Lionel Garnery

Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data.

A total of 738 colonies from 64 localities along the African continent have been analysed using the DraI RFLP of the COI–COII mitochondrial region. Mitochondrial DNA of African honeybees appears to be composed of three highly divergent lineages. The African lineage previously reported (named A) is present in almost all the localities except those from north-eastern Africa. In this area, two newly described lineages (called O and Y), putatively originating from the Near East, are observed in high proportion. This suggests an important differentiation of Ethiopian and Egyptian honeybees from those of other African areas. The A lineage is also present in high proportion in populations from the Iberian Peninsula and Sicily. Furthermore, eight populations from Morocco, Guinea, Malawi and South Africa have been assayed with six microsatellite loci and compared to a set of eight additional populations from Europe and the Middle East. The African populations display higher genetic variability than European populations at all microsatellite loci studied thus far. This suggests that African populations have larger effective sizes than European ones. According to their microsatellite allele frequencies, the eight African populations cluster together, but are divided in two subgroups. These are the populations from Morocco and those from the other African countries. The populations from southern Europe show very low levels of ‘Africanization’ at nuclear microsatellite loci. Because nuclear and mitochondrial DNA often display discordant patterns of differentiation in the honeybee, the use of both kinds of markers is preferable when assessing the phylogeography of Apis mellifera and to determine the taxonomic status of the subspecies.

Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis

Variability of mitochondrial DNA (mtDNA) of the honey bee Apis mellifera L. has been investigated by restriction and sequence analyses on a sample of 68 colonies from ten different subspecies. The 19 mtDNA types detected are clustered in three major phylogenetic lineages. These clades correspond well to three groups of populations with distinct geographical distributions: branch A for African subspecies (intermissa, monticola, scutellata, andansonii and capensis), branch C for North Mediterranean subspecies (caucasica, carnica and ligustica) and branch M for the West European populations (mellifera subspecies). These results partially confirm previous hypotheses based on morphometrical and allozymic studies, the main difference concerning North African populations, now assigned to branch A instead of branch M. The pattern of spatial structuring suggests the Middle East as the centre of dispersion of the species, in accordance with the geographic areas of the other species of the same genus. Based on a conservative 2% divergence rate per Myr, the separation of the three branches has been dated at about 1 Myr BP.

A simple test using restricted PCR-amplified mitochondrial DNA to study the genetic structure ofApis mellifera L

The COI-COII intergenic region ofApis mellifera mitochondrial DNA contains an important length polymorphism based on a variable number of copies of a 192–196 bp sequence (Q) and the completer or partial deletion of 67 pb sequence (Po). This length variability has been combined with a restriction site polymorphism to produce a rapid and simple test for the characterization of mtDNA haplotypes. This test included the amplification by the polymerase chain reaction of the COI-COII region followed by aDraI restriction of the amplified fragment. In a survey of 302 colonies belonging to 12 subspecies, 21 different haplotypes have been found which have been unambiguously allocated to one of the 3 mtDNA lineages of the species. Although all colonies of lineage C exhibit the same pattern (C1), each one of lineages A and M presents up to 10 different haplotypes, opening the way to studies on the genetic structure and the evolution of a large fraction of the species. This test also differentiates southern Spanish and South African colonies, which can be of great interest for the Africanized bee problem.

THE ORIGIN OF WEST EUROPEAN SUBSPECIES OF HONEYBEES (APIS MELLIFERA): NEW INSIGHTS FROM MICROSATELLITE AND MITOCHONDRIAL DATA

Apis mellifera is composed of three evolutionary branches including mainly African (branch A), western and northern European (branch M), and southeastern European (branch C) populations. The existence of morphological clines extending from the equator to the Polar Circle through Morocco and Spain raised the hypothesis that the branch M originated in Africa. Mitochondrial DNA analysis revealed that branches A and M were characterized by highly diverged lineages implying very remote links between both branches. It also revealed that mtDNA haplotypes from lineages A coexisted with haplotypes M in the Iberian Peninsula and formed a south‐north frequency cline, suggesting that this area could be a secondary contact zone between the two branches. By analyzing 11 populations sampled along a France‐Spain/Portugal‐Morocco‐Guinea transect at 8 microsatellite loci and the DraI RFLP of the COI‐COII mtDNA marker, we show that Iberian populations do not present any trace of “africanization” and are very similar to French populations when considering microsatellite markers. Therefore, the Iberian Peninsula is not a transition area. The higher haplotype A variability observed in Spanish and Portuguese samples compared to that found in Africa is explained by a higher mutation rate and multiple and recent introductions. Selection appears to be the best explanation to the morphological and allozymic clines and to the diffusion and maintenance of African haplotypes in Spain and Portugal.

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.

Mitochondrial DNA variation in Moroccan and Spanish honey bee populations

The mitochondrial DNAs of 192 Moroccan and 173 Spanish honey bee colonies were characterized by a rapid test involving the restriction by DraI of a PCR‐fragment of the COI‐COII region. In Morocco, we found eight haplotypes, all characteristic of the African (A) lineage, suggesting that most if not all the maternal lineages of the colonies repeatedly imported from Europe over the last 150 years have not contributed mitochondrial genomes to the local population. Using two new genetic distances analogous to the shared allele distance defined for nuclear genes, we showed that Morocco was most probably colonized by two sublineages, one from the north‐east and the other one from the south of the country and that the contact zone between them extends along both sides of the Atlas range. In Spain, we found eight haplotypes characteristic of lineage A (six in common with Morocco) and four of lineage M (the West European lineage). The distribution of haplotypes of both lineages forms a gradient with c. 10% of lineage M in the south of Spain (Seville) and up to 100% in the north (San Sebastian). Three hypotheses are presented to explain the large differences of haplotype frequencies between Moroccan and lineage A Spanish colonies: a non‐Moroccan origin of lineage A in Spain, an ancient Moroccan origin or a recent Moroccan origin with a rapid shift of haplotype frequencies due to a founder effect.

Relatedness among honeybees (Apis mellifera) of a drone congregation

The honeybee (Apis mellifera) queen mates during nuptial flights, in the so–called drone congregation area where many males from surrounding colonies gather. Using 20 highly polymorphic microsatellite loci, we studied a sample of 142 drones captured in a congregation close to Oberursel (Germany). A parentage test based on lod score showed that this sample contained one group of four brothers, six groups of three brothers, 20 groups of two brothers and 80 singletons. These values are very close to a Poisson distribution. Therefore, colonies were apparently equally represented in the drone congregation, and calculations showed that the congregation comprised males that originated from about 240 different colonies. This figure is surprisingly high. Considering the density of colonies around the congregation area and the average flight range of males, it suggests that most colonies within the recruitment perimeter delegated drones to the congregation with an equal probability, resulting in an almost perfect panmixis. Consequently, the relatedness between a queen and her mates, and hence the inbreeding coefficient of the progeny, should be minimized. The relatedness among the drones mated to the same queen is also very low, maximizing the genetic diversity among the different patrilines of a colony.

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

We present a population genetic study focused on the two subspecies of the M evolutionary lineage, A. m. mellifera and A. m. iberiensis. Nuclear and mtDNA variation was analysed in 27 bee populations from the Iberian Peninsula, France and Belgium. Microsatellite data provides compelling evidence of a barrier to neutral gene flow at the Pyrenees. In addition, they suggest isolation by distance between populations of the M lineage. Mitochondrial data support the hypothesis that the Iberian Peninsula served as glacial refugia for the honeybees of western Europe. They show two paths of post-glacial re-colonization in the extremes of the Pyrenees and suggest that the western path was more significant in the post-glacial re-colonization process. Thus, we report here on three main factors for mellifera and iberiensis subspecies differentiation: the Pyrenean barrier, isolation by distance and the post-glacial re-colonization process.ZusammenfassungDie Unterarten der Honigbiene (Apis mellifera) werden in die 5 evolutionäre Linien A (African), C (northern Mediterranean), M (western Europe), O (Oriental) and Y (Yemenitica) gruppiert. Die in dieser Studie untersuchte evolutionäre Linie M enthält zwei Unterarten: A. m. mellifera wird von Frankreich bis zu den Bergen des Ural gefunden und A. m. iberiensis ist auf der iberischen Halbinsel verbreitet. Obwohl allgemein angenommen wird, dass die Pyrenäen ein bedeutendes Hindernis für den Genfluss zwischen A. m. mellifera und A. m. iberiensis darstellt, wurde ein solcher Barriereeffekt bislang nicht nachgewiesen. Die Existenz genetischer Gefälle vom Süden der Iberischen Halbinsel bis zum nördlichen Europa und eine ungewisse taxonomische Zuordnung einiger Populationen in den Pyrenäen tragen zu der Unsicherheit bezüglich der Rolle der Pyrenäen als genetische Barriere bei. Andererseits wird seit längerem angenommen, dass die Iberische Halbinsel während der Eiszeit als Refugium für die westliche Honigbiene diente (Ruttner, 1952, 1988) und eine nacheiszeitliche Wiederbesiedlung von Nordeuropa wird von mehreren Autoren unterstützt (Garnery et al., 1998a,b; Franck et al., 1998, 2000b). Gegenstand dieser Untersuchung war es, einen potentiell isolierenden Effekt der Pyrenäen nachzuweisen, neue Daten zu dem Differenzierungsprozess der zwei Unterarten beizutragen und den Ablauf der Wiederbesiedlung zu untersuchen. Wir untersuchten 1398 Völker aus 27 Populationen der Iberischen Halbinsel sowie aus Frankreich und Belgien auf Variation an 10 Mikrosatellitenloci und der COI-COII intergenischen Region der mtDNA. Wir verwendeten verschiedene Arten statistischer Analysen wie die DA genetische Distanzmatrix, neighbor-joining trees, Korrelationen erster Ordnung und partielle Korrelationen, AMOVA, Analyse räumlicher Autokorrelationen und COCOPAN für Mikrosatellitendaten. Die Ergebnisse zeigten eine Isolation zwischen den verschiedenen Populationen der M Linie durch die Entfernung auf und lieferten sehr deutliche Hinweise auf eine Barriere für den neutralen Genfluß bei den Pyrenäen. Die Verteilung der mtDNA Haplotypen bestätigte das Iberische Refugium der Westeuropäischen Honigbiene in der Eiszeit. Wir konnten zwei verschiedene Wege der nacheiszeitlichen Wiederbesiedlung von der Iberischen Halbinsel aus an den beiden Enden der Pyrenäen ableiten. Es gab deutliche Unterschiede in der Verteilung der Mitotypen zwischen den westlichen und östlichen Enden der Pyrenäen, diese legten nahe, dass der westliche Weg für den nacheiszeitlichen Widerbesiedlungsprozess wichtiger war. Nach der in dieser Untersuchung beobachteten hohen Variabilität der M Mitotypen südlich der Pyrenäen könnten diese eine nützliche genetische Ressource für die Konservation der Westeuropäischen Honigbienen darstellen. Der nacheiszeitliche Wiederbesiedlungsverlauf, die Isolation durch die Entfernung und die von den Pyrenäen gebildete Verbreitungsbarriere sind Einflüsse, die zu der Ausbildung der Unterarten A. m. mellifera und A. m. iberiensis beigetragen haben.

Both geometric morphometric and microsatellite data consistently support the differentiation of the Apis mellifera M evolutionary branch

Traditional morphometrics, allozymes, and mitochondrial data have supported a close relationship between the M branch subspecies A. m. iberiensis and the North African subspecies (A branch). However, studies using nuclear DNA markers have revealed a clear distinction between the latter and the two European M branch subspecies. In help resolve this paradox, we analyzed 663 colonies from six European and African subspecies. A geometric morphometrics approach was applied to the analysis of wing shape, and the results were compared with data of six microsatellite loci. Both data sets were found to be highly consistent and corroborated a marked divergence of West European subspecies from North African ones. This supports the hypothesis that the presence of the African lineage mitotype in Iberian honey bee populations is likely the consequence of secondary introductions, with a minimal African influence within the current Iberian genetic background. Wing geometric morphometrics appears more appropriate than mitochondrial DNA analysis or traditional morphometrics in the screening and identification of the Africanization process.

Microsatellite analysis of sperm admixture in honeybee.

Sperm usage was investigated in an instrumentally inseminated honeybee queen. Her progeny were examined in the first 3 months of the egg‐laying period using a microsatellite marker. Frequencies of different subfamilies differed significantly from one month to another. However, there was no evidence for sperm displacement or sperm precedence of a specific male in the worker progeny. The variance of subfamily proportions decreased over time suggesting that sperm admixture in the spermatheca was incomplete at the beginning of the egg‐laying period of the queen and improved progressively during the first months after mating.