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Title: Genetic diversity and structuring of the grey wolfpopulation from the Central Balkans based on mitochondrialDNA variation
Author: Mihajla Djan Vladimir Maletic Igor Trbojevic DunjaPopovic Nevena Velickovic Jelena Burazerovic Dusko Cirovic
PII: S1616-5047(14)00023-8DOI: http://dx.doi.org/doi:10.1016/j.mambio.2014.03.001Reference: MAMBIO 40662
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Received date: 21-8-2013Revised date: 28-2-2014Accepted date: 11-3-2014
Please cite this article as: Djan, M., Maletic, V., Trbojevic, I., Popovic, D., Velickovic, N.,Burazerovic, J., Cirovic, D.,Genetic diversity and structuring of the grey wolf populationfrom the Central Balkans based on mitochondrial DNA variation, Mammalian Biology(2014), http://dx.doi.org/10.1016/j.mambio.2014.03.001
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Genetic diversity and structuring of the grey wolf population from the Central 1
Balkans based on mitochondrial DNA variation2
3
Mihajla Djan1, Vladimir Maletić2, Igor Trbojević3, Dunja Popović1, Nevena Veličković1, 4
Jelena Burazerović4, Duško Ćirović45
1University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg 6
Dositeja Obradovica 2, 21000 Novi Sad, Serbia; 2University of Kiril and Metodij, Faculty 7
of Forestry, Bul. Aleksandar Makedonski bb, 1000 Skopje, FRY Macedonia; 3University 8
of Banja Luka, Faculty of Science, Mladena Stojanovića 2, 51000 Banja Luka, Bosnia 9
and Herzegovina; 4University of Belgrade, Faculty of Biology, Studentski Trg 16, 11000 10
Belgrade, Serbia11
12
Corresponding author:13
Mihajla Djan, Professor Assistant14
University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology15
Trg Dositeja Obradovica 216
21000 Novi Sad, Serbia17
tel: +381 21 485 279918
mob: +381 63 810248819
fax:+381 21 450 62020
e-mail: [email protected]
Word count: 443322
Running title: Genetic diversity of grey wolves from the Central Balkans23
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Abstract25
26
The Dinaric-Balkan grey wolf population used to be at a border between the 27
large remaining Eastern European populations and the largely eradicated Western28
European populations. During the last few decades we have witnessed the Western 29
European wolf population recovery. Substantial genetic variation has previously been 30
reported in the Balkan wolf population, but rigorous genetic characterization has not been31
done for its central parts. The aims of this research were to determine genetic diversity 32
based on mtDNA sequence variability, to infer possible population structuring, to find 33
genetic signals of population expansions or bottlenecks and to evaluate phylogenetic 34
position of the grey wolf population from the Central Balkans. Six haplotypes were 35
detected, of which three have only been found in the Balkan region. These haplotypes 36
belong to both haplogroups previously determined in Europe. Based on our mtDNA 37
sequence analyses, the Dinaric-Balkan wolf population is vertically differentiated into 38
"western" (Croatia/Bosnia and Herzegovina) and "eastern" (Serbia/Macedonia) 39
subpopulations. None of the results support assumption of population expansion. Instead, 40
significantly positive values for Tajima's D and Fu's Fs may suggest recent population 41
bottleneck. Obtained data may be helpful in observation to which extent gene pool from 42
the Balkans contribute to newly founded populations in Western Europe.43
44
Key words: grey wolf; Balkans; mtDNA; population structuring45
46
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The grey wolf (Canis lupus) was abundant and widely distributed over Eurasia, 47
North America and North Africa until the end of the 19th and beginning of 20th century 48
(Boitani, 2000). Widespread destruction of the wolf’s habitats, direct eradication, and the 49
decrease of natural prey led to their disappearance from Central and Western Europe 50
(Delibes, 1990; Randi et al., 2000; Randi, 2011). Only two isolated populations survived, 51
one in Italy (Boitani, 1992) and one in Iberia (Delibes, 1990), while larger populations 52
remained in the Balkans and Eastern Europe (Boitani, 2000; Lucchini et al., 2004; 53
Gomerčić et al., 2010). The Balkan grey wolf population represented the border between 54
the Eastern European population and the largely extinct Western European population.55
Legal protection, together with high dispersal and breeding potential of the 56
wolves, led to European wolf recovery in the last few decades (Boitani, 1992; Salvatori 57
and Linnell, 2005; Hausknecht et al., 2010). Wolves have expanded rapidly along the 58
Apennine ridge, recolonizing the West Italian and French Alps (Valière et al., 2003; 59
Lucchini et al., 2004). Genetic monitoring of those populations suggested that wolves in 60
Italy are partially isolated from other populations in Europe. Lucchini et al (2004) stated 61
that wolves with distinct genotypes from the east expanded from Slovenia towards the 62
Italian border in the eastern Alps (Lucchini et al., 2004). Several previous genetic studies 63
suggest that wolf population(s) in the Balkan region have retained a significant portion of 64
historical variation on the pan-European scale (Randi et al., 2000; Lucchini et al., 2004; 65
Gomerčić et al., 2010). No wide-ranging genetic characterization of grey wolf 66
populations from the Central Balkan area has been done so far. Only some individuals 67
from this region have been included in previous population genetic studies (Vilà et al., 68
1999; Randi et al., 2000; Pilot et al., 2010). Milenković (1997) suggested, based on 69
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morphometric analyses, that biogeographic features of the Central Balkan region, 70
specifically Morava-Vardar valley, influence the Dinaric-Balkan grey wolf population 71
structure.72
According to the recent estimates, there are now ~700-800 wolves in Serbia, over 73
1000 in Macedonia, around 400 in Bosnia and Herzegovina (Boitani, 2000; Milenković et 74
al., 2007) and around 250 in Croatia (Fabbri et al., 2013). Wolves underwent severe 75
declines in Croatia during the 20th century, but since then the population has grown to its 76
current estimated size during the last two decades (Kusak and Huber 2010; Fabbri et al. 77
2013). Population decline has also been reported in Bosnia and Herzegovina (Boitani, 78
2000). On the contrary, in Serbia and Macedonia the wolf populations are growing 79
(Boitani, 2000; Milenković et al., 2007).80
Wolves in Europe don't have large-scale phylogeographical structure (Vilà et al., 81
1999; Randi et al., 2000; Pilot et al., 2010). The genealogic network of Eastern European 82
wolf populations does not seem to exhibit an explicit geographical pattern in mtDNA 83
haplotypes distribution (Hausknecht et al., 2010); neither does in the Dinaric-Balkan 84
population (Gomerčić et al., 2010).85
The main aims of this research were to: (1) analyse mtDNA sequence variability 86
of grey wolf population from the Central Balkans, (2) to infer possible population 87
structuring of the Dinaric Balkan grey wolf population caused by biogeographical 88
features of the Central Balkans (e.g. Morava-Vardar valley), (3) to study the demographic 89
history and to find genetic signals of population expansions or bottlenecks and (4) to 90
evaluate the phylogenetic position of Central Balkan wolves.91
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The Dinaric-Balkan grey wolves’ dataset consisted of 192 mtDNA control region 92
sequences. Muscle tissue samples of 87 grey wolves from Serbia (53), Macedonia (18), 93
and Bosnia and Herzegovina (16) were collected during legal hunts, during winter 94
seasons (1997-2010) and analysed in this research (Fig. 1). Remaining 114 sequences 95
were retrieved from GenBank (Gomerčić et al., 2010; Vilà et al., 1999; Randi et al., 96
2000) and a respective number of individuals per haplotype were inferred from the 97
original reference. Haplotype lu9 reported by Vilà et al. (1999) and found for one 98
individual from Croatia has not been uploaded to the GenBank and therefore was not 99
included in our dataset. 100
Fig. 1.101
Total DNA was extracted from ethanol preserved muscle tissue samples using 102
standard phenol chloroform isoamylalcohol extraction with proteinase K digestion 103
(Sambrook and Russel, 2001). Partial fragment of mitochondrial control region was 104
amplified with CR1 and CR2R primers published by Palomares et al. (2002) with a target 105
sequence length of 280bp. Approximately 100ng of genomic DNA was amplified in a 106
total volume of 25μl containing 0.2mM dNTP, 0.5 μM of each primer, 3mM MgCl2, 1U 107
Taq polymerase and 1x reaction buffer. PCR amplification conditions were set as 108
follows: initial step of denaturation at 95oC for 5 min, followed by 35 cycles of 109
amplification – each cycle being 94oC for 40 s, 55oC for 50s and 72oC for 1min – and a 110
final extension step at 72oC for 10 min. The PCR products were purified using QIAquick 111
PCR Purification Kit (QIAGENE). Sequencing was conducted on an ABI3730xl genetic 112
analyzer (Applied Biosystems).113
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The sequences were aligned using the Clustal W algorithm (Thompson et al., 114
1994) implemented in BioEdit 7.0.9.0. (Hall, 1999), and final adjustments were done by 115
eye. The length of analysed sequences after alignment was 261bp. DNA polymorphism 116
(h-haplotype diversity, π-nucleotide diversity, k-mean number of pairwise differences), 117
parameters of overall genetic variability, haplotype frequencies and distances between 118
haplotypes were calculated using ARLEQUIN 3.5.1.2 (Excoffier and Lischer, 2010). 119
Nucleotide diversity for total dataset was calculated under the Kimura 2P (Kimura, 1980) 120
model of nucleotide substitution with Gamma correction for among-site variation in 121
substitution rates (γ=0.05) as suggested by a model test in MEGA version 5 (Tamura et 122
al., 2011). 123
During the initial analyses samples were organized into seven sampling groups 124
based on their geographic proximity and biogeographic features of the sampling areas:125
Western Serbia, Eastern Serbia, Southern Serbia, Eastern Macedonia, Western 126
Macedonia, Bosnia and Herzegovina and Croatia. Basic genetic indices for each 127
sampling group, analysis of molecular variance (AMOVA) among and within analyzed 128
groups and calculation of pairwise Φst values among seven sampling groups were 129
calculated using ARLEQUIN 3.5.1.2. The pairwise Φst values were used for the 130
construction of UPGMA tree in MEGA version 5.131
Our initial analyses clearly supported the presence of two distinctive genetic 132
groups or subpopulations within the Dinaric-Balkan wolf population (Table 1). Therefore 133
above-mentioned and further analyses were conducted for these subpopulations 134
separately as well as for the whole dataset. Basic genetic indices for each subpopulation 135
were calculated and analysis of molecular variance among and within subpopulations was 136
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done in ARLEQUIN 3.5.1.2. To gain insight into the possible historical changes in 137
demography of grey wolves from the Central Balkans, mismatch distribution analyses 138
were carried out, under the null hypothesis that the observed data fit the sudden 139
expansion model. The mismatch analysis was performed in ARLEQUIN 3.5.1.2. The 140
significance of the fit of observed mismatch distribution to the expected was estimated by 141
means of the sum of the squared deviations (SSD). Furthermore, two neutrality tests, 142
often used to investigate demographic changes, were performed in DnaSP v5 (Librado 143
and Rozas, 2009). The mismatch distribution analysis and Fs test were run with a 144
transition–transversion weight ratio of 1:1.145
To perform an analysis of phylogenetic relationships among wolf mtDNA 146
haplotypes, we collected all available haplotypes from the GenBank and combined them 147
with our Dinaric-Balkan dataset. This dataset comprised of 84 haplotypes and the final 148
alignment of this dataset was 223bp. Few previously published haplotypes collapsed 149
together in this fragment alignment (for details see Table S1). To avoid further 150
complications with haplotype designation, we adopted the designation presented by Pilot 151
et al. (2010) in our study. For the analysis on phylogenetic relationships of our combined 152
dataset a median-joining (MJ) network (Bandelt, et al., 1999) was constructed with the 153
software Network 4.6.0.0 (available at http://www.fluxus-engineering.com/sharenet.htm). 154
Network approaches are more suitable in determining the relationships among haplotypes 155
in intraspecific studies as they allow for the presence of ancestral haplotypes in a sample 156
(Posada and Crandall, 2001; Hausknecht et al., 2010; Zachos et al., 2010).157
The analysis conducted on 192 mtDNA control region sequences from the 158
Dinaric-Balkan grey wolf population, with a total length of 261 nucleotides revealed six 159
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different haplotypes (Table 1). There were in total 12 polymorphic sites, of which all 160
were parsimoniously informative transitions. Haplotype diversity value was 0.775±0.014, 161
nucleotide diversity (π) was 0.020±0.011 and the average number of nucleotide 162
differences (k) was 5.444±2.632. We observed a relatively high amount of mtDNA 163
variation in the Dinaric-Balkan grey wolf population. Genetic diversity levels of other 164
Balkan wolves at mtDNA control region were also high (Randi et al., 2000; Pilot et al., 165
2010; Gomerčić et al., 2010; Moura et al. 2013; Fabbri et al., 2013) as compared with 166
other European wolf populations (Randi et al., 2000; Valière et al., 2003; Ellegren et al., 167
1996; Hausknecht et al., 2010; Sastre et al., 2011). The high genetic variability found in 168
the Balkans might have originated from a past continuous large grey wolf population, that 169
has retained despite human and environmental influences, as it was indicated for 170
Bulgarian grey wolves (Randi et al., 2000), Croatian wolves (Gomerčić et al., 2010; 171
Fabbri et al., 2013), and all Balkan populations (Pilot et al., 2010). Furthermore, the 172
employment of microsatellite markers in population genetic studies of grey wolves also 173
detected the highest diversity in samples from the Balkan wolves (Lucchini et al., 2004; 174
Moura et al. 2013).175
Tab. 1176
177
No support for the presumed subdivision of the population along the Morava-178
Vardar valley was obtained based on mtDNA variation (Table S2) despite the observed 179
morphometric differences (Milenković 1997). Pairwise Φst values were significant180
between two sampling groups (Bosnia and Herzegovina and Croatia) and any other 181
sampling group (not shown). The constructed UPGMA tree based on pairwise Φst values 182
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of the seven grey wolf sampling groups from the Central Balkans separated grey wolves 183
from Bosnia and Herzegovina and Croatia into one cluster from all others that made a 184
second branch, with a significant percent of variation among subpopulations (Fig. S1).185
We detected a clear genetic subdivision within the Dinaric-Balkan wolf 186
population and those two clusters were recognized as two subpopulations and were 187
named “western” and “eastern” subpopulation. Basic genetic indices for those two 188
subpopulations were calculated (Table 1). “Eastern” subpopulation has lower diversity 189
than “western” subpopulation. “Eastern” subpopulation has one dominating haplotype 190
(BLK1) and four rare haplotypes, while “western” subpopulation has three haplotypes 191
with intermediate frequencies and one haplotype with lower frequency. Furthermore, 192
Fu’s Fs test was significant in both subpopulations and total sample (Table 1), reflecting 193
a recent population bottlenecks. Positive Tajima D values also suggest a decline in 194
population sizes, although this value was not statistically significant in “eastern” 195
subpopulation. The Fu’s Fs test of neutrality is based on the distribution of haplotype 196
frequencies and very sensitive to demographic changes (Fu, 1997), while Tajima’s D-test 197
of neutrality (Tajima, 1989) is less powerful than FS, and is based on the distribution of 198
mutation frequencies. Analysis of molecular variance (AMOVA) showed higher genetic 199
variability among subpopulations than among sampling groups within subpopulations, 200
and Φst value among subpopulations was significant (Table 2). Several causes might lead 201
to the cryptic genetic structure on fine geographical scale. Wolves within Europe show 202
genetic structuring over relatively short distances (Pilot et al., 2006; Stronen et al., 2012; 203
Hindrikson et al. 2013; Moura et al., 2013). We might argue that possibly river Drina 204
(which is also the border between Serbia and Bosnia and Herzegovina) acts as a fine 205
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barrier separating to a certain degree a Peridinaric region subpopulation on the east, but it 206
is unlikely since the river is not obvious movement barrier. We have no data on possible 207
limited female dispersal and no registered anthropogenic influence that can limit wolf 208
migration. Several processes might be expected to cause this kind of genetic differences 209
within such short geographic distances in the Dinaric-Balkan grey wolves. Published data 210
suggest that hunting and eradication programmes in the 20th century caused severe 211
population size decline in Croatia which is now recovering and decline has been 212
registered in Bosnia and Herzegovina (Boitani, 2000). Observed multimodal mismatch 213
distribution and positive values of neutrality tests may point to these past and recent 214
events in “western” subpopulation. We have found genetic signal of population 215
bottleneck in “western” subpopulation and our results clearly reflect the 1980s decline in 216
population size. For Serbian and Macedonian (“eastern” subpopulation) wolves there are 217
no available data on remarkable decline of population size, but we have detected genetic 218
signal of population bottleneck in “eastern” subpopulation as well. The different 219
demographic histories of these subpopulations may explain this result. It seems that both 220
subpopulations have gone through bottlenecks, but their timing might differ and/or the221
population size decline was not as severe in “eastern” subpopulation as it was in 222
“western” subpopulation. The excess of haplotypes with intermediate frequencies in 223
“western” subpopulation might be a consequence of genetic drift after severe population 224
bottleneck or haplotype admixture from genetically differentiated populations. Wolf 225
population from this region always remained and still is connected with the neighboring226
small wolf population in Slovenia (Štrbenac et al., 2005, 2008; Gomerčić et al., 2010; 227
Fabri et al., 2013), and continuous gene flow was presumed among all other regional 228
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groups on the Eastern Balkans (Gomerčić et al., 2010). One dominant haplotype and 229
several rare in “eastern” subpopulation, together with neutrality test results, might 230
indicate that population bottleneck has happened earlier or was not remarkable, and 231
might present genetic signal of population growth. The test for a mismatch distribution 232
(Ssd=0.044; p=0.020) and Fu’s Fs and Tajima’s D tests of neutrality did not support 233
sudden expansion model of the total analyzed population, or in “western” and “eastern” 234
subpopulations (Table 1; Fig. S2). Ragged mismatch distribution in Dinaric-Balkan 235
population and both defined subpopulations also point to recent population decline (Fig. 236
S2). No available data indicate recent population size decline in “eastern” subpopulation 237
and the constant population size increase is reported for grey wolves in Serbia and 238
Macedonia (Boitani, 2000; Milenković, et al., 2007). Only registered recent population 239
size decline (although not as severe as in “western” population) has happened in the mid 240
20th century in Serbia and Macedonia, due to the poisoning (Milenković, 1997). This 241
event may have resulted in a bottleneck, as suggested by our results.242
243
Tab. 2244
245
Similar subdivision between the Western and Eastern samples was reported for 246
Bulgarian wolves using mtDNA control region marker (Moura et al., 2013), which 247
possibly reflects fragmentation during the period of lower population size and local 248
environmental differences. Pilot et al. (2006) also stated that wolf populations in Eastern 249
Europe show a non-random spatial genetic structure and that ecological differences play 250
an important role in population structuring. However, structuring of the Dinaric-Balkan 251
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wolf population due to local ecological characteristics is unlikely. Hindrikson et al. 252
(2013) discovered cryptic population structure in the Estonian and Latvian wolf 253
population which was unexpected and proposed that strong hunting pressure is the most 254
likely the major factor that could maintain the population sub-structuring. Wolf hunting 255
has never been banned in Serbia and Macedonia, and is still allowed year round. Year256
round wolf hunting is allowed in Bosnia and Herzegovina for males, and is restricted for 257
females. The Croatian wolf population became legally protected in 1995 and a National 258
Wolf Management Plan was implemented in 2005 (Gomerčić et al., 2010). Therefore, 259
these differences in hunting pressure can not been excluded as possible cause of 260
population fragmentation. However, further speculations on the reasons for the observed 261
clustering are prevented due to limitation of making inferences based on the single locus 262
marker and further studies with more variable genetic markers are needed to confirm this 263
result and to gain more detail information on Dinaric-Balkan grey wolf population 264
structure.265
Our comprehensive phylogenetic analysis of wolf mtDNA sequences (Fig. 2) 266
indicated that the six haplotypes found in this study have previously been described267
(Table S1). Three of these haplotypes seem to be characteristic for the Balkans region 268
(Fig. 2). Comparing to the so far the most extensive study of phylogeographic history of 269
grey wolves in Europe (Pilot et al., 2010), one new haplotype was detected (BLK2). This 270
haplotype is unique for the Balkans and the network analysis grouped it into haplogroup 271
II, as well as BLK1 haplotype. Haplotypes BLK3, BLK4, BLK5 and BLK6, were all in 272
haplogroup I. Since haplogroup II is evolutionary older and largely replaced by 273
haplogroup I (Pilot et al., 2010), we may conclude that in the analysed Central Balkan 274
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population the mixture of old and new haplotypes was detected. Found haplotypes from 275
the Central Balkan populations have not contributed to defining a clear phylogeographic 276
pattern, as it was suggested earlier that they might contribute (Randi et al., 2000; Pilot et 277
al., 2010). For all European grey wolves it was shown that haplotypes representing two 278
haplogroups overlap geographically, but differ in frequency between populations from 279
South-western and Eastern Europe (Pilot et al., 2010).280
281
Fig. 2282
283
Our analyses of mtDNA variation within Dinaric-Balkan wolves revealed 284
relatively high genetic diversity. We detected a clear genetic divergence on a west-east 285
axis dividing the population into two subpopulations; samples from Serbia and 286
Macedonia into one ("eastern") and samples from Croatia and Bosnia and Herzegovina 287
into another ("western"). None of the results support assumption of population expansion, 288
but suggest recent population bottlenecks. We have found profound genetic differences 289
between “eastern” and “western” subpopulations of the Dinaric-Balkan grey wolves, 290
which may reflect the different demographic histories or might be consequence of 291
differences in hunting pressure. Fine scale differences detected may help in conservation 292
and management strategy for the Dinaric-Balkan population, and the European grey wolf 293
populations as well. This data may be helpful in tracing gene flow between populations in 294
Europe and in the process of recolonization of the Alps, to observe to which extent gene 295
pool from the Balkans contribute to new founded populations in this area. In order to get 296
even better insight in actual gene pool variability of these populations we think that the 297
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analyses of microsatellite markers in grey wolves from the Balkans might be a useful 298
next step. 299
300
Acknowledgements301
302
The authors thank all the contributors who helped in the collection of samples for 303
this work. This work was financially supported by the Ministry of Science, Republic of 304
Serbia, Grant No. 43002 and the Provincial Secretariat for Science and Technological 305
Development, Grant No. 114-457-2173/2011-01.306
307
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426
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426
Figure 1. Geographic positions of sampled localities in this study. Localities were 427
organized in six sampling groups according to geographic location: Bosnia and 428
Herzegovina, Western Serbia, Eastern Serbia, Southern Serbia, Eastern Macedonia and 429
Western Macedonia. Number of sampled individuals per locality is given in brackets (N). 430
431
Figure 2. Median-joining network of control region mtDNA haplotypes based on 223bp 432
sequence. Haplotypes detected in this study are designated by BLK 1-6. Details on433
haplotypes w1-75 are given in Supplementary Material, Table S1.434
435
436
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Table 1. Haplotype distribution and molecular indices for western and eastern subpopulations of Dinaric-Balkan grey wolf population
Western
subpopulation
Eastern
subpopulation
Total
Total number of samples 114 78 192
Total number of haplotypes 4 5 6
BLK1 (% of total sample) 7.02% 73.08% 65 (33.85%)
BLK2 (% of total sample) 22.81% / 26 (13.54%)
BLK3 (% of total sample) / 11.54% 9 (4.69%)
BLK4 (% of total sample) / 12.82% 10 (5.21%)
BLK5 (% of total sample) 34.21% 1.28% 40 (20.83%)
BLK6 (% of total sample) 35.96% 1.28% 42 (21.88%)
No. of polymorphic sites 10 12 12
No. of transitions 10 12 12
No. of transversions / / 0
Haplotype diversity (h) 0.703±0.016 0.442±0.063 0.775±0.014
Nucleotide diversity (π) 0.020±0.011 0.014±0.008 0.020±0.011
Average number of nucleotide
differences (k)
5.479±2.656 3.763±1.918 5.444±2.632
Ssd (p) 0.124 (0.020) 0.198 (0.020) 0.044 (0.020)
Tajima’s D 3.281** 0.889 2.272*
Fu’s Fs 11.320** 4.910* 8.450**
* p<0.05; **p<0.01
Table
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Table 2. AMOVA between “western” and “eastern” subpopulations. “Western” subpopulation
consists of Croatia and Bosnia and Herzegovina sampling groups, and “eastern” subpopulation
consists of all other sampling groups
Source of variation Percentage of variation Φst p
Among subpopulations 21.43 0.214 0.037
Among sampling groups
within subpopulation
1.36 0.017 0.137
Within sampling groups 77.21 0.227 0.001
Table
