The western honey bee,
Turkey is in the contact zone of the different lineages and was first proposed to be O lineage, but then studies revealed it to be mostly C-lineage (Smith et al., 1997; Palmer et al., 2000; Kandemir et al., 2006; Özdil et al., 2009). Within Turkey, wide ranges of climates and habitats are found, and several honey bee subspecies and ecotypes have been described. According to Ruttner (1988),
Within the Balkan Peninsula very near to the Thrace region, several honey bee subspecies and ecotypes including
In this study, a detailed morphological and genetic characterization of honey bees from the Thrace and western Anatolian region of Turkey was surveyed. We made a comprehensive sampling and used forty-one morphological characteristics and sequenced the tRNAleu-Cox2 intergenic region to identify the honey bee (
The aim of this study was threefold: (i) to provide a detailed morphological characterization of the Thrace and the western Anatolian region of Turkey, with a large sampling size through the analysis of morphological characteristics; (ii) to reveal the different haplotypes after the completion of the survey of mtDNA variation of the tRNAleu-cox2 region through the sequencing of a large collection of individuals sampled across the entire Thrace and western Anatolian region and to revise the C lineage haplotypes which have already been deposited to the GenBank database; (iii) to determine whether the dominant Thrace region ecotype belonged to
The worker bees were collected between May 2014 and September 2015 for morphological and mitochondrial surveys. Honey bees were sampled from five different localities; three provinces in the Thrace region: Tekirdağ, Kırklareli, Edirne provinces; the Çanakkale province: (Gallipoli Peninsula and western Anatolian side); the island of Gökçeada on the Marmara Sea near Gallipoli Peninsula (Fig. 1). A total of 1650 worker bee samples (110 colonies from twenty-three apiaries, fifteen repetitions per colony) were evaluated for forty-one morphological characteristics. On the other hand, 217 worker bees, including morphologically measured 110 samples, from 133 apiaries in seventy-nine different localities with the two of each reference samples (
Sampling locations of honey bees in European part of Turkey-Thrace region (Tekirdağ, Kırklareli and Edirne), Island of Gökçeada and Çanakkale province (both Gallipoli Peninsula and western Anatolian side).
The worker bee samples were collected and stored in 72% ethyl alcohol until preparation. Each part of the samples was fixed on the slide with chloral hydrate-free Hoyer’s liquid. Each of the 110 colonies consisted of fifteen worker bees, so a total of 1650 (15x110) worker bees were used for morphological measuring. The right forewings of the fifteen worker bees from each colony were mounted on glass slides with Hoyer’s liquid. In each sample, the standard and most commonly used anterior wing vein angles A4, B4, D7, E9, G18, J10, J16, K19, L13, N23 and O26 were measured biometrically with the use of the software available in the Olympus trinocular stereo microscope (SZ61) (Ruttner et al., 1978; Moritz, 1991). Forty-one morphological characters given in Tab. 1 and Tab. 2 were measured as described by Moritz (1991), Güler (2010) and Güler et al. (2010). Measurements were made with the Olympus SZ61 trinocular stereo microscopewith the computer program (cellSens standart 1.8).
The mean and standard errors of the directly measured morphological characters of the Thrace, Çanakkale and island Gökçeada worker bee samples
Character | Regions |
X±Sx | ||||
---|---|---|---|---|---|---|
Tekirdağ | Kırklareli | Edirne | Çanakkale | Gökçeada | ||
LH*** | 0.238±0.004c | 0.228± 0.003c | 0.231±0.002c | 0.270±0.003b | 0.283±0.004a | 0.247±0.003 |
WTa*** | 1.117±0.022b | 1.100±0.015bc | 1.051±0.010c | 1.127±0.227b | 1.186±0.015a | 1.109±0.009 |
WTb* | 0.300±0.012ab | 0.316±0.011ab | 0.305±0.011ab | 0.336±0.012a | 0.296±0.013b | 0.311±0.006 |
LPr** | 6.247±0.077b | 6.316±0.072ab | 6.319±0.023ab | 6.511±0.068a | 6.422±0.108ab | 6.351±0.030 |
LFNS | 2.621±0.016 | 2.637±0.011 | 2.622±0.008 | 2.631±0.020 | 2.663±0.016 | 2.633±0.006 |
LT* | 3.144±0.019ab | 3.145±0.016ab | 3.116±0.011b | 3.145±0.023ab | 3.178±0.016a | 3.143±0.008 |
LM* | 2.044±0.013b | 2.092±0.013a | 2.078±0.009ab | 2.073±0.013ab | 2.092±0.013a | 2.074±0.006 |
WM* | 1.168±0.008ab | 1.162±0.009b | 1.190±0.006a | 1.169±0.009ab | 1.155±0.008b | 1.170±0.004 |
WT3*** | 1.896±0.007b | 1.869±0.008b | 1.877±0.007b | 1.961±0.013a | 1.984±0.015a | 1.910±0.006 |
WT4*** | 1.846±0.007c | 1.806±0.008d | 1.822±0.007cd | 1.920±0.012b | 1.953±0.014a | 1.859±0.006 |
WS3* | 2.811±0.012b | 2.804±0.014b | 2.792±0.009b | 2.912±0.076a | 2.823±0.013ab | 2.825±0.015 |
LWM*** | 1.481±0.020b | 1.421±0.009bc | 1.404±0.006c | 1.664±0.043a | 1.639±0.009a | 1.505±0.014 |
WWMNS | 2.346±0.012 | 2.361±0.013 | 2.342±0.011 | 2.396±0.057 | 2.342±0.013 | 2.357±0.012 |
DWM*** | 0.315±0.005b | 0.325±0.005b | 0.317±0.005b | 0.349±0.011a | 0.352±0.006a | 0.329±0.003 |
LS6U* | 2.606±0.012b | 2.633±0.014ab | 2.640±0.010ab | 2.642±0.031ab | 2.679±0.017a | 2.637±0.008 |
WS6* | 3.119±0.018b | 3.137±0.018b | 3.128±0.015b | 3.161±0.041ab | 3.215±0.016a | 3.146±0.011 |
LFW*** | 8.333±0.022b | 8.343±0.036b | 8.362±0.018b | 8.546±0.016a | 8.486±0.028a | 8.402±0.014 |
WFW** | 2.856±0.009b | 2.866±0.015ab | 2.878±0.008ab | 2.902±0.008ab | 2.893±0.019a | 2.877±0.006 |
LCa** | 0.491±0.004ab | 0.504±0.005a | 0.502±0.004a | 0.488±0.005ab | 0.484±0.005b | 0.495±0.002 |
LCb*** | 0.218±0.003ab | 0.212±0.006bc | 0.201±0.002c | 0.230±0.003a | 0.226±0.003a | 0.216±0.002 |
CT2*** | 6.981±0.105a | 6.788±0.105ab | 6.597±0.103b | 5.994±0.153c | 6.615±0.125b | 6.621±0.060 |
CT3** | 6.763±0.111a | 6.403±0.092b | 6.389±0.078b | 6.099±0.207b | 6.787±0.108a | 6.479±0.059 |
CT4*** | 4.200±0.069a | 4.365±0.056a | 4.372±0.064a | 3.580±0.103c | 3.831±0.108b | 4.113±0.045 |
CSc** | 0.853±0.147a | 0.451±0.078bc | 0.325±0.059c | 0.863±0.109a | 0.666±0.135ab | 0.618±0.052 |
Length of hairs (LH), Width tomentums a (WTa), Width tomentum b (WTb), Length of proboscis (LPr), Length of femur (LF), Length of tibia (LT), Length of metatarsus (LM), Width of metatarsus (WM), , Width of tergite 3 (WT3), Width of tergite 4 (WT4), Width of sternit 3 (WS3), Length of wax mirror, (LWM), Width of wax mirror (WWM), D. Between mirrors (DWM), Length of sternum 6 (LS6), Width of sternum 6 (WS6), Length of forewing (LFW), Width of forewing (WFW), Length of cubital a (LCa), Length of cubital b (LCb), Colour of tergit 2 (CT2), colour of tergit 3 (CT3), Colour of tergit 4 (CT4), Colour of scutelum (CSc)
NS, *, **, *** non significant and significant at 0.05, 0.01, and 0.001 respectively.
The mean and standard errors of some calculated and direct measured morphological characters of the Thrace, Çanakkale and Gökçeada regions worker bee samples
Character | Regions |
X±Sx | ||||
---|---|---|---|---|---|---|
Tekirdağ | Kırklareli | Edirne | Çanakkale | Gökçeada | ||
TI* | 4.284±0.232ab | 3.887±0.148b | 3.809±0.165b | 3.952±0.234b | 4.683±0.231a | 4.080±0.093 |
LHLNS | 7.809±0.045 | 7.871±0.033 | 7.816±0.027 | 7.833±0.054 | 7.934±0.043 | 7.846±0.018 |
CI*** | 2.302±0.043b | 2.471±0.044a | 2.552±0.051a | 2.153±0.044b | 2.188±0.048b | 2.365±0.025 |
T3+T4*** | 3.737±0.015c | 3.675±0.017d | 3.695±0.014cd | 3.868±0.031b | 3.937±0.026a | 3.765±0.013 |
MI*** | 57.206±0.387a | 55.716±0.331bc | 57.394±0.317a | 56.471±0.496ab | 55.236±0.194c | 56.508±0.180 |
S6INS | 83.398±0.490 | 84.056±0.296 | 84.581±0.327 | 83.681±0.499 | 83.452±0.301 | 83.875±0.182 |
A4*** | 32.109±0.206bc | 30.970±0.242d | 31.769±0.249c | 32.532±0.193ab | 32.866±0.320a | 31.953±0.122 |
B4*** | 104.037±0.543ab | 105.727±0.598a | 103.399±0.660bc | 101.882±0.517cd | 101.027±0.802d | 103.474±0.312 |
D7*** | 100.857±0.344a | 99.495±0.377b | 99.413±0.355b | 101,121±0.376a | 102.036±0.527a | 100.432±0.193 |
E9*** | 20.392±0.153bc | 21.013±0.153a | 20.693±0.252ab | 19.855±0.134c | 19.158±0.294d | 20.336±0.105 |
G12** | 88.837±0.430bc | 88.360±0.348bc | 87.933±0.348c | 90.190±0.331a | 89.480±0.436ab | 88.857±0.184 |
J10** | 52.605±0.398b | 53.989±0.358a | 54.274±0.443a | 53.876±0.499a | 52.536±0.520b | 53.521±0.206 |
J16*** | 90.103±0.385bc | 91.953±0.357a | 90.859±0.401ab | 91.927±0.405a | 89.436±0.434c | 90.936±0.196 |
K19NS | 74.372±0.367 | 75.440±0.416 | 75.356±0.291 | 75.615±0.428 | 74.662±0.499 | 75.104±0.179 |
L13*** | 16.145±0.119b | 15.556±0.143cd | 15.390±0.112d | 17.759±0.290a | 15.989±0.273bc | 16.112±0.112 |
N23*** | 88.683±0.402cd | 91.161±0.263b | 89.854±0.405bc | 92.905±1.066a | 88.180±0.408d | 90.212±0.288 |
O26* | 34.496±0.420b | 35.195±0.456ab | 34.147±0.444b | 34.537±0.602b | 36.538±0.807a | 34.862±0.239 |
Tomentum Index (TI), Length of Hind Leg (LHL), Cubital Index (CI), Body size (T3+T4), Metatarsal Index (MI), Sternum 6 Index (S6I), Veinal angles A4, B4, D7, E9, G12; J10, J16, K19, L13, N23, O26
NS, *, **, *** non significant and significant at 0.05, 0.01, and 0.001 respectively.
The morphometrics data were analysed through ANOVA, and the differences between the means were compared with the Student Newman Keuls (SNK) post hoc tests (SPSS, 2004). In addition, the Multivariate Discriminant Stepwise Analysis Method (MDSAM), which determines the differences and the grouping levels in terms of the morphological characteristics between more than two biological sources, was used to determine the Standard Multivariate Canonical Discriminant Function and Constant Descriptive Coefficients (SMCDFCDC) of the five different regions from Thrace and western Anatolia (Cooley & Lohnes, 1971). The territorial regions of the groups in a Coordinate system were determined and standardized with the use of the SMCDFCDC (Fig. 2).
Discriminant Analysis of honey bee (
The worker bees were individually placed in the Eppendorf tubes containing 95% ethanol and transported to the Molecular Genetics Laboratory, the Department of Agricultural Biotechnology, Tekirdağ Namık Kemal University. Total genomic DNA was extracted from thoraces according to the phenol-chloroform extraction method (Sambrook & Russel, 2001). The genomic DNA concentration was quantified with a Qubit™ 2.0 Fluorometer (ThermoFisher Scientific), and 20–30 ng of genomic DNA was used for the PCR.
The tRNAleu-cox2 gene region was amplified according to Garnery et al. (1992) with E2 and H2 primers. The amplified PCR products were sequenced on an Applied Biosystems 3500XL Genetic Analyzer (Applied Biosystems, USA) in order to verify the nucleotide variations. The sequences were assembled with ChromasPro version 1.7.6 and aligned with the BioEdit Sequence Alignment Editor with Clustal W multiple alignment modules (Hall, 1999). Phylogenetic trees were generated with the use of the neighbor-joining algorithm in MEGA 6 (Tamura et al., 2013). The SplitsTree was also used to construct a network from the distance matrices based on allele-sharing distances (Huson & Bryant, 2006).
The worker-bee samples belonging to the five locations were found to be similar in only five characters but different from one another (P<0.001) in thirty-six characters (Tab. 1 and Tab. 2).
The Discriminant Analysis Stepwise method showed that the hair length (HL), wing L13 veinal angle, fourth tergite width (T4), wing N23 veinal angle, metatarsal index (MI), wing E9 veinal angle, second tergite color (CT2), scutellum color (CSC), both wing O26 and D7 veinal angles and wing length (WL) characters determined that the worker-bee samples represented the different (P<0.001) bee populations respectively (Tab. 1 and Tab. 2).
The method determined 94.5% to be the proper grouping level of 110 worker bee samples representing these five different locations. Grouping in different areas indicated that these bees come from different genetic sources in terms of morphological structure Tab. 3.
Grouping levels of 110 worker bee samples collected from bee genotypes in five different areas and the number and ratios of the genotype groups each sample represents
Groups Tekirdağ Kırklareli | Estimation group memberships |
Total | ||||||
---|---|---|---|---|---|---|---|---|
Edirne | Çanakkale | Gökçeada | ||||||
Tekirdağ | 23 | 2 | 25 | |||||
Kırklareli | 23 | 2 | 25 | |||||
Number | Edirne | 2 | 23 | 25 | ||||
Çanakkale | 20 | 20 | ||||||
Orijinal Groups | Gökçeada | 15 | 15 | |||||
Tekirdağ | 92.0 | 8.0 | 100.0 | |||||
Kırklareli | 92.0 | 8.0 | 100.0 | |||||
% | Edirne | 8.0 | 92.0 | 100.0 | ||||
Çanakkale | 100.0 | 100.0 | ||||||
Gökçeada | 100.0 | 100.0 |
Morphological differences according to the discriminant functions (F1) were found among the worker bee samples of the Thrace Region (Kırklareli, Tekirdağ and Edirne), Çanakkale and Gökçeada Island (Fig. 2). The Çanakkale and Gökçeada samples formed a cluster in completely different areas from one another and from samples of the Thrace Region (100%). Therefore, three different populations were formed in terms of the morphological structure: one on the continent of the Europe-Thrace region, one in the Çanakkale province on Asian/Anatolian side of the Turkey and one Gökçeada Island. The Sea of Marmara and Gallipoli Peninsula, important barriers in separating Asian and European continents, have also played an important role in forming this genetic variation.
The populations of the Thrace region (Tekirdağ, Edirne and Kırklareli) were found similar to one another in relation to the morphological structure. The samples of the region were overlapped at certain levels. For example, two samples (8%) from Tekirdağ were placed within the samples from Kırklareli and two samples (8%) from Kırklareli in Edirne, and two samples (8%) from Edirne in Kırklareli. The overlapped clustering (Fig. 2) in this region was the result of a similar morphological structure. In fact, the worker bee samples in the Thrace region were found to be similar to one another in some of the morphological characteristics (HL, WT3, WTb, WS3, DWM, LS6, WS6, LFW, and CT4) (Tab. 1 and Tab. 2). On the other hand, the most important difference between the bee samples of the Thrace region was seen in the wing veinal angles.
The analysis of the sequence data at the tRNAleu-cox2 region produced twenty-two different haplotypes, of which sixteen were novel), and all of them were ascribable to the East European C-lineage and characterized by the presence of a single Q sequence corresponding to the predicted composition of this region. The mtDNA fragment corresponded to the positions 3363–3935 bp (Crozier & Crozier, 1993). The polymorphic sites of the intergenic tRNAleu-cox2 region of the previous C2 haplotypes and the haplotypes that are found in this study, GenBank accession numbers, and the variations with the nucleotide positions were summarized in Tab. 4. Sixteen polymorphic sites were detected within the sequenced 571–573 bp mtDNA fragment, and the sequences were deposited in the GenBank database with accession numbers MH939332-MH939353 (Tab. 4). In our study, 104 samples out of 217 (~48%) were found to be exactly the same as the published C2d haplotype in the NCBI Genbank database (accession numbers FJ824584, FJ824585, FJ037777, JQ977701, JF723977). C2d was found as a common haplotype all over the Thrace region, especially in Tekirdağ (0.78), Kırklareli (0.24), Edirne (0.67) and Gökçeada Island (0.50) (Tab. 4 and Tab. 5). In this study, the C2d sequence was deposited to the GenBank database with the accession number MH939332. Along with the C2d haplotype, previously published C2s, C2v, C2i, C2j, and C2h haplotypes were also observed in the Thrace region less frequently, forty-four samples out of 217 (20.27%). The DNA sequences of the
The polymorphic sites of the intergenic tRNAleu-cox2 region of the previous C2 haplotypes and the haplotypes that are found in this study, NCBI GenBank accession umbers and the citing references are given. The mtDNA fragment corresponds to the positions 3363–3935 bp published by Crozier & Crozier (1993).
Haplotypes | bp | 3406 | 3410* | 3424* | 3425* | 34281 | 3442 | 3449 | 3474 | 3488* | 3512* | 3514 | 3535 | 3536* | 3567 | 3569 | 3573* | 3575 | 3576* | 3588 | 3589 | 3605 | 3634 | 3664* | 3670 | 3735 | 3769 | 3771 | References |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
JQ977700_C2c ( |
572 | T | A | A | T | - | T | T | T | A | T | T | A | A | A | A | A | - | C | G | - | . | T | T | T | T | C | C | Suśnik et al., 2004; Muñoz et al., 2009, 2012 |
JQ977701 / FJ824585_C2d ( |
572/526. | . | . | . | - | . | . | - | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Suśnik et al., 2004; Muñoz et al., 2009, 2012 |
JQ977702_C2e ( |
571 | . | . | . | . | - | . | . | - | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Kozmus et al., 2007; Muñoz et al., 2009 |
FJ357806_C2f ( |
571 | . | . | . | . | - | - | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Özdil et al., 2009 |
FJ357807_C2g ( |
571 | . | . | . | . | - | - | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | . | T | . | Özdil et al., 2009 |
FJ357808_C2h ( |
514 | . | . | . | . | - | . | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | . | T | T | Özdil et al., 2009 |
FJ447491_C2i ( |
517 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | A | - | . | C | . | . | . | T | . | Nedić et al., 2009 |
JF723978_C2j ( |
572 | . | . | . | . | - | . | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | . | T | . | Nedić et al., 2009; Coroian et al., 2014 |
GQ433624_C2k ( |
401 | . | . | . | . | - | . | . | - | . | . | . | . | . | . | . | . | - | . | A | - | . | C | . | . | . | T | . | Razpet at al., 2009 unpublished |
GQ433625_C2l ( |
401 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | - | . | - | . | . | - | . | . | . | . | . | . | . | Razpet at al., 2009 unpublished |
GQ433626_C2m ( |
402 | C | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | . | . | . | . | Razpet at al., 2009 unpublished |
GQ433627_C2n ( |
402 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | C | . | . | . | Razpet at al., 2009 unpublished |
JQ977704_C2o ( |
571 | . | . | . | . | - | . | . | - | . | . | A | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Muñoz et al., 2012 |
JQ977705_C2p ( |
571 | . | . | . | . | - | . | C | - | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Muñoz et al. 2012; Coroian et al. 2014 |
HM117905_C2q ( |
549 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Muñoz 2013/Coroian et al. 2014 |
HM117906_C2r ( |
546 | . | . | . | . | - | . | . | . | . | . | . | T | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Coroian et al. 2014 |
JF723979_C2s | 572 | . | . | . | . | - | . | . | . | . | . | A | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | Muñoz 2013 |
JQ973663_C2t | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | A | T | . | Muñoz 2013 |
JQ973664_C2v | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | . | . | T | . | Muñoz 2013 |
JQ754649 C2x | 573 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | A | . | A | - | . | C | . | . | . | T | . | Muñoz 2013/Coroian et al. 2014 |
JQ754650_C2y | 571 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | - | . | . | . | . | . | . | Muñoz 2013; Coroian et al. 2014 |
JQ754648_C2z | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | . | . | . | . | Muñoz 2013/; Coroian et al. 2014 |
MH939332_C2d | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939333_C2d1 | 571 | . | . | . | . | - | . | . | . | . | . | . | . | - | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939334_C2s | 572 | . | . | . | . | - | . | . | . | . | . | A | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939335_C2v | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | . | . | T | . | |
MH939336_C2v1 | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | A | - | . | . | . | . | . | T | . | |
MH939337_C2v2 | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | T | . | - | . | . | . | . | . | T | . | |
MH939338_C2d2 | 572 | . | . | . | . | - | . | . | . | . | C | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939339_C2i | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | A | - | . | C | . | . | . | T | . | |
MH939340_C2i2 | 572 | . | T | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939341_C2i3 | 571 | . | T | - | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939342_C2s1 | 572 | . | T | . | . | - | . | . | . | . | . | A | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939343_C2c1 | 572 | . | T | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | . | . | . | . | . | . | |
MH939344_C2i1 | 572 | . | T | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | A | - | . | C | . | . | . | T | . | |
MH939345_C2j | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939346_C2h | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | T | . | . | - | . | . | - | . | C | . | . | . | T | T | |
MH939347_C2d3 | 572 | . | . | . | C | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | . | |
MH939348_C2d4 | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | T | - | . | . | - | . | C | . | . | . | T | . | |
MH939349_C2h1 | 572 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | T | |
MH939350_C2h2 | 573 | . | . | . | . | - | . | . | . | . | . | . | . | . | . | . | . | - | . | . | G | . | C | . | . | . | T | T | |
MH939351_C2h3 | 571 | . | . | . | . | - | . | . | . | - | . | . | . | . | . | . | . | - | . | . | - | . | C | . | . | . | T | T | |
MH939352_C2s2 | 572 | . | . | . | . | - | . | . | . | . | . | A | . | . | . | . | . | - | . | . | - | . | C | C | . | . | T | . | |
MH939353_C2s3 | 571 | . | . | . | . | - | . | . | . | . | . | . | . | - | . | . | . | - | . | . | - | . | C | C | . | . | T | . |
The positions that are indicated with asterics are novel polymorphisms, character “-” shows deletions found.
Number of analyzed colonies (n), haplotype distributions in the honey bee colonies from Thrace and the reference populations
Location | n | C2d | C2d1 | C2s | C2v | C2v1 | C2v2 | C2d2 | C2i | C2i1 | C2i2 | C2i3 | C2s1 | C2c1 | C2j | C2h | C2d3 | C2d4 | C2h1 | C2h2 | C2h3 | C2s2 | C2s3 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Tekirdağ | 50 | 39 | 2 | 5 | 1 | 2 | 1 | ||||||||||||||||
Kırklareli | 67 | 16 | 1 | 8 | 2 | 1 | 2 | 3 | 22 | 6 | 2 | 3 | 1 | ||||||||||
Edirne | 49 | 33 | 1 | 2 | 5 | 4 | 2 | 2 | |||||||||||||||
Çanakkale | 31 | 6 | 8 | 3 | 5 | 2 | 2 | 2 | 3 | ||||||||||||||
Gökçeada | 20 | 10 | 1 | 4 | 1 | 1 | 3 | ||||||||||||||||
217 | 104 | 10 | 12 | 18 | 3 | 2 | 2 | 6 | 3 | 22 | 6 | 2 | 3 | 6 | 2 | 2 | 3 | 2 | 2 | 3 | 1 | 3 | |
2 | 2 | ||||||||||||||||||||||
2 | 2 | ||||||||||||||||||||||
2 | 2 |
The phylogeny of the haplotypes based on the tRNAleu-cox2 intergenic region in this study with the published Genbank reference haplotypes revealed mainly two, indeed three different clusters (Fig 3); C2c, C2l, C2m, C2n C2v, C2y, C2z and novel haplotypes found in this study C2c1, C2v1, C2v2 were clustered together. These novel haplotypes were only observed in the European part of Turkey-Thrace region. The second cluster was comprised of C2t, C2j, C2g, and C2h and novel C2h1, C2h2, C2h3 haplotypes, which had been previously defined as
Neighbor-joining tree based on intergenic tRNAleu-cox2 region sequences performed on nucleotide distances computed by Kimura’s (1980) formula through MEGA6 (Tamura et al., 2013).
▪Indicates novel haplotypes that were found in this study.
In this study, a NeighborNet network was constructed by the SplitsTree4 (version 4.1) program with the published and newly found haplotypes (Fig. 4). According to the NeighborNet network, all haplotypes were split into three main groups, which had been found in agreement with the morphometric and phylogenetic studies.
A NeighborNet Network constructed from SplitsTree4 (version 4.1)
**Novel haplotypes found in this study are written in blue.
In the present study, we wanted to reveal the morphometric and genetic structure of the honey bees found in the European Part of Turkey, the Thrace region, located in the southeast of the Balkan Peninsula. Previous morphometric studies by Ruttner (1988), classified Thrace honey bees of Turkey as
According to previous morphological and molecular studies, the honey bee populations of the Thrace region can be considered as an ecotype of
Although Ruttner (1988) stated that the Thrace honey bee populations were morphologically similar to the
Due to the ecological differences (flora, climatic, etc.) in the Balkan Peninsula, different ecotypes of the Carniolan subspecies have been originated, and the Thrace honey bees can be one of them because of the high variation that was observed between the populations of
On the other hand, the Gökçeada Island samples were clustered in a narrow space in the coordinate system (Fig. 2). The most important reason for that, the island is a completely isolated area and it maintains its natural position. The island worker bee samples were found to be completely different from the others in terms of seventeen characters. For example, the island bee has the longest hair (HL) and the largest body size (T3+T4) structures, which showed that its populations maintained their morphological structure compared to the previous studies (Güler & Kaftanoğlu, 1999). It is emphasized that the island’s cold and continuous winds were considered to be the main reason for these morphological differences.
In this study, we also provide the most comprehensive survey of DNA sequencing of the tRNAleu-cox2 region whichever reported for the European part of Turkey-Thrace region, Gökçeada Island and Çanakkale province (Gallipoli Peninsula and west Anatolian side). The C1/C2 haplotypes of the tRNAleu-cox2 region were first reported by Franck et al., (2000) and the only difference between the C1 and C2 was a single C nucleotide deletion (h) at the 3428th position of the mitochondrial genome. Franck et al. (2000) reported the C1, C2a and C2b haplotypes in their study, and the C2c and C2d haplotypes were observed by Sušnik et al. (2004) in Slovenia, Croatia (
The genetic origin of honey bees found in the European part of the Turkey-Thrace region has been a mystery for a long time. First, Thrace honey bees were classified as