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Detecting population admixture in honey bees

of Serbia

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Journal of Apicultural Research 53(2): 303-313 (2014) © IBRA 2014
DOI 10.3896/IBRA.1.53.2.12

ORIGINAL RESEARCH ARTICLE

Detecting population admixture in honey bees

of Serbia

Nebojsa Nedić1, Roy Mathew Francis2, Ljubiša Stanisavljević3, Ivan Pihler4, Nikola Kezić5, Christian
Bendixen6 and Per Kryger2*

1
Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia.
2
Department of Agroecology, University of Ǻrhus, Forsøgsvej 1, Slagelse 4200, Denmark.
3
Center for Biology of Bees, Faculty of Biology, University of Belgrade, Serbia.
4
Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia.
5
Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia.
6
Dept. of Molecular Biology and Genetics, University of Ǻrhus, Blichers Allé 20, Tjele 8830, Denmark.

Received 17 November 2013, accepted subject to revision 28 March 2014, accepted for publication 23 April 2014.

*Corresponding author: Email: per.kryger@agrsci.dk

Summary
Honey bee workers were sampled across the Serbian territory during 2009-2010 from mostly non-migratory apiaries to determine the population
structure of these bees using morphometric, genetic, and spatial information. A total of 134 bees were sampled, of which 77 were analysed
using classical wing morphometrics and 122 bees were successfully analysed using 24 DNA microsatellite markers. A combination of methods
including multivariate statistics and assignment tests (frequency-based and Bayesian) revealed the honey bees of this region to resemble the
subspecies Apis mellifera macedonica, Apis mellifera carnica or hybrids of these two subspecies. Based on Bayesian assignment (‘Structure’)
and spatial PCA, honey bees within the Serbian territory were composed of 56%-58% A. m. carnica and 42%-44% A. m. macedonica. Spatial
analysis showed the existence of a north-west to south-east cline in genetic differentiation. The bees in the north-west resemble A. m. carnica,
while the bees in the south-east of the country are more similar to A. m. macedonica. Thus, the extent of A. m. macedonica within Serbia was
greater than previously estimated. We define a line of hybridisation between A. m. carnica and A. m. macedonica within our study area. The
cline of differentiation was still evident using a combination of genetic and spatial information, in spite of beekeeping activities including
transhumance and breeding efforts.

Detección de la hibridación en poblaciones de la abeja de la

miel de Serbia
Resumen
Se muestrearon abejas obreras procedentes en su mayoría de colmenares no trashumantes a través del territorio de Serbia durante los años
2009 y 2010 para determinar la estructura poblacional de estas abejas utilizando datos morfométricos, genéticos y de información espacial. Se
muestrearon un total de 134 abejas, de las cuales 77 fueron analizadas utilizando la morfometría clásica del ala y las otras 122 abejas fueron
analizadas utilizando 24 marcadores microsatélites de ADN. Una combinación de métodos que incluyen la estadística multivariante y pruebas
de asignación (basada en la frecuencia y el método bayesiano) reveló que las abejas de la miel de esta región se parecen a las subespecies
Apis mellifera macedonica, Apis mellifera carnica o a híbridos entre estas dos subespecies. En base a la asignación bayesiana ('Structure') y el
PCA espacial, se observó que las abejas de la miel en el territorio de Serbia estaban compuestas entre un 56%-58% de A. m. carnica y entre
un 42%-44% de A. m. macedonica. El análisis espacial mostró la existencia de una variación clinal de la diferenciación genética en dirección
noroeste a sureste. Las abejas del noroeste del país se asemejan a A. m. carnica, mientras que las abejas del sureste del país son más similares
a A. m. macedonica. Por lo tanto, el rango de distribución de A. m. macedonica dentro de Serbia fue mayor que el estimado previamente.
304 Nedić et al.

Definimos una línea de hibridación entre A. m. carnica y A. m. macedonica dentro de nuestra área de estudio. La distribución clinal de la
diferenciación genética es aún evidente usando una combinación de las herramientas genéticas y espaciales, a pesar de la actividad apícola y
de los esfuerzos de la cría de abejas y la trashumancia.

Keywords: DNA microsatellites, classical wing morphometry, population structure, adegenet, spatial PCA, admixture, Apis mellifera L., Serbia

C2a occurs in Croatian bees (Franck et al., 2000a), C2b occurs in A. m.

Introduction
caucasica (Franck et al., 2000b; Kandemir et al., 2006), C2c dominates
In the former Yugoslavia, beekeeping was characterized by long- in bees from Slovenia and Croatia (Munoz et al., 2009) and C2d in
distance mobility covering large spatial territory. During this time, the Greek bees (Sušnik et al., 2004), C2e occurs in Croatian (Munoz et al.,
Carniolan honey bee (Apis mellifera carnica Pollman) was the preferred 2009) and Serbian bees (Kozmus et al., 2007; Nedić et al., 2009), C2f
bee, and breeding organisations were established. From 1990, as a occurs in cypriotic bees (Kandemir et al., 2006), C2g and C2h occurs
result of new political boundaries, beekeeping activities have been in bees from western Turkey (Ozdil et al., 2009), C2i occurs in Greek
restricted to smaller areas. The breeding programme of Serbian bee- bees (Munoz et al., 2009), C2i and C2j occurs in Serbian bees (Nedić
keepers is mostly concerned with the propagation of A. m. carnica. et al., 2009) and most recently, C2o and C2p were reported from
Based on Ruttner’s morphometric analyses (Ruttner, 1988), A. m. carnica south-west and north Serbia (Muñoz et al., 2012). Most C-lineage
is native in south eastern Austria, Slovenia, Slovakia, Croatia, Bosnia mitotypes have been obtained from sequence data rather than
and Herzegovina, Serbia, Montenegro, Hungary, Albania, and parts of restriction digestion.
Romania, whilst A. m. macedonica covers northern Greece, Bulgaria, Recent mitochondrial studies in the Serbian territory indicate the
and parts of Romania and Ukraine. Although, our study area is known presence of both A. m. macedonica and A. m. carnica haplotypes. A
to be near the distribution limit of A. m. carnica, the presence of schematic representation of C2 mitotypes previously described from
A. m. macedonica remains unclear. within Serbia is shown in Fig. 1. Kozmus et al. (2007) described C2e in
Živanović (1893) described this region’s black bees and yellow bees Serbian bees from north-west, east and south-east. Nedić et al, (2009)
as two different varieties. Based on morphological comparisons of honey suggested C2e in the north-east, east, south-east and south-west.
bee samples from north-east Serbia (Banat) with A. m. carnica, A. m. They also described two new C2 haplotypes C2i in the south and C2j
ligustica, A. m. cypria and A. m. syriaca, (Grozdanić, 1926) suggested in the east. A gene flow of A. m. macedonica into Serbia was suggested
the separation of the bees from Banat into a new subspecies A. m. by Kozmus et al. (2007) and Muñoz et al. (2012). Stevanović et al. (2010)
banatica. However, the Banat bees merely show a higher frequency of indicated the presence of A. m. macedonica in east, south, south-west
yellow colour, but otherwise resemble the Carniolan bee (Ruttner, 1988). Serbia and Republic of Macedonia while northern Serbia, Bosnia and
Recent morphometric studies of honey bees from the Serbian Herzegovina probably contain A. m. carnica populations.
territory indicate the existence of local morphotypes (Jevtić et al., 2007; Microsatellite DNA studies have been used to study population
Nedić et al., 2011). Nedić et al. (2011) analysed forewing and cubital structure, genetic identity, and introgression patterns. In honey bees,
indices on 70 adult worker bees sampled from seven beekeeping regions microsatellite markers have been used since the 1990s (Estoup et al.,
in the area. The highest morphological difference was observed between 1993; Estoup et al., 1995; Solignac et al., 2003; Jensen et al., 2005).
honey bees from the north of the country (Sivac) and bees originating Due to their codominant inheritance and high variability, they are
from the south (Zubin Potok). However, morphological characteristics suitable for measuring gene flow and admixture rates in local and
are not suitable to determine areas of hybridisation, since little is known introduced honey bee populations (De la Rúa et al., 2001a,b). Several
about the heritability of the various morphological traits. In addition, studies have employed DNA microsatellites to elucidate population
morphologic traits may be sensitive to selection (convergence) and structure of honey bees (Garnery et al., 1998; De la Rúa et al., 2001b;
environmental conditions. Chromosomal investigation of honey bees Franck et al., 2001). Based on microsatellite DNA analysis, bees from
from three regions of Serbia namely north (Banat), east (Timočka), Slovenia, which is within the modern range of A. m. carnica, are closely
and south-west (Syenichko-Peshterski) points to the existence of three related to bees from Croatia, whilst bees originating from Greece (A. m.
distinct karyotypes (Stanimirović et al., 2005). The authors suggested macedonica) formed a separate population (Sušnik et al., 2004).
all karyotypes to be local ecotypes of the Carniolan bee, with the The object of this study was to determine the distribution of A.
greatest difference between the north (Banat) and south (Syenichko- mellifera subspecies in Serbia using DNA microsatellite markers and
Peshterski). classical wing morphometry. Analysis approaches used are frequency-
The highly variable COI-COII region of the honey bee mitochondrial based assignment, Bayesian assignments and spatial analysis. The
DNA is a widely accepted marker to study the phylogeny of honey bees ‘adegenet’ package in ‘R’ software (R Development Core Team, 2011)
(Garnery et al., 1992; Meixner et al., 2013). Within the C lineage, at is particularly suited to understanding cryptic population structures
least 13 mitotypes (C1, C2a to C2j, C2o, and C2p) have been described which otherwise may be undetected using classical methods (Jombart
so far. C1 was identified as dominant in Italian bees (Munoz et al., 2009), et al., 2008).
Admixture in Serbian honey bees
Fig. 1. Overview of the seven C2 mitotypes (C2c, C2d, C2e, C2i, C2j,
C2o and C2p) within the Serbian territory as reported from previous
studies. Most studies have focussed on six regions in Serbia: A (Srem); E9, L13, J10, J16, N23, K19, G18 and O26. In addition, the Cubital
B (Banat); C (Timočka); D (Timočka); E (Raska); F (Pcinja). Mitotypes Index (CI) was also measured. Principal Component analysis was
identified at each location is shown along with superscripts indicating
study. The studies are as follows: 1 (Muñoz et al., 2012); 2 (Nedić et
al., 2009); 3 (Kozmus et al., 2007); 4 (Stevanović et al., 2010).
Material and method
Sampling
Honey bee workers were collected from 58 localities across Serbian
territory (Fig. 4) in 2009 and 2010. A total of 134 colonies from mostly
non-migratory apiaries were sampled, two workers per colony. The
geographical coordinates of the sampled locality was recorded. Samples add-in to ‘Excel’ (Microsoft) was used to organise the data, calculate
were preserved in absolute ethanol and stored at -20°C prior to analysis. frequency-based statistics, population assignment and to export to
The complete list of samples and coordinates are provided as
supplementary data Table S1.
[http://www.ibra.org.uk/downloads/20140430/download]
Wing morphometry (Classical)
A subset of 77 Serbian bees (from the 134 bees sampled) were clipped Bayesian method (Rannala and Mountain, 1997) and a Bayesian MCMC
at the base and mounted on glass photographic frames. For morpho-
metric comparison, 15 A. m. carnica bees from Slovenia and 15 A. m.
macedonica bees from Macedonia were used as reference samples.
Measurements were taken using Leica XTL-3400D microscope and
305
software package ’IL1009’ in accordance with the standard method by
Ruttner et al. (1978). Eleven angles were measured namely A4, B4, D7,
performed on the dataset using statistical software ‘R’.
DNA extraction and genotyping
All colonies sampled were used for DNA microsatellite analysis (one
worker per colony). For genetic comparison, 107 additional bee samples
from Albania, northern Greece, Republic of Macedonia, and Bulgaria
were obtained to represent the A. m. macedonica subspecies, along
with 58 bee samples obtained from Croatia and Slovenia to represent
the A. m. carnica subspecies.
DNA Extraction was carried out using DNeasyTM 96 Blood & Tissue
Kit (Qiagen). The thoracic muscles of each bee were separated using
clean forceps into Dneasy 96 tubes. DNA was extracted according to
the kit instructions. DNA was eluted into 100 μl sterile water and
stored at -80°C.
Four separate PCR reactions were carried out with fluorescent-
tagged primer pairs with six to nine primers multiplexed per reaction.
In total, 28 loci were genotyped using standard primers (Table 1)
(Estoup et al., 1995; Solignac et al., 2003). For locus A014, the primer
set was redesigned to simplify the scoring, thereby reducing the length
of the amplicon from 230 bp to 117 bp. The new primers used were
F: ACG CGG CGA TCG TAA AA and R: CCA CCG TGC GAT GAC G.
Genotyping was carried out on a 96 capillary ABI 3730xl Sequencer
(Applied Biosystems). Alleles were initially scored by ‘GeneMapper’
software and verified twice by eye and corrected manually if required.
Only 24 polymorphic loci were used in subsequent analyses. Loci AC306,
AP238 and AT005 were removed due to greater than 50% missing data.
The primer pairs AC087 and AC088 amplify the same locus and only
the AC088 was included in the analyses. Of the 134 bees genotyped,
12 bees were removed due to greater than 25% missing data and the
remaining 122 bees were used in all subsequent genetic analyses.
Non-spatial analysis
The ‘GenAlEx’ 6.4/6.5 package (Peakall and Smouse, 2006, 2012), an
various formats. Frequency-based statistics and assignment were
computed on 287 bees as three declared populations (122 Serbian
bees, 58 A. m. carnica and 107 A. m. macedonica reference bees).
Individual assignment test was carried out in software ‘GeneClass’
2.0.h (Piry et al., 2004) using Nei’s standard distance (Nei, 1972), a
method (Cornuet et al., 1999) as the criteria for computation.
The current dataset along with reference data were analysed using
the ‘Structure’ 2.3.3 software (Pritchard et al., 2000), which implements
a Bayesian method using Monte Carlo Markov Chain (MCMC) approach
306 Nedić et al.

Table 1. List of genotyped loci and primers. All standard primers were used (Estoup et al., 1995; Solignac et al., 2003), except locus A014.
The groups denote primers which were multiplexed together as a single reaction. The fluorescent dye attached to each primer is indicated as
Label. *denotes loci discarded from the analyses. **denotes use of modified primers.

No. Locus Label Forward primer (labelled) Reverse Primer

1 A008 NED GAAGGTAAGGTAAATGGAAC TTGGCGGTTAAAGTTCTGG
2 A079 FAM GAAGGTTGCGGAGTCCTC GTCGTCGGACCGATGCG
3 A088 VIC CGAATTAACCGATTTGTCGTG GATCGCAATTATTGAAGGAGTA
4 AC011 NED TTACGCCAATCTCTCCACG CGGTTAATTTCGTTTCTCGC
5 AC088 VIC CGAGGCAAGTGTTCGCTG ATCATCATTCCCAAGGTGAC
6 AP224 FAM AAGCGCACCGGAATGACG TATGTTCGGCCAGGCGGA
7 AP249 PET TCGCGCGACGACGAAATG TCCTTTGATTCGCGCTACC
8 AP274 PET ATCCCGGTGGCCACGTC GTCGCCACGCATAACTCG
9 A113 PET CTCGAATCGTGGCGTCC CTGTATTTTGCAACCTCGC
10 AC306* FAM GAATATGCCGCTGCCACC TTTTCGTTGCATCCGAGCG
11 AP090 PET GCGAAAATTGCCGGGTTATA GCAACTTTATCGTTTCGACGT
12 AP223 PET AATCGTACAACGTCGCGC CCGCTCGCCTGTATCTG
13 AP226 NED TAACGGTGTTCGCGAAACG AGCCAACTCGTGCGGTCA
14 Ap288 FAM GTTAGTTCGTCGTCGACCG TCTTAGCTTTATAACGAGCACG
15 A014** PET ACGCGGCGATCGTAAAA CCACCGTGCGATGACG
16 A029 NED AAAACAGTACATTTGTGACCC ATCAACTTCAACTGAAATCCG
17 AC087* FAM TCGAGGCTCGAGGCAAG ACATTGGCCAAGCGAACG
18 AP085 PET ATCAAACACACAAACGAAAGC ACCGGAAGCCTAATCAAGG
19 AP273 VIC GATCTTGTGTTAAACAGCCG ATCTCTGGCAGACGAAGAG
20 AT005* NED ATCCACCGGTATCCACCG TAATCGGTTTCCACTGGCC
21 AT163 FAM GCATTAGCATATACACGAGG TCGGGTCTCGCAGTAACG
22 AT188 VIC CGCATTTAGCCAACGATTAC GAACTTTACCTCACGAACACGA
23 A024 VIC TACACAAGTTCCAACAATGC GCACATTGAGGATGAGCG
24 A043 FAM CACCGAAACAAGATGCAAG CCGCTCATTAAGATATCCG
25 AC139 NED CCAGTGTTCACGGTAAACG ATCATAGAGTACGCGCAAAG
26 AP015 VIC GGGGGTAACGGAGAGAGG TGTACGAGGCACGCAATC
27 AP068 PET GTCTGCCCTCCTCTCTGTT ACACATCGAGCGAGAAGGC
28 AP238* NED GTCTCGTGCGTGCGAATG CATCATGTTCTCAAATTTCTTTG

to cluster individuals. The MCMC was run using admixture model for Geographic coordinates for the sampling points were converted from
1,000,000 iterations with a 100,000 burn-in period, with the number longitude-latitude to UTM (Universal Transverse Mercator) coordinates
of clusters (K) ranging from 1-7. At least two repeats were performed for zone 34. Duplicate coordinates were shifted randomly by a factor
to check the convergence of likelihood value for each K. Ten repeats of 10 (approx. 100 m to avoid zero distances between samples).
were performed for the most likely value of K. Values which maximized Spatial Principal Component Analysis (sPCA) was carried out using
the likelihood of the data for each K was taken as the most probable Delaunay triangulation as the connection network. The variance was
clustering and visualized as proposed by (Evanno et al., 2005). plotted against spatial autocorrelation (Moran’s I) to produce a screeplot
In addition to ‘Structure’, the data were analysed using R-package which was used to visually estimate if existence of general spatial
‘adegenet’ (Jombart, 2008) in statistical software ‘R’ 2.14 (R Development structures could be inferred. Monte Carlo tests were performed using
Core Team, 2011). The number of clusters was also estimated in ‘R’ genetic data and spatial weights for 10000 iterations to statistically test
package ‘adegenet’. Sixty principal components were retained for the the presence of global and local spatial structure. Mantel test was run
analysis. Principal Component Analysis (PCA) was carried out using R- using function mantel.randtest() from R-package ‘ade4’. Detailed
packages ‘adegenet’ and ‘ade4’ (Dray, 2007). explanations for sPCA methods are covered in (Jombart et al., 2008).
Background maps of political boundaries were obtained from Global
Spatial analysis Administrative Areas database (GADM v1.0, 2011). Background maps
Spatial data analysis was carried out in ‘R’ using package ‘adegenet’ were plotted using R-package ‘PBSmapping’ (Schnute, 2010). The
and ‘PBSmapping’ (Schnute, 2010). The 122 Serbian samples were eigenvectors for each eigenvalue scores (retained scores 1 to 5) obtained
used for spatial analysis without the reference populations. Missing from the sPCA analysis were mapped out to geographical coordinates.
genotype values were replaced by mean values for each locus. These eigenvectors at each location were smoothed by taking an average
Admixture in Serbian honey bees 307

Table 2. Summary of classical wing morphometry analysis of 77 bees from Serbian territory in comparison to reference bees. The four blocks
of rows show the sampled test bees (Test), reference bees in this study (Ref), Data from Ruttner (1988) (Rut) and reference data from
Oberursel data bank, Germany (Obr). Columns show dataset, subspecies, n (number of specimens analysed), 11 wing landmarks and cubital
index. The mean (top value) and standard deviation (bottom value) of the measurements are shown. The sampled test bees were split into
A. m. carnica and A. m. macedonica bees based on sPCA results. Car – A. m. carnica, Mac – A. m. macedonica, CI – cubital index.

Pop n A4 B4 D7 E9 G18 J10 J16 K19 L13 N23 O26 CI

Serb Car 47 30.0 110.2 96.6 23.0 91.8 53.3 91.4 78.6 13.5 95.4 39.2 2.5
2.2 4.9 3.1 1.8 3.2 3.8 4.3 2.7 1.6 3.6 3.4 0.3
Mac 30 30.5 108.9 96.7 23.3 91.5 55.3 90.5 78.7 14.1 95.8 38.5 2.5
1.6 4.1 3.4 1.7 3.0 3.7 2.8 2.2 1.6 2.2 4.2 0.4
Ref Car 15 29.7 110.4 99.4 23.7 93.6 52.3 91.1 80.3 13.9 94.3 34.0 2.6
1.6 4.3 2.1 2.0 1.8 2.2 3.3 2.3 1.0 2.5 3.0 0.4
Mac 15 31.1 106.0 101.2 22.1 92.3 55.2 85.9 78.1 14.0 88.3 36.6 2.3
2.0 7.2 3.4 1.6 2.3 3.8 3.6 3.0 0.8 3.0 3.6 0.2
Rut Car 21 23.1 93.1 52.2 12.5 2.6
2.1 3.3 3.5 3.0 0.4
Mac 20 22.1 94.1 54.8 14.2 2.6
1.5 3.2 3.6 1.6 0.4
Obr Car 20 28.6 112.9 98.6 23.6 92.0 52.7 95.5 79.1 13.4 94.4 37.1 2.7
1.1 2.9 1.7 0.8 1.9 1.8 1.8 1.8 0.9 1.4 1.9 0.2
Mac 10 29.6 108.6 98.6 21.9 94.7 55.0 95.1 79.0 13.8 95.3 37.5 2.8
0.8 3.0 2.1 0.5 1.5 1.5 1.8 1.7 0.8 1.9 1.5 0.3

of the value to its immediate neighbours. The smoothed eigenvectors data were discarded. Four loci were also discarded due to bad quality
was used to create a 2-colour gradient interpolated map within the data or redundant data. Therefore, 122 individuals for 24 loci were
sampling region using R-package ‘akima’ (Akima et al., 2013). used in subsequent analyses. The genetic results for Serbian bees and
To further verify the geographical cline trend in Serbian bees, reference populations are summarized in Table 3. All loci were found
correlations tests were carried out. For each sample, its corresponding to be polymorphic in all three populations. The average observed
latitude was divided by its longitude to produce a number which we heterozygosity (Ho) for the all three populations were similar to the
refer to as the geographical index. The geographical index in essence, expected heterozygosities (He). The number of alleles detected in the
corresponds to a north-west to south-east spatial distribution of sample. studied Serbian population ranged from three (loci AC88, AP274, AP288)
The geographical index is also more representative of the shape of the to 21 (locus A29), while those in the A. m. macedonica and A. m.
country than latitudes or longitudes. The cluster probabilities from carnica populations ranged from 2 alleles (locus AP274) to 25 alleles
‘Structure’ were correlated to the geographical index, latitude and (locus A29).
longitude using spearman correlation.
Population structure
Assignment test on 122 Serbian bees with reference samples were
carried out in GeneClass 2.0 (Piry et al., 2004). Comparing Bayesian
Results
(Rannala and Mountain, 1997) and Nei’s standard distance (Nei, 1972),
Morphometric analysis we were able to allocate 49% of the bees as A. m. macedonica, 36%
All measurements from the wing morphometry yielded usable data. A as A. m. carnica and 15% were ambiguous. Results were declared
summary of the wing data of the 77 measured bees in comparison to ambiguous if the two methods yielded conflicting assignment. Bayesian
reference bees is shown in Table 2. The data was used to perform a MCMC (Cornuet et al., 1999) allocated 70% of the bees as A. m.
PCA along with the reference bees. Morphometrically, the 77 measured macedonica, 30% as A. m. carnica with only one ambiguous individual.
bees were most similar to the A. m. carnica reference bees, but distant This one ambiguous individual was assigned to either subspecies with
from A. m. macedonica reference bees (Fig. 2A.). The A. m. carnica equal probability.
reference bees were tightly clustered and may not represent the full Population genetic structure was inferred through the Bayesian
variance of the subspecies. clustering method incorporated in software ‘Structure’. The program
was run for K = 1 to K = 7 and the real number of clusters (K) was
Microsatellite analysis calculated according by (Evanno et al., 2005). The Log likelihood was
All samples used for DNA microsatellite analysis were genotyped the best with two clusters (K = 2), which appeared to be the optimum
successfully. Twelve samples with greater than 25% missing allelic sub-division of our data. In the Structure results, cluster 1 contained
308 Nedić et al.

Fig. 2A. Scatterplot of Principal Component Analysis results (Wing morphometric data) of 77 sampled bees in comparison to 30 reference
bees (15 A. m. carnica and 15 A. m. macedonica bees). Axis represent PC1 and PC2. The sampled bees show some resemblance to reference
A. m. carnica, but cluster differently from reference A. m. macedonica bees which may be a result of breeding activity. B. Scatterplot of PCA
results (DNA microsatellite data) of 122 bees from the study area with reference populations (58 A. m. carnica and 107 A. m. macedonica
bees). Axes represent PC1 and PC2. The ellipses show 67% spread of data. The sampled bees can be clearly seen to be admixed between the
reference populations. The barplots at bottom left corner shows the relative variance explained by the first ten principal components.

Table 3. Summary of frequency-based statistics. The populations shown are reference A. m. carnica and A. m. macedonica bees and sampled
test bees. The test bees were then split into A. m. carnica or A. m. macedonica based on sPCA results. Ho – Observed hetrozygosity, He –
Expected heterozygosity.

No. of
Population Sample Size No. of alleles Ho He Fixation Index
effective alleles
Mean 56.1 7.0 2.7 0.45 0.45 0.012
A. m. carnica
SE 1.0 0.9 0.6 0.05 0.05 0.020
Mean 103.8 7.4 2.9 0.49 0.50 0.023
A. m. macedonica
SE 0.8 1.0 0.6 0.05 0.05 0.017
Mean 115.7 7.7 2.7 0.48 0.48 -0.003
Test bees
SE 2.0 0.8 0.5 0.05 0.05 0.015

Test Car Mean 67.2 7.0 2.8 0.49 0.47 -0.023

SE 1.3 0.8 0.5 0.06 0.05 0.023
Test Mac Mean 48.5 6.1 2.5 0.47 0.46 -0.012
SE 0.8 0.7 0.4 0.05 0.05 0.018

57 of the 58 reference bees from Slovenia and Croatia, defining this shown in Fig. 3. The two reference populations can be distinctly identified
cluster as the A. m. carnica population. In cluster 2, the majority of and the Serbian population appears to be admixed between the two
the bees (95 of 107) from the reference samples from Republic of populations. The Serbian bees are sorted north-west to south-east
Macedonia, Bulgaria, Greece and Albania were contained, defining this (based on geographical index) so that the cline is discernable. The
as the A. m. macedonica population. The remaining 12 bees were find.clusters() function in the R-package ‘adegenet’ also gave the
hybrids showing substantial admixture with A. m. carnica bees. Of the lowest BIC (Bayesian Information Criterion) for K = 2. In ‘adegenet’,
test samples, 55% were assigned to cluster 1 (A. m. carnica) and 45% sPCA global score 1 assigned the Serbian bees as follows: 58% to A. m.
were assigned to cluster 2 (A. m. macedonica). However, large individual carnica and 42% to A. m. macedonica.
variation exists for the Serbian population. The ‘Structure’ results are
Admixture in Serbian honey bees 309

Fig. 3. Barplot of Bayesian population assignment probabilities from ‘Structure’ when K = 2. Each vertical line represents an individual bee
sample. The colour represents the probability with which an individual belongs to a population. The figure shows from left to right: reference
populations A. m. carnica and A. m. macedonica followed by the Serbian bees sorted by geographical index (refer section 2.5) north-east to
south-west (left to right). The reference populations produce distinct clusters discriminating A. m. carnica from A. m. macedonica while the
Serbian bees show admixture. Locations of reference populations are indicated as (SL-Slovenia, HR-Croatia, MK-Republic of Macedonia, GR-
Greece, AL-Albania, BG-Bulgaria and RS-Serbia).

Fig. 4A. Spatial distribution and genetic background of honey bees sampled from the Serbian territory based on spatial Principal Component
Analysis (sPCA global score 1). The black and white squares represent two distinct clusters. The size of the squares denotes the probability
with which the sample belongs to the given cluster. Here, black squares represent A. m. carnica subspecies while white squares represent A. m.
macedonica subspecies. Inset A figure at top right corner shows location of the study area in Europe. Dotted line indicates the disputed political
border between Serbia and Kosovo. B. Interpolated gradient map showing the cline for the probability of A. m. carnica (blue colour) from high
in the north to low in the south and vice-versa for A. m. macedonica (dark red). A few samples in the north-west and elsewhere resemble A. m.
macedonica bees and vice-versa which may be due to human activity. Black points denote sampling sites and samples used in genetic analyses.
Points with circles denote samples which where morphometrically analysed. The dotted crosshatch area shows mountainous region revealing
that the native extent of A. m. carnica and A. m. macedonica subspecies approximately follow the natural topographical boundaries in this
region. Inset B figure at top right corner shows the number of samples (n = 122) assigned to A. m. carnica (Car) and A. m. macedonica (Mac)
populations using ‘sPCA’ and ‘Structure’ methods.
310 Nedić et al.

Spatial analysis techniques over the years. The results have not been conclusive.
A total of 122 bees from the study area were spatially analysed. The Ruttner (1988), based on morphometric data was the first to conclusively
Spatial PCA was carried out using Delaunay triangulation as connection point out the presence of A. m. carnica in Yugoslavia and the north-
network. The screeplot() revealed (result not shown) that the first east range of A. m. macedonica up to Yugoslavia. High frequency of
score could be well distinguished from other eigenvalues suggesting yellow banded bees in the Banat region of Serbia led (Grozdanic, 1926)
the possibility of a spatial pattern. A global Monte Carlo test (10000 to define a new subspecies ‘Apis mellifera banatica’. However, Ruttner,
iterations) indicated significant (P < 0.05) global spatial structure. The based on a wider comparison concluded that these bees are A. m.
global spatial structure as the name suggests is aimed to reveal carnica, but show a higher frequency of yellow colouration. Morpho-
population structure on the full spatial scale of the data. Clustering is metric and karyotypic studies have reported possible regional ecotypes
based on similarities between individuals. Each global score reveals and distinct differences between the northern and southern regions of
only two populations. A combination of positive scores can be used to the country. Yet, these studies have failed to assign these ecotypes to
elucidate multiple populations. In this dataset, no obvious clusters specific subspecies. Mitochondrial DNA has been used in a few studies
could be identified above the first global score. The local Monte Carlo with results suggesting a possible presence of A. m. macedonica
test (10000 iterations) showed no significant (P = 0.624) local structure. population in Serbia. These mtDNA studies (Kozmus et al., 2007; Nedić
Local scores are based on dissimilarities between individuals and more et al., 2009; Stevanović et al., 2010; Muñoz et al., 2012) have
suited to detect localised spatial structures confined to smaller spatial demonstrated the presence of mitotypes typical of A. m. carnica in the
areas. northern and western part of Serbia and mitotypes typical of A. m.
The eigenvectors of the first global score were plotted to the macedonica in the southern and eastern regions. Yet, the extent of
geographical coordinates (Fig. 4A.). Positive values are shown as black A. m. macedonica inside Serbia remained unclear.
squares while negative values are shown as white squares with black Comparing the morphometric and genetic PCA results, we find
outlines. As seen in the figure, the white squares dominate the southern pronounced differences. Based on the morphometric PCA, the Serbian
region of the study area, where the influence of A. m. macedonica is bees show some resemblance to A. m. carnica reference bees, and in
strongest. The black squares dominate the northern region of Serbia general are quite different from A. m. macedonica bees (Fig. 2A.). In
connected to areas of A. m. carnica origin. The majority of samples in Table 2, we have listed the means and standard deviations for Serbian
the north-west tend to be population one (A. m. carnica) while most bees, split into A. m. carnica and A. m. macedonica bees based on the
samples in the south-west belong to population two (A. m. macedonica). sPCA analysis, compared to three different reference sources. There is
There are several outliers, which could be due to transhumance of substantial difference between the measurements of the reference
honey bees. Monte-Carlo Mantel test (100,000 iterations) showed material, either reflecting differences in the applied methodology or
significant correlation (P < 0.05) between geographic and genetic uncertainty of the purity of the reference material. Therefore, direct
distances. comparison between datasets is difficult. The PCA based on genetic
The predicted probabilities (sPCA) at the spatial locations were data reflects the hybrid nature of the Serbian bees with most of the
interpolated to better visualise the cline and overall spatial trend (Fig. Serbian samples having an intermediate position between the A. m.
4B.). This map shows a general population cline from north-west to carnica and A. m. macedonica reference samples (Fig. 2B.). We
south-east of the study area. ‘Structure’ assignment probabilities hypothesise that the discrepancy between the genetic and morphometric
2
correlated significantly with latitude (R = 0.43, P < 0.0005), longitude analysis may result from extensive breeding programme of Serbian
(R2 = 0.44, P < 0.0005) and geographical index (R2 = 0.52, P < 0.0005). bees based on morphometric resemblance to A. m. carnica. The use
The low Spearman’s R2 value is due to the presence of several outliers. of morphometry as a selection criterion can result in bees converging
The maps were overlaid with political boundaries to show actual toward A. m. carnica appearance, despite different genetic background,
geographic locations. This shows that the study area is situated at the as demonstrated previously in Bavaria, Germany (Moritz, 1991). In
boundary of these two populations. ‘Structure’ assignment probabilities addition to the effect of breeding, the relatively poor resolution of the
when plotted out to geographical coordinates were also able to reveal morphometric method seen in this study may also be due to low
the north-east to south-west cline of genetic differentiation (results not precision measurement and insufficient replicates (Meixner et al., 2013).
shown). Nevertheless, the subset of samples used for morphometrics was
representative of the full sample set and was not genetically or spatially
skewed (Fig. 4B.). A subset of bees were analysed since the bees were
obtained from different sources.
Discussion The combined analyses including deterministic assignment, Bayesian
The geographical position of the Serbian territory has made it difficult assignment, PCA and spatial PCA, demonstrate that the Serbian honey
to identify the bee populations, despite several studies using various bees are composed of two discrete populations and their hybrids. Based
Admixture in Serbian honey bees
on our reference populations, we can infer that a population of A. m.
carnica dominates in the north-west of Serbia, while a population of
A. m. macedonica exists in the south-east of the country. The Bayesian
methods implemented in GeneClass estimated the presence of A. m.
macedonica (49%-70%) over A. m. carnica (30%-36%). ‘Structure’
and sPCA, on the other hand, estimated 56%-58% of bees to resemble
A. m. carnica while 42%-44% resemble A. m. macedonica. Based on
sPCA, Structure correlations and Mantel test, the spatial distribution of
these two populations is clearly along a north-east to south-west cline.
In other words, the honey bee population in our study is situated along
part of the boundary between these two subspecies. Thus our results
support the hypothesis of Ruttner (1988) on the geographic location
of the boundaries of A. m. macedonica and A. m. carnica subspecies.
The midpoint of the cline is not situated in the geographic centre of the
Serbia but rather more to the South (Fig. 4B.). The cline is concomitant
with the mountain ranges (Dinaric Alps and the Balkan mountains) that CORNUET, J-M; PIRY, S; LUIKART, G; ESTOUP, A; SOLIGNAC, M (1999)
cross through the southern region of Serbia coinciding with the currently
disputed political boundary between Serbia and Kosovo. This shows
that, despite the transhumance of bees, we are able to detect the
native distribution and the natural boundary of A. m. carnica and A. m.
macedonica populations in this region.
The cline is concomitant with the mountain ranges (Dinaric Alps
and the Balkan mountains) that cross through the southern region of
Serbia coinciding with the currently disputed political boundary between
Serbia and Kosovo. To unravel the influence from transhumance of
bees in relation to the original distributions between the two subspecies
is difficult to quantify, given the lack of documentation of hive movement. DRAY, S; DUFOUR, A B (2007) The ade4 package: implementing the
The current political situation in the area may decrease the trade in bees,
but it will take time before an effect of this on the population structure
can be demonstrated. Our results show that, despite the transhumance ESTOUP, A; GARNERY, L; SOLIGNAC, M; CORNUET, J M (1995)
of bees, we are able to detect the native distribution and the natural
boundary of A. m. carnica and A. m. macedonica populations in this
region. On the Iberian peninsula, where large scale movement of honey
bees is well documented, there still exists clear north-south and east-
west clines of genetic differentiation (Chávez-Galarza et al., 2013).
Acknowledgements
This study was funded by project numbers III 46008, III 46009, III
43001 from the Ministry of Education, Science and Technological
Development of the Republic of Serbia. We wish to thank the bee-
keepers who contributed bee material towards this study. We wish to
thank Bente Flügel Majgren for genotyping of samples, and Lene
Hasmark Andersen for assistance with DNA extractions. We wish to
thank Aleksandar Uzunov, Aleš Gregorc, Evgeniya Ivanova and Irena
Dzimrevska for contributing samples. We wish to thank Stefan Fuchs
and Marina Meixner for the morphometric reference data from the
Institut für Bienenkunde, Oberursel, Germany. We wish to thank all
311
beekeepers who contributed samples. Finally, we wish to thank the
referees for their valuable comments.
References
AKIMA, H; GEBHARDT, A; PETZOLDT, T; MAECHLER, M (2013) Akima:
interpolation of irregularly spaced data [Online]. Available:
http://CRAN.R-project.org/package=akima
CHÁVEZ-GALARZA, J; HENRIQUES, D; JOHNSTON, J S; AZEVEDO, J C;
PATTON, J C; MUÑOZ, I; DE LA RÚA, P; PINTO, M A (2013)
Signatures of selection in the Iberian honey bee (Apis mellifera
iberiensis) revealed by a genome scan analysis of single nucleotide
polymorphisms. Molecular Ecology 22(23): 5890-5907.
http://dx.doi.org/10.1111/mec.12537
New methods employing multilocus genotypes to select or exclude
populations as origins of individuals. Genetics 153(4): 1989-2000.
DE LA RÚA, P; GALIAN, J; SERRANO, J; MORITZ, R F A (2001a) Genetic
structure and distinctness of Apis mellifera L. populations from the
Canary Islands. Molecular Ecology 10(7): 1733-1742.
http://dx.doi.org/10.1046/j.1365-294X.2001.01303.x
DE LA RÚA, P; GALIAN, J; SERRANO, J; MORITZ, R F A (2001b)
Molecular characterization and population structure of the honey
bees from the Balearic islands (Spain). Apidologie 32(5): 417-427.
http://dx.doi.org/10.1051/apido:2001141
duality diagram for ecologists. Journal of Statistical Software 22(4):
1-20.
Microsatellite variation in honey bee (Apis mellifera L.) populations
- hierarchical genetic structure and test of the infinite allele and
stepwise mutation models. Genetics 140(2): 679-695.
ESTOUP, A; SOLIGNAC, M; HARRY, M; CORNUET, J-M (1993)
Characterization of (Gt)n and (Ct)n microsatellites in 2 insect species
- Apis mellifera and Bombus terrestris. Nucleic Acids Research 21(6):
1427-1431. http://dx.doi.org/10.1093/nar/21.6.1427
EVANNO, G; REGNAUT, S; GOUDET, J (2005) Detecting the number
of clusters of individuals using the software STRUCTURE: a
simulation study. Molecular Ecology 14(8): 2611-2620.
http://dx.doi.org/10.1111/j.1365-294X.2005.02553.x
FRANCK, P; GARNERY, L; CELEBRANO, G; SOLIGNAC, M; CORNUET, J-M
(2000a) Hybrid origins of honey bees from Italy (Apis mellifera
ligustica) and Sicily (A. m. sicula). Molecular Ecology 9(7): 907-921.
http://dx.doi.org/10.1046/j.1365-294x.2000.00945.x
FRANCK, P; GARNERY, L; LOISEAU, A; OLDROYD, B P; HEPBURN, H R;
SOLIGNAC, M; CORNUET, J-M (2001) Genetic diversity of the
honey bee in Africa: microsatellite and mitochondrial data. Heredity
86: 420-430. http://dx.doi.org/10.1046/j.1365-2540.2001.00842.x
312
FRANCK, P; GARNERY, L; SOLIGNAC, M; CORNUET, J-M (2000b)
Molecular confirmation of a fourth lineage in honey bees from the
Near East. Apidologie 31(2): 167-180.
http://dx.doi.org/10.1051/apido:2000114
GADM V1.0 (2011) Global Administrative Areas [Online]. Available at:
http://www.gadm.org/
GARNERY, L; CORNUET, J-M; SOLIGNAC, M (1992) Evolutionary history
of the honey bee Apis mellifera inferred from mitochondrial DNA
analysis. Molecular Ecology 1(3): 145-154.
http://dx.doi.org/10.1111/j.1365-294X.1992.tb00170.x
GARNERY, L; FRANCK, P; BAUDRY, E; VAUTRIN, D; CORNUET, J-M;
SOLIGNAC, M (1998) Genetic diversity of the west European honey NEDIĆ, N; JEVTIĆ, G; JEŽ, G; ANDJELKOVIĆ, B; MILOSAVLJEVIĆ, S;
bee (Apis mellifera mellifera and A. m. iberica). II. Microsatellite loci.
Genetics Selection Evolution 30: S49-S74.
http://dx.doi.org/10.1051/gse:19980703
GROZDANIC, S D (1926) “Žuta” banatska pčela. Glasnik Entomološkog NEDIĆ, N; STANISAVLJEVIĆ, L J; MLADENOVIĆ, M; STANISAVLJEVIĆ, J
Društva, 1: 1-16.
JENSEN, A B; PALMER, K A; BOOMSMA, J J; PEDERSEN, B V (2005)
Varying degrees of Apis mellifera ligustica introgression in protected
populations of the black honey bee, Apis mellifera mellifera, in
northwest Europe. Molecular Ecology 14(1): 93-106.
http://dx.doi.org/10.1111/j.1365-294X.2004.02399.x
JEVTIĆ, G; MLADENOVIĆ, M; LUGIĆ, Z; SOKOLOVIĆ, D (2007)
Morphological and production characteristics of carniolan honey
bee (Apis mellifera carnica Poll.) from different parts of Serbia.
Biotechnology in Animal Husbandry 23(5-6-1): 609-617.
http://dx.doi.org/10.2298/bah0701609j
JOMBART, T (2008) Adegenet: an R package for the multivariate
analysis of genetic markers. Bioinformatics 24(11): 1403-1405.
http://dx.doi.org/10.1093/bioinformatics/btn129
JOMBART, T; DEVILLARD, S; DUFOUR, A B; PONTIER, D (2008)
Revealing cryptic spatial patterns in genetic variability by a new
multivariate method. Heredity 101(1): 92-103.
http://dx.doi.org/10.1038/hdy.2008.34
KANDEMIR, I; KENCE, M; SHEPPARD, W S; KENCE, A (2006)
Mitochondrial DNA variation in honey bee (Apis mellifera L.)
populations from Turkey. Journal of Apicultural Research 45(1):
33-38. http://dx.doi.org/10.3896/ibra.1.45.1.08
KOZMUS, P; JEVROSIMA, S; STANIMIROVIC, Z; STOJIC, V; KULISIC, Z;
MEGLIC, V (2007) Analysis of mitochondrial DNA in honey bees
(Apis mellifera) from Serbia. Acta Veterinaria 57(5-6): 465-476.
http://dx.doi.org/10.2298/Avb0706465k
MEIXNER, M D; PINTO, M A; BOUGA, M; KRYGER, P; IVANOVA, E;
FUCHS, S (2013) Standard methods for characterising subspecies
and ecotypes of Apis mellifera. In V Dietemann; J D Ellis; P Neumann
(Eds) The COLOSS BEEBOOK, Volume I: standard methods for
Apis mellifera research. Journal of Apicultural Research 52(4):
http://dx.doi.org/10.3896/IBRA.1.52.4.05
Nedić et al.
MORITZ, R F A (1991) Limitations of biometric control on pure race
breeding in Apis mellifera. Journal of Apicultural Research 30(2):
54-59.
MUÑOZ, I; DALL'OLIO, R; LODESANI, M; DE LA RUA, P (2009)
Population genetic structure of coastal Croatian honey bees (Apis
mellifera carnica). Apidologie 40(6): 617-626.
http://dx.doi.org/10.1051/apido/2009041
MUÑOZ, I; STEVANOVIĆ, J; STANIMIROVIĆ, Z; DE LA RÚA, P (2012)
Genetic variation of Apis mellifera from Serbia inferred from
mitochondrial analysis. Journal of Apicultural Science 56(1): 59-69.
http://dx.doi.org/10.2478/v10289-012-0007-9
KOSTIĆ, M (2011) Forewing differentiation of the honey bees from
Serbia. Biotechnology in Animal Husbandry 27(3): 1387-1394.
http://dx.doi.org/10.2298/bah1103387n
(2009) Molecular characterization of the honey bee Apis mellifera
carnica in Serbia. Archives of Biological Science 61(4): 587-598.
http://dx.doi.org/10.2298/abs0904587n
NEI, M (1972) Genetic distance between populations. American
Naturalist 106(949): 283-292. http://dx.doi.org/10.2307/2459777
OZDIL, F; YILDIZ, M A; HALL, H G (2009) Molecular characterization
of Turkish honey bee populations (Apis mellifera) inferred from
mitochondrial DNA RFLP and sequence results. Apidologie 40(5):
570-576. http://dx.doi.org/10.1051/apido/2009032
PEAKALL, R; SMOUSE, P E (2006) GENALEX 6: genetic analysis in
Excel. Population genetic software for teaching and research.
Molecular Ecology Notes 6(1): 288-295.
http://dx.doi.org/10.1111/j.1471-8286.2005.01155.x
PEAKALL, R; SMOUSE, P E (2012) GenAlEx 6.5: genetic analysis in
Excel. Population genetic software for teaching and research - an
update. Bioinformatics 28(19): 2537-9.
http://dx.doi.org/10.1093/bioinformatics/bts460
PIRY, S; ALAPETITE, A; CORNUET, J-M; PAETKAU, D; BAUDOUIN, L;
ESTOUP, A (2004) GENECLASS 2: a software for genetic assignment
and first-generation migrant detection. Journal of Heredity 95(6):
536-9. http://dx.doi.org/10.1093/jhered/esh074
PRITCHARD, J K; STEPHENS, M; DONNELLY, P (2000) Inference of
population structure using multilocus genotype data. Genetics 155
(2): 945-959.
R DEVELOPMENT CORE TEAM (2011) R: A language and environment
for statistical computing. R Foundation for Statistical Computing
[Online]. Vienna. Available at: http://www.R-project.org/
RANNALA, B; MOUNTAIN, J L (1997) Detecting immigration by using
multilocus genotypes. Proceedings of the National Academy of
Sciences 94(17): 9197-9201.
http://dx.doi.org/10.1073/pnas.94.17.9197
Admixture in Serbian honey bees
RUTTNER, F (1988) Biogeography and taxonomy of honey bees.
Springer-Verlag; Berlin, Germany.
SCHNUTE, J T; BOERS, N M; HAIGH, R; COUTURE-BEIL, A (2010)
PBSmapping: Mapping fisheries data and spatial analysis tools
[Online]. Available: http://CRAN.R-project.org/package=PBSmapping
SOLIGNAC, M; VAUTRIN, D; LOISEAU, A; MOUGEL, F; BAUDRY, E;
ESTOUP, A; GARNERY, L; HABERL, M; CORNUET, J-M (2003) Five
hundred and fifty microsatellite markers for the study of the honey
bee (Apis mellifera L.) genome. Molecular Ecology Notes 3(2): 307 SUŠNIK, S; KOZMUS, P; POKLUKAR, J; MEGLIČ, V (2004) Molecular
-311. http://dx.doi.org/10.1046/j.1471-8286.2003.00436.x
313
STANIMIROVIĆ, Z; STEVANOVIĆ, J; ANDJELKOVIĆ, M (2005)
Chromosomal diversity in Apis mellifera carnica from Serbia.
Apidologie 36(1): 31-42. http://dx.doi.org/10.1051/apido:2004067
STEVANOVIĆ, J; STANIMIROVIĆ, Z; RADAKOVIĆ, M; KOVAČEVIĆ, S R
(2010) Biogeographic study of the honey bee (Apis mellifera L.)
from Serbia, Bosnia and Herzegovina and Republic of Macedonia
based on mitochondrial DNA analyses. Russian Journal of Genetics
46(5): 603-609. http://dx.doi.org/10.1134/S102279541005014
characterisation of indigenous Apis mellifera carnica in Slovenia.
Apidologie 35(6): 623-636. http://dx.doi.org/10.1051/apido:2004061
ŽIVANOVIĆ (1893) Srpski pčelar. Knjižara Luke Jocića Novi Sad, 298.