Molecular confirmation of a fourth lineage in honeybees from the Near East

Apidologie Molecular confirmation of a fourth lineage in honeybees from the Near East FRANCK 1 l GARNERY 0 l SOLIGNAC 0 Jean-Marie CORNUET 1 0 Laboratoire Population, Génétique et Évolution , CNRS, Gif-sur-Yvette , France 1 Laboratoire de Modélisation et Biologie Evolutive, URLB-INRA , 488 rue Croix de Lavit, 34090 Montpellier , France - The mitochondrial DNA (mtDNA) from 75 honeybee colonies from the Lebanon was characterized by DraI restriction fragment length polymorphism (RFLP) of the COI-COII intergenic region. The seven observed haplotypes were different enough from all haplotypes already known in Apis mellifera to justify their assignment to a fourth mtDNA lineage. The nucleotide sequence of a 380 base pair (bp) fragment of the NADH2 gene was determined for two haplotypes, which showed a high similarity with two published sequences from A. m. lamarkii and A. m. meda. A microsatellite analysis of a large Lebanese population sample (50 colonies, 8 loci) suggests that Near East populations are also differentiated at the nuclear level from the three previously characterized evolutionary branches of the species A. mellifera. 1. INTRODUCTION The evolutionary history of the species Apis mellifera was first addressed on morphometric grounds. Based on a multivariate analysis of an extensive set of samples, Ruttner et al. [30] proposed the first scenario in 1978. This hypothesis was long considered the standard in the field. According to these authors, the western honeybee originated in Asia and invaded Africa and Europe in three distinct evolutionary branches, a South and Central African branch (A), a North African and West European branch (M) and a North Mediterranean branch (C). This scenario was further refined by the addition of a fourth evolutionary branch, called O, which included the Near and Middle Eastern subspecies (anatoliaca, adami, cypria, syriaca, meda, caucasica and armeniaca) [29]. This scenario received support from several morphometrical studies performed on local populations of West European and North African subspecies [5–7, 25]. Morphological characters are not well suited to phylogeographical studies because they can be sensitive to environmental selection pressures. In that respect, mitochondrial DNA (mtDNA) is a much better genetic marker [2]. The first studies on mtDNA variation globally agreed with Ruttner’s scenario [ 4, 16, 31, 32 ] in that they showed the existence of three mitochondrial lineages corresponding roughly to three of the evolutionary branches (A, M and C). These three evolutionary branches were also supported by a microsatellite analysis of nine populations belonging to seven different subspecies from Europe and Africa [ 11 ]. The main difference provided by molecular markers was the inclusion of North African subspecies (A. m. intermissa and sahariensis) in the African branch instead of the West European branch [ 16 ]. The first clue to a possible fourth lineage was the deep branching of an Egyptian sample in a general phylogenetic tree of mtDNA haplotypes from many subspecies of honeybees [31]. Note that in Ruttner’s classification [29, 30], the Egyptian geographic race, A. m. lamarkii, was included in the branch A and not in the branch M. A second and more convincing clue to a fourth lineage was given by Arias and Sheppard who sequenced approximately 700 base pairs (bp) in the NADH dehydrogenase 2 region of the mtDNA from 14 different honeybee subspecies [1]. Two sequences, one common to two lamarckii colonies from Assiout (Egypt) and the other from a single meda colony from Latakya (Mediterranean coast of Syria) clustered together and away from all other sequences, supporting the hypothesis of a fourth lineage. Note also here that the sequence of a second meda colony (located a few hundred kilometers eastward from the former) unambiguously belonged to lineage C. Another subspecies considered by Ruttner as a member of branch O is A. m. anatoliaca which is distributed over most of the Turkish territory and comes into contact with meda in South Eastern Turkey. A survey of Turkish colonies showed that all of them also had mtDNA haplotypes belonging to lineage C [33] (see also [23] for isozyme analysis). A similar conclusion had already been reached from samples of the subspecies caucasica [ 16, 31 ]. Considering the very small number of colonies supporting the hypothesis of a fourth mtDNA lineage and their scattered and rather unexpected geographical distribution, further studies were needed to answer the following questions: i) how real is this putative fourth mtDNA lineage; and ii) if definite proof of its existence can be given, how does it relate to Ruttner’s branch O? In this paper, we analyzed the mtDNA variation of 75 Lebanese colonies (subspecies syriaca) with a test previously shown to be powerful in various honeybee population surveys [ 13, 18–20 ]. This test consists of analyzing the COI-COII intergenic region which is composed of a var (...truncated)