Pr oce eed din ngs

ww ww.huntssymposiium.com m

“M MOD DERN N ASP PECT TS OF F SUSTAIN NABL LE MA ANAG GEME ENT OF GAME G E PO OPULA LATIO ON”

Proceeed din ngs No ovi O ber, 20013. Saad, Serrbia, 17-20 Octob

U University y of Novi Sad, Facculty of Agricultur A re Novvi Sad, Seerbia

2nd INTERNATIONAL SYMPOSIUM ON HUNTING “MODERN ASPECTS OF SUSTAINABLE MANAGEMENT OF GAME“

PROCEEDINGS

Novi Sad, Serbia, 17-20 October, 2013.

University of Novi Sad, Faculty of Agriculture Novi Sad, Serbia

ISBN: 978-86-7520-279-0 UDC: 639.1(082) International symposium on hunting “Modern aspects of sustainable management of game population” Publisher University of Novi Sad, Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000, Novi Sad, Serbia Phone: +38121 485 35 00; www.polj.uns.ac.rs Editor in chief Prof. dr Miloš Beuković, University of Novi Sad, Faculty of Agriculture, Serbia Editors Prof. dr Zoran Popović, University of Belgrade, Faculty of Agriculture, Zemun, Serbia Prof. dr Nenad Đorđević, University of Belgrade, Faculty of Agriculture, Zemun, Serbia Prof. dr Milutin Đorđević, University of Belgrade, Faculty of Veterinary Medicine, Serbia Prof. dr Mihajla Đan, University of Novi Sad, Faculty of Sciences, Serbia mr Dejan Beuković, University of Novi Sad, Faculty of Agriculture, Serbia MScEF Vukan Lavadinović, Albert Ludwig University of Freiburg, Faculty of Environment and Natural Resources, Germany

Paper Review All papers reviewed by the international Board of Reviewers Cover mr Dejan Beuković Printed by „Stojkov” Novi Sad Number of copies 100 copies

SUPPORTED BY

Organizing Board: President Prof. dr Miloš BEUKOVIĆ ~ University of Novi Sad, Faculty of Agriculture, Serbia Prof. dr Zoran POPOVIĆ ~ University of Belgrade, Faculty of Agriculture, Zemun, Serbia Prof. dr Nenad ĐORĐEVIĆ ~ University of Belgrade, Faculty of Agriculture, Zemun, Serbi Prof. dr Milutin ĐORĐEVIĆ ~ University of Belgrade, Faculty of Veterinary Medicine, Serbia Prof. dr Mihajla ĐAN ~ University of Novi Sad, Faculty of Sciences, Serbia

Symposium secretary: mr Dejan BEUKOVIĆ ~ University of Novi Sad, Faculty of Agriculture, Serbia MScEF Vukan LAVADINOVIĆ ~ Albert Ludwig University of Freiburg, Faculty of Environment and Natural Resources, Germany M.Sc Nevena VELIČKOVIĆ ~ University of Novi Sad, Faculty of Sciences, Serbia M.Sc Nikola PUVAČA ~ University of Novi Sad, Faculty of Agriculture, Serbia

Organizers University of Novi Sad, Faculty of Agriculture, Serbia

University of Novi Sad, Faculty of Sciences, Serbia

University of Belgrade, Faculty of Agriculture, Zemun, Serbia

University of Belgrade, Faculty of Veterinary Medicine, Serbia „Ss Cyril and Methodius“ University in Skopje, Faculty of Forestry, F.Y. Republic of Macedonia “Albert Ludwig” University of Freiburg, Faculty ofj Environment and Natural Resources, Germany

kj

Honorary Board: Prof dr. Dragan GLAMOČIĆ Dipl. ecc Danilo GOLUBOVIĆ Prof. dr Radivoje MITROVIĆ mr Nenad KATANIĆ Prof. dr Dragoslav PETROVIĆ Prof. dr Miroslav VESKOVIĆ Prof. dr Milan POPOVIĆ Prof. dr Milica PETROVIĆ Prof. dr Vlado TEODOROVIĆ Prof.dr Neda Mimica DUKIĆ Prof. dr Milan MEDAREVIĆ dr. Al Mahmoud Daghistani MASSOUD Dipl ecc. Igor BRAUNOVIĆ dipl ing. Milan PAŽIN

Ministry of Agriculture, Trade, Forestry and Water management, Department of Forestry Government of Republic of Serbia – Minister Ministry of Agriculture, Trade, Forestry and Water management, Department of Forestry Government of Republic of Serbia – State secretary Ministry of Education and Science Development, Government of Republic of Serbia – State secretary Ministry of Agriculture, Trade, Forestry and Water management, Department of Forestry Government of Republic of Serbia – Deputy Minister Provincial Secretariat for Science And Technological Development, Autonomous Province of Vojvodina University of Novi Sad, Rector of the University University of Novi Sad, Faculty of Agriculture, Serbia, Dean University of Belgrade, Faculty of Agriculture, Serbia, Dean University of Belgrade, Faculty of Veterinary Medicine, Serbia, Dean University of Novi Sad, Faculty Science, Serbia, Dean Faculty of Forestry, University of Belgrade, Serbia, Dean General secretary, Kennel Club of Republic Serbia State Enterprise for Forest Management "Srbijašume" Belgrade., General Manager Hunt Association of Vоjvodina, Novi Sad, Serbia

Symposium supported by: • • •

Ministry of Agriculture, Trade, Forestry and Water management Ministry of Education and Science Provincial Secretariat for Science And Technological Development, Autonomous Province of Vojvodina

Scientific Board: Prof. dr Miloš BEUKOVIĆ

(Serbia)

Prof. dr Rory PUTMAN, professor emeritus

(United Kingdom)

Prof. dr Jon SWENSON

(Norway)

Prof. dr Zoran POPOVIĆ

(Serbia)

Prof. dr Milutin ĐORĐEVIĆ

(Serbia)

Prof. dr Nenad ĐORĐEVIĆ

(Serbia)

Prof. dr Vladimir MALETIĆ

(F.Y. Republic of Macedonia)

Prof. dr Algimantas PAULAUSKAS

(Lithuania)

Prof. dr Ulrich SCHRAML

(Germany)

Prof. dr Ladislav PAULE

(Slovakia)

Prof. dr Sándor CSÁNYI

(Hungaria)

Prof. dr Dragan GVOZDIĆ

(Serbia)

Prof. dr Duško ĆIROVIĆ

(Serbia)

Prof. dr Ivan KOS

(Slovenia)

Prof. dr Tihomir FLORIJANČIĆ

(Croatia)

Prof. dr Hristo MIHAILOV

(Bulgaria)

Prof. dr Mile POČUČA

(Serbia)

Prof. dr Rajko TEPAVAC

(Serbia)

Prof dr Zoran RISTIĆ

(Serbia)

Prof. dr Zdravko JANICKI

(Croatia)

Doc. Ing. Jaroslav SLAMEČKA, CSc

(Slovakia)

Asst. prof. dr. Yusuf Ziya OGRAK

(Turkey)

Asst. prof. dr. Mihajla ĐAN

(Serbia)

Asst. prof.dr. Ivan STANČIĆ

(Serbia)

Symposium supported by: • • •

Kennel Club of Serbia State Enterprise for Forest Management "Srbijašume" Vectornic Aerospace

2nd International Symposium on Hunting, » Мodern aspects of sustainable management of game populations« Novi Sad, Serbia, 17 – 20. October, 2013. ___________________________________________________________________________________________

PLENARY LESTURES POTENTIAL EVOLUTIONARY CONSEQUENCES OF HUNTING BROWN BEARS Swenson, J. E., Zedrosser, A., Bischof, R.____________________________________________ 1-4

1. MANAGEMENT OF GAME POPULATIONS THE BROWN BEAR IN EUROPEAN RUSSIA: HISTORY AND CURRENT STATUS Vaisfeld, M., Pushkaryov, S._______________________________________________________ 5-12 GOLDEN JACKAL: OPINION VERSUS FACTS - EXPERIENCES FROM SERBIA AND HUNGARY Heltai, M., Ćirović, D., Szabó, L., Penezić, A., Nagyapáti, N., Kurys, A., Lanszki, J.______________________________________________________________________ 13-20 CONSERVATION AND MANAGEMENT OF LARGE CARNIVORES IN SERBIA – CAN A PASSIVE APPROACH LEAD TO A POSITIVE OUTCOME? Paunović, M., Milenković, M._______________________________________________________ 21-28 SUSTAINABLE MANAGEMENT IN HARE POPULATIONS IN VOJVODINA IN THE YEAR 2012, ALONG WITH A REVIEW OF THE LAST TEN YEARS Beuković M., Popović Z., Beuković, D.___________________________________________________________ 29-38 ANALYSIS OF THE STATE OF THE HARE POPULATION IN THE HUNTING GROUNDS OF CENTRAL SERBIA Popović, Z., Beuković, М., Milošević, G._______________________________________________ 39-47 ACTUAL STATUS AND DEVELOPMENT OF EUROPEAN BROWN HARE (LEPUS EUROPAEUS) POPULATION IN SLOVAKIA Slamečka, J., Sládeček, T., Gašparík, J., Jurčík, R., Paule, L.______________________________ 48-53 DENSITY OF BROWN HARE (LEPUS EUROPAEUS PALLAS, 1778) IN THE PLAIN HABITATS OF BULGARIA Zhelev C., Ninov N., Mihaylov H., Gruychev G., Stoyanov S., Mirchev R.____________________ 54-59 THE CALCULATION OF LOSSES IN HARES IN VOJVODINA FOR THE PERIOD FROM 1967. TO 2011 Ristić A. Z., Nađ I., Urošević I.M., Matejević M., Sajko, G.______________ 60-66 BROWN HARE SPOTLIGHT CENSUS: ALTERNATIVE VS. TRADITIONAL METHOD Lavadinović, V., Beuković, M., Beuković, D., Namestovski Z., Friđeš, R._____________________ 67-72 CORRELATION OF HUNTING MASS WITH REPRODUCTIVE POTENTIAL OF FEMALE HARE (LEPUS EUROPAEUS P.) Beuković M., Stančić I,. Božić A., Beuković D., Djan, M., Velićković, N.______________________ 73-78 ANALYSES OF TROPHY WORTHINESS OF RED DEER'S (Cervus elaphus L.) ANTLERS IN THE BARANJA REGION (EASTERN CROATIA)Tot, Z., Degmečić, D., Pintur, K., Florijančić, T., Ozimec, S., Bošković, I._______________________________________ 79-83


DETERMINATION OF REAL GROWTH AND TROPHY STRUCTURE OF DEER GAME IN THE HUNTING GROUNDS OF VOJVODINA Ristić A. Z., Nađ I., Urošević I.M., Matejević M., Sajko, G.________________________________________________ 84-90 THE IMPORTANCE OF LONG-TERM AND LARGE-SCALE DATA SETS IN THE EVALUATION OF RED DEER MANAGEMENT Csányi, S._____________________________ 91-99 WINTER MORTALITY IN THE POPULATION OF GREY PARTRIGES (Perdix perdix L.) Maletić, V., Stojčevski, D., Lavadinović, V.__________________________________________ 100-105 EURASIAN LYNX IN THE CENTER OF EUROPEAN RUSSIA IN THE BEGINNING OF THIS CENTURY: DISTRIBUTION, NUMBER AND HARVEST Vaisfeld, M., Pushkaryov, S., Gubar, Yu.____________________________________________ 106-1113 ABUNDANCE DYNAMICS OF GAMEBIRDS (ANSERIFORMES, CHARADRIIFORMES) IN THE REGION OF LAGOONS OF THE WESTERN COAST OF THE MIDDLE CASPIAN SEA Vilkov, Ye.V.______________________________________________________ 114-122 HABITAT SELECTION OF EURASIAN WOODCOCK SCOLOPAX RUSTICOLA URING THE SPRING MIGRATION PERIOD IN HUNGARY Schally, G., Katona, K., Bleier, N., Szemethy, L.____________________________________ 123-129 PLANNED HARMONIZATION OF GAME MANAGEMENT AND FOREST MANAGEMENT Medarević, M., Šljukić, B., Obradović, S., Ćirović, P. ___________________ 130-137 REVIEW OF METHODS FOR ESTIMATING POPULATION NUMBERS AND DENSITY OF WILD UNGULATES IN POLAND Bobek, B., Bieniek, M., Bek, M., Borowska, A._________________________________________ 138-143 WILDLIFE MANAGEMENT FROM PREDATOR-POOR TO PREDATOR-RICH SYSTEMS, A PERSPECTIVE FROM MONTANA USA Alt, K., Hamlin, K., Kujala, Q.____________________________________________________ 144-149 HUNTING AND FISHING AREAS INTEGRAL MANAGEMENT Šarčević, B., Obradović, S., Đekić, V., Šekler, M., Marković, M., Živkov-Baloš, M., Stefanović, R.___________________________________________________ 150-154 CONTEMPORARY CONDITION AND THE THREATS TO POPULATION OF WOLVERINE GULO GULO IN THE EUROPEAN RUSSIA Emelyanova , L., Gubar, Y._______________________________________________________ 155-157

CYCLES OF ABUNDANCE DYNAMICS OF SQUIRREL (Sciurus vulgaris, L.) IN TRANSBAIKALIA, RUSSIA Agafonov, G.M. , Yerdakov, L.N. _________________________ 153-161

2. GAME NUTRITITION


IS SUPPLEMENTARY FEEDING ESSENTIAL FOR RED DEER (CERVUS ELAPHUS) IN HUNGARY? Katona, K., Terhes, A., Bartucz, K., Szemethy, L.______________________________________ 162-168 SEASONAL DIET COMPOSITION OF RED DEER (CERVUS ELAPHUS) IS AFFECTED BY GAME PRESERVATION Szemethy, L., Katona, K., Mátrai, K., Sonkoly, K., Szabó, L., Schally, G., Galló, J., Bleier, N.________________________ 169-172 PHEASANT CHICKS MORTALITY DEPENDING ON DIET ON THE FARM AND YEAR Đorđević, N., Bojović, B., Popović, Z., Beuković, M., Beuković, D., Đorđević, M.____________________________________________ 173-177 PRESENCE OF AFLATOXIN B1 IN LIVER OF BROWN HARE Beuković, D., Krstović, S., Beuković, M., Jajić, I., Popović, Z., Đorđević, N.________________ 178-184 DIFFERENT ORGANIC SELENIUM CONCENTRATION EFFECT ON COMMON PHEASANT (Phasianus colchicus) PRODUCTION CHARACTERISTICS Šarčević, B., Obradović, S., Đekić, V., Šekler, M., Marković, M., Živkov-Baloš, M., Stefanović, R.____________________________ 185-190

3. GENETIC AND GAME CONSERVATION BROWN HARES IN HUNTING AREAS OF VOJVODINA: GENETIC DIVERSITY AS REVEALED BY mtDNA SEQUENCES Đan, M., Veličković, N., Obreht, D., Stefanović, M., Beuković, D., Beuković, M. _____________________________________________________ 191-196 GENETIC DIVERSITY OF CAPERCAILLIE (Tetrao urogallus L.) ALONGTHE CARPATHIANS Paule, L., Klinga, P., Mikoláš, M., Zhelev, P. .________________________________________ 197-203

ESTIMATON OF GENETIC VARIABILITY OF WILD BOAR POPULATIONS IN VOJVODINA BASED ON MICROSATELLITES Veličković, N., Đan, M., Ferreira, E., Obreht, D., Beuković, M., Fonseca, C.______________ 204-208

4. HEALTH CARE OF GAME DISINFECTION AS A BIOSAFETY MEASURE IN PHEASENT FARMS Đorđević, M., Pesić, B., Radanović, O.______________________________________________ 209-214

ROLE OF FOXES (VULPES VULPES L.) IN THE EPIDEMIOLOGY OF HELMINTH ZOONOSES IN URBAN ENVIRONMENT CONDITION Pavlović, I., Đorđević, M., Pavlović, M., Kulišić, Z., Ćirović, D._________________________ 215-219


THE BACTERIAL INFECTIONS OF RESPIRATORY TRACT OF WILD BOARS Prodanov-Radulović, J., Došen, R., Pušić, I., Stojanov, I., Petrović, T., Urošević, M._________ 220-224 HEALTH MONITORING OF FREE-RANGING WOLVES (Canis Lupus) IN GERMANY Szentiks, C. A., Lesniak, I., Fritsch, G., Galateanu, G., Grobbel, M., Reinhardt, I., Kluth, G., Ellerbroek, L., Nöckler, K., Müller, T., Aue, A., Wittstatt, U., Hildebrandt, T.B., Krone, O.________________________ 225-228

TICK SPECIES (ACARI: IXODIDAE) OF RED FOXES (VULPES VULPES) IN SERBIA Tomanović, S., Radulović, Ž., Ćakić, S., Mihaljica, D., Sukara, R., Penezić, A., Burazerović, J., Ćirović, D._____________________________________________ 229-235 ACTIVITY CONCENTRATION OF RADIONUCLIDES IN VARIUS GAME MEAT SPECIES IN SERBIA Mitrović, B., Vitorović, G., Lazarević-Macanović, M., Krstić, N., Andrić, V., Grdović S., Vitorović, D._________________ 236-238 ENVIRONMENTAL LEVEL OF MERCURY AND CADMIUM IN DEERS AND WILD BOARS FROM DIFFERENT SERBIAN HUNTING AREAS Petrović, Z., Vranić, D., Đinović-Stojanović, J., Milićević, D., Lukić, M., Nikolić, D.__________ 239-244 IMAGE-GUIDED VIRTUAL BALLISTIC FINDINGS APPLIED FOR MONITORING OF FREE-RANGING WOLVES (CANIS LUPUS) IN GERMANY Galateanu, G., Fritsch, G., Szentiks, C.A., Krone, O., Hildebrandt, T. B.__________________________________________________________ 245-248 CONFIRMATION OF CARBOFURAN POISONING OF WILDLIFE Mihaljev, Ž., Marić, B., Ratajac, R., Živkov-Baloš, M., Jakšić, S._________________________249-253 SEROPREVALENCE OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) INFECTION IN WILD SWINE IN SERBIA Stojanac, N., Stevančević, O., Potkonjak, A., Lupulović, D., Stančić, I.______________________________________________ 254-257

5. HUNTING LEGISLATION, TOURISM AND HUMAN DIMENSION IN WILDLIFE COMPARISON OF THE FAT AND PROTEIN CONTENT IN THE RED DEER HIND MEAT FROM CONFINED AND OPEN AREAS Bleier, N., Galló, J., Szabó, L., Balázs, B., Tóth, B., Biró, Zs., Heltai, M., Szemethy, L.______________ 258-264

TREND IN GAME MEAT PRODUCTION AND CONSUMPTION IN HUNGARY OVER THE PERIOD 1970-2012 Bleier, N., Biró, Zs., Galló, J., Szemethy, L., Csányi, S.______________________________ 265-271

2nd International Symposium on Hunting, » Мodern aspects of sustainable management of game populations« Novi Sad, Serbia, 17 – 20. October, 2013. ___________________________________________________________________________________________ Original scientific paper UDC: 639.112

BROWN HARES IN HUNTING AREAS OF VOJVODINA: GENETIC DIVERSITY AS REVEALED BY mtDNA SEQUENCES Đan, M., 1 Veličković, N.,1 Obreht, D., 1 Stefanović, M.,1 Beuković, D. 2 , Beuković, M. 2

Summary: Brief introduction: Brown hare (Lepus europaeus Pallas) represents one of the most important game species in hunting grounds in Vojvodina. Remarkable decline of brown hare populations has been registered in several last decades all around Europe which led to efforts to evaluate genetic diversity of brown hare populations, with main goal to contribute to maintenance of genetic resources and sustainable management of brown hare populations. Previous studies of genetic variability of brown hare populations in Vojvodina showed moderate levels of intra- and inter-population diversity. The main aim of this study was to determine genetic variability of brown hare populations in Vojvodina, after remarkable decline registered before last hunting season, using mitochondrial genome variability, as a part of continuous monitoring of this game species. Materials and methods: Total number of 90 brown hare individuals sampled from 10 localities across Vojvodina during hunting season 2012/13 was included in this study. Hypervariable domain 1 of the mtDNA control region was amplified and sequenced, and sequence variability was analysed using standard population-genetic software. Results and conclusions: Total number of 32 mtDNA haplotypes was found. Haplotype diversity value was Hd=0.938±0.012, while nucleotide diversity was π=0.0128 and average number of nucleotide differences k=5.342. Three genetically different groups were defined in the study area (Fct=0.201, p=0.013), even though no geographical barriers were determined (r=0.042, p=0.421). Detected inter-population diversity points that controlled translocations are required in order to maintain present level of genetic variability. Genetic monitoring represents one of necessary evaluation procedures for sustainable management of brown hare populations in studied hunting grounds. Key words: brown hare, mtDNA variability, Vojvodina

Introduction The brown hare (Lepus europaeus Pallas) is one of the most important and most hunted game species in Vojvodina (Popović et al., 2012). This species occurs throughout large parts of Europe, and it is the only Lepus species that can be found in Balkan Peninsula (Suchentrunk et al., 2000; Mamuris et al., 2001; Kasapidis et al., 2005; Alves et al., 2008). The brown hare’s habitats include agricultural areas, open woodland and grassland up to 1500m (Mitchell-Jones et al., 1999; Modesto et al., 2011). Several decades ago, remarkable decline of brown hare populations has been registered in whole area of Europe, most likely as a consequence of anthropogenic effects on habitat (Vapa and Šelmić, 1997; Fickel et al., 2005). The main factors that influence brown hare populations decline in Vojvodine likely to be increasing of agricultural areas, development of agricultural techniques, urbanization, and lack of high quality diet (Beuković et al., 2012). As a consequences of these influences to genetic variability, higher inbreeding coefficients were registered, as well as local fixation of certain alleles, reduced gene flow and genetic drift, which led to genetic variability decrease (Đan et al., 2012).

Mihajla Đan, PhD, associate professor, Nevena Veličković, MSc, teaching assistant, Dragana Obreht, PhD, associate professor, Milomir Stefanović, BSc, research contributor, University of Novi Sad, Faculty of Sciences, Novi Sad, Serbia; ² Dejan Beuković, MSc., research contributor, Miloš Beuković, PhD, full professor, University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia; Corresponding author: Mihajla Đan, University of Novi Sad, Faculty of Sciences, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia; E-mail: [email protected]; Phone: +381 21 485 2799. 1

191


Considering continuous environmental changes for hares and their regional population declines in large parts of Europe, maintenance of genetic resources of locally adapted populations is considered important for the long-term development of this species (Vapa and Šelmić, 1997). Population genetic structure has important ecological and evolutionary consequences and disappearance of certain genes significantly reduces the chance of future genetic variability as well as the chance of adaptations to changes in the environment. Hares do not only play an important role for the hunting economy of the Vojvodina, but they also represent a significant prey species particularly in the agrosystem of the region, and they contribute to the flow of organic matter and nutrients (Vapa et al., 2007). Due to these facts the continual monitoring of brown hares is crucial in order to obtain knowledge of populations’ genetic diversity. Brown hare populations from Vojvodina were first genetically characterized using allozymes (Vapa et al., 1999; Vapa et al., 2002; Davidović, 2003; Vapa et al., 2007), and these analyses revealed shallow gene pool divergence among populations. Microsatellite variability analyses revealed higher level of substructuring among populations (Đan, 2008). Mitochondrial DNA markers were also analyzed, primary for phylogenetic analyses, but also as additional marker for population genetic study (Djan et al., 2006), and confirmed moderate levels of intra- and inter-population genetic variability, with high number of unique control region haplotypes typical for the Balkan Peninsula. The aim of this study was to determine genetic variability of brown hare populations in Vojvodina, after remarkable decline registered before last hunting season, using mitochondrial genome variability, and to contribute to continuous monitoring of this game species in hunting grounds of Vojvodina.

Material and Methods In total, 90 brown hare individuals were collected from the 10 localities in Vojvodina: Aradac (5), Bačka Palanka (6), Bačka Topola (10), Gložan (10), Kraljevci (9), Margita (10), Novo Miloševo (10), Petrovci (10), Sonta (10) and Voganj (10) (number of samples per locality is given in parenthesis). Muscle tissue of each individual was sampled and frozen at −20°C immediately after the death of the animal. Total DNA was extracted using a modified method of Kocher et al. (1989). Hypervariable domain of the mitochondrial control region (CR-1) was amplified following Kasapidis et al. (2005). Approximately 100ng of genomic DNA were amplified in a total volume of 25μl containing 0.2mM dNTP, 0.5 μM of each primer, 3mM Mg(OAc2), 0.5U Taq polymerase (Fermentas) and 1x reaction buffer. PCR amplification conditions were as follows: initial step of denaturation at 95oC for 5 min, followed by 35 cycles of amplification – each cycle being 94oC for 60 s, 50oC for 45s and 72oC for 45s – and a final extension step at 72oC for 5 min. The PCR products were purified following the ExoSAP protocol (Fermentas) and sequencing was conducted on an ABI3730xl DNA Analyzer using BigDyeTerminator v3.1 Cycle Sequencing Kit. Obtained sequences were aligned using the Clustal W algorithm implemented in BioEdit v7.0.5.3 (Hall 1999). After alignment dataset comprised 90 sequences of total length of 417 nucleotides. Basic indices of genetic diversity, number and haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (k) were calculated in DnaSP v.5.10.01 (Librado and Rosas, 2009) and Arleuqin v3.5 (Excoffier and Lischer, 2010). Same programs were used for the calculations of pairwise Fst and Kxy values where ten sampling localities were treated as populations. Neighbour joining dendograms were constructed in MEGA v5.2 (Tamura et al., 2011) using nucleotide substitution model TN93 (Tamura and Nei, 1993), as the best model as estimated in MEGA. Possible isolation-by-distance was estimated by correlation between Fst and geographic distance in IBD v3.23 (Jensen et al., 2005). Defining of groups of populations that are geographically homogeneous and maximally differentiated from each other was done in SAMOVA v1.0 (Dupanloup et al., 2002).

192


Results and Discussion The CR-1 mtDNA sequence variability analysis revealed the presence of 32 haplotypes. Total number of polymorphic sites was 48, while 34 were parasimony informative. Average haplotype diversity value was Hd=0.938±0.012, while nucleotide diversity was π=0.0128 and average number of nucleotide differences k=5.342. The values of basic genetic variability indices per each population are given in Table 1. Table 1. The values of basic genetic variability indices in ten brown hare populations from Vojvodina Populations I II III IV V VI VII VIII IX X Number of 5 6 10 10 9 10 10 10 10 10 samples Number of 3 4 6 7 5 6 5 7 7 7 haplotypes Unique 1 1 1 2 1 1 2 4 5 3 haplotypes Number of 3 7 10 6 15 8 9 11 20 23 transitions Number of 0 0 0 0 2 0 0 5 2 3 transversions Indels 0 0 0 0 1 0 0 0 1 1 Hd 0.700 0.867 0.844 0.911 0.861 0.866 0.800 0.911 0.933 0.933 π 0.003 0.007 0.009 0.004 0.014 0.007 0.005 0.015 0.021 0.016 k 1.400 2.867 3.778 1.800 5.833 2.867 2.133 6.355 8.755 6.755 I - Aradac, II - Bačka Palanka, III - Bačka Topola, IV - Gložan, V - Kraljevci, VI - Margita, VII - Novo Miloševo, VIII - Petrovci, IX – Sonta, X – Voganj; Hd – haplotype diversity; π – nucleotide diversity; k – average number of nucleotide differences Found haplotype and nucleotide diversity in brown hares from Vojvodina as revealed by mtDNA variability is at the moderate level and corresponds to previous data for Central European populations (Kasapidis et al., 2005; Stamatis et al., 2009). Comparison with previously published data for brown hares from Serbia (Đan, 2008) reveals lower values of basic parameters of genetic diversity. Unique haplotypes were found in each population, which provides additional evidence that genetic differentiation of brown hare populations is mainly maternal (Fickel et al., 2005; Đan, 2008; Stamatis et al., 2009). Genetic differentiation among populations was determined by Fst values and neighbor joining dendogram was constructed (Figure 1). The graphical presentation indicates grouping of populations from Bačka and Srem regions, comparing to populations from Banat region. Generally, higher values of genetic differentiations were detected between populations comparing to previously published data (Đan, 2008), but this difference may be due to the fact that single locus marker was employed in this study, and additional nuclear markers were analysed in previous study (Đan, 2008). Statistically significant pairwise Fst value was found between Gložan and Novo Miloševo (Fst=0.567), Bačka Palanka and Novo Miloševo (Fst=0.432), and Bačka Topola and Novo Miloševo (Fst=0.450).

193


Gložan Bačka Palanka Voganj Bačka Topola Aradac Kraljevci Petrovci Sonta Novo Miloševo Margita 0.02

Figure 1. Neighbor joining dendogram clustering ten brown hare populations from Vojvodina based on pairwise Fst values Spatial analysis of molecular variance (SAMOVA) revealed the presence of three different groups: first Sonta, second Novo Miloševo and third which comprises all other populations (Fct=0.201, p=0.013). Even though the percentage of variation among groups is lower than within population, it is statistically significant (Table 2). Table 2. SAMOVA results defining three genetically differentiated groups of brown hare populations in Vojvodina Source of variation Percentage of variation Fixation indices Among groups 20.17 0.201* Among populations within 6.94 0.271 groups Within populations 72.89 0.087* *p