Determination of genetic variations between

Turkish Journal of Zoology
Turk J Zool
(2015) 39: 630-642
© TÜBİTAK
doi:10.3906/zoo-1405-75
http://journals.tubitak.gov.tr/zoology/
Research Article
Determination of genetic variations between Apodemus mystacinus populations
distributed in Turkey inferred from mtDNA PCR–RFLP
1,
1
2
2
Gül OLGUN KARACAN *, Reyhan ÇOLAK , Ercüment ÇOLAK
Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey
2
Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey
Received: 30.05.2014
Accepted/Published Online: 27.02.2015
Printed: 30.07.2015
Abstract: The rocky mouse, Apodemus mystacinus, is a rodent species distributed in Anatolia. A total of 108 specimens from 19 localities
in Turkey were collected to study the molecular variation of A. mystacinus inferred from RFLP of mtDNA cytochrome b (cytb) and
D-loop. MboI, HaeIII, and RsaI from cytb digestion and MboI, BfaI, and HinfI from D-loop digestion showed differentiation among the
studied specimens. The UPGMA dendrogram of the combined data of cytb and D-loop indicated 3 main clades, A. m. euxinus, A. m.
mystacinus, and A. m. smyrnensis, distributed in Turkey. The haplotype diversity observed in the present study was high. Furthermore,
the fixation index values (FST) ranged from 0.75093 to 0.83384, which indicated high diversity among the clades. These 3 A. mystacinus
clades of Turkey were also supported by analysis of molecular variance (AMOVA) results, by revealing that genetic diversity was higher
among groups (79.55%) than within the groups.
Key words: Apodemus mystacinus, mtDNA, RFLP, cytochrome b, D-loop, Turkey
1. Introduction
The rocky mouse, Apodemus mystacinus, is a rodent
species distributed in the eastern Mediterranean region.
This species lives in varied vegetation, including rocky
and stony surfaces covered with forests, small bushes, or
cultivated areas up to an elevation of 2700 m (Vohralik
et al., 2002; Çolak et al., 2004). A. mystacinus has four
controversial subspecies: A. m. mystacinus Danford
and Alston (1877) from Sebil, Turkey; A. m. smyrnensis
Thomas (1903) from western Turkey; A. m. rhodius Festa
(1914) from Rhodes, Crete, and southwestern Turkey; and
A. m. euxinus Allen (1915) from northern or northeastern
Turkey. A. epimelas Nehring (1902), formerly described
as a subspecies of A. mystacinus, is distributed in the
Balkan region (former Yugoslavia, Greece, and Bulgaria).
This subspecies has been raised to the level of species on
the basis of morphometric studies using body size and
dental characters (Spitzenberger, 1973; Mezhzherin, 1997;
Vohralik et al., 2002), a paleontological approach (Storch,
1977), allozyme research (Filippucci et al., 2002), and DNA
level (Michaux et al., 2002, 2005; Bugarski-Stanojević et
al., 2011).
Apart from the fact that A. mystacinus and A. epimelas
can be easily separated from each other, these species
are differentiated from the other Sylvaemus species. This
*Correspondence: glolgn@gmail.com
630
theory is supported by using morphological characters
such as body size (Rietschel and Storch, 1973; Storch, 1974)
and cranial differentiations (Kuncová and Frynta, 2009),
enzymatic polymorphism studies (Filippucci et al., 2002),
and DNA polymorphisms (Martin et al., 2000; Michaux
et al., 2002; Bellinvia, 2004). Thus, A. mystacinus and A.
epimelas belong to Karstomys, which is a controversial
subgenus of Apodemus.
The validity of A. m. smyrnensis is controversial; some
authors have claimed that this subspecies is synonymous
with A. m. mystacinus (Ellerman, 1948; Ellerman and
Morrison-Scott, 1951; Çolak et al., 2004), while others have
revealed that A. m. smyrnensis lives in the Taurus Mountains
(Neuhäuser, 1936) and western Turkey (Çolak et al., 2007;
Olgun et al., 2009). Similarly, there is a disagreement
about the taxonomic status of A. m. euxinus (Allen, 1915)
at the level of distribution and validity. Neuhäuser (1936)
claimed that northern Turkey is the distribution area of this
subspecies, whereas Çolak et al. (2007) showed differences
in the Artvin (northeastern Turkey) populations from
the other northern populations. Furthermore, studies
including morphological, biometrical, karyological,
bacular, and phallic differentiations (Çolak et al., 2004) and
RAPD variations (Olgun et al., 2009) have indicated the
homogeneity of the eastern and southeastern populations.
OLGUN KARACAN et al. / Turk J Zool
However, multivariate procedures based on molar, skull,
and body measurements did not confirm the validity of
this subspecies (Vohralik et al., 2002).
When all of these studies are evaluated, it is obvious
that the results are still controversial regarding the validity
of the subspecies of the rocky mouse. While morphological
studies have been especially insufficient in determining
the variations of subspecies, a molecular viewpoint has
been more effective in determining the lineages of species.
In particular, DNA phylogenies may adduce the lineages,
subspecies, and species (Hewitt, 1996). In many different
applications, Cytb is one the most preferred proteinencoding regions of mtDNA to detect intra- or interspecific
genetic variability (Deffontaine et al., 2005; Michaux et al.,
2005; Hürner et al., 2010; Kryštufek et al., 2012; Zhang et
al., 2013). Another frequently used mtDNA marker is the
control region known as D-loop, which might be useful in
taxonomic studies, since it is affected by selective pressure
because of its noncoding and more variable nature (Matson
and Baker, 2001; Bellinvia, 2004; Michaux et al., 2005;
Macholán et al., 2007; Zhang et al., 2013). Polymerase
chain reaction–restriction fragment length polymorphism
(PCR–RFLP) of specific mtDNA has been a useful tool to
infer genetic structure and differentiation at the species
and subspecies levels of different organisms (Marchesin et
al., 2008; Gonzalez et al., 2009; Presti et al., 2010; Suzuki et
al., 2010; Darvish et al., 2012; Gherman et al., 2012; Rahim
et al., 2013; Sanra et al., 2013).
Therefore, in this study, we used mtDNA cytb and
D-loop regions to investigate the genetic diversity and
population structure of A. mystacinus distributed in
Turkey, inferred by the PCR–RFLP technique. These
findings would be useful to determine the subspecies of A.
mystacinus and also to discover a subspecies-specific RFLP
marker.
2. Materials and methods
2.1. Sample collection
To study the molecular variation of A. mystacinus, 108
specimens (Table 1) from 19 localities in Turkey (Figure
1) were collected. Moreover, 11 Sylvaemus samples (4
specimens belonging to Apodemus uralensis, 4 specimens
belonging to Apodemus witherbyi, and 3 specimens
belonging to Apodemus flavicollis) were used as the outgroup in this study.
2.2. DNA isolation and PCR amplification
DNA was extracted from kidney tissue by using
cetyltrimethylammonium bromide (CTAB), using Doyle
and Doyle’s (1991) isolation protocol with modifications. The
cytb gene was amplified using the universal primers L14724a
(5’-CGAAGCTTGATATGAAAAACCATCGTTG-3’) and
H15915a
(5’-AACTGCAGTCATCTCCGGTTTACAA
GAC-3’) (Irwin et al., 1991). Amplification reactions
consisted of an initial denaturation at 94 °C for 5
min, 35 cycles of denaturation at 93 °C for 1 min,
annealing at 46 °C for 1 min, extension at 65 °C
for 1 min 30 s, and a 10-min final extension at 65
°C. D-loop sequence was amplified using L15926
(5’-TCAAAGCTTACACCAGTCTTGTAAACC-3’) and
HN00651
(5’-TAACTGCAGAAGGCTAGGACCAAA
CCT-3’) (Matson and Baker, 2001). Reactions for D-loop
were carried out following this protocol: 40 cycles of
denaturation at 95 °C for 30 s, annealing at 45 °C for 1 min,
extension at 65 °C for 1 min, and a 5-min final extension at
65 °C. The reactions were performed in a THERMO PX2
thermal cycler (Thermo Scientific Hybaid). The PCR was
prepared in 25 µL of a reaction mixture containing 80 ng of
the DNA samples; 10X reaction buffer (750 mM Tris-HCl
pH: 8.8, 200 mM (NH4)2SO4, 0.1%, Tween 20; Thermo);
1.5 u of Taq DNA polymerase (500 units, Thermo); 0.2
mM of each deoxynucleotide triphosphate (100 mM of
dNTP set solutions, Thermo); 2 mM MgCl2; and 20 pmol
of each primer (Thermo).
2.3. Restriction digestion
Afterwards, only one PCR sequence from each mtDNA
region was sequenced. NEBcutter Version 2.0 (Vincze
et al., 2003) was utilized in order to determine which
enzymes could be used for digestion. Therefore, we
digested cytb with the HaeIII, MboI, RsaI, HinfI, BamHI,
and TaqI restriction enzymes (RE), and D-loop with the
MboI, HinfI, TaqI, BfaI, and NdeI enzymes (Table 2).
As the next step, 0.1 µg of PCR products were digested
with RE (Thermo Scientific) according to their incubation
conditions. The restriction fragments were visualized
on 2% agarose gel after staining with ethidium bromide.
Fragment lengths were determined using a 100-bp ladder
(Thermo).
2.4. Statistical analyses
Restriction fragments were scored as the presence (1)
and the absence (0) of bands. The UPGMA tree for RFLP
fragments was constructed based on the binary data using
PAST software (Hammer et al., 2001) by Jaccard similarity
coefficient. The robustness of the tree was determined by
bootstrap resampling (1000 replicates) (Felsenstein, 1985).
Furthermore, Numerical Taxonomy and Multivariate
Analysis System-pc (NTSYS) version 2.2 (Exeter Software,
Setauket, NY, USA) and Statistical Package for the Social
Sciences version 20 (SPSS) (IBM Corp., Armonk, NY,
USA) software were used together for principal coordinate
analyses (PCoA). NTSYS was run to calculate the
eigenvalues, and the PCoA graphic was visualized with
SPSS using these eigenvalues.
Analysis of molecular variance (AMOVA) was
performed to detect the genetic variation within and among
the clades using the Arlequin version 3.5 (Excoffier and
Lischer, 2010) software package with 10,100 permutations.
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OLGUN KARACAN et al. / Turk J Zool
Table 1. Number of specimens belonging to localities and their distribution by region.
Species
A. mystacinus
Locality
Number of specimens
ARTVİN
9
RİZE
6
TRABZON
6
ORDU
2
KASTAMONU
1
ZONGULDAK
8
DÜZCE
1
BALIKESİR
12
İZMİR
3
AYDIN
2
MUĞLA
11
BURDUR
1
KONYA
3
ANTALYA
9
MERSİN
8
ADANA
7
KAHRAMANMARAŞ
8
ERZİNCAN
9
ADIYAMAN
2
Sylvaemus (outgroup) A. flavicollis
A. uralensis
A. witherbyi
EASTERN BLACK SEA
CENTRAL BLACK SEA
WESTERN BLACK SEA
WESTERN TURKEY
SOUTHWESTERN TURKEY
SOUTHERN TURKEY
EASTERN TURKEY
MUĞLA
1
ADANA
2
ARDAHAN
1
ARTVİN
1
TRABZON
2
İZMİR
1
ANKARA
2
KONYA
1
To determine the genetic differentiation among clades, the
pairwise fixation index (FST) was evaluated with Arlequin.
Slatkin’s linearized FST values (Slatkin, 1995) were also used
to estimate the gene flow (Nm), with the formula Nm =
(1 – FST)/4 FST.
Tajima’s D (Tajima, 1989) and Fu’s Fs (Fu, 1997)
values were calculated using Arlequin, as well as with
the bootstrap method, to introduce the population
demographic expansion, bottleneck, and neutrality,
using 10,000 permutations. Haplotype diversities (h) and
pairwise differences, which gave information about the
structure of the clades, were also evaluated with Arlequin.
3. Results
In total, the cytb and D-loop regions of the 108 specimens
obtained from the 19 localities were analyzed both
632
Region
separately and combined. Each mtDNA region has nearly
1100 base pairs.
3.1. Restriction patterns obtained from the cyt b and
D-loop regions
Three of six restriction enzymes produced different band
profiles among the A. mystacinus populations inferred
from cytb: MboI, HaeIII, and RsaI (Figure 2). Five
restriction enzymes were used to digest the D-loop region,
and while two (MboI and BfaI) exhibited intrapopulation
variable patterns, one gained population-specific profiles
(HinfI) (Figure 3). Each RE pattern is represented with a
letter (A–J) and each individual is described through the
specific combination of 6 RFLP patterns (6 letters). There
were 24 haplotypes out of 108 individuals (Table 3).
The UPGMA dendrogram was constructed using
binary matrices (1/0) obtained from the haplotypes of the
OLGUN KARACAN et al. / Turk J Zool
Figure 1. Sampling localities of A. mystacinus specimens.
Table 2. Restriction enzymes and their digestion sites with reaction procedures.
Restriction enzyme
BamHI
BfaI
HaeIII
HinfI
MboI
NdeI
RsaI
TaqI
Restriction site
5’...G↓G A T C C...3’
3’...C C T A G↑G...5’
5’...C↓T A G...3’
3’...G A T↑C...5’
5’...G G↓C C...3’
3’...C C↑G G...5’
5’...G↓A N T C...3’
3’...C T N A↑G...5’
5’...↓G A T C ...3’
3’... C T A G↑...5’
5’...C A↓T A T G...3’
3’...G T A T↑A C...5’
5’...G T↓A C...3’
3’...C A↑T G...5’
5’...T↓C G A...3’
3’...A G C↑T...5’
Reaction degree
Reaction time
37 °C
16 h
37 °C
15 min
37 °C
15 min
37 °C
15 min
37 °C
16 h
37 °C
15 min
37 °C
16 h
65 °C
16 h
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OLGUN KARACAN et al. / Turk J Zool
Figure 2. Restriction patterns of MboI, HaeIII, and RsaI inferred from cytb digestion (M: Marker–100bp DNA Ladder,
1. Ordu, 2. Trabzon, 3. Rize, 4. Artvin, 5. Erzincan, 6. Kahramanmaraş, 7. Adıyaman, 8. Adana, 9. Muğla, 10. Burdur,
11. Konya, 12. Antalya, 13. Mersin, 14. Kastamonu, 15. Zonguldak, 16. Düzce, 17. Balıkesir, 18. İzmir, 19. Aydın, 20. A.
uralensis, 21. A. witherbyi, 22. Cytb PCR product).
combined data, and this dendrogram indicated 3 main
clades of A. mystacinus distributed in Turkey (Figure 4).
Furthermore, PcoA exhibited the segregation of these 3
clades clearly (Figure 5).
Clade 1 included specimens from the eastern Black
Sea (Rize–Artvin–Trabzon), the central Black Sea (Ordu),
and eastern (Erzincan–Adıyaman) and southern Anatolia
(Kahramanmaraş–Adana). This clade had 2 subgroups,
Clade 1a (Rize, Artvin, and the other specimens in this
clade) and Clade 1b (only specimens from Rize and Artvin).
The other main clade was Clade 2, which branched from
the same node as Clade 1. Clade 2 included the specimens
from the southwest (Muğla) and the rest of southern
Anatolia (Burdur–Konya–Antalya–Mersin). The western
634
Anatolian specimens (Kastamonu–Zonguldak–Düzce–
Balıkesir–İzmir–Aydın) constituted Clade 3, which was
separate from the other clades (Figures 4–5).
A total of 24 combined haplotypes were detected in
the 3 clades, and are listed in Table 3. It seemed that there
were no common haplotypes among the clades according
to these results; Hap01–Hap10 were distinctive for Clade
1, while Clade 2 comprised Hap11–Hap18, and Hap19–
Hap24 were representative for Clade 3. The haplotype
diversity (h) in each A. mystacinus clade ranged between
0.8401 and 0.5988, and the highest haplotype diversity
was calculated in Clade 1 (0.8401) while the lowest was in
Clade 2 (0.5988) (Table 3).
OLGUN KARACAN et al. / Turk J Zool
Table 3. Distribution of composite haplotypes among 3 A. mystacinus clades.
Haplotype
compositions*
Haplotypes
Hap_01
Clade 1
Clade 2
Clade 3
(N = 49)
(N = 32)
(N = 27)
Specimens
Artvin–Trabzon
AAAAAA
0.286
0
0
Ordu
Kahramanmaraş
Hap_02
AAAABA
0.0816
0
0
Kahramanmaraş
Hap_03
AAABAA
0.0408
0
0
Rize–Adana
Hap_04
AAACAA
0.245
0
0
Hap_05
AAACBA
0.0612
0
0
Hap_06
AAAEAA
0.0612
0
0
Hap_07
ABAAAA
0.0612
0
0
Artvin
Hap_08
ABABAA
0.0204
0
0
Artvin
Hap_09
ABACAA
0.122
0
0
Rize
Hap_10
AEAAAA
0.0204
0
0
Artvin
Hap_11
CAAGBA
0
0.625
0
Hap_12
CAAHBA
0
0.0312
0
Muğla
Hap_13
CAAIBA
0
0.0312
0
Mersin
Hap_14
CAAJBA
0
0.125
0
Mersin
Hap_15
CACGBA
0
0.0312
0
Muğla
Hap_16
CACGBB
0
0.0312
0
Muğla
Hap_17
CACHBB
0
0.0312
0
Muğla
Hap_18
CDAGBA
0
0.0938
0
Konya
Hap_19
CCBDBA
0
0
0.037
Düzce
Hap_20
CCBEBA
0
0
0.296
Zonguldak
Hap_21
CCBEBB
0
0
0.519
Hap_22
CCBFBA
0
0
0.037
Balıkesir
Hap_23
CCBGBA
0
0
0.037
Balıkesir
Hap_24
DCBEBA
0
0
0.0741
Balıkesir
Haplotype diversity
0.8401
0.5988
0.6581
Standard deviation
± 0.0302
± 0.0951
± 0.0721
Adana–Erzincan
Adıyaman
Erzincan
Kahramanmaraş
Erzincan–Adana
Muğla–Burdur
Antalya–Mersin
Kastamonu–Balıkesir
İzmir–Aydın
*A single capital letter (A–J) was assigned to each of 6 PCR–RFLP profiles in cytb and D-loop.
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OLGUN KARACAN et al. / Turk J Zool
Figure 3. Restriction patterns of HinfI inferred from D-loop digestion (M: Marker–100bp DNA Ladder,
1. Ordu, 2. Trabzon, 3. Rize, 4. Artvin, 5–6. Erzincan, 7–8. Kahramanmaraş, 9. Adıyaman, 10–11. Adana,
12. Muğla, 13. Burdur, 14. Konya, 15. Antalya, 16. Mersin, 17. Kastamonu, 18. Zonguldak, 19. Düzce, 20.
Balıkesir, 21. İzmir, 22. Aydın, 23. A. uralensis, 24. A. witherbyi, 25. D-loop PCR products).
AMOVA was executed to determine the genetic
variation rate; 79.55% (P < 0.01) of the total variation
explained the correlation within groups, while 20.45% (P
< 0.01) of the correlation was among groups (Table 4).
Genetic differentiations among clades were obtained
from the pairwise FST values: the highest was between
Clades 2 and 3 (0.83384), the lowest was between Clades
1 and 2 (0.75093) (Table 5). The Nm inferred from the FST
values was higher between Clades 1 and 2 (0.08292) than
that from among other clades (Table 5).
Tajima’s D and Fu’s Fs tests were performed to present
the demographic structure as the expansion, bottleneck,
or selective effects of the A. mystacinus clades. If the
population began to expand generally after a bottleneck,
the D and Fs values would be negative due to the loss of
mutations, unlike the positive values (Tajima, 1989; Fu,
1997). Moreover, the negative values were not sufficient
to remark about the demographics of the populations
636
separately and so P was a value to measure the significance
of D and Fs; if P was below 0.05, the sums of D and Fs
would be evaluated as significant. In our results, Clade 2
had the highest negative for Tajima’s D and Fu’s Fs values
as –0.89106 and –1.98851, respectively. However, none of
the results obtained from any of the clades were significant
at P > 0.05 (Table 6).
4. Discussion
In this study, mtDNA sequences from both cytb and D-loop
regions were used to exhibit the genetic differentiation
among A. mystacinus populations. A total of 24 composite
haplotypes, obtained from restriction profiles of cytb
and D-loop, were specified for each individual and clade
(Table 3). Diversities among the clades were supported by
various analyses, including haplotype diversity, molecular
variance, and neutrality tests.
OLGUN KARACAN et al. / Turk J Zool
Figure 4. UPGMA dendrogram of the composite data by combining cytb and D-loop regions.
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OLGUN KARACAN et al. / Turk J Zool
Figure 5. PCoA analysis of A. mystacinus clades. The scatter plot is of the scores of three
principal eigenvalues inferred from NTSYS software. Each scatter point represents a specimen
of A. mystacinus.
Table 4. AMOVA of mtDNA composite haplotypes inferred from cytb and D-loop among A. mystacinus groups.
Source of variation
DF*
Sum of squares
Variance components
Percentage of variation (%)
Among groups
2
364.348
5.20118Va
79.55
Within groups
105
140.355
1.33672Vb
20.45
Total
107
504.704
6.53790
0.79554
(P = 0.000)
Fixation index
FST
*DF: Degrees of freedom.
Table 5. Pairwise FST (below diagonal) and Nm (above diagonal) values among A. mystacinus clades.
Population
Clade 1
Clade 2
Clade 3
Clade 1
-
0.08292
0.0589
Clade 2
0.75093 (0.00000)*
-
0.0498
Clade 3
0.80924 (0.00000)*
0.83384 (0.00000)*
-
* P values are given in parentheses.
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OLGUN KARACAN et al. / Turk J Zool
Table 6. Neutrality tests of A. mystacinus clades.
Neutrality tests
Tajima’s D
Fu’s Fs
No. of specimens
CLADE 1
CLADE 2
CLADE 3
No. of specimens
49
32
27
D
1.33034
–0.89106
–0.3941
P
0.919
0.216
0.398
49
32
27
Fs
0.34907
–1.98851
0.628
P
0.595
0.126
0.649
The haplotype diversity observed in the three clades
was high in the present study (Table 3). While Clade 1
had the highest haplotype diversity (0.8401), Clade 2 had
the lowest (0.5988). The high haplotype diversity might
indicate a bottleneck (Michaux et al., 2004; Liu et al., 2008;
Xiao et al., 2008), where many species were affected by
Pliocene and early Pleistocene events (Grant and Bowen,
1998). Similarly, Michaux et al. (2004) found that the high
haplotype diversity of A. flavicollis in Turkey indicated the
expansion of this population. This study supported both the
recent expansion and the bottleneck of A. mystacinus clades
in Turkey according to the haplotype diversities. While
Clade 1 might indicate a bottleneck scenario, Clade 2 might
tend to indicate a recent expansion after a bottleneck.
The pairwise FST statistics (Table 5) and AMOVA (Table
4) were studied to examine the genetic differentiation
among the A. mystacinus groups. The FST values ranged
between 0.75093 and 0.83384, which indicated high
diversity among the clades, i.e. values greater than 0.25
were evaluated as substantially different (Nei, 1987). This
diversity was also supported by the Nm obtained from the
FST values, which indicated genetic distance when the Nm
was <1 (Wright, 1951). High diversities among subspecies
could also lead to speciation by decreasing the gene flow
over time. The highest genetic differentiation, between
Clades 2 and 3 (FST = 0.83384), also indicated the lowest
gene flow between these two clades (Nm = 0.0498) (Table
5). It could be said that there were more effective factors
inhibiting the genetic breeding between these clades than
that of the others. These diversities were supported by RAPD
data (Olgun et al., 2009) and esterase variations (Çolak et
al., 2007). Contrary to our findings, morphometric studies
using the body and skull measurements showed that the
western specimens (indicated as Clade 3 in our study)
were closer to the southeastern specimens (Clade 1 in our
study) (Çolak et al., 2004).
These three A. mystacinus clades of Turkey were also
supported by AMOVA results, which showed that the
genetic diversity was higher among the groups (79.55%)
than within the groups (20.45%). These genetic structures
among the groups and within the groups were similar
to those of the other Apodemus species, i.e. A. sylvaticus
(Michaux et al., 2003) and A. flavicollis (Michaux et al.,
2004).
Despite the fact that Clade 2 was negative for Tajima’s
D and Fu’s Fs, indicating recent expansion after a
bottleneck, none of the results were significant at P < 0.05
in this study (Table 6). Moreover, although Clade 1 for D
and Clade 1 and 3 for Fs had positive values that did not
indicate a recent expansion, P was again not significant.
Similarly, Gündüz et al. (2007) studied a small rodent
species, Spermophilus taurensis, distributed in the Taurus
Mountains and southwestern Anatolia. They concluded
that the Taurus Mountains were a glacial refugium, based
on positive values for both Tajima’s D and Fu’s Fs. The
Taurus refugium was also supported by Çıplak (2003),
who studied grasshoppers in that region. In our study,
the specimens from the Taurus Mountains were collected
from the Antalya region and were in Clade 2. Contrary to
the results of Gündüz et al. (2007) and Çıplak (2003), this
clade had the highest negative values in neutrality tests,
and low haplotype diversity indicated that this region was
not a refugium for A. mystacinus populations.
All of these findings indicate that there are 3 subspecies
of A. mystacinus. The specimens from the eastern Black
Sea region and eastern and southeastern Anatolia that
were grouped together as Clade 1 may refer to A. m.
euxinus, which was also supported by Çolak et al. (2004)
and Olgun et al. (2009). This group was also separated
into 2 subgroups; the Artvin and Rize specimens were
situated in both of these 2 subgroups (Clade 1a and
Clade 1b). Doğramacı (1972) suggested, according to the
morphological data, that Artvin was a hybrid zone between
A. m. mystacinus and A. m. euxinus; this was supported
by Çolak et al. (2007) using esterase variations. Similarly,
the hierarchy in Clade 1 also may indicate that the region
including the Artvin and Rize specimens is a hybrid zone
for A. m. euxinus in our study.
639
OLGUN KARACAN et al. / Turk J Zool
The southern Anatolia populations, including the type
locality of A. mystacinus (Sebil–Mersin), constituted Clade
2. Differentiations between A. m. mystacinus and A. m
euxinus were supported by the morphological data: the
ears were longer in A. m. mystacinus than in A. m. euxinus
(Çolak et al., 2004).
Furthermore, a separate lineage that included the
western Anatolia and western Black Sea specimens
(Clade 3) corresponded to A. m. smyrnensis, which had
been confirmed in previous studies (Çolak et al., 2007;
Olgun et al., 2009). Neuhäuser (1936) reported that A. m.
smyrnensis was distributed in western Anatolia and in the
Taurus Mountains. Similarly, Ellerman (1948) claimed that
A. m. smyrnensis was synonymous with A. m. mystacinus.
We have presented here a different lineage that places A.
m. smyrnensis in western Anatolia. Similarly, Çolak et al.
(2004) proved the validity of A. m. smyrnensis using body
and skull measurements (the condylobasal lengths, hind
foot, and ear). However, Michaux et al. (2005) reported
a different lineage in southwestern Turkey that was
synonymous with A. m. rhodius, using DNA analysis. This
has also been supported by RAPD markers (Olgun et al.,
2009). However, our findings disagree, as we did not find
this subspecies.
The possible reasons for subspeciation of the A.
mystacinus populations might be the geographic structure
of Anatolia and the effects of climatic fluctuations during
the Pleistocene. The Kızılırmak River and its valley may
be a barrier between A. m. smyrnensis (distributed in
northwestern and western Anatolia) and the northeastern
specimens of A. m. euxinus. A similar case might be possible
for A. m. mystacinus and A. m. euxinus populations, due to
the influence of the rivers in the southeastern region (i.e.
Seyhan River, Ceyhan River); the results showed that the
Nm between these two clades was higher than the others,
indicating the permanence of the gene flow. Furthermore,
southwestern Anatolia seems to have been a barrier
separating A. m. smyrnensis and A. m. mystacinus, perhaps
due to the collapsing of this region below sea level during
the Pleistocene (Görür et al., 1995). The lowest Nm value
between A. m. smyrnensis and A. m. mystacinus in this
study also supports the reduction of gene flow between
these two clades.
In conclusion, we confirm that RFLP markers could be a
useful tool to determine the geographical differentiation of
A. mystacinus. However, the RFLP data were not sufficient
to discover the possible reasons for this differentiation, or
to display the speciation tendency of A. mystacinus clades.
Therefore, further analysis using more efficient markers
such as sequence analyses or microsatellites should be
used to clarify the speciation of A. mystacinus subspecies
in Turkey.
Acknowledgments
We would like to thank Lisa Anne Meredith for the English
editing, Dr Tamer Çırak for image editing, and the two
anonymous referees and the editor, who provided valuable
comments.
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