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Article

Mitochondrial DNA Suggests the Introduction of Honeybees of African Ancestry to East-Central Europe

by
Andrzej Oleksa
1,*,
Szilvia Kusza
2 and
Adam Tofilski
3,*
1
Department of Genetics, Faculty of Biological Sciences, Kazimierz Wielki University, Powstańców Wielkopolskich 10, 85-090 Bydgoszcz, Poland
2
Centre for Agricultural Genomics and Biotechnology, University of Debrecen, 4032 Debrecen, Hungary
3
Department of Zoology and Animal Welfare, University of Agriculture in Krakow, Adama Mickiewicza 24/28, 30-059 Kraków, Poland
*
Authors to whom correspondence should be addressed.
Insects 2021, 12(5), 410; https://doi.org/10.3390/insects12050410
Submission received: 24 March 2021 / Revised: 28 April 2021 / Accepted: 30 April 2021 / Published: 2 May 2021
(This article belongs to the Section Insect Ecology, Diversity and Conservation)
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Abstract

:

Simple Summary

In Europe, a well-known threat to the conservation of honeybee diversity is the loss of genetic uniqueness of local populations due to beekeepers’ preference for a few genetic lineages. However, due to climate change and large-scale ongoing movement of breeding individuals, the expansion of bees of African origin could represent another threat. This issue has not yet been recognised in detail, although bees bearing African mitochondrial DNA occur in South-West and South Europe due to natural gene flow. Here, we determine the diversity of mitochondrial DNA in honey bees from East-Central Europe. We sequenced the COI-COII region in 427 bees sampled along two 900 km transects (17.5° N and 23° E). We found that 1.64% of bees (95% CI: 0.66–3.35%) had African mitochondrial DNA. It is unlikely that their presence in the area resulted from natural migration but instead human-driven introductions of hybrids of African ancestry. This expansion deserves more attention, as it may contribute to the dissemination of undesirable traits, parasites and diseases.

Abstract

In Europe, protecting the genetic diversity of Apis mellifera is usually perceived in the context of limiting the spread of the evolutionary C-lineage within the original range of the M-lineage. However, due to climate change and large-scale ongoing movement of breeding individuals, the expansion of bees from the African A-lineage could represent another threat. This issue has not yet been investigated in detail, although A-mitotypes occur in South-West and South Europe due to natural gene flow. Here, we determine the diversity of mtDNA in honey bees from East-Central Europe. We sequenced the COI-COII region in 427 bees sampled along two 900 km transects (17.5° N and 23° E). We found that 1.64% of bees (95% CI: 0.66–3.35 %) had A-mitotypes. It is unlikely that their presence in the area resulted from natural migration but instead human driven introductions of hybrids of African ancestry. This expansion deserves more attention, as it may contribute to the dissemination of undesirable traits, parasites and diseases.

1. Introduction

There is a growing concern that the genetic variability of the western honey bee, Apis mellifera, is under serious threat due to human activities. Some studies have suggested that this reduced variability may contribute to the worrying global decline of bees [1,2,3]. In general, a decrease in genetic variation may result from two types of mechanisms. Firstly, selective breeding usually leads to reduced genetic diversity in domesticated animals [4,5]. Secondly, the human-mediated transportation of bees (including transhumance of colonies and trade of queen bees) may result in the loss of differences between locally adapted populations–in other words, a decrease in between-population variation [6], despite this potentially being accompanied by an increase of within-population genetic diversity [7]. As a result, many local varieties (subspecies and ecotypes) are on the path towards loss of genetic uniqueness due to replacement or hybridisation with introduced bees [8,9,10,11,12]. Because admixture is an increasingly severe threat to maintaining the diversity of honey bees, the need to better understand the spread of non-native subspecies of A. mellifera is gaining in urgency.
The global diversity of A. mellifera is well recognised, as the honey bee has been the subject of numerous population genetic surveys using maternal and biparental genetic markers [13,14,15,16,17,18], along with morphometry [19,20,21,22,23]. These studies reveal high diversification within the species, with approximately 30 described subspecies grouped into five major evolutionary lineages: A (African), C (subspecies from east and south of the Alps), M (subspecies ranging from western and northern Europe to central Asia in the east), O (Oriental, subspecies from the Middle East), Y (subspecies from Arabian Peninsula and Ethiopia) [15,17,22,24,25]. Since the natural range of the honey bee covers diverse environments, from tropical to temperate biomes [22,26], local populations of the species have adapted to unique ecological conditions. For that reason, indigenous bee populations are essential reservoirs of local adaptations [10,27]. Bee colonies of local provenance survive for longer in their original environmental conditions than imported bees [28]. Despite this, in Europe and worldwide, most honey bee queen breeders use only two of more than thirty subspecies: the Italian A. m. ligustica, and the Carniolan honey bee A. m. carnica, which are among the favourite subspecies kept by beekeepers [1]. Both belong to the evolutionary C-lineage, whose natural distribution encompasses Southeastern Europe, including Italy and the Balkans, and the adjacent part of Central Europe. Italian and Carniolan queens are massively imported to areas originally inhabited by other subspecies. In vast parts of Europe, this has resulted in the replacement or loss of genetic uniqueness of the European dark bee A. m. mellifera [8,9,10].
Aside from the massive importation of bees of the C-lineage, another phenomenon strongly affecting the honey bee’s genetic distribution is the spread of genes of African origin. The best described example of this is the expansion of ‘Africanised’ bees within the New World, where African bees (A. m. scutellata) were introduced and created highly invasive hybrids with bees of European origin such as the European dark bee A. m. mellifera, the Italian bee A. m. ligustica and the Iberian bee A. m. iberiensis [29]. Elsewhere, bees with mitochondrial haplotypes typical of the A-lineage spread through Southern Europe as a result of natural migration events. Outside of Africa, bees with A-lineage haplotypes have also been reported in the Iberian Peninsula [30,31,32,33,34,35], the adjacent area of Gascony in South-West France [36], Mediterranean islands (Sicily [37], Malta [38], Baleares [39]) and Macaronesia [40,41]. We are not aware of any reports of the presence of African mitotypes elsewhere in Europe or in the Middle East.
In this study, we aimed to investigate the mitochondrial DNA diversity of honey bees in East-Central Europe. In this part of the continent, the Carpathian mountain range has been the main biogeographical barrier for many organisms. These mountains have also constituted a putative border between the M- and C-lineages, i.e., between West- and North-European dark bees (A. m. mellifera) and Carniolan bees (A. m. carnica) from the Pannonian Basin and adjacent areas [22]. However, in recent decades, the border between these evolutionary lineages has become blurred, as beekeepers have increasingly preferred the latter subspecies. Significant numbers of Carniolan queens are imported north of the Carpathians, changing the mitochondrial gene pool of the honey bee population [42]. A. m. mellifera does not enjoy similar popularity among beekeepers [43], and is not intensively traded, so we did not expect it to occur frequently in the native area of A. m. carnica. Thus, we hypothesised that (1) bees in East-Central Europe typically have mitotypes derived from the C- or M-lineages, (2) in the area north of the Carpathians, native honey bees of the M-lineage have to a large extent been replaced by the imported C-lineage, and (3) the frequency of M-lineage mtDNA is increasing northwards. Surprisingly, we found evidence that almost 2% of bees in East-Central Europe can be considered African by matrilineal descent.

2. Materials and Methods

We investigated the maternal origin of honey bees in Central-Eastern Europe, considering an area encompassing northern Poland, Hungary and Romania (Figure 1g). The northern part of the study area (most of Poland) is part of the North European Lowland, while the southern zone (the Carpathians and Pannonian Basin) covers the Alpine system of young mountains and basins north of the Mediterranean. The Carpathians were the most likely geographical barrier to admixture between A. m. mellifera in the north and A. m. carnica in the south, and the exact location of the hybridization zone between subspecies was not thoroughly investigated before human activity blurred the boundaries of these subspecies [22,44,45,46].
For this study, we sampled bees on both sides of Carpathians (n = 153 on the southern side and n = 274 on the northern side of the mountain range, Figure 1g). Foraging worker bees were captured with an entomological net from flowers in 2017–2018, immediately preserved in 90% ethanol, and stored in a −20 °C freezer until DNA extraction. Samples were collected along two latitudinal transects in Poland: (1) along the eastern border of the country, approximately along the 18° E meridian (N = 163) and (2) in the middle of Poland, roughly along the 23° E meridian (N = 111). Samples from Hungary, together with a few bees from the adjacent part of Romania, were collected from more dispersed random locations, N = 58 and N = 95 from the east and west, respectively (Figure 1). Overall we believe that both sampling designs (linear and scattered) were equally efficient in providing us with an adequate representation of bees living in the study area.
Genomic DNA was extracted from the bee thoraces using the Insect Easy DNA Kit (EZNA) (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s recommended procedure.
From each location in Hungary, only one worker was sampled, while in Poland up to 4 workers per site were sampled and genotyped. However, the distances between neighbouring sampling sites in Poland were greater than in Hungary (median distance between closest sampling sites was 4.4 km in Poland and 2.4 km in Hungary). To make sure that bees were unrelated and represented independent data points, we genotyped them using 13 microsatellite loci (detailed information on loci used in [47]), estimated relatedness among pairs of individuals sampled at distance <5 km (Queller–Goodnight estimator, computed with R-package ‘related’ ver. 0.8 [48]) and left only single individuals from each pair sharing the same mtDNA haplotype and relatedness significantly exceeding zero (lower 9% confidence interval of r > 0).
Samples were analysed to specifically genotype the mitochondrial region located between the tRNAleu and COII genes (originally named COI-COII intergenic region) [49,50]. PCR amplification of the sequence was performed using E2 (5′-GGCAGAATAAGTGCATTG-3′) and H2 (5′-CAATATCATTGATGACC-3′) primers following the method described in [51]. We chose the COI-COII region due to its extensive prior use in studies of A. mellifera. Its amplification, followed by restriction with DraI enzyme, has been used since the 1990s as the most popular PCR-RFLP assay to discriminate the maternal origin of honey bees [51,52]. The COI-COII region has variability which results from the presence or lack of two types of repeated, non-coding sequences, named P and Q (Figure 2).
The PCR reaction was set up in a 50 μL volume, containing 25 μL 2× Multiplex PCR Master Mix (QIAGEN, Hilden, Germany), 1 μM of each primer, 5 μL (10 ng/μL) of the template DNA and ddH20 to complete the final volume. Amplifications were conducted with PTC200 thermal cycler (MJ Research, Waltham, MA, USA) using the following profile: initial denaturation at 95 °C for 15 min, followed by 29 cycles of {a denaturation step at 94 °C for 30 s, an annealing step at 50 °C for 45 s, and an extension step at 64 °C for 1 min}, and a final extension step at 72 °C for 10 min.
The PCR products were purified using a PCR/DNA Clean-Up Purification Kit (EURx) and then sequenced bidirectionally using the Sanger method on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Forward and reverse reads were assembled into contigs using BioEdit version 7.2.5 [53]. The sequences were aligned with the MAFFT online service version 7 at https://mafft.cbrc.jp, accessed on 20 March 2021, using the FFT-NS-i algorithm [54].
The number of unique mitochondrial haplotypes (mitotypes), their frequency and genealogical relationships (based on a statistical parsimony haplotype network with a 95% connection limit and simple indel coding method [55]) were estimated using the R package ‘mitotypes’ v.1.0 [56]. Additionally, we constructed maximum-likelihood trees using MEGA X [57]. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The substitution model (Hasegawa-Kishino-Yano model, HKY [58]) applied in the analysis was selected on the basis of the Akaike information criterion with IQ-TREE 2 [59].
95% confidence limits around observed proportions of mitotypes were calculated using ‘binom.confint’ function with methods = ‘exact’ from the R package ‘binom’ [60].
The obtained sequences were compared with the GenBank database using the online version of BLAST (available at http://blast.ncbi.nlm.nih.gov, accessed on 20 March 2021). Unique mitotypes were submitted to GenBank under accession numbers MW939577-MW939620.
The relationship between the presence of a particular mitotype and geographic coordinates was explored using logistic regression (i.e., generalised linear models with a logit link function). In models, we coded mitotypes as binary variables (for example, A-lineage mitotype or non-A-lineage mitotype). Because we did not have observations for the full range of longitude, we included this variable as a categorical variable (‘transect’) with two levels (with 20° E as the demarcation value for samples from west and east).

3. Results

We successfully amplified and obtained sequences from 444 samples. Seventeen samples were rejected by microsatellite relatedness analysis, as workers could be mothered by same queen, resulting in 427 workers for further examination. In these studied samples, we identified 45 unique mitotypes (Figure 2 and Figure 3, Supplementary Material S1 and S2). The detected mitotypes could be grouped into three evolutionary lineages, depending on the presence and number of repeat motifs P, P0 and Q [49,50]: the Southeast European C-lineage (Q, N = 376); the Western and Northern European M-lineage (PQ, N = 1; PQQ, N = 36; PQQQ, N = 7); and the African A-lineage (P0Q, N = 2; P0QQ, N = 5) (Table 1, Figure 3).
The most abundant C-mitotypes accounted for 88.06% of all mitotypes (with 95% bootstrap CIs from 84.60% to 90.98%), while M-mitotypes were significantly less numerous (10.30%, 95% CIs from 7.59% to 13.59%) and occurred only north of the Carpathians (Figure 1c). The frequency of M-mitotypes decreased from north to south, while C-mitotypes showed the opposite trend (Figure 1). Moreover, logistic regression (Table 2) indicated that latitude was a significant predictor of M mitotype presence (p < 0.001). M-mitotypes were not present to the south of the Carpathians, and they occurred slightly more often in eastern Poland, where they also ranged further south (Figure 1c). The categorical west/east variable ‘transect’, however, did not prove to be a highly significant predictor for M mitotype presence (p = 0.08).
African mitotypes were found in 1.64% of studied bees (95% CIs from 0.66% to 3.35%). Bees bearing A mitotypes were detected in Romania (n = 1), Hungary (n = 3) and Poland (n = 3), without a statistically significant spatial tendency (Figure 1b,e).

4. Discussion

Our results show for the first time that almost 2% of honey bees in East-Central Europe have mitochondrial DNA of African origin. As far as we know, this type of introgression has only previously been reported in Europe in the southern-western Iberian Peninsula [30,31,32,33,34,35], Sicily [37,61], the Balearic Islands [39], Malta [38] and France [36,62].
The presence of African mitotypes in the Iberian Peninsula and Mediterranean Islands can be explained by natural gene flow due to the similarity of their current climates and the existence of land bridges connecting these regions during ice ages [30,31,32,33,34]. In France, on the other hand, African mitotypes were probably introduced by humans, as in 1990s studies of the region, these haplotypes were not detected [31,51]. Moreover, haplotype A4, which is recorded in France, has not been identified in Spain [33,34].
The presence of African haplotypes in East-Central Europe is more likely to be caused by trading. In earlier studies, no African haplotypes were found in Europe to the north and east of France [8,9,11,63]. Neither of the three African haplotypes reported here (A1e, A4 and A4s) were recorded in Iberian Peninsula [32,33,35]. Moreover, haplotype A2, which is most common in Spain [34], was not recorded in this study. Two of the African haplotypes reported here (A4, A1e) were recorded previously in Africa [64] and in Africanized bees in North America [65] and South America [66]. The third African haplotype (A4s) was most similar to one recorded again in South America [66]. Thus, the likely origin of the A-lineage bees found in our study is Sub-Saharan Africa. It is also possible that genetic material was first introduced to the Americas and later from there to Europe.
In Eastern Europe, subspecies from lineages M and C are separated from African subspecies by subspecies from lineage O [22,67]. Moreover, there is no clear pattern in the geographic distribution of the African mitotypes in the study area. Therefore, it is unlikely that natural processes caused the presence of African mitotypes in Eastern and Central Europe. It is more likely that the presence of African mitotypes in this region is related to human activities. In a few cases, African subspecies were imported to Europe for experimental purposes [68,69,70,71]. However, as far as we know, the number of colonies imported for this purpose was small and they did not survive very well [71]; therefore, it is unlikely that the African haplotypes observed here originated from those bees.
The African haplotype introgression observed in this study was most likely caused by beekeeping practices, including importation of non-native subspecies and their intensive breeding by honey bee queen breeders. Some of these breeding lines, in particular breeding lines referred to as “Buckfast” can be hybrids containing genes of African ancestry. It is generally assumed that Buckfast bees represent C-lineage [72,73,74]. However, some reports reveal that two African subspecies, A. m. sahariensis and A. m. monticola, were used in crosses to obtain the Buckfast line [75,76,77]. It has been confirmed that more than 2% of Buckfast bees contain African haplotype A1 [78]. Both A. m. sahariensis and A. m. monticola contain haplotypes A1 and A4 [51] which were recorded in this study. However, it is worth noting that Buckfast bees can be very heterogeneous and the share of African ancestry can vary greatly, as there is no central supervision over the breeding program of this strain–any beekeeper can produce hybrids and distribute them as “Buckfast bees”. The increasing popularity of Buckfast bees has been reported in Slovakia [79] and Great Britain [80], and they have also been introduced in other European countries including Poland and Hungary [81].
So far, most of the growing concern regarding the conservation of bee genetic resources in Europe has been focused on A. m. mellifera [8,9,11,82]. The predominant subspecies of the evolutionary C-lineage, especially the Italian A. m. ligustica and Carniolan bee A. m. carnica, which are strongly preferred by beekeepers, have seemed less vulnerable, and in some cases, were even perceived as a threat to other subspecies or local ecotypes of honey bees [61,83,84]. Our study, which was carried out in the northern part of the native range of the Carniolan bee, shows that this species, highly appreciated by beekeepers, may also be threatened by hybridisation with bees from A-lineage.
Bees of African origin may pose a threat, not only to genetic diversity, but also to beekeeping and public health in Europe. They are less suitable for beekeeping, because of their propensity for absconding [85]. Moreover, one of the African subspecies, A. m. capensis, can produce females from unfertilized eggs, and clones of this subspecies have caused substantial losses for the beekeeping industry in South Africa [86]. African subspecies host parasites and pathogens which are not present in most of Europe, including small hive beetle [87]. Although the imported queens are screened for diseases, some as-yet-undescribed pathogens, for example, viruses, may go undetected. In general, it is believed that Africanized bees have caused significant problems related to beekeeping in the New World ([88,89,90,91], but see [92]).
The importation of African subspecies also poses a risk to public health. It is well known that Africanised bees are more defensive in comparison to European subspecies [93]; hence, they pose a higher risk of stinging incidents and related health issues among members of the general public [94].
African honey bee subspecies have proved to be invasive in the Americas [95,96,97]. Therefore, the USA and some other countries have introduced certification procedures to detect and control the spread of Africanized bees [98,99]. In consequence, no African mitotypes have been detected in queen breeding populations from the USA [73]. As far as we know, such control was not implemented in the European Union, where it is permitted to import queens from some non-EU countries [80]. Only the health of the bees, and not their genetic origin, is verified (Commission Regulation (EU) 206/2010). Among other origins, honey bee queens are imported to the EU from Argentina [42] where Africanized bees are present [100].
It can be argued that the climate in Europe is not suitable for Africanized bees. However, some other countries with similar or colder climates have banned the import of Africanized bees [101]. For example, in Canada, imported queens need a certificate to state that they are not Africanized [102]. Moreover, a warming climate may increase the likelihood of African hybridization in Europe [103].
From an economics perspective, Africanized bees have some advantages over European subspecies, as they tolerate parasitic mite-Varroa destructor [104,105,106,107]. This tolerance can be related to both a higher resistance of bees and a lower virulence of mites [108]. It was demonstrated that tolerance could depend on the environment [87,109]. Therefore, it is possible that varroa-tolerance of Africanized bees will be less effective in the European environment. Moreover, colonies tolerating varroa are also present in Europe [110]. In general, varroa-tolerant bees, either of African or European origin, are not used on a large scale because they are less suitable for beekeeping, especially as the mite tolerance mechanism is usually due to smaller colony size and a greater tendency to swarm [110].
It should be stressed that African hybridization was detected here using mitochondrial DNA. It remains to be clarified what the proportion of African genes in the nuclear genome is. Even if the nuclear genes are largely of European origin, the presence of African mitotypes indicates a large scale import of non-native subspecies, which needs to be monitored.

5. Conclusions

The presence of African mitotypes in honey bees from East-Central Europe deserves more attention, as it may contribute to the dissemination of undesirable traits, parasites and diseases. Importation and spread of alien honey bees should be better monitored as this could be a serious threat to the sustainable apiculture.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/insects12050410/s1, Table S1: Honeybee mitotypes found in this study, File S2: Aligned sequences in FASTA format.

Author Contributions

Conceptualization, A.O. and A.T.; methodology, A.O.; validation, A.O., S.K. and A.T.; investigation, A.O., S.K. and A.T.; writing—original draft preparation, A.O.; writing—review and editing, A.T., A.O. and S.K.; visualization, A.O.; project administration, A.O.; funding acquisition, A.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science Centre in Poland, grant number UMO-2015/19/B/NZ9/03718 and by the Polish Minister of Science and Higher Education under the program “Regional Initiative of Excellence” in 2019–2022, grant number 008/RID/2018/19 (to A.O.).

Institutional Review Board Statement

Ethical review and approval were waived for this study. The study complies with the applicable laws of the countries in which the research was conducted.

Data Availability Statement

Data were submitted to the NCBI Nucleotide database at https://www.ncbi.nlm.nih.gov/nuccore/, accessed on 15 April 2021.

Acknowledgments

The authors would like to thank Katarzyna Meyza and Ewa Sztupecka for their laboratory work.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. De la Rúa, P.; Jaffé, R.; Dall’Olio, R.; Muñoz, I.; Serrano, J. Biodiversity, conservation and current threats to European honeybees. Apidologie 2009, 40, 263–284. [Google Scholar] [CrossRef] [Green Version]
  2. vanEngelsdorp, D.; Meixner, M.D. A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. J. Invertebr. Pathol. 2010, 103. [Google Scholar] [CrossRef]
  3. Zayed, A. Bee genetics and conservation. Apidologie 2009, 40, 237–262. [Google Scholar] [CrossRef] [Green Version]
  4. Taberlet, P.; Coissac, E.; Pansu, J.; Pompanon, F. Conservation genetics of cattle, sheep, and goats. Comptes Rendus Biol. 2011, 334, 247–254. [Google Scholar] [CrossRef]
  5. Groeneveld, L.F.; Lenstra, J.A.; Eding, H.; Toro, M.A.; Scherf, B.; Pilling, D.; Negrini, R.; Finlay, E.K.; Jianlin, H.; Groeneveld, E.; et al. Genetic diversity in farm animals—A review. Anim. Genet. 2010, 41, 6–31. [Google Scholar] [CrossRef] [Green Version]
  6. De la Rúa, P.; Jaffé, R.; Muñoz, I.; Serrano, J.; Moritz, R.F.A.A.; Kraus, F.B. Conserving genetic diversity in the honeybee: Comments on Harpur et al. (2012). Mol. Ecol. 2013, 22, 3208–3210. [Google Scholar] [CrossRef]
  7. Harpur, B.A.; Minaei, S.; Kent, C.F.; Zayed, A. Management increases genetic diversity of honey bees via admixture. Mol. Ecol. 2012, 21, 4414–4421. [Google Scholar] [CrossRef]
  8. Jensen, A.B.; Palmer, K.A.; Boomsma, J.J.; Pedersen, B.V. Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee, Apis mellifera mellifera, in northwest Europe. Mol. Ecol. 2005, 14, 93–106. [Google Scholar] [CrossRef] [PubMed]
  9. Pinto, M.A.; Henriques, D.; Chávez-Galarza, J.; Kryger, P.; Garnery, L.; van der Zee, R.; Dahle, B.; Soland-Reckeweg, G.; de la Rúa, P.; Dall’ Olio, R.; et al. Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: A genome-wide assessment using SNPs and mtDNA sequence data. J. Apic. Res. 2014, 53, 269–278. [Google Scholar] [CrossRef] [Green Version]
  10. Strange, J.P.; Garnery, L.; Sheppard, W.S. Persistence of the Landes ecotype of Apis mellifera mellifera in southwest France: Confirmation of a locally adaptive annual brood cycle trait. Apidologie 2007, 38, 259–267. [Google Scholar] [CrossRef] [Green Version]
  11. Oleksa, A.; Chybicki, I.; Tofilski, A.; Burczyk, J. Nuclear and mitochondrial patterns of introgression into native dark bees (Apis mellifera mellifera) in Poland. J. Apic. Res. 2011, 50, 116–129. [Google Scholar] [CrossRef] [Green Version]
  12. Parejo, M.; Wragg, D.; Gauthier, L.; Vignal, A.; Neumann, P.; Neuditschko, M. Using Whole-Genome Sequence Information to Foster Conservation Efforts for the European Dark Honey Bee, Apis mellifera mellifera. Front. Ecol. Evol. 2016, 4. [Google Scholar] [CrossRef] [Green Version]
  13. Arias, M.C.; Sheppard, W.S. Molecular phylogenetics of honey bee subspecies (Apis mellifera L.) inferred from mitochondrial DNA sequence. Mol. Phylogenet. Evol. 1996, 5, 557–566. [Google Scholar] [CrossRef] [PubMed]
  14. Garnery, L.; Cornuet, J.M.; Solignac, M. Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis. Mol. Ecol. 1992, 1, 145–154. [Google Scholar] [CrossRef]
  15. Franck, P.; Garnery, L.; Loiseau, A.; Oldroyd, B.P.; Hepburn, H.R.; Solignac, M.; Cornuet, J.M. Genetic diversity of the honeybee in Africa: Microsatellite and mitochondrial data. Heredity (Edinb) 2001, 86, 420–430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Wallberg, A.; Han, F.; Wellhagen, G.; Dahle, B.; Kawata, M.; Haddad, N.; Simões, Z.L.P.; Allsopp, M.H.; Kandemir, I.; De la Rúa, P.; et al. A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera. Nat. Genet. 2014, 46, 1081–1088. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Garnery, L.; Franck, P.; Baudry, E.; Vautrin, D.; Cornuet, J.-M.; Solignac, M. Genetic diversity of the west European honey bee (Apis mellifera mellifera and A. m. iberica) II. Microsatellite loci. Genet. Sel. Evol. 1998, 30, 49–74. [Google Scholar] [CrossRef]
  18. Tihelka, E.; Cai, C.; Pisani, D.; Donoghue, P.C.J. Mitochondrial genomes illuminate the evolutionary history of the Western honey bee (Apis mellifera). Sci. Rep. 2020, 10, 1–10. [Google Scholar] [CrossRef]
  19. Kandemir, İ.; Özkan, A.; Fuchs, S. Reevaluation of honeybee (Apis mellifera) microtaxonomy: A geometric morphometric approach. Apidologie 2011, 42, 618–627. [Google Scholar] [CrossRef] [Green Version]
  20. Francoy, T.M.; Wittman, D.; Drauschke, M.; Muller, S.; Bezerra-Laure, M.A.F.; De Jong, D.; Goncalves, L.S. Identification of Africanized honey bees through wing morphometrics: Two fast and efficient procedures. Apidologie 2008, 39, 488–494. [Google Scholar] [CrossRef] [Green Version]
  21. Tofilski, A. Using geometric morphometrics and standard morphometry to discriminate three honeybee subspecies. Apidologie 2008, 39, 558–563. [Google Scholar] [CrossRef] [Green Version]
  22. Ruttner, F. Biogeography and Taxonomy of Honeybees; Springer: Heidelberg/Berlin, Germany; New York, NY, USA, 1988; ISBN 3-540-17781-7. [Google Scholar]
  23. Meixner, M.D.; Pinto, M.A.; Bouga, M.; Kryger, P.; Ivanova, E.; Fuchs, S. Standard methods for characterising subspecies and ecotypes of Apis mellifera. J. Apic. Res. 2013, 52, 1–28. [Google Scholar] [CrossRef]
  24. Sheppard, W.S.; Meixner, M.D. Apis mellifera pomonella, a new honey bee subspecies from Central Asia. Apidologie 2003, 34, 367–375. [Google Scholar] [CrossRef] [Green Version]
  25. Meixner, M.D.; Leta, M.A.; Koeniger, N.; Fuchs, S. The honey bees of Ethiopia represent a new subspecies of Apis mellifera—Apis mellifera simensis n. ssp. Apidologie 2011, 42, 425–437. [Google Scholar] [CrossRef]
  26. Hepburn, H.R.; Radloff, S.E. Honeybees of Africa; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2013. [Google Scholar]
  27. Parker, R.; Melathopoulos, A.P.; White, R.; Pernal, S.F.; Guarna, M.M.; Foster, L.J. Ecological Adaptation of Diverse Honey Bee (Apis mellifera) Populations. PLoS ONE 2010, 5, e11096. [Google Scholar] [CrossRef]
  28. Büchler, R.; Costa, C.; Hatjina, F.; Andonov, S.; Meixner, M.D.; Le Conte, Y.; Uzunov, A.; Berg, S.; Bienkowska, M.; Bouga, M.; et al. The influence of genetic origin and its interaction with environmental effects on the survival of Apis mellifera L. colonies in Europe. J. Apic. Res. 2014, 53, 205–214. [Google Scholar] [CrossRef] [Green Version]
  29. Pinto, M.A.; Sheppard, W.S.; Johnston, J.S.; Rubink, W.L.; Coulson, R.N.; Schiff, N.M.; Kandemir, I.; Patton, J.C. Honey Bees (Hymenoptera: Apidae) of African Origin Exist in Non-Africanized Areas of the Southern United States: Evidence from Mitochondrial DNA. Ann. Entomol. Soc. Am. 2007, 100, 289–295. [Google Scholar] [CrossRef] [Green Version]
  30. Smith, D.R.; Palopoli, M.F.; Taylor, B.R.; Garnery, L.; Cornuet, J.-M.; Solignac, M.; Brown, W.M. Geographical Overlap of Two Mitochondrial Genomes in Spanish Honeybees (Apis mellifera iberica). J. Hered. 1991, 82, 96–100. [Google Scholar] [CrossRef]
  31. Miguel, I.; Iriondo, M.; Garnery, L.; Sheppard, W.S.; Estonba, A. Gene flow within the M evolutionary lineage of Apis mellifera: Role of the Pyrenees, isolation by distance and post-glacial re-colonization routes in the western Europe. Apidologie 2007, 38, 141–155. [Google Scholar] [CrossRef]
  32. Cánovas, F.; De la Rúa, P.; Serrano, J.; Galián, J.; Rúa, P.D. La Geographical patterns of mitochondrial DNA variation in Apis mellifera iberiensis (Hymenoptera: Apidae). J. Zool. Syst. Evol. Res. 2007, 46, 24–30. [Google Scholar] [CrossRef]
  33. Chávez-Galarza, J.; Garnery, L.; Henriques, D.; Neves, C.J.; Loucif-Ayad, W.; Jonhston, J.S.; Pinto, M.A. Mitochondrial DNA variation of Apis mellifera iberiensis: Further insights from a large-scale study using sequence data of the tRNAleu-cox2 intergenic region. Apidologie 2017, 48, 533–544. [Google Scholar] [CrossRef] [Green Version]
  34. Garnery, L.; Mosshine, E.H.; Oldroyd, B.P.; Cornuet, J.M. Mitochondrial DNA variation in Moroccan and Spanish honey bee populations. Mol. Ecol. 1995, 4, 465–472. [Google Scholar] [CrossRef]
  35. Pinto, M.A.; Henriques, D.; Neto, M.; Guedes, H.; Muñoz, I.; Azevedo, J.C.; de la Rúa, P. Maternal diversity patterns of Ibero-Atlantic populations reveal further complexity of Iberian honeybees. Apidologie 2013, 44, 430–439. [Google Scholar] [CrossRef] [Green Version]
  36. Benstead, E. Genetic composition and phenology of mating drone congregations in the honey bee Apis melllfera. Rev. d’Ecologie (La Terre la Vie) 2009, 64, 343–350. [Google Scholar]
  37. Franck, P.; Garnery, L.; Celebrano, G.; Solignac, M.; Cornuet, J.-M. Hybrid origins of honeybees from Italy (Apis mellifera ligustica) and Sicily (A. m. sicula). Mol. Ecol. 2000, 9, 907–921. [Google Scholar] [CrossRef] [PubMed]
  38. Zammit-Mangion, M.; Meixner, M.; Mifsud, D.; Sammut, S.; Camilleri, L. Thorough morphological and genetic evidence confirm the existence of the endemic honey bee of the Maltese Islands Apis mellifera ruttneri: Recommendations for conservation. J. Apic. Res. 2017, 56, 514–522. [Google Scholar] [CrossRef]
  39. De La Rúa, P.; Galián, J.; Serrano, J.; Moritz, R.F.A. Molecular characterization and population structure of the honeybees from the balearic islands (Spain). Apidologie 2001, 32, 417–427. [Google Scholar] [CrossRef] [Green Version]
  40. De la Rúa, P.; Serrano, J.; Galián, J. Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.). Mol. Ecol. 1998, 7, 1543–1547. [Google Scholar] [CrossRef] [PubMed]
  41. de la Rúa, P.; Galián, J.; Pedersen, B.V.; Serrano, J. Molecular characterization and population structure of Apis mellifera from Madeira and the Azores. Apidologie 2006, 37, 699–708. [Google Scholar] [CrossRef] [Green Version]
  42. Lodesani, M.; Costa, C. Bee breeding and genetics in Europe. Bee World 2003, 84, 69–85. [Google Scholar] [CrossRef]
  43. Meixner, M.D.; Costa, C.; Kryger, P.; Hatjina, F.; Bouga, M.; Ivanova, E.; Büchler, R. Conserving diversity and vitality for honey bee breeding. J. Apic. Res. 2010, 49, 85–92. [Google Scholar] [CrossRef]
  44. Bornus, I.; Demianowicz, A.; Gromisz, M. Morfometryczne badania krajowej pszczoly miodnej. Pszczel. Zesz. Nauk. 1966, 10, 1–46. [Google Scholar]
  45. Meixner, M.D.; Worobik, M.; Wilde, J.; Fuchs, S.; Koeniger, N. Apis mellifera mellifera in eastern Europe—morphometric variation and determination of its range limits. Apidologie 2007, 38, 191–197. [Google Scholar] [CrossRef]
  46. Péntek-Zakar, E.; Oleksa, A.; Borowik, T.; Kusza, S. Population structure of honey bees in the Carpathian Basin (Hungary) confirms introgression from surrounding subspecies. Ecol. Evol. 2015, 5, 5456–5467. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Oleksa, A.; Tofilski, A. Wing geometric morphometrics and microsatellite analysis provide similar discrimination of honey bee subspecies. Apidologie 2015, 46, 49–60. [Google Scholar] [CrossRef] [Green Version]
  48. Pew, J.; Wang, J.; Muir, P.; Frasier, T. Related: An R package for Analyzing Pairwise Relatedness Data Based on Codominant Molecular Markers. R Package Version 0.8/r2. 2014. Available online: https://R-Forge.R-project.org/projects/related/ (accessed on 20 March 2021).
  49. Cornuet, J.M.; Garnery, L.; Solignac, M. Putative origin and function of the intergenic region between COI and COII of Apis mellifera L. mitochondrial DNA. Genetics 1991, 128, 393–403. [Google Scholar] [CrossRef]
  50. Cornuet, J.M.; Garnery, L. Mitochondrial DNA variability in honeybees and its phylogeographic implications. Apidologie 1991, 22, 627–642. [Google Scholar] [CrossRef] [Green Version]
  51. Garnery, L.; Solignac, M.; Celebrano, G.; Cornuet, J.M. A simple test using restricted PCR-amplifled mitochondrial DNA to study the genetic structure of Apis mellifera L. Experientia 1993, 49, 1016–1021. [Google Scholar] [CrossRef]
  52. Rortais, A.; Arnold, G.; Alburaki, M.; Legout, H.; Garnery, L. Review of the DraI COI-COII test for the conservation of the black honeybee (Apis mellifera mellifera). Conserv. Genet. Resour. 2011, 3, 383–391. [Google Scholar] [CrossRef]
  53. Hall, T.T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  54. Katoh, K.; Rozewicki, J.; Yamada, K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2017. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Simmons, M.P.; Ochoterena, H. Gaps as Characters in Sequence-Based Phylogenetic Analyses. Syst. Biol. 2000, 49, 369–381. [Google Scholar] [CrossRef] [Green Version]
  56. Aktas, C. Package “haplotypes”: Haplotype Inference and Statistical Analysis of Genetic Variation; R Package ver. 1.0. R Foundation for Statistical Computing: Vienna, Austria, 2015. Available online: https://cran.r-project.org/web/packages/haplotypes/ (accessed on 20 March 2021).
  57. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  58. Hasegawa, M.; Kishino, H.; Yano, T. aki Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 1985, 22, 160–174. [Google Scholar] [CrossRef] [PubMed]
  59. Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; von Haeseler, A.; Lanfear, R. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Dorai-Raj, S. binom: Binomial Confidence Intervals for Several Parameterizations. R Package Version 1.1-1. 2014. Available online: https://CRAN.R-project.org/package=binom (accessed on 20 March 2021).
  61. Muñoz, I.; Dall’olio, R.; Lodesani, M.; De la Rúa, P. Estimating introgression in Apis mellifera siciliana populations: Are the conservation islands really effective? Insect Conserv. Divers. 2014. [Google Scholar] [CrossRef]
  62. Bertrand, B.; Alburaki, M.; Legout, H.; Moulin, S.; Mougel, F.; Garnery, L. MtDNA COI-COII marker and drone congregation area: An efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres. Mol. Ecol. Resour. 2015, 15, 673–683. [Google Scholar] [CrossRef]
  63. Božič, J.; Kordiš, D.; Križaj, I.; Leonardi, A.; Močnik, R.; Nakrst, M.; Podgoršek, P.; Prešern, J.; Sušnik Bajec, S.; Zorc, M.; et al. Novel aspects in characterisation of Carniolan honey bee (Apis mellifera carnica, Pollmann 1879). Acta Agric. Slov. 2016, 5 (Suppl. 5), 18–27. [Google Scholar]
  64. Eimanifar, A.; Kimball, R.T.; Braun, E.L.; Ellis, J.D. Mitochondrial genome diversity and population structure of two western honey bee subspecies in the Republic of South Africa. Sci. Rep. 2018, 8, 1333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Szalanski, A.L.; Magnus, R.M. Mitochondrial DNA characterization of Africanized honey bee (Apis mellifera L.) populations from the USA. J. Apic. Res. 2010, 49, 177–185. [Google Scholar] [CrossRef] [Green Version]
  66. Collet, T.; Ferreira, K.M.; Arias, M.C.; Soares, A.E.E.; Del Lama, M.A. Genetic structure of Africanized honeybee populations (Apis mellifera L.) from Brazil and Uruguay viewed through mitochondrial DNA COI–COII patterns. Hered (Edinb) 2006, 97, 329–335. [Google Scholar] [CrossRef] [Green Version]
  67. Han, F.; Wallberg, A.; Webster, M.T. From where did the Western honeybee (Apis mellifera) originate? Ecol. Evol. 2012, 2, 1949–1957. [Google Scholar] [CrossRef] [PubMed]
  68. Moritz, R.F.A.; Mautz, D. Development of Varroa jacobsoni in colonies of Apis mellifera capensis and Apis mellifera carnica. Apidologie 1990, 21, 53–58. [Google Scholar] [CrossRef] [Green Version]
  69. Köppler, K.; Vorwohl, G.; Koeniger, N. Comparison of pollen spectra collected by four different subspecies of the honey bee Apis mellifera. Apidologie 2007, 38, 341–353. [Google Scholar] [CrossRef] [Green Version]
  70. Woyke, J. Experiences with Apis mellifera adansonii in Brazil and in Poland. Apiacta 1973, 8, 115–116. [Google Scholar]
  71. Woyke, J.; Jasiński, Z.; Smagowska, B. Badania nad międzyrasowymi mieszańcami pszczoły miodnej. II. Porównanie organów rozrodczych oraz efektów sztucznego i naturalnego unasieniania pszczół różnych ras i ich mieszańców. Pszczel. Zesz. Nauk. 1974, 18, 53–75. [Google Scholar]
  72. De La Rúa, P.; Jiménez, Y.; Galián, J.; Serrano, J. Evaluation of the biodiversity of honey bee (Apis mellifera) populations from eastern Spain. J. Apic. Res. 2004, 43, 162–166. [Google Scholar] [CrossRef]
  73. Magnus, R.M.; Tripodi, A.D.; Szalanski, A.L. Mitochondrial DNA diversity of honey bees, Apis mellifera L. (Hymenoptera: Apidae) from queen breeders in the United States. J. Apic. Sci. 2011, 55, 37–47. [Google Scholar]
  74. Susnik, S.; Kozmus, P.; Poklukar, J.; Meglic, V. Molecular characterisation of indigenous Apis mellifera carnica in Slovenia. Apidologie 2004, 35, 623–636. [Google Scholar] [CrossRef] [Green Version]
  75. Kralj, J. Selection of Honey Bees with Rapid Development as a Component of Varroa Mite Resistance; University of Guelph: Guelph, ON, Canada, 1998. [Google Scholar]
  76. Österlund, E. Exploring Monticola—Efforts to find an acceptable Varroa-resistant honey bee. Am. Bee J. 1991, 131, 49–56. [Google Scholar]
  77. Okuyama, H.; Hill, J.; Martin, S.J.; Takahashi, J. The complete mitochondrial genome of a Buckfast bee, Apis mellifera (Insecta: Hymenoptera: Apidae) in Northern Ireland. Mitochondrial DNA Part B 2018, 3, 338–339. [Google Scholar] [CrossRef] [Green Version]
  78. Strange, J.P.; Garnery, L.; Sheppard, W.S. Morphological and molecular characterization of the Landes honey bee (Apis mellifera L.) ecotype for genetic conservation. J. Insect Conserv. 2008, 12, 527–537. [Google Scholar] [CrossRef]
  79. Šťastný, M.; Gasper, J.; Bauer, M. Genetic structure of Apis mellifera carnica in Slovakia based on microsatellite DNA polymorphism. Biologia (Bratisl) 2017, 72, 1341–1346. [Google Scholar] [CrossRef]
  80. Learner, J. Imports and Exports of Honey Bees. Bee Cr. 2017, 99, 7–10. [Google Scholar]
  81. Bieńkowska, M.; Wilde, J.; Panasiuk, B.; Gerula, D. Bee breeding activity in Poland. In Proceedings of the SICAMM 2018 Conference, Mustiala, Finland, 13–15 July 2018; Organized by the Finnish Beekeepers’ Association at HAMK, Häme University of Applied Sciences. p. 6. [Google Scholar]
  82. Soland-Reckeweg, G.; Heckel, G.; Neumann, P.; Fluri, P.; Excoffier, L. Gene flow in admixed populations and implications for the conservation of the Western honeybee, Apis mellifera. J. Insect Conserv. 2008, 13, 317–328. [Google Scholar] [CrossRef] [Green Version]
  83. Techer, M.A.; Clémencet, J.; Simiand, C.; Turpin, P.; Garnery, L.; Reynaud, B.; Delatte, H. Genetic diversity and differentiation among insular honey bee populations in the southwest Indian Ocean likely reflect old geographical isolation and modern introductions. PLoS ONE 2017, 12, e0189234. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. De la Rúa, P.; Serrano, J.; Galián, J. Biodiversity of Apis mellifera populations from Tenerife (Canary Islands) and hybridisation with East European races. Biodivers. Conserv. 2002, 11, 59–67. [Google Scholar] [CrossRef]
  85. Winston, M.L.; Otis, G.W.; Taylor, O.R. Absconding Behaviour of the Africanized Honeybee in South America. J. Apic. Res. 1979, 18, 85–94. [Google Scholar] [CrossRef]
  86. Martin, S.; Wossler, T.; Kryger, P. Usurpation of African Apis mellifera scutellata colonies by parasitic Apis mellifera capensis workers. Apidologie 2002, 33, 215–232. [Google Scholar] [CrossRef] [Green Version]
  87. Muli, E.; Patch, H.; Frazier, M.; Frazier, J.; Torto, B.; Baumgarten, T.; Kilonzo, J.; Kimani, J.N.; Mumoki, F.; Masiga, D.; et al. Evaluation of the Distribution and Impacts of Parasites, Pathogens, and Pesticides on Honey Bee (Apis mellifera) Populations in East Africa. PLoS ONE 2014, 9, e94459. [Google Scholar] [CrossRef]
  88. McDowell, R. The Africanized Honey Bee in the United States: What will Happen to the US Beekeeping Industry? Agricultural Economic Report; United States Dept. of Agriculture (USA): Washington, DC, USA, 1984.
  89. Taylor, O.R. African Bees: Potential Impact in the United States. Bull. Entomol. Soc. Am. 1985, 31, 15–24. [Google Scholar] [CrossRef]
  90. Winston, M.L. Killer Bees. The Africanized Honey Bee in the Americas; Harvard University Press: Cambridge, MA, USA, 1992; ISBN 067450352X. [Google Scholar]
  91. Byatt, M.A.; Chapman, N.C.; Latty, T.; Oldroyd, B.P. The genetic consequences of the anthropogenic movement of social bees. Insectes Soc. 2016, 63, 15–24. [Google Scholar] [CrossRef]
  92. Livanis, G.; Moss, C.B. The effect of Africanized honey bees on honey production in the United States: An informational approach. Ecol. Econ. 2010, 69, 895–904. [Google Scholar] [CrossRef]
  93. Villa, J.D. Defensive Behaviour of Africanized and European Honeybees at two Elevations in Colombia. J. Apic. Res. 1988, 27, 141–145. [Google Scholar] [CrossRef]
  94. Ferreira, R.S.; Almeida, R.A.M.B.; Barraviera, S.R.C.S.; Barraviera, B. Historical Perspective and Human Consequences of Africanized Bee Stings in the Americas. J. Toxicol. Environ. Health Part B 2012, 15, 97–108. [Google Scholar] [CrossRef] [PubMed]
  95. Moritz, R.F.A.; Härtel, S.; Neumann, P. Global invasions of the western honeybee (Apis mellifera) and the consequences for biodiversity. Écoscience 2005, 12, 289–301. [Google Scholar] [CrossRef]
  96. Smith, D.R. African bees in the Americas: Insights from biogeography and genetics. Trends Ecol. Evol. 1991, 6, 17–21. [Google Scholar] [CrossRef] [Green Version]
  97. Pinto, M.A. Africanization in the United States: Replacement of Feral European Honeybees (Apis mellifera L.) by an African Hybrid Swarm. Genetics 2005, 170, 1653–1665. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  98. Sylvester, H.A.; Rinderer, T.E.; Shimanuki, H. Certification options for dealing with Africanized bees. Am. Bee J. 1992, 132, 182–184. [Google Scholar]
  99. Page, R.E.; Erickson, E.H. Identification and Certification of Africanized Honey Bees. Ann. Entomol. Soc. Am. 1985, 78, 149–158. [Google Scholar] [CrossRef]
  100. Abrahamovich, A.H.; Atela, O.; De la Rúa, P.; Galián, J. Assessment of the mitochondrial origin of honey bees from Argentina. J. Apic. Res. 2007, 46, 191–194. [Google Scholar] [CrossRef]
  101. Winston, M.L.; Mitchell, S.R.; Punnett, E.N. Feasibility of Package Honey Bee (Hymenoptera: Apidae) Production in Southwestern British Columbia, Canada. J. Econ. Entomol. 1985, 78, 1037–1041. [Google Scholar] [CrossRef]
  102. Armitage, P. Regulatory Framework for the Importation of Honey Bees in Canada. BeesCene 2018, 34, 42–47. [Google Scholar]
  103. Harrison, P.A.; Berry, P.M.; Butt, N.; New, M. Modelling climate change impacts on species’ distributions at the European scale: Implications for conservation policy. Environ. Sci. Policy 2006, 9, 116–128. [Google Scholar] [CrossRef]
  104. Moretto, G.; Gonçalves, L.S.; De Jong, D.; Bichuette, M.Z. The effects of climate and bee race on Varroa jacobsoni Oud infestations in Brazil. Apidologie 1991, 22, 197–203. [Google Scholar] [CrossRef] [Green Version]
  105. Arechavaleta-Velasco, M.E.; Guzmán-Novoa, E.; Guzman-Novoa, E. Relative effect of four characteristics that restrain the population growth of the mite Varroa destructor in honey bee (Apis mellifera) colonies. Apidologie 2001, 32, 157–174. [Google Scholar] [CrossRef] [Green Version]
  106. Guzman-Novoa, E.; Emsen, B.; Unger, P.; Espinosa-Montaño, L.G.; Petukhova, T. Genotypic variability and relationships between mite infestation levels, mite damage, grooming intensity, and removal of Varroa destructor mites in selected strains of worker honey bees (Apis mellifera L.). J. Invertebr. Pathol. 2012, 110, 314–320. [Google Scholar] [CrossRef]
  107. Rosenkranz, P. Honey bee (Apis mellifera L.) tolerance to Varroa jacobsoni Oud. in South America. Apidologie 1999, 30, 159–172. [Google Scholar] [CrossRef]
  108. Carreck, N.L. Breeding honey bees for varroa tolerance. In Varroa-Still a Problem in the 21th Century; Carreck, N.L., Ed.; International Bee Research Association: Cardiff, UK, 2011; pp. 60–66. [Google Scholar]
  109. Corrêa-Marques, M.H.; De Jong, D.; Rosenkranz, P.; Gonçalves, L.S. Varroa-tolerant Italian honey bees introduced from Brazil were not more efficient in defending themselves against the mite Varroa destructor than Carniolan bees in Germany. Genet. Mol. Res. 2002, 1, 153–158. [Google Scholar] [PubMed]
  110. Locke, B. Natural Varroa mite-surviving Apis mellifera honeybee populations. Apidologie 2016, 47, 467–482. [Google Scholar] [CrossRef] [Green Version]
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Figure 1. (a–c) Distribution of bees with mtDNA from C-, A- and M-lineages in the studied area and (d–f) logistic regression curves showing relationships between mitotypes occurrence and latitude. (g) General location of the study area in Europe; numbers indicate the sample sizes obtained from different parts of study area (western and eastern transect, i.e., west or east to 20° E meridian; north and south of the Carpathians).
Figure 1. (a–c) Distribution of bees with mtDNA from C-, A- and M-lineages in the studied area and (d–f) logistic regression curves showing relationships between mitotypes occurrence and latitude. (g) General location of the study area in Europe; numbers indicate the sample sizes obtained from different parts of study area (western and eastern transect, i.e., west or east to 20° E meridian; north and south of the Carpathians).
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Figure 2. Schematic representation of COI-COII intergenic region of the mtDNA examined in this study. It is composed of partial sequences of tRNAleu and COII genes, and a variable number of non-coding sequences P and Q. Bees of C-lineage have only one Q-sequence and no P-sequences, while A- and M-lineages have up to three Q-sequences and one P-sequence, which occurs in two variants, i.e., P0 in A-lineage, and P in M-lineage.
Figure 2. Schematic representation of COI-COII intergenic region of the mtDNA examined in this study. It is composed of partial sequences of tRNAleu and COII genes, and a variable number of non-coding sequences P and Q. Bees of C-lineage have only one Q-sequence and no P-sequences, while A- and M-lineages have up to three Q-sequences and one P-sequence, which occurs in two variants, i.e., P0 in A-lineage, and P in M-lineage.
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Figure 3. (a) The haplotype network of the COI-COII region in the mtDNA, based on a statistical parsimony with a 95% connection limit and simple indel coding method [56]. The area of a circle is proportional to the logarithm of the number of observed individuals. (b) Unrooted tree of 45 unique COI-COII sequences, inferred by using the Maximum Likelihood method and Hasegawa–Kishino–Yano model [58]. The tree with the highest log likelihood (−1348.20) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2079)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 67.94% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st + 2nd + 3rd + Noncoding. There were a total of 1042 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [57].
Figure 3. (a) The haplotype network of the COI-COII region in the mtDNA, based on a statistical parsimony with a 95% connection limit and simple indel coding method [56]. The area of a circle is proportional to the logarithm of the number of observed individuals. (b) Unrooted tree of 45 unique COI-COII sequences, inferred by using the Maximum Likelihood method and Hasegawa–Kishino–Yano model [58]. The tree with the highest log likelihood (−1348.20) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2079)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 67.94% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were 1st + 2nd + 3rd + Noncoding. There were a total of 1042 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [57].
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Table
Table 1. The proportion of honeybees carrying mitotypes of the three evolutionary lineages: south-eastern European (C), African (A) and West and North European (M), estimated based on total sample of 427 individuals.
Table 1. The proportion of honeybees carrying mitotypes of the three evolutionary lineages: south-eastern European (C), African (A) and West and North European (M), estimated based on total sample of 427 individuals.
LineageSequence TypeEstimated Frequency (95% Confidence Intervals)
CQ0.881 (0.846–0.910)
all C0.881 (0.846–0.910)
AP0Q0.005 (0.001–0.017)
P0QQ0.012 (0.004–0.027)
all A0.016 (0.007–0.033)
MPQ0.002 (0.000–0.013)
PQQ0.084 (0.060–0.115)
PQQQ0.016 (0.007–0.033)
all M0.103 (0.076–0.136)
Table
Table 2. Results of logistic regression models between mitotypes and location. Dependent variables (mitotypes from C-, A- and M-lineages) were coded as binary variables (yes or no). Transect was a factor variable with two levels (western vs. eastern). Values indicate regression coefficients and their standard errors are shown in brackets.
Table 2. Results of logistic regression models between mitotypes and location. Dependent variables (mitotypes from C-, A- and M-lineages) were coded as binary variables (yes or no). Transect was a factor variable with two levels (western vs. eastern). Values indicate regression coefficients and their standard errors are shown in brackets.
C-LineageA-LineageM-Lineage
Latitude−0.383 ***−0.0440.535 ***
(0.079)(0.136)(0.108)
Transect #0.3400.962−0.615 +
(0.319)(0.852)(0.356)
Constant21.578 ***−2.441−29.638 ***
(4.155)(6.947)(5.681)
Model fit χ2 (2)35.48 ***1.6848.17 ***
Pseudo-R2 (McFadden)0.1140.0240.170
# western in relation to eastern transect, *** p < 0.001, + 0.05 < p < 0.1.
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Oleksa, A.; Kusza, S.; Tofilski, A. Mitochondrial DNA Suggests the Introduction of Honeybees of African Ancestry to East-Central Europe. Insects 2021, 12, 410. https://doi.org/10.3390/insects12050410

AMA Style

Oleksa A, Kusza S, Tofilski A. Mitochondrial DNA Suggests the Introduction of Honeybees of African Ancestry to East-Central Europe. Insects. 2021; 12(5):410. https://doi.org/10.3390/insects12050410

Chicago/Turabian Style

Oleksa, Andrzej, Szilvia Kusza, and Adam Tofilski. 2021. "Mitochondrial DNA Suggests the Introduction of Honeybees of African Ancestry to East-Central Europe" Insects 12, no. 5: 410. https://doi.org/10.3390/insects12050410

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