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Apidologie 37 (2006) 547–555 547
c
INRA/DIB-AGIB/EDP Sciences, 2006
DOI: 10.1051/apido:2006029 Original article
Genetic characterization of honey bee (Apis mellifera cypria)
populations in northern Cyprus*
Irfan Ka,b,MarinaD.Mb,AycaOa,WalterS.Sb
aDepartment of Biology, Zonguldak Karaelmas University, Incivez 67100 Zonguldak, Turkey
bDepartment of Entomology, Washington State University, Pullman 99164-6382 WA, USA
Received 15 April 2005 – revised 16 December 2005 – accepted 22 December 2005
Abstract – The variability of the honey bees of northern Cyprus was investigated using morphometric,
mitochondrial DNA (mtDNA) and microsatellite analyses. Morphometric analysis resulted in a clear classi-
fication of the Cyprus bees as Apis mellifera cypria, but showed the influence of imported A. m. anatoliaca
in some areas. In eastern Cyprus, several samples showed a similarity to A. m. meda, possibly corroborating
a published report of similarity between A. m. cypria and Mediterranean A. m. meda. However, the importa-
tion of A. m. meda into Cyprus could not be ruled out. MtDNA analysis showed that most Cyprian samples
belonged to the mitochondrial C lineage, but a small proportion of samples displayed restriction patterns
typical for the mitochondrial O lineage. Population differentiation between Cyprus and honey bees from ad-
jacent mainland populations was low, but the northwestern Cyprus population appeared to be introgressed
to a larger extent by alleles from the Turkish mainland.
Apis mellifera cypria /mtDNA /microsatellites /morphometry /Cyprus
1. INTRODUCTION
Traditionally, subspecific classification and
phylogeographic inferences in Apis mellifera
L. have been based on the variation of behav-
ior and morphology within the endemic range
of the species. Using morphometric analy-
ses, Ruttner (1988, 1992) hypothesized the ex-
istence of four evolutionary lineages within
the species: M in northern and western Eu-
rope, A in Africa, C in southeastern Europe,
and O in western Asia. Subsequent studies,
based on variation of mitochondrial DNA,
confirmed Ruttner’s hypotheses about the phy-
logeographic structure of Apis mellifera to a
large extent (Garnery et al., 1992, 1993; Arias
and Sheppard, 1996; Franck et al., 2000a).
The most widely used marker in these stud-
ies was variation in the intergenic region be-
tween the COI and the COII gene in Apis mel-
Corresponding author: I. Kandemir,
ikandemir@gmail.com
* Manuscript editor: Stefan Fuchs
lifera mtDNA, as determined by sequencing
or restriction analysis (Garnery et al., 1992,
1993, 1998; Franck et al., 1998). However, us-
ing these methods, the morphological C and
O branches were undistinguishable and were
subsumed into a single mitochondrial lineage
(C).
Recently, Franck et al. (2000a) reported the
existence of a previously unknown mtDNA re-
striction enzyme pattern in honey bees sam-
pled from Lebanon and inferred the existence
of a fourth mitochondrial lineage of honey
bees (‘mitochondrial O’). This lineage may
be analogous to the mtDNA lineage hypothe-
sized based on restriction enzyme data (Palmer
et al., 2000) and mitochondrial ND2 gene se-
quences (Arias and Sheppard, 1996). The dis-
tribution of the mitochondrial O lineage re-
mains unknown, but may extend from Syria to
Egypt (Arias and Sheppard, 1996).
The island of Cyprus is situated at the east-
ern end of the Mediterranean Sea, south of
Turkey (75 km), west of Syria and Lebanon
Article published by EDP Sciences and available at http://www.edpsciences.org/apidoor http://dx.doi.org/10.1051/apido:2006029
548 I. Kandemir et al.
Figure 1. Map indicating sampling lo-
cations in Cyprus.
Table I . Sampling locations, geographical positions, and number of colonies sampled for this study.
Location Geographical position # Colonies sampled year
1-Omorfo 35o12’N 32o59’E 5 2000
2-Lefke 35o06’N 32o51’E 4 2000
3-Gaziveren 35o11’N 33o01’E 6 2000
4-Kalecik 35o20’N 34o00’E 3 2000
5-Iskele 35o16’N 33o54’E 3 2000
6-Ardahan 35o21’N 33o52’E 47 2000, 2002
7-Yedikonuk 35o24’N 34o01’E 10 2002
8-Taslica 35o23’N 34o04’E 5 2002
9-Kantara 35o23’N 33o53’E 3 2002
10-Mersinlik 35o24’N 33o55’E 4 2002
11-Kaplica 35o23’N 33o54’E 5 2002
12-Girne 35o19’N 33o19’E 6 2004
(105 km) and north of Egypt (380 km). The
honey bees of Cyprus were described as a
separate subspecies, A. m. cypria, by Pollman
(1879) and shown by Ruttner (1988) to be-
long to the morphological O lineage of Apis
mellifera. While other island populations and
subspecies of honey bees in the Mediterranean
have received more scientific interest (Crete:
Ruttner, 1980; Sicily: Badino et al., 1985;
Sinacori et al., 1998; Franck et al., 2000b;
Malta: Sheppard et al., 1997; Balearics: De la
Rúa et al., 2001, 2003), very little is known
about the honey bee of Cyprus. The geo-
graphic location of Cyprus positions A. m.
cypria in close proximity to subspecies to both
the mitochondrial C and O lineages and the
geographic region of transition between them.
In this paper we report the results of an ex-
tensive morphometric and genetic analysis of
the honey bees of Cyprus and compare their
morphometric and genetic variability to that of
neighboring subspecies.
2. MATERIALS AND METHODS
2.1. Collection of bee samples
A total of 101 colonies were sampled from 12
locations in northern Cyprus in the years 2000 (40),
2002 (55), and 2004 (6) (Fig. 1, Tab. I). Samples
were stored in 90% ethanol (2000, 2004) or in dry
ice (2002).
2.2. Morphometric analysis
A total of 18 colonies (3 from each location of
the 2000 collection) were subjected to morphome-
tric analysis. Between 11–15 worker bees per sam-
ple were dissected and measured for 39 morphome-
tric characters according to methods of Ruttner et al.
Genetic characterization of Apis mellifera cypria 549
(1978) and Ruttner (1988, 1992). Characters of pi-
losity and pigmentation were assessed with a stere-
omicroscope and an ocular micrometer. All other
characters were measured with a CCD camera com-
bined with a morphometric measurement program
(Bee2, c
Meixner, 2004). Reference data of honey
bee subspecies of the eastern Mediterranean region
were obtained from the database of the Institut für
Bienenkunde, Oberursel. These included A. m. car-
nica (20 samples), A. m. macedonica (10), A. m. ce-
cropia (10), A. m. anatoliaca (13), A. m. syriaca (9),
A. m. adami (5), and A. m. meda (25). Reference
data for A. m. meda came from samples of A. m.
meda collected in Turkey and Syria (Ruttner, 1988;
Ftayeh et al., 1994). Data were subjected to prin-
cipal component analysis and discriminant analysis
using the SPSS 12.0.1 statistical software.
2.3. Restriction and sequence analysis
of mitochondrial DNA
Total nucleic acids of one bee per sample were
isolated with a modified phenol-chloroform ex-
traction (Arias and Sheppard, 1996) or a modi-
fied CTAB extraction protocol (Doyle and Doyle,
1987). A mitochondrial fragment containing the
intergenic region between the tRNAleu gene and
the second subunit of the cytochrome oxidase
gene was amplified using the primer pair E2-H2
(Garnery et al., 1993): E2: 5’-GGC AGA ATA
AGT GCA TTG-3’, H2: 5’-CAA TAT CAT TGA
TGA CC-3’. The 25 µL reaction mix consisted
of 0.8 µM of each primer, 0.2 mM of PCR Nu-
cleotide mix (Boehringer Mannheim), 1.5 mM
MgCl2(Promega), 1X Reaction Buffer (Promega),
1UTaq Polymerase (Promega) and 1 µLoftem-
plate. The amplification cycle consisted of an initial
denaturation step of 2 min at 92 ◦C,followedby35
cycles of 30 s at 92 ◦C, 30 s at 47 ◦Cand2minat
63 ◦C, followed by a final extension step of 10 min
at 63 ◦C. Five µL of the PCR products were run
on a 1.5% agarose gel, stained with ethidium bro-
mide and photographed under UV illumination. A
20 µL aliquot of each positive reaction was digested
with the restriction enzyme DraIat37◦C overnight.
Restriction fragments were separated on 10% poly-
acrylamide gels, stained with ethidium bromide and
photographed under UV illumination.
Among the samples expressing restriction pro-
files of the C and O mitochondrial lineages, we se-
quenced the COI-COII region of one sample and
the NADH dehydrogenase subunit 2 gene of two
samples each, using a cycle sequencing protocol
(Craxton, 1991) and an ABI 377 automated se-
quencer. The ND2 sequences were aligned with
corresponding published sequence data from other
Apis mellifera subspecies (Arias and Sheppard,
1996) using Clustal X (Thompson et al., 1997).
Phylogenetic analyses using both neighbor-joining
and parsimony methods were performed with
MEGA 3.1 (Kumar et al., 2004). Sequences were
deposited in GenBank under the accession numbers
AY618919–AY618921.
2.4. Microsatellite analysis
The samples were analyzed for nine microsatel-
lite loci: A7, A24, A28, A88, A113, B124 (Estoup
et al., 1995), Ap55, Ap66, and Ap81 (Garnery et al.,
unpubl. data). Amplifications were performed in
10 µL reactions containing 1 µL extracted DNA, 1X
reaction buffer, 3 mM dNTPs, 0.001 mg BSA, 1–
4 mM of respective primers and 1.5 units Ta q poly-
merase. Microsatellite primers were combined into
two multiplex reactions with optimized concentra-
tions of MgCl2: 1.2 mM for A7, A113, Ap55 and
Ap81; and 1.5 mM for loci A24, A28, A88, Ap66
and B124. The PCR reaction conditions were iden-
tical for all loci and consisted of 7 min at 95 ◦C,
followed by 30 cycles of 95 ◦C(30s),54◦C(30s),
72 ◦C (30 s), and a final 60 min cycle at 72 ◦C. For-
ward primers were fluorescent labeled and amplifi-
cation products were separated on an ABI 3730 au-
tomatic sequencer. The resulting electropherograms
were analyzed using GeneMapper Software (Ap-
plied Biosystems).
For analysis, the microsatellite data were com-
bined with unpublished reference data from popula-
tions in Turkey (n =47), Syria (n =22) and Iran (n =
43). Exact tests for genetic structure and genetic dif-
ferentiation between populations using unbiased es-
timates of Fst were calculated using the Genepop
package version 3.4 (Raymond and Rousset, 1995).
A neighbor-joining tree based on the microsatellite
data and the chord distance of Cavalli-Sforza and
Edwards was constructed using the Phylip program
package (Felsenstein, 2005) with bootstrap values
computed over 2000 replications.
3. RESULTS
3.1. Morphometry
In a principal component analysis based
on three factors describing 38.8%, 11.9% and
550 I. Kandemir et al.
Figure 2. Discriminant analysis con-
taining the samples from Cyprus and
reference samples of subspecies belong-
ing to the morphological O-branch. X-
axis: discriminant function 1, Y-axis:
discriminant function 3. The ellipses of
confidence (75%) for each group are in-
cluded. The confidence ellipse of the
new Cyprus samples was constructed
excluding the three samples not classi-
fied as A. m. cypria.
7.9% of the morphological variation, respec-
tively, the Cyprus samples mainly fell within
the range previously published for A. m. cypria
within the morphological O lineage (Ruttner,
1988). Two samples occupied positions away
from the A. m. cypria cluster and appeared to
be associated with A. m. anatoliaca. No rela-
tionship with A. m. carnica or other subspecies
of the morphological C lineage was observed
(plot not shown). The allocation of our sam-
ples to reference data of A. m. cypria,A. m.
anatoliaca,A. m. meda,orA. m. syriaca was
examined further using discriminant analysis.
In this analysis (Fig. 2), 11 of the samples were
clearly identified as A. m. cypria with proba-
bility scores of P>0.99, while four samples
were assigned to A. m. cypria with scores of
0.85 ≥P≥0.97. Two samples (both from the
same location) were identified as A. m. anato-
liaca, and one sample (from a collection site
in the east of Cyprus) was assigned to A. m.
meda.
3.2. Mitochondrial DNA
Restriction enzyme digestion of the mito-
chondrial fragment containing the intergenic
region with DraI resulted in two different pat-
terns assignable to the C and O mitochondrial
lineages as described by Garnery et al. (1993)
and Franck et al. (2000a). The majority (99 of
101) of our samples displayed the C2 mito-
chondrial haplotype previously reported from
Italy, Greece and Iran (Garnery et al., 1993),
and Turkey (Kandemir et al., 2006). Two sam-
ples from the eastern part of Cyprus displayed
the O1b haplotype known to occur in honey
bees of Lebanon (Franck et al., 2000a) and the
western part of Syria (Meixner et al., unpubl.
data).
Inclusion of mitochondrial ND2 sequence
data from C2 or O1b haplotypes in the phy-
logenetic analyses of subspecies consistently
clustered the C2 sample with subspecies from
the C lineage branch. The O1b sample clus-
tered with the bees sampled from Egypt
and Syria, previously hypothesized to form
a fourth mitochondrial lineage (Arias and
Sheppard, 1996) (tree not shown).
3.3. Microsatellite analysis
Heterozygosity estimates of microsatellite
loci in the Cyprus populations ranged from
0.286 (Ap81) to 0.857 (A113) with a mean
across loci of 0.553 ±0.26 for northwest-
ern Cyprus and 0.554 ±0.22 for northeast-
ern Cyprus. All loci were in Hardy-Weinberg
Genetic characterization of Apis mellifera cypria 551
equilibrium with respect to the populations
studied. The number of alleles, the allele size
range in bp and the expected and observed het-
erozygosities (H. exp. and H. obs.) and the al-
lele frequencies for each individual locus are
presented in Table II. The results of pairwise
population comparisons using multilocus F-
statistics between the northwestern and north-
eastern Cyprus populations and the reference
populations were low and ranged between
0.003 (northwestern Cyprus, Turkey) and
0.081 (northeastern Cyprus, Syria) (Tab. III).
The populations of northwestern and north-
eastern Cyprus showed significant differences
in their microsatellite variability (P<0.001,
Fisher exact test). When compared to sur-
rounding mainland populations, the allelic dis-
tribution of the northwestern Cyprus popula-
tion was not significantly different from the
population of Turkey, but different from Syria
and Iran (P<0.001). The bees of eastern
Cyprus differed significantly from all adja-
cent mainland honey bee populations (Turkey,
Syria, Iran) (P<0.001).
A neighbor-joining tree based on the
Cavalli-Sforza and Edwards chord distance re-
sulted in low resolution between the popu-
lations from Iran and the branch combining
the other groups from the Near East. Within
this branch, the honey bee populations from
Cyprus were incorporated into a subcluster
with Syria (Fig. 3).
4. DISCUSSION
Several different subspecies of honey bees
belonging to two different evolutionary lin-
eages (C and O) come together in the eastern
Mediterranean and the Near East. Although
these two evolutionary lineages are distin-
guishable by morphological methods, the
delineation based on restriction analysis of mi-
tochondrial DNA is incongruent and seem-
ingly confusing. The honey bee subspecies
of the entire Near East, including Turkey,
morphologically belong to the O evolution-
ary lineage sensu Ruttner (1988). However,
in the southern portion of this range a divi-
sion between mitochondrial lineages C (sensu
Garnery et al., 1993) and O (sensu Franck
et al., 2000a) occurs further south and east.
Thus, C mitochondrial haplotypes occur in
many subspecies belonging to the O morpho-
logical lineage sensu Ruttner, including most
of the honey bees of Turkey (Kandemir et al.,
2006) and those that occur east into Iran and
Central Asia at the eastern edge of the Apis
mellifera range (unpublished data; Sheppard
and Meixner, 2003). Further south, extending
from southern Turkey (Kandemir et al., 2006)
through Lebanon (Franck et al., 2000a), Syria
and Egypt (Arias and Sheppard, 1996; unpubl.
data), honey bee populations are characterized
by haplotypes belonging to the (perhaps unfor-
tunately named) mitochondrial lineage O (as
described and named by Franck et al., 2000a).
Our results show that the contemporary
honey bee population of (northern) Cyprus re-
tain A. m. cypria characteristics as described
by Ruttner (1988), although in some areas
the influence of other subspecies, especially
A. m. anatoliaca, can be detected. Beekeepers
in Cyprus predominantly use primitive hives,
but the use of modern equipment, migratory
beekeeping and commercial pollination prac-
tices are increasing (Kandemir, 2003). The two
Cyprian samples that were morphometrically
classified as A. m. anatoliaca and the one with
an intermediate score between A. m. anatoli-
aca and A. m. cypria all came from modern
beekeeping operations involved in citrus pol-
lination (located in northwestern Cyprus) and
may reflect past or recent importation of A.
m. anatoliaca queens. In contrast, three other
samples with intermediate scores showed an
affinity to A. m. meda and, together with the
one sample classified as A. m. meda, originated
from the eastern part of the island where tra-
ditional beekeeping in trunk hives is still pre-
dominant.
Mitochondrial analysis predominantly
placed our Cyprus collection into the mito-
chondrial C lineage, but also showed a small
proportion of restriction profiles characteristic
for the mitochondrial O lineage. Whether
this observation reflects a mixed ancestry
of the Cyprus population or a more recent
introduction of honey bees from the eastern
shore of the Mediterranean is unknown. While
O mitochondrial lineage haplotypes might be
a remnant of the Pleistocene fauna of Cyprus
552 I. Kandemir et al.
Table II. Genetic parameters: sample size, allele size range, number of alleles, expected heterozygosity (H. exp.), expected heterozygosity unbiased estimate (H.
n.b.), and observed heterozygosity (H. obs.) (Nei, 1978) of nine microsatellite loci for northeast and northwest Cyprus. The allele frequencies for each locus are
available in Appendix of the online version.
Regions in
northern
Cyprus
Genetic Parameters Locus
A7 A24 A28 A88 Ap66 B124 A113 Ap55 Ap81
Northwest
Sample size 14 20 17 17 16 17 14 14 14
Allele size range (bp) 87–155 94–106 127–139 130–148 85–135 212–230 216–237 153–177 135–139
Numberofalleles1454787963
H exp. 0.9158 0.6592 0.3460 0.6107 0.7 0.7284 0.8393 0.6224 0.2526
H n.b. 0.9497 0.6791 0.3565 0.6292 0.722 0.7504 0.8704 0.6455 0.2619
H obs. 0.7143 0.6471 0.4118 0.6471 0.563 0.7647 0.8571 0.6429 0.2857
Northeast
Sample size 69 74 72 74 71 75 58 54 64
Allele size range (bp) 78–159 94–106 101–139 133–145 93–137 218–238 210–237 171–185 125–145
Number of alleles 29 5 9 6 7 8 13 7 9
H exp. 0.8951 0.7218 0.3440 0.6179 0.566 0.5189 0.8353 0.5485 0.2834
H n.b. 0.9016 0.7267 0.3464 0.6221 0.570 0.5224 0.8426 0.5537 0.2857
H obs. 0.7826 0.7432 0.3750 0.7838 0.549 0.5733 0.7414 0.5185 0.1406
Genetic characterization of Apis mellifera cypria 553
Table III. Fst results from Genepop.
Northwest Northeast
Cyprus Cyprus
Northeast Cyprus 0.018
Iran 0.021 0.048
Turkey 0.003 0.050
Syria 0.069 0.081
Figure 3. Neighbor-joining tree based on the
Cavalli-Sforza and Edwards chord distance be-
tween populations (based on nine microsatellite
loci). Bootstrap values are based on 2000 replica-
tions.
that contained African elements such as dwarf
hippos and elephants (Schüle, 1993), it is more
likely that the presence of these O lineage
colonies in Cyprus resulted from more recent
human-mediated introductions. For example,
it is known that extensive importations of
honey bee queens were made from present
day Syria and Lebanon to Cyprus in the 19th
century (Strange, 2001).
In contrast to the high differentiation
observed using mitochondrial markers, mi-
crosatellite analysis indicated a relatively low
level of differentiation among the Near East-
ern populations studied, irrespective of their
assignment to mitochondrial lineages C or O.
While overall Fst values between Cyprus and
all reference populations were low, the honey
bee population of northwestern Cyprus was
introgressed to a larger extent by microsatel-
lite alleles from Turkey, suggesting the role
of queen importation from the Turkish main-
land. Thus, while our results confirm the dis-
tinctness of A. m. cypria as island subspecies
of Cyprus, they also show that importation of
bees from adjacent mainland areas may be-
come a threat to its conservation in the future.
ACKNOWLEDGEMENTS
This project was partially supported by USDA-
IFAFS grant 2001-52103-11417 to WSS, and ZKU-
2003-13-06-04, TUBITAK-TBAG 2403 grants
and TUBITAK-BAYG 2219 scholarship to IK.
We gratefully acknowledge the Institut für Bi-
enenkunde in Oberursel, Germany, for use of mor-
phometric reference data. We thank Stefan Fuchs,
Lionel Garnery and three anonymous reviewers for
comments on earlier versions of the manuscript.
Résumé –Caractérisation génétique des popu-
lations d’abeilles domestiques (Apis mellifera cy-
pria)àChypre. L’île méditerranéenne de Chypre
possède sa propre sous-espèce d’abeille domes-
tique, Apis mellifera cypria Pollmann 1879, mais
on connaît peu de choses concernant sa variabilité
génétique et ses relations avec les sous-espèces voi-
sines. De par la position géographique de Chypre,
A. m. cypria avoisine directement les sous-espèces
des lignées mitochondriales C et O. Nous avons
étudié la variabilité des abeilles de Chypre par les
méthodes morphométriques et par des analyses de
l’ADN mitochondrial et des microsatellites. Au to-
tal 101 échantillons ont été prélevés dans 12 loca-
lités du nord de Chypre ; 18 d’entre eux ont fait
l’objet d’une analyse morphométrique. Les mesures
de 39 caractères ont été analysées par les méthodes
de statistique multivariées. Des échantillons de réfé-
rence provenant des régions continentales voisines
ont également été analysés. Un fragment mitochon-
drial contenant la région intergénique entre le gène
ARNtleu et la seconde sous-unité du gène de la
cytochrome oxydase a été amplifié et digéré par
l’enzyme de restriction DraI. Le fragment conte-
nant la région intergénique et un fragment conte-
nant l’ARNt pour l’isoleucine et une partie du Gène
mitochondrial ND2 ont été séquencés pour deux
échantillons représentant chacun des haplotypes ob-
servés dans l’analyse de restriction. Pour l’analyse
de la variabilité des microsatellites neuf locus diffé-
rents ont été amplifiés avec des amorces marquées
par une substance fluorescente et analysés dans un
séquenceur automatique.
L’analyse morphométrique a nettement classé les
abeilles de Chypre comme étant A.m.cypria,mais
a montré également l’influence dans certaines ré-
gions des importations d’A.m.macedonica.Dans
la partie orientale de Chypre plusieurs échantillons
présentaient des similitudes avec A. m. meda et là
non plus l’importation d’A. m. meda à Chypre n’a
pu être écartée. Les analyses de restriction comme
celles de la séquence de l’ADNmt ont montré que
la plupart des échantillons chypriotes appartenaient
à la lignée mitochondriale C, mais une petite pro-
portion d’échantillons présentait des profils de res-
triction typiques de la lignée mitochondriale O.
D’après la fréquence allélique des microsatellites
554 I. Kandemir et al.
la différenciation entre les échantillons chypriotes
et les populations voisines du continent était faible.
La population du nord-ouest de Chypre semble
avoir subi une large introgression par des allèles
venant du continent turque. Ainsi, alors que nos
résultats confirment la particularité d’A. m. cypria
comme sous-espèce de l’île de Chypre, ils montrent
aussi que l’importation d’abeilles du continent voi-
sin peut devenir une menace pour sa conservation à
l’avenir.
Apis mellifera cypria /ADNmt /microsatellite /
morphométrie /Chypre
Zusammenfassung –Genetische Charakterisie-
rung von Populationen der Honigbiene in Nord-
zypern (Apis mellifera cypria). Die Mittelmeerin-
sel Zypern besitzt ihre eigene Unterart der Honig-
biene, Apis mellifera cypria Pollmann 1879, aber
bisher ist noch wenig über ihre genetische Varia-
bilität und ihre Beziehungen zu benachbarten Un-
terarten bekannt. Bedingt durch die geographische
Lage Zyperns ist A. m. cypria sowohl zu Unter-
arten der mitochodrialen C als auch der O Linie
direkt benachbart. Wir untersuchten die Variabili-
tät von A. m. cypria sowohl mit morphometrischen
Methoden als auch mit Analysen der mitochondria-
len DNA und von neun Mikrosatellitenloci. Insge-
samt wurden 101 Proben an 12 Orten in Nordzy-
pern gesammelt, wovon 18 einer morphometrischen
Analyse unterzogen wurden. Es wurden 39 mor-
phometrische Merkmale gemessen und mit multiva-
riaten statistischen Methoden analysiert, wobei Re-
ferenzproben von umliegenden Festlandspopulatio-
nen einbezogen wurden. Ein mitochondriales Frag-
ment, das die nichtkodierende Region zwischen
dem tRNAleu Gen und dem CytochromoxidaseII
Gen enthält, wurde amplifiziert und mit dem Re-
striktionsenzym DraI verdaut. Für jeweils zwei Pro-
ben mit den im Restriktionsversuch beobachteten
verschiedenen Haplotypen wurde das Fragment mit
der nichtkodierenden Region sowie ein Fragment
das die tRNAileu sowie einen Teil des mitochon-
drialen ND2 Gens enthält, sequenziert. Für die Un-
tersuchung der Mikrosatellitenvariabilität wurden
neun verschiedenen Loci mit Fluoreszenzfarbstoff
markierten Primern amplifiziert und in einem auto-
matischen Sequenzierer analysiert.
Die morphomterische Analyse ergab eine eindeuti-
ge Zuordnung der Proben aus Zypern zu A. m. cy-
pria, aber in einigen Gegenden war auch ein Ein-
fluss von importierter A. m. anatoliaca deutlich. Im
östlichen Zypern zeigten einige Proben Ähnlichkeit
mit A. m. meda, aber auch hier kann ein Import von
A. m. meda nach Zypern nicht ausgeschlossen wer-
den. Sowohl Restriktions- als auch Sequenzanalyse
der mtDNA ergaben, dass die meisten zyprischen
Proben zur mitochondrialen C Linie gehören, je-
doch wies ein kleiner Prozentsatz der Proben typi-
sche Haplotypen der O Linie auf. Auf der Basis der
Allelfrequenzen der Mikrosatelliten war die Diffe-
renzierung zwischen den Proben aus Zypern und
den benachbarten Festlandspopulationen gering, je-
doch erschien die Population aus Nordwestzypern
stärker von Allelen des türkischen Festlands beein-
flusst. Damit bestätigen unsere Ergebnisse zwar die
Besonderheit von A. m. cypria als Unterart der In-
sel Zypern, sie zeigen aber auch, dass Importe von
Bienen vom benachbarten Festland eine potentielle
Bedrohung für die Erhaltung dieser Biene darstel-
len.
Apis mellifera cypria /mtDNA /Morphometrie /
Zypern /Mikrosatelliten
REFERENCES
Arias M.C., Sheppard W.S. (1996) Molecular phyloge-
netics of honey bee subspecies (Apis mellifera L.)
inferred from mitochondrial DNA sequence, Mol.
Phyl. Evol. 5, 557–566.
Badino G., Celebrano G., Manino A., Longo S. (1985)
Enzyme polymorphism in the Sicilian honeybee,
Experientia 41, 752–754.
Bee2 Morphometric Software c
1997–2005, A.
Meixner and M.D., Frankfurt, Germany.
Craxton M. (1991) Linear amplification sequencing: A
powerful method for sequencing DNA, Methods
3, 20–24.
De la Rúa P., Galian J., Serrano J., Moritz R.F.A.
(2001) Molecular characterization and population
structure of the honeybees from the Balearic is-
lands (Spain), Apidologie 32, 417–427.
De la Rúa P., Galian J., Serrano J., Moritz R.F.A.
(2003) Genetic structure of Balearıc honeybee
populations based on microsatellite polymor-
phism, Genet. Sel. Evol. 35, 339–350.
Doyle J.J., Doyle L.L. (1987) A rapid DNA isolation
procedure for small quantities of fresh leaf tissue,
Phytochem. Bull. 19, 11–15.
Estoup A., Garnery L., Solignac M., Cornuet J.-M.
(1995) Microsatellite variation in honey bee (Apis
mellifera L.) populations: hierarchical genetic
structure and test of the infinite allele and stepwise
mutation models, Genetics 140, 679–695.
Felsenstein J. (2005) Phylip (Phylogeny Interference
Package) version 3.6, Distributed by the
author, Department of Genome Sciences,
University of Washington, Seattle, USA.
http://evolution.genetics.washington.edu/phylip.html
(accessed on 13 April 2006).
Franck P., Garnery L., Solignac M., Cornuet J.-M.
(1998) The origin of west European subspecies of
honey bees (Apis mellifera): New insights from
microsatellite and mitochondrial data, Evolution
52, 1119–1134.
Genetic characterization of Apis mellifera cypria 555
Franck P., Garnery L., Solignac M., Cornuet J.M.
(2000a) Molecular confirmation of a fourth lin-
eage in honeybees from the Near East, Apidologie
31, 167–180.
Franck P., Garnery L., Celebrano G., Solinac M.,
Cornuet J.M. (2000b) Hybrid origins of the Italian
honeybees, Apis mellifera ligustica and A. m. sic-
ula, Mol. Ecol. 9, 907–923.
Ftayeh A., Meixner M., Fuchs S. (1994)
Morphometrical investigation in Syrian hon-
eybees, Apidologie 25, 396–401.
Garnery L., Cornuet J.-M., Solignac M. (1992)
Evolutionary history of the honey bee Apis mel-
lifera inferred from mitochondrial DNA analysis,
Mol. Ecol. 1, 145–154.
Garnery L., Solignac M., Celebrano G., Cornuet J.-
M. (1993) A simple test using restricted PCR-
amplified mitochondrial DNA to study the ge-
netic structure of Apis mellifera L., Experientia 49,
1016–1021.
Garnery L., Franck P., Baudry E., Vautrin D., Cornuet
J.-M., Solignac M. (1998) Genetic biodiversity of
the west European honey bee (Apis mellifera mel-
lifera and A. m. iberica). I. Mitochondrial DNA,
Genet. Sel. Evol. 30, 31–47.
Kandemir I. (2003) Beekeeping experience and devel-
opments in Turkey and in Northern Cyprus, Am.
Bee J. 143, 464–467.
Kandemir I., Kence M., Sheppard W.S., Kence A.
(2006) Mitochondrial DNA variation in honey bee
(Apis mellifera L.) populations from Turkey, J.
Apic. Res. and Bee World 45, 33–38.
Kumar S., Tamura K., Nei M. (2004) MEGA3:
Integrated software for Molecular Evolutionary
Genetics Analysis and sequence alignment,
Briefings in Bioinformatics 5, 150–163.
Nei M. (1978) Estimation of average heterozygosity
and genetic distance from a small number of indi-
viduals, Genetics 89, 583–590.
Palmer M.R., Smith D.R., Kaftanoglu O. (2000)
Turkish Honey bees: Genetic variation and
evidence for a fourth lineage of Apis mellifera
mtDNA, J. Hered. 91, 42–46.
Pollmann A. (1879) Wert der verschiedenen
Bienenrassen und deren Varietäten, 2nd ed.,
Voigt, Berlin Leipzig (1st ed. with description of
A. m. carnica).
Raymond M., Rousset F. (1995) GENEPOP (Version
1.2): Population genetics software for exact tests
and ecumenicism, J. Hered. 86, 248–249.
Ruttner F. (1980) Apis mellifera adami (n. ssp.), die
kretische Biene, Apidologie 11, 385–400.
Ruttner F. (1988) Biogeography and Taxonomy of
Honey bees, Springer-Verlag, Berlin, Heidelberg.
Ruttner F. (1992) Naturgeschichte der Honigbienen,
Ehrenwirth Verlag, München.
Ruttner F., Tassencourt L., Louveaux J. (1978)
Biometrical-statistical analysis of the geographic
variability of Apis mellifera L. 1. Materials and
Methods, Apidologie 9, 363–381.
Schüle W. (1993) Mammals, vegetation and the initial
human settlement of the Mediterranean islands: a
paleoecological approach, J. Biogeogr. 20, 399–
411.
Sheppard W.S., Arias M.C., Grech A., Meixner M.D.
(1997) Apis mellifera ruttneri, a new honeybee
subspecies from Malta, Apidologie 28, 287–293.
Sheppard W.S., Meixner M.D. (2003) Apis mellif-
era pomonella, a new honey bee subspecies from
Central Asia, Apidologie 34, 367–375.
Sinacori A., Rinderer T.E., Lancaster V., Sheppard
W.S. (1998) A morphological and mitochondrial
assessment of Apis mellifera from Palermo, Italy,
Apidologie 29, 481–592.
Strange J.P. (2001) “A severe stinging and much fa-
tigue” – Frank Benton and his 1881 search for Apis
dorsata, Am. Entomol., Summer 2001, 112–116.
Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin
F., Higgins D.G. (1997) The ClustalX windows
interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools, Nucleic
Acids Res. 24, 4876–4882.
