S rRNA gene employing
four different optimality criteria yielded very slightly different
topologies, and only the BI tree is shown in Figure 2.
Table 1. List of the samples used for P. siculus for 16S rRNA and cytb
Genbank Accession no
Sample no Locality 16S rRNA cytb
1-1 Çanakkale-Gelibolu MF187695 MF187722
1-2 Çanakkale-Gelibolu MF187696 MF187723
2-1 İstanbul-Reşadiye MF187685 MF187710
2-2 İstanbul-Reşadiye MF187685 MF187709
3İstanbul-Tuzla MF187685 –
4İstanbul-Başakşehir MF187686 MF187711
5İstanbul-Beykoz MF187685 MF187709
6İstanbul-Samandıra MF187686 MF187712
7İstanbul-Üsküdar MF187686 MF187711
8İstanbul-Belgrad Ormanları–MF187710
9-1 İstanbul MF187686 MF187711
9-2 İstanbul MF187685 –
10-1 Kocaeli-Gebze Orman İşletme MF187685 MF187707
10-2 Kocaeli-Gebze Orman İşletme MF187685 MF187708
10-3 Kocaeli-Gebze Orman İşletme MF187685 MF187708
10 Kocaeli-Gebze –MF187709
11-1 Kocaeli-Darıca MF187692 MF187711
11-2 Kocaeli-Darıca MF187691 MF187717
12-1 Kocaeli-Maşukiye MF187688 MF187714
12-2 Kocaeli-Maşukiye MF187688 MF187720
12-3 Kocaeli-Maşukiye MF187692 MF187721
13 Kocaeli-Yanıkköy MF187687 MF187713
14-1 Sakarya-Arifiye MF187693 MF187718
14-2 Sakarya-Arifiye MF187691 MF187719
15 Sakarya-Dörtyol MF187686 MF187712
16-1 Sakarya-AdapazarıMF187686 MF187711
16-2 Sakarya-AdapazarıMF187686 MF187711
16-3 Sakarya-AdapazarıMF187686 MF187711
17-1 Düzce MF187698 MF187724
17-2 Düzce MF187699 MF187724
18-1 Zonguldak-Ereğli MF187689 MF187715
18-2 Zonguldak-Ereğli MF187686 MF187711
19-1 Zonguldak-Filyos MF187690 MF187716
19-2 Zonguldak-Filyos MF187691 MF187717
20-1 Zonguldak-Çaycuma MF187692 MF187721
20-2 Zonguldak-Çaycuma MF187694 MF187718
21 Zonguldak-Devrek MF187691 MF187709
22-1 Samsun-Atakum MF187697 MF187724
22-2 Samsun-Atakum MF187693 MF187725
Table 2. List of the samples used for P. tauricus for 16S rRNA and cytb
Genbank Accession no
Sample no Locality 16S rRNA cytb
1 Edirne-Enez MF187700 MF187684
2 Edirne-Uzunk€
opr€
u MF187706 MF187684
3-1 Edirne-B€
uy€
ukd€
oll€
uk MF187700 MF187684
3-2 Edirne-B€
uy€
ukd€
oll€
uk MF187700 MF187684
3-3 Edirne-B€
uy€
ukd€
oll€
uk –MF187684
4-1 Kırklareli-Vize-Sergen MF187700 MF187684
4-2 Kırklareli-Vize-Sergen MF187700 MF187683
4-3 Kırklareli-Vize-Sergen MF187700 MF187684
4-4 Kırklareli-Vize-Sergen MF187701 MF187684
4-5 Kırklareli-Vize-Sergen MF187700 MF187683
4-6 Kırklareli-Vize-Sergen MF187700 MF187683
5Kırklareli-Vize-Evrencik MF187700 MF187684
6-1 Tekirda
g-Saray MF187700 MF187684
6-2 Tekirda
g-Saray MF187700 MF187684
7-1 _
Istanbul-A
gva MF187700 MF187684
7-2 _
Istanbul-A
gva MF187702 MF187684
7-3 _
Istanbul-A
gva MF187703 MF187684
8-1 Kocaeli-Gebze-Balc¸ık MF187703 MF187684
8-2 Kocaeli-Gebze-Balc¸ık MF187703 MF187684
8-3 Kocaeli-Gebze-Balc¸ık MF187703 MF187684
8-4 Kocaeli-Gebze-Balc¸ık MF187700 MF187684
9 Kocaeli-Gebze-Mollafenari MF187700 MF187684
10-1 Kocaeli-K€
orfez-Belen MF187700 MF187684
10-2 Kocaeli-K€
orfez-Belen MF187700 MF187684
10-3 Kocaeli-K€
orfez-Belen MF187700 MF187684
11 Koceli-K€
orfez-Derek€
oy MF187704 MF187684
12-1 Kocaeli-K€
orfez-Sipahiler MF187700 MF187684
12-2 Kocaeli-K€
orfez-Sipahiler MF187700 MF187684
12-3 Kocaeli-K€
orfez-Sipahiler MF187700 MF187684
13 Kocaeli-Kandıra MF187700 MF187684
14 Kocaeli-C¸ubuklu MF187700 MF187684
15 Kocaeli-Yassıba
g MF187705 MF187684
16-1 Kocaeli-G€
olc€
uk MF187700 MF187684
16-2 Kocaeli-G€
olc€
uk MF187700 MF187684
17 Sakarya-Serdivan-Esentepe MF187700 –
18-1 Sakarya-Serdivan-Derek€
oy MF187700 MF187684
18-2 Sakarya-Serdivan-Derek€
oy MF187700 MF187684
19-1 D€
uzce-Y€
or€
ukk€
oy MF187700 MF187684
19-2 D€
uzce-Y€
or€
ukk€
oy MF187700 MF187684
19-3 D€
uzce-Y€
or€
ukk€
oy MF187700 MF187684
19-4 D€
uzce-Y€
or€
ukk€
oy MF187700 MF187684
MITOCHONDRIAL DNA PART A 667
Anatolian populations of P. siculus formed three clades
(Clades A–C) for 16 S rRNA.
The main relationships were as follows for 16 S rRNA:
Clade A consists of a haplotype (ada1), (NJ, ML and MP
BS¼100, 99 and 100, respectively and BPP¼1.0).
Clade B consists of 14 haplotypes (goi1, yan, ere3, fil1, fil4,
dar1, geli1, cayc2, masuk, geli2, ata1, arif1, duz1 and duz2)
from Turkey (NJ, ML and MP BS¼100, 99 and 100,
respectively and BPP¼1.0). Clade B is divided into two sub-
clades (Subclade B1 and Subclade B2). Subclade B1 consists
of goi1 haplotype and Subclade B2 has 13 remain haplotypes
(NJ, ML and MP BS¼73, 64 and 88, respectively and
BPP¼0.9). The relationships in the B1 clade were partially
resolved. Subclade B2 is divided into three lineages (Lineage
B2-1, Lineage B2-2 and Lineage B2-3). Lineage B2-1 has two
haplotype (duz1 and duz2), Lineage B2-2 has a haplotype
Figure 2. Bayesian tree of a 501-bp sequence of 16 S rRNA for P. siculus. Numbers above branches represent bootstrap support for NJ/ML/MP (1000 replicates) inher-
ence, and numbers below branches indicate Bayesian Posterior Probabilities.
Figure 3. Bayesian tree of a 469-bp sequence of cytb for P. siculus. Numbers above branches represent bootstrap support for NJ/ML/MP (1000 replicates) inherence,
and numbers below branches indicate Bayesian Posterior Probabilities.
668 H. KOC¸ ET AL.
(arif1) and Lineage B2-3 has remaining haplotypes (NJ, ML
and MP BS¼73, 64 and 88, respectively, and BPP¼0.9). The
relationships in the B2 Clade were unresolved.
The p-distances were as follows for 16 S rRNA:
The p-distances among the populations of P. siculus in Turkey
were very low and they were ranged from 0.2 (goi1 and
ada1; goi1 and arifl; yan and ere3; yan and fil1; mas
and geli1; mas and geli2; mas and cayc2; ere3 and dar1; fil1
and geli1; dar1 and fil4; cayc2 and fil4; geli1 and fil4) to 1.6%
(goi1 and ere3; dar1 and duz1) in 16 S rRNA (Table 3).
The main relationships were as follows for cytb:
Clade A includes three haplotypes (sak1 and ada2) from
Sakarya and Clade B consists of 17 haplotypes (mas2, ere3,
belg, goi1, geb1, goi2, duz2, ata1, arif1, fil4, geli1, mas, geli2,
mas1, arif2, fil1 and yan) for cytb (NJ, ML and MP BS¼-, 100
and -, respectively and BPP¼1.0). Subclade B1 consists of two
subclades (Subclades B1 and Subclade B2). Subclade B1 has a
haplotype (yan) and Subclade B2 has 16 remain haplotypes
(BPP¼0.6). The relationships in the B Clade were unresolved
for cytb (Figure 3).
The p-distances were as follows for cytb:
The values of p-distances were ranged from 0.0% (fil4 and
arif1; fil1 and araif2) to 3.3% (sak1 and mas) in cytb and the
p-distances in cytb among the populations of P. siculus in
Turkey were relatively higher than the distances in 16 S rRNA
(Table 3).
The main relationships were as follows for combining
data:
Clade A consists of two haplotypes (ada2 and sak1) and
Clade B includes 21 ones (dar1, ere3, mas2, yan, res1, goi1,
res3, goi2, duz1, duz2, ata1, ata2, arif1, geli1, geli2, cayc2,
mas, fil1, fil4, mas1 and arif2) (NJ, ML and MP BS¼100, - and
100, respectively and BPP¼1.0). Clade B consists of three sub-
clades (Subclade B1, Subclade B2 and Subclade B3). Subclade
B1 has a haplotype (res3), Subclade B2 has a haplotype (res1)
and Subclade B3 has 19 remaining in NJ and ML (NJ, ML and
MP BS¼100, - and 100, respectively, and BPP¼0.9). The rela-
tionships in the B Clade were unresolved for combine data.
The interrelationships among these groups are rather
ambiguous, showing a monophyly for 16 S rRNA, cytb and
combining data according to all phylogenetic analyses (NJ,
ML, MP and BI).
Podarcis tauricus
Phylogenetic analyses: sequence variation
A total of 497 homologous base pairs of the 16 S rRNA
sequences and 412 homologous base pairs of the cytb
sequences for 38 individuals were obtained, respectively.
Table 3. Comparison of uncorrected p-distance (in %) for fragments of 16S rRNA and cytb among haplotypes of P. siculus in Turkey
123456789101112131415161718
16S rRNA
goi1
ada1 0.2
yan 1.4 1.2
mas 0.8 1.0 0.6
ere3 1.6 1.4 0.2 0.8
fil1 1.2 1.4 0.2 0.4 0.4
fil4 1.2 1.4 0.6 0.4 0.4 0.4
dar1 1.4 1.2 0.4 0.6 0.2 0.6 0.2
arif1 0.2 0.4 1.2 0.6 1.4 1.0 1.0 1.2
cayc2 1.0 1.2 0.8 0.2 0.6 0.6 0.2 0.4 0.8
geli1 1.0 1.2 0.4 0.2 0.6 0.2 0.2 0.4 0.8 0.4
geli2 0.6 0.8 0.8 0.2 1.0 0.6 0.6 0.8 0.4 0.4 0.4
ata1 0.6 0.8 0.8 0.6 1.0 0.6 1.0 1.2 0.4 0.8 0.8 0.4
duz1 0.6 0.8 1.2 0.6 1.4 1.0 1.0 1.2 0.4 0.8 0.8 0.4 0.8
duz2 0.6 0.8 1.2 1.0 1.4 1.0 1.4 1.6 0.4 1.2 1.2 0.8 0.4 0.4
cytb
goi1
goi2 0.2
geb1 0.4 0.2
belg 0.2 0.4 0.2
ada2 1.5 1.3 1.1 1.3
sak1 1.3 1.5 1.3 1.1 0.2
yan 1.3 1.1 0.9 1.1 0.2 0.4
mas 0.6 0.4 0.6 0.9 1.7 1.9 1.5
ere3 1.3 1.1 1.3 1.5 0.6 0.9 0.4 1.5
fil1 0.9 0.6 0.9 1.1 1.9 2.1 1.7 0.6 1.3
fil4 0.6 0.4 0.6 0.9 1.7 1.9 1.5 0.4 1.1 0.6
arif1 0.9 0.6 0.9 1.1 1.5 1.7 1.3 0.6 0.9 0.9 0.2
arif2 1.1 0.9 1.1 1.3 1.7 1.9 1.5 0.9 1.1 0.2 0.9 0.6
mas1 1.3 1.1 1.3 1.5 1.9 2.1 1.7 0.6 1.3 0.4 0.6 0.4 0.2
mas2 1.5 1.3 1.5 1.7 0.9 1.1 0.6 1.3 0.2 1.5 0.9 0.6 1.3 1.1
geli1 0.9 0.6 0.9 1.1 1.9 2.1 1.7 0.2 1.3 0.4 0.2 0.4 0.6 0.4 1.1
geli2 0.9 0.6 0.9 1.1 1.9 2.1 1.7 0.6 1.3 0.4 0.2 0.4 0.6 0.4 1.1 0.4
ata1 0.4 0.2 0.4 0.6 1.5 1.7 1.3 0.6 0.9 0.4 0.2 0.4 0.6 0.9 1.1 0.4 0.4
ata2 0.4 0.2 0.4 0.6 1.5 1.7 1.3 0.6 0.9 0.4 0.2 0.4 0.6 0.9 1.1 0.4 0.4 0.0
MITOCHONDRIAL DNA PART A 669
There was 3 bp-deletion in 16 S rRNA gene, while there was
4 bp-deletion in cytb gene. In total, 7 mitochondrial haplo-
types for 16 S rRNA gene were identified and 2 haplotypes
for cytb gene were recognized. When we combined both the
16 S rRNA and cytb sequences, we identified 9 haplotypes. In
the NJ, ML and MP the best fit model selected by MEGA 6.0
v (Tamura et al. 2013), HKY þGþI (Kishino and Hasegawa
1989) for 16 S rRNA and HKY þG (Kishino and Hasegawa
1989) for 1st, 2nd and 3rd codon positions of cytb. Because
of the best fit model similarity, the sequences of 16 S rRNA
and cytb were combined and HKY þGþI (Kishino and
Hasegawa 1989) model was selected for the combined
sequences. In BI, the likelihood settings for the best-fit model
were selected as HKY (Kishino and Hasegawa 1989) in for
16 S rRNA and cytb genes. Because of the best fit model simi-
larity, the sequences of 16 S rRNA and cytb were combined.
HKY þGþI (Kishino and Hasegawa 1989) model was selected
for the combined data.
Phylogenetic relationships: genetic distances
NJ, MP, ML and BI phylogenetic analyses of each of the
studied genes and the combined dataset gave very similar
results and they showed only minor differences, mainly con-
cerning relationships between these groups and their support
values. Combining tree and the tree belonging to 16 S rRNA
were almost similar. Because of this reason, only BI trees for
16 S rRNA and cytb are shown in Figures 4 and 5.
The main relationships were as follows for 16 S rRNA, cytb
and combine data:
All analyses shown that P. tauricus populations in Turkey
have only one clade (Clade A). Clade A consists of seven hap-
lotypes (ser2, ser6, agva2, bal1, kor, uzun and yas2) (NJ, ML
and MP BS¼100, 100 and 100, respectively, and BPP¼1.0) for
16 S rRNA while it consists of two haplotypes (ser2 and ser6),
(NJ, ML and MP BS¼100, 100 and 100, respectively, and
BPP¼1.0). For combine data, Clade A consists of nine haplo-
types (ser2, ser3, ser6, agva2, bal1, kor, uzun, gol1 and yas2)
(NJ, ML and MP BS¼100, 100 and 100, respectively, and
BPP¼1.0).
The p-distances were as follows for 16 S rRNA and cytb:
The p-distances among the populations of P. tauricus in
Turkey were very low and they were ranged from 0.0 (ser2
and agva2; ser2 and bal1; ser2 and yas2; agva2 and bal1;
agva2 and yas2 and bal1 and yas2) to 0.6% (ser6 and uzun)
in 16 S rRNA. On the other hand, the p-distances in cytb
among the populations of P. siculus were 0.2% in Turkey
(Table 4).
The basic topology of the trees derived from NJ, ML, MP
and BI of the 16 S rRNA, cytb and combined data set shows a
monophyletic relationship for both P. siculus and P. tauricus
specimens in Turkey.
Discussion
The results of the present study identified a number of haplo-
type clades which based on the observed levels of sequence
divergence representing long-separated lineages and diverse
evolutionary histories within P. siculus and P. tauricus.
According to current literature, there are many contradict-
ory hypotheses on the taxonomic relationships for Podarcis
genus. Oliverio et al. (2009) reported morphologically recog-
nized three groups of the species: the first group consisted
muralis, tiliguerta, filfolensis, wagleriana and milensis (Bedriaga
1882); the second one consisted the Iberian and Madeiran
Figure 4. Bayesian tree of a 497-bp sequence of 16 S rRNA for P. tauricus.
Numbers above branches represent bootstrap support for NJ/ML/MP (1000 repli-
cates) inherence, and numbers below branches indicate Bayesian Posterior
Probabilities.
Figure 5. Bayesian tree of a 412-bp sequence of cytb for P. tauricus. Numbers
above branches represent bootstrap support for NJ/ML/MP (1000 replicates)
inherence, and numbers below branches indicate Bayesian Posterior
Probabilities.
Table 4. Comparison of uncorrected p-distance (in %) for fragments of 16S
rRNA and cytb among haplotypes of P. tauricus in Turkey
123456
16S rRNA
ser2
ser6 0.4
agva2 0.2 0.6
bal1 0.2 0.2 0.4
kor 0.2 0.6 0.4 0.4
yas2 0.2 0.6 0.0 0.4 0.4
uzun 0.6 1.0 0.4 0.8 0.4 0.4
123456
cytb
ser2
ser6 0.2
670 H. KOC¸ ET AL.
species and the last one consisted siculus, melisellensis and
two eastern Mediterranean species (Lanza and Cei 1977).
Based on the phylogenetic relationships, the Podarcis genus
was separated into four geographic groups [the Western
island group, the Balkan group (including P. tauricus), the
Italian group (including P. siculus) and the Southwestern
group] but relationships were mainly unresolved (Harris and
Arnold 1999; Oliverio et al. 2000). On the other hand, Oliverio
et al. (2009) reported that the Italian group of the genus was
split into three groups: the first comprised P. filfolensis,
P. melisellensis. P. wagleriaria, P. muralis, and P. raffonei, the
second group was P. siculus with its various subspecies and
the third one was composed of P. tiliguerta.
In order to estimate the times of lineage splitting from
sequence divergence data, a known rate belonging to cold-
blooded vertebrates is used. In particular, mitochondrial ribo-
somal genes are considered as more rate-homogeneous
(Oliverio et al. 2009). As it was explained in the study of
Oliverio et al. (2009), the rate of 0.38% sequence divergence
per MY for mitochondrial ribosomal genes derived for
European newts by Caccone et al. (1997) and the splitting of
the Podarcis from other groups would date to ca. 35 MY BP.
Furthermore, P. siculus would have diverged ca 17-15 MY BP.
The specimens of P. siculus in Turkey are mainly distrib-
uted from Marmara Region to Central Black Sea Region.
Although the mountain ranges in the Western Black Sea
Region, which lies between the Marmara Region and the
Central Black Sea Region, may consitute a barrier, the p-dis-
tances among the specimens from these regions were very
low (ranged from 0.2 to 1.6%) for 16 S rRNA. Similarly, the p-
distances among these populations were not high (maximum
3.3%) for cytb. These low genetic distances and tree topolo-
gies belonging to two genes showed that P. siculus had only
one lineage in Turkey.
If we roughly apply the rate of 0.38% sequence divergence
per MY for mitochondrial ribosomal genes, the splitting of
our haplotypes and Italian populations (AY770920.1 and
AY770919.1 haplotypes in Genbank) of the P. s. siculus would
date to ca 5.2 MY. In addition, the maximum divergence
between our haplotypes and Spain populations (HM746963.1
and HM746964.1 haplotypes in Genbank) of the same sub-
species was 3.4%. They would have been divergenced each
other for ca 8.9 MY. When we compared our specimens to
populations of Croatia (EU362073.6 and EU362074.1 haplo-
types in Genbank) representing other subspecies (P. s. cam-
pestris), we found that the maximum divergence was 4.4%.
They would have been separated from each other for ca 11.5
MY. Our findings are consistent with the results of Oliverio
et al. (2009).
On the other hand, Oliverio et al. (2009) did not state the
cytb p-distance values on their study. However, cytb
sequence data were also utilized for estimating the genetic
variability of the sampled populations, population genetic
structure and genetic differentiation among populations
(Giovannotti et al. 2010). In the present study, the p-distance
in the cytb gene was 12.1% between our samples and a
haplotype (JX072953.1 in Genbank) from a Portugal popula-
tion of the P. s. siculus. In addition, the p-distances between
our haplotypes and Italy (KF372034.1 and AY770895.1) and
Spain (JX072939.1 and JX072941.1) haplotypes of the same
subspecies in Genbank were 11.8% and 9.2%, respectively.
On the other hand, the genetic distance between our haplo-
types and two haplotypes (AY770882.1 and AY770892.1 in
Genbank) of P. s. campestris from Crotia was 9.8%.
When we compared our specimens with the haplotypes in
Genbank (based on the phylogenetic trees and p-distances
for 16 S rRNA and cytb genes), we considered that Turkish
populations of the Italian wall lizard represent the P. s. siculus
(Supplemental Figures 1 and 2).
In the present study, the basic intraspecific phylogeo-
graphical pattern of tauricus’s populations is characterized by
the existence of only one main lineage. This lineage, which
forms a monophyletic unit, corresponds to the populations of
P. tauricus in Turkey. Our mtDNA data and results previous
studies based on mtDNA (Harris and Arnold 1999; Oliverio
et al. 2000, Poulakakis et al. 2005 b) are consistent with the
morphological classification of the species.
Considering the distribution of P. tauricus in Turkey it
appears that there is no significant geographical barrier
among populations of the species. Conformable, we found
very low p-distances (maximum 0.6% for 16 S rRNA and 0.2%
for cytb).
The Balkan species are divided into two subgroups, the
first subgroup is P. tauricus and the second one is P. erhardii.
The subgroup of P. tauricus consists of P. tauricus, P. milensis,
P. gaigeae and perhaps P. melisellensis) and the subgroup of
P. erhardii consists of P. erhardii and P. peloponnesiaca
(Poulakakis et al. 2005 b). The distribution of the P. tauricus
subgroup (P. tauricus, P. milensis, P. gaigeae and perhaps P.
melisellensis) mainly on the Balkan Peninsula and its absence
from the rest of Europe, suggest that the ancestral species of
this group originated somewhere in the Balkan Peninsula and
expanded in this area. This information fits well with the
divergence time estimated in study of Poulakakis et al.
(2005 b) for the beginning of the diversification of P. tauricus
subgroup. According to date estimation of divergence events
(0.46% sequence divergence per MY for mitochondrial riboso-
mal genes and 1.55% for cytb), Poulakakis et al. (2005 b)
reported the separation time of P. tauricus subgroup as 10.8
and 10.5 million years for 16 S rRNA and cytb, respectively.
As in the case of P. siculus, if we apply the rate of 0.38%
sequence divergence per MY by Caccone et al. (1997) for P.
tauricus, the splitting of our haplotypes and Greece popula-
tions (AY768728.1 and AY768727.1 haplotypes in Genbank) of
the P. t. tauricus would date to ca 14.2 MY for 16 S rRNA
gene (Table 3). On the other hand, the p-distance between
our samples and Albanian specimens (KX658351.1 in
Genbank) of the other subspecies, P. t. ionicus was 4.8%. They
would have divergenced each other for ca 12.6 MY.
Because we had only two haplotypes for cytb gene, we
did not perform a comparison between our haplotypes and
the Genbank haplotypes. Based on our phylogenetic analyses
and haplotypes in Genbank belonging to the specimens from
European populations of the species, we considered that
Turkish populations of the Crimean wall lizard represent the
P. t. tauricus (Supplemental Figure 3).
Our phylogenetic analyses of the two genes employing
four different optimality criteria and low p-distances in these
MITOCHONDRIAL DNA PART A 671
genes could be explained either by high levels of gene flow
among the respective populations of both species (P. siculus
and P. tauricus) in Turkey, as implied in the study of Kornilios
et al. (2011).
In conclusion, the monophyly of P. siculus and P. tauricus
was strongly supported by the cladistic analysis of Turkish
populations.
Acknowledgements
The animals were treated in accordance with the guidelines of the local
ethics committee (KTU.53488718-567/2015/39).
Disclosure statement
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Funding
This study was supported financially by the Karadeniz Technical
University, Scientific Researches Unit (Project Code: 9734 and Project ID:
190)
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