Geographical overlap of two mitochondrial genomes in Spanish honeybees (Apis mellifera iberica)

Restriction enzyme cleavage maps of mitochondrial DNA from the Spanish honeybee, Apis mellifera iberica (Hymenoptera: Apidae), were compared with those from the European subspecies A. m. mellifera, A. m. ligustica, and A. m. carnica, and the African subspecies A. m. intermissa and A. m. scutellata. The mitochondrial DNA (mtDNA) of the two African subspecies can be distinguished by restriction fragment polymorphisms revealed by Hinf I digests. Two distinct mtDNA types were found among Spanish honeybees: a west European mellifera-like type, which predominates in the north of Spain, and an African intermissa-like type, which predominates in the south. Spain appears to be a region of contact and hybridization between the two subspecies A. m. intermissa and A. m. mellifera, which respectively represent African and west European honeybee lineages. This natural boundary between European and African honeybee populations in the Old World may provide a model for predicting the eventual outcome of the colonization of North America by introduced African honeybees.     

Hybrid origins of honeybees from italy (Apis mellifera ligustica) and sicily (A. m. sicula)

The genetic variability of honeybee populations Apis mellifera ligustica, in continental Italy, and of A. m. sicula, in Sicily, was investigated using nuclear (microsatellite) and mitochondrial markers. Six populations (236 individual bees) and 17 populations (664 colonies) were, respectively, analysed using eight microsatellite loci and DraI restriction fragment length polymorphism (RFLP) of the cytochrome oxidase I (COI)-cytochrome oxidase II (COII) region. Microsatellite loci globally confirmed the southeastern European heritage of both subspecies (evolutionary branch C). However, A. m. ligustica mitochondrial DNA (mtDNA) appeared to be a composite of the two European (M and C) lineages over most of the Italian peninsula, and only mitotypes from the African (A) lineage were found in A. m. sicula samples. This demonstrates a hybrid origin for both subspecies. For A. m. ligustica, the most widely exported subspecies, this hybrid origin has long been obscured by the fact that in the main area of queen production (from which most of the previous ligustica bee samples originated) the M mitochondrial lineage is absent, whereas it is present almost everywhere else in Italy. This presents a new view of the evolutionary history of European honeybees. For instance, the Iberian peninsula was considered as the unique refuge for the M branch during the quaternary ice periods. Our results show that the Apennine peninsula played a similar role. The differential distribution of nuclear and mitochondrial markers observed in Italy seems to be a general feature of introgressed honeybee populations. Presumably, it stems from the social nature of the species in which both genome compartments are differentially affected by the two (individual and colonial) reproduction levels.     

Use of a PCR method for controlling pure-breeding of honeybees Apis mellifera mellifera L. in the southern Urals

A key problem of honeybee (Apis mellifera mellifera) breeding in the Southern Urals is its cross-breeding with the Caucasian honeybee Apis mellifera caucasica. Mitochondrial DNA (mtDNA) in these subspecies differ in the length of a fragment localized between genes CO-I and CO-II, which can be used as a marker. A pair of 20-mer primers for PCR was chosen by means of computer design in order to determine the fragment size in both of the subspecies. The amplified fragment was shown to have a length of 350 bp in A. m. caucasica and 600 bp in A. m. mellifera. The difference in length results from the different ratio between two main elements P and Q, which comprise a major part of this sequence in these subspecies: a copy of P element and two copies of Q element in A. m. mellifera, and a copy of Q element only in A. m. caucasica. This sharply defined distinction allows us to use PCR for differentiating the subspecies, estimating the heterogeneity in the colonies, and rejecting queens in the selection process because of the maternal inheritance of the studied character. The nucleotide sequence of the amplified mtDNA fragment of A. m. mellifera was determined.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

Genetic structure and distinctness of Apis mellifera L. populations from the Canary Islands

The genetic structure of Apis mellifera populations from the Canary Islands has been assessed by mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu-COII region) and nuclear (microsatellites) studies. These populations show a low level of genetic variation in terms of average number of alleles and degree of heterozygosity. Significant differences in the distribution of alleles were found in both data sets, confirming the genetic differentiation among some of the islands but not within them. Two mitochondrial haplotypes characteristic of the Canary Islands are found at high frequencies, although populations are introgressed by imported honeybees of eastern European C lineage. This introgression is rather high on Tenerife and El Hierro and low on Gran Canaria and La Gomera, whereas on La Palma it has not been recorded. The finding of microsatellite alleles characteristic of the eastern European lineage corroborates the genetic introgression. Phylogenetic analyses indicate that the Canarian honeybees are differentiated from other lineages and provide genetic evidence of their African origin.     

Apis mellifera evolutionary lineages in Northern Africa: Libya, where orient meets occident

Abstract  The distribution of various evolutionary lineages of Apis mellifera subspecies in Africa is still controversial. We sampled honeybees from eight coastal locations and three Saharan oases in Libya and analyzed mtDNA variability with restriction fragment length polymorphisms and the sequence of the tRNAleu-cox2 intergenic region. Haplotypes belonging to the oriental O evolutionary lineage, including four which are newly described, were detected in all investigated locations. Haplotypes belonging to the European M lineage were rarely detected, probably reflecting the effect of sporadic importations. Honeybees belonging to the A lineage were detected in Al Aziziyah and Zlitan close to the Tunisian border. The distribution of the O lineage extends westward up to the border between Libya and Tunisia, a contact area between the O and A lineages. Various Libyan honeybee populations in Saharan oases are characterized by novel and unique haplotypes (O4, O5, O5′ and O5″). These might be natural relic populations that became isolated when the North African Sahara desert was still grassland (0.126–0.168 Myr ago).     

A microsatellite DNA toolkit for studying population structure in Apis mellifera

We present a set of 18 microsatellite DNA markers that can be run in two multiplex polymerase chain reactions as standard tool for assessing molecular ecological problems in honeybees (Apis mellifera). In addition to a set of six unlinked loci testing for classical population genetic parameters, we present three sets of four tightly linked loci, each located on three different chromosomes. These linked markers are useful for determining the number of colonies in a population as well as the parentage of drones and workers. Moreover, the tool kit can test for various modes of natural selection in honeybee populations.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

Biodiversity of Apis mellifera populations from Tenerife (Canary Islands) and hybridisation with East European races

The biodiversity of honeybee (Apis mellifera) populations from Tenerife (Canary Islands, Spain) has been assessed by restriction analysis of a mitochondrial non-coding intergenic region. Seventy-nine colonies were analysed from thirteen apiaries in six populations that have been kept from recent queen introduction. The length and restriction pattern of the PCR amplified products of the intergenic region identified four mitochondrial haplotypes. One of these haplotypes shows the same restriction pattern and composition of the intergenic region carried by honeybees belonging to the African lineage. Two haplotypes are characterised by a particular intergenic region found with high frequency in the Canarian populations. The haplotype representative of the East European honeybee lineage shows a frequency of 35%, thus indicating introduction of queen honeybees. The finding of this haplotype in Canarian honeybees suggests that hybridisation between the endemic Apis mellifera populations and imported bees is occurring in Tenerife.     

Monthly changes in various drone characteristics of Apis mellifera ligustica and Apis mellifera syriaca

This study was conducted to investigate drone rearing activity and semen production of Apis mellifera ligustica and Apis mellifera syriaca . Tendency of worker bees of both subspecies towards egg laying under semiarid conditions were also monitored in the experiments. Differences were not observed in drone brood production between both honeybee subspecies throughout the investigation. Worker bees of both subspecies needed a significantly shorter time to start egg laying during February and March in comparison with the time those workers needed for laying eggs during the remaining months of the study. Syrian bee workers started egg laying earlier than Italian bee workers. Drones from laying workers were much smaller and produced less sperms with more abnormalities than normal drones. Drones produced from queens in May were heavier and produced more sperms with less abnormalities than those produced during the other months. The drone brood rearing of both subspecies tended to follow the same general cycle in 2005 and 2006. The study suggests that virgin queens have a better chance to receive adequate viable sperm amounts from drones in April and May in semiarid Mediterranean conditions.     

Molecular genetic analysis of five extant reserves of black honeybee <Emphasis Type="Italic">Apis melifera melifera</Emphasis> in the Urals and the Volga region

Local populations of the black honeybee Apis mellifera mellifera from the Urals and the Volga region were examined in comparison with local populations of southern honeybee subspecies A. m. caucasica and A. m. carpatica from the Caucasus and the Carpathians. Genetic analysis was performed on the basis of the polymorphism of nine microsatellite loci of nuclear DNA and the mtDNA COI–COII locus. On the territory of the Urals and the Volga region, five extant populations (reserves) of the black honeybee A. m. mellifera were identified, including the Burzyanskaya, Tatyshlinskaya, Yuzhno-Prikamskaya, Visherskaya, and Kambarskaya populations. These five populations are the basis of the modern gene pool of the black honeybee A. m. mellifera from the Urals and the Volga region. The greatest proportion of the remaining indigenous gene pool of A. m. mellifera (the core of the gene pool of the population of A. m. mellifera) is distributed over the entire territory of Perm krai and the north of the Republic of Bashkortostan. For the population of A. m. mellifera from the Urals and the Volga region, the genetic standards were calculated, which will be useful for future population studies of honeybees.     

Discrimination of Iranian honeybee populations (Apis mellifera meda) from commercial subspecies of Apis mellifera L. using morphometric and genetic methods

The morphological characters of honeybees have an important role for discriminating honeybee subspecies. In the present research, Iranian populations of honeybee (Apis mellifera) were collected from 19 areas in Iran. The samples were collected from stationary beekeeping sites. Moreover, pictures of honeybee forewings held in the Bee Data Bank in Oberursel were compared with Iranian honeybee populations. 19 morphological characters were measured for each forewing of worker honeybee to evaluate differentiation of Iranian honeybee populations from the commercial honeybee subspecies A. m. mellifera, A. m. carnica, A. m. caucasica and A. m. ligustica. Additionally, part of the tRNA^leu gene, an intergenic region and part of COII was used to confirm differentiation of the commercial subspecies from Iranian honeybee populations. Results of the cluster analyses showed that 19 morphological characters of forewings differentiated Iranian populations from the commercial subspecies. Moreover, the phylogenetic tree of part of the tRNA^leu gene, an intergenic region and part of COII differentiated the commercial subspecies from Iranian honeybee populations. Results of the discriminant function analyses (DFA) indicated that the references samples of A. m. meda overlapped with Iranian populations.     

Fertility and reproductive rate of Varroa mite,Varroa destructor,in native and exotic honeybee,Apis mellifera L., colonies under Saudi Arabia conditions

Varroa mite is the most destructive pest to bee colonies worldwide. In Saudi Arabia, preliminary data indicated high infestation levels in the exotic honeybee colonies; such as Apis mellifera carnica and Apis mellifera ligustica, compared to native honeybee subspecies Apis mellifera jemenitica, which may imply higher tolerance to Varroasis. In this study, fertility and reproductive rate of Varroa mite, Varroa destructor, in capped brood cells of the native honeybee subspecies were investigated and compared with an exotic honeybee subspecies, A. m. carnica. Mite fertility was almost alike (87.5% and 89.4%) in the native and craniolan colonies respectively. Similarly, results did not show significant differences in reproduction rate between both subspecies (F = 0.66, Pr > F = 0.42). Number of adult Varroa daughters per fertile mother mite was 2.0 and 2.1 for native and craniolan honeybee subspecies respectively. This may indicate that mechanisms of keeping low infestation rates in the native honeybee colonies are not associated with Varroa reproduction. Therefore, potential factors of keeping lower Varroa infestation rates in native honey bee subspecies should be further investigated.     

Gene flow in admixed populations and implications for the conservation of the Western honeybee, Apis mellifera

Anthropogenic activity, especially modern apiculture, has considerable impact on the natural distribution of the Western honeybee, Apis mellifera, leading to the spread, replacement and fragmentation of many subspecies. This creates demand for the conservation of some subspecies, in particular, Apis mellifera mellifera, which once was widely distributed in Western Europe and nowadays is endangered through habitat loss and fragmentation. Moreover, A. m. mellifera may be further endangered by hybridisation in populations that now occur in artificial sympatry with other subspecies. Here, we quantify and compare individual hybridisation between sympatric and allopatric honeybee populations of A. m. mellifera and A. m. carnica using microsatellite markers and a Bayesian model-based approach. We had a special focus on pure breeding populations, which are a major tool in honeybee conservation. Our results demonstrate that subspecies are still highly differentiated, but gene flow is not prevented by the current management strategies, creating urgent demand for an improved conservation management of A. m. mellifera. However, the occurrence of a high number of pure individuals might suggest that some sort of hybrid barrier acts against the complete admixture of the two subspecies.     

Thelytokous parthenogenesis in unmated queen honeybees (Apis mellifera capensis): central fusion and high recombination rates

The subspecies of honeybee indigenous to the Cape region of South Africa, Apis mellifera capensis, is unique because a high proportion of unmated workers can lay eggs that develop into females via thelytokous parthenogenesis involving central fusion of meiotic products. This ability allows pseudoclonal lineages of workers to establish, which are presently widespread as reproductive parasites within the honeybee populations of South Africa. Successful long-term propagation of a parthenogen requires the maintenance of heterozygosity at the sex locus, which in honeybees must be heterozygous for the expression of female traits. Thus, in successful lineages of parasitic workers, recombination events are reduced by an order of magnitude relative to meiosis in queens of other honeybee subspecies. Here we show that in unmated A. m. capensis queens treated to induce oviposition, no such reduction in recombination occurs, indicating that thelytoky and reduced recombination are not controlled by the same gene. Our virgin queens were able to lay both arrhenotokous male-producing haploid eggs and thelytokous female-producing diploid eggs at the same time, with evidence that they have some voluntary control over which kind of egg was laid. If so, they are able to influence the kind of second-division meiosis that occurs in their eggs post partum.     

Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae)

Similar microsatellite electromorphs (PCR products of the same size) can arise from independent mutational events. Such alleles are not identical by descent. This phenomenon, termed size homoplasy, was studied by sequencing electromorphs of two microsatellite loci in which the stretch of basic repeats is interrupted by different short (1-2 bp) DNA motifs. The number and position of these interruptions were established for electromorphs from closely and distantly related populations of honeybees and bumblebees. No sequence difference was found when electromorphs came from the same subspecies or from closely related subspecies, suggesting that they were probably identical by descent. In contrast, sequence differences were often detected in distantly related subspecies, showing that size homoplasy frequently occurs at this level of population differentiation. Size homoplasy is increased by limits to free length variation of alleles, a phenomenon that seems to act on interrupted microsatellites when comparing distantly related taxa, that is, honeybee subspecies from different evolutionary lineages. Electromorph sequences suggest that, within the scope of these limits, large mutation events have occurred frequently at both interrupted loci studied. In good agreement with the molecular data, computations based on the observed heterozygosity and number of electromorphs and simulation studies showed that neither locus fits the one-step stepwise mutant model (SMM). We speculate that interrupted microsatellites in general could be characterized by a higher variance in repeat number and consequently a lower homoplasy rate than pure ones. Hence, interrupted microsatellites should be most appropriate for investigating population differentiation and evolutionary relationship between relatively distant populations.      

MtDNA COI‐COII marker and drone congregation area: An efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile‐de‐France, France. CANIF's honeybee colonies were intensively studied over a 3‐year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI‐COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.     

Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana

Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species.     

引入西方蜜蜂对中蜂的危害及生态影响

作者阐述自1896年中国引进西方蜜蜂Apis melliferaL.的优良品种如意大利蜂Apis mellifera ligusticaSpinola和喀尼阿兰蜂Apis mellifera Carnica Pollmann以来,使当地的东方蜜蜂Apis cerana F.受到严重危害,其分布区域缩小75%以上,种群数量减少80%以上。使山林植物授粉总量减少,导致植物多样性减少。文中提出: 建立自然保护区保存本地蜜蜂遗传特性,和采用基因转移等技术,培育具有西方蜜蜂优良生产性能的中蜂新品种,逐步恢复中蜂的种群数量。     

Evaluation of the efficacy of mitochondrial ATP 6 and 8, Cyt b and 16S rDNA genes for differentiation of Iranian honeybees (Apis mellifera meda) from commercial subspecies of Apis mellifera L. and comparison with geometric morphometric method

Mitochondrial DNA sequence variations and the geometric morphometric method can be used to differentiate honeybee subspecies and evolutionary lineages. Molecular markers are powerful tools for discriminating honeybee subspecies. In this study, 19 beekeeping sites were selected to collect Iranian honeybee samples. The honeybee forewing images stored at Oberursel (the Bee Data Bank) were used to compare with those of Iranian honeybees using the geometric morphometric method. Furthermore, the abilities of DNA markers to differentiate Iranian honeybees (A. m. meda) from the most common commercial subspecies (A. m. carnica and A. m. ligustica) were assessed. In the present research, 16S rDNA (Mitochondrial 16S rDNA Region) showed greater ability in differentiating Iranian honeybees from other subspecies compared with ATP 6 and 8 and Cyt b. The phylogenetic tree derived from 16S rDNA differentiated A. m. carnica and A. m. ligustica from Iranian honeybees. Principle component analysis (PCA) discriminated C lineage and Z subgroup from A and M lineages using 16S rDNA. In addition, the phylogenetic tree of the 16S rDNA affirmed the findings of the cluster analysis derived from the geometric morphometric method in differentiating A. m. carnica and A. m. ligustica from Iranian honeybees. The cluster analysis grouped reference subspecies of A. m. meda with Iranian honeybees. Moreover, the Discriminant Function Analyses (DFA) differentiated Iranian honeybees from A. m. ligustica and A. m. carnica.