Michel Solignac

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.

Precise Assessment of the Number of Patrilines and of Genetic Relatedness in Honeybee Colonies

Sociobiologists have long sought to estimate precisely the relatedness among members of social insect colonies because of the central significance of kinship in evolutionary and behavioural studies. By using microsatellites, we directly identified the 7-20 subfamilies (patrilines) present in five honeybee colonies belonging to three different subspecies (Apis mellifera mellifera, A. m. carnica and A. m. ligustica). By focusing further investigations on one A. m. mellifera colony, we showed that the genetic structure remained largely unchanged over time as long as the colony is headed by the same queen. The genetic diversity within the colony also provided a good estimate of the genetic diversity of the local honeybee population. The distribution of patrilines was significantly different in swarming workers compared with contemporaneous pupae of the original colony, probably due to unequal propensities for swarming among patrilines. Conversely, no bias in the relative proportions between worker and queen brood could be detected.

Genetic differentiation of continental and island populations of Bombus terrestris (Hymenoptera: Apidae) in Europe.

Ten microsatellite loci and a partial sequence of the COII mitochondrial gene were used to investigate genetic differentiation in B. terrestris, a bumble bee of interest for its high‐value crop pollination. The analysis included eight populations from the European continent, five from Mediterranean islands (six subspecies altogether) and one from Tenerife (initially described as a colour form of B. terrestris but recently considered as a separate species, B. canariensis). Eight of the 10 microsatellite loci displayed high levels of polymorphism in most populations. In B. terrestris populations, the total number of alleles detected per polymorphic locus ranged from 3 to 16, with observed allelic diversity from 3.8 ± 0.5 to 6.5 ± 1.4 and average calculated heterozygosities from 0.41 ± 0.09 to 0.65 ± 0.07. B. canariensis showed a significantly lower average calculated heterozygosity (0.12 ± 0.08) and observed allelic diversity (1.5 ± 0.04) as compared to both continental and island populations of B. terrestris. No significant differentiation was found among populations of B. terrestris from the European continent. In contrast, island populations were all significantly and most of them strongly differentiated from continental populations. B. terrestris mitochondrial DNA is characterized by a low nucleotide diversity: 0.18%± 0.07%, 0.20%± 0.04% and 0.27%± 0.04% for the continental populations, the island populations and all populations together, respectively. The only haplotype found in the Tenerife population differs by a single nucleotide substitution from the most common continental haplotype of B. terrestris. This situation, identical to that of Tyrrhenian islands populations and quite different from that of B. lucorum (15 substitutions between terrestris and lucorum mtDNA) casts doubts on the species status of B. canariensis. The large genetic distance between the Tenerife and B. terrestris populations estimated from microsatellite data result, most probably, from a severe bottleneck in the Canary island population. Microsatellite and mitochondrial DNA data call for the protection of the island populations of B. terrestris against importation of bumble bees of foreign origin which are used as crop pollinators.

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.

Monoandry and polyandry in bumble bees (Hymenoptera; Bombinae) as evidenced by highly variable microsatellites

Highly variable microsatellites enabled a precise assessment of the number of queen matings in the colonies of five bumble bee species. Fifteen of the sixteen microsatellites initially cloned from B. terrestris had flanking regions similar enough to allow PCR amplification on the other Bombus species analysed. The microsatellites selected for intracolony study (four per species) were characterized by a high heterozygosity (0.58–0.93) and a large number of alleles (3–18) in the local populations from which the colonies originated. A single male appeared to have inseminated the queens in the colonies of four species, B. terrestris, B. lucorum, B. lapidarius and B. pratorum, which belong to three subgenera, whereas two of the three analysed colonies of B. hypnorum were polyandrous (minimum number of two and four patrilines, respectively).

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.

Mitochondrial DNA evolution in themelanogaster species subgroup ofDrosophila

SummaryDetailed restriction maps (40 cleavage sites on average) of mitochondrial DNAs (mtDNAs) from the eight species of themelanogaster species subgroup ofDrosophila were established. Comparison of the cleavage sites allowed us to build a phylogenetic tree based on the matrix of nucleotide distances and to select the most parsimonious network. The two methods led to similar results, which were compared with those in the literature obtained from nuclear characters. The three chromosomally homosequential speciesD. simulans, D. mauritiana, andD. sechellia are mitochondrially very related, but exhibit complex phylogenetic relationships.D. melanogaster is their closest relative, and the four species form a monophyletic group (theD. melanogaster complex), which is confirmed by the shared unusual length of their mt genomes (18–19 kb). The other four species of the subgroup (D. yakuba, D. teissieri, D. erecta, andD. orena) are characterized by a much shorter mt genome (16–16.5 kb). The monophyletic character of theD. yakuba complex, however, is questionable. Two species of this complex,D. yakuba andD. teissieri, are mitochondrially indistinguishable (at the level of our investigation) in spite of their noticeable allozymic and chromosomal divergence. Finally, mtDNA distances were compared with the nuclear-DNA distances thus far established. These sequences seem to evolve at rather similar rates, the mtDNA rate being barely double that of nuclear DNA.

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.