MEC1274.fm Page 1347 Monday, May 7, 2001 4:46 PM Molecular Ecology (2001) 10, 1347–1355 OriginBlackwell Science, Ltd of honeybees (Apis mellifera L.) from the Yucatan peninsula inferred from mitochondrial DNA analysis KYLEA E. CLARKE,* BENJAMIN P. OLDROYD,* J. JAVIER, G. QUEZADA-EUÁN† and THOMAS E. RINDERER‡ *School of Biological Sciences A12, University of Sydney, NSW 2006, Australia, †Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Apdo. Postal 4–116, Mérida, Yucatán 97100, México, ‡Honey-Bee Breeding, Genetics and Physiology Research Laboratory, USDA-ARS, 1157 Ben Hur Road, Baton Rouge, LA 70820, USA Abstract Honeybees (Apis mellifera L.) sampled at sites in Europe, Africa and South Ámerica were analysed using a mitochondrial DNA restriction fragment length polymorphism (RFLP) marker. These samples were used to provide baseline information for a detailed analysis of the process of Africanization of bees from the neotropical Yucatan peninsula of Mexico. Radical changes in mitochondrial haplotype (mitotype) frequencies were found to have occurred in the 13-year period studied. Prior to the arrival of Africanized bees (1986) the original inhabitants of the Yucatan peninsula appear to have been essentially of southeastern European origin with a smaller proportion having northwestern European ancestry. Three years after the migration of Africanized bees into the area (1989), only very low levels of maternal gene flow from Africanized populations into the resident European populations had occurred. By 1998, however, there was a sizeable increase in the proportion of African mitotypes in domestic populations (61%) with feral populations having 87% of mitotypes classified as African derived. The results suggest that the early stages of Africanization did not involve a rapid replacement of European with African mitotypes and that earlier studies probably overestimated the prevalence of African mitotypes. Keywords: Africanized bee, Apis mellifera, gene flow, honeybee, hybridization, mitochondrial RFLP Received 2 November 2000; revision received 14 November 2000; accepted 14 November 2000 presumably resulted in a strong correlation between the racial Introduction origin of the bees and the nationality of the settlers. Hence, The honeybee, Apis mellifera L., occurs naturally in Europe, in the South American continent, we would expect that many the Middle East and Africa (Ruttner 1988). This diverse of the early introductions would have been from Portugal range of habitats has required adaptation to a variety of and Spain, probably A.m. iberica and A.m. mellifera. More ecological and climatic conditions and historical separa- recent introductions have included commercially import- tion has caused the evolution of over 24 named subspecies ant subspecies from Italy (A.m. ligustica), Yugoslavia (A.m. (Ruttner 1988). On the basis of morphology, these sub- carnica) and the Near East (A.m. caucasica). Infamously, the species have been grouped into four distinct evolutionary African subspecies, A.m. scutellata, was released in Brazil branches, namely the African (A), western and northern in the 1950s in an attempt to improve honey production. European (M), southeastern European (C) and Near Eastern European honeybees do not flourish in the tropics, prob- (O) branches (Ruttner 1988), and molecular analyses have ably because of differences in environmental conditions to broadly supported this classification (e.g. Garnery et al. 1992; those in which they evolved (Kerr 1967; Rinderer et al. Franck et al. 2000). 1993). Populations of ‘Africanized’ honeybees have since Honeybees have also been disseminated around the world expanded from Brazil at an estimated rate of 100–300 km by humans. Initial introductions by European settlers annually. At present, African-derived populations can be found across south and central South America and in the Correspondence: K. E. Clarke. Fax: 61-29351-4771; E-mail: southern USA (Rinderer et al. 1993). A.m. scutellata displays [email protected] certain undesirable characteristics such as extreme defensive © 2001 Blackwell Science Ltd MEC1274.fm Page 1348 Monday, May 7, 2001 4:46 PM 1348 K. E. CLARKE ET AL. behaviour and excessive swarming (Rinderer et al. 1993). bees are thought to have been comprised largely of the For this reason there has been considerable interest in subspecies A.m. caucasica and later A.m. ligustica (Quezada- the migration and hybridization of these Africanized bee Euán & Hinsull 1995). Due to the importance of the bee- populations. keeping industry in the area, a large population of domestic Whether the European-derived and African-derived European bees has been established (17 colonies/km2), subspecies of honeybees are hybridizing on a massive although feral bees were apparently rare prior to the scale, or whether the Africanized honeybees are essen- appearance of Africanized bees (Quezada-Euán et al. 1996). tially maintaining their genetic integrity and thereby largely Thus in this region, low levels of European markers could displacing previously established European populations, not be due to unequal population sizes. remains controversial, as does the underlying means of Africanized bees are considered to have arrived in the dispersal of Africanized bees. Conflicting findings seem to Yucatan region in 1986 (Fierro et al. 1987). Rinderer et al. be dependent on the nature of the markers used, the clim- (1991) showed that hybridization was occurring in the atic and ecological conditions of the sampling region, and Yucatan in 1989, but that the process was by no means com- the numbers of European bees that are encountered by plete. The possibility remained that over time, postulated incoming Africanized bees. mitochondrial fitness differences (Harrison & Hall 1993) Several researchers have reported populations of African- or selection against late generation hybrids (Hall 1991) ized bees that are comprised almost exclusively of African- could result in a much more Africanized population than derived mitochondria (Hall & Muralidharan 1989; Smith was found in 1989. Furthermore, the 1989 study used a et al. 1989; Hall & Smith 1991). Nuclear markers show either mitochondrial marker that is now known to be ambiguous considerable mixing of European and Africanized popula- for African mitotypes, because some bees of western and tions (Lobo et al. 1989; Rinderer et al. 1991; Quezada-Euán northern European origin could be classified as African & Hinsull 1995; Quezada-Euán et al. 1996) or limited admix- (Smith et al. 1991). ture with only a very low frequency of European alleles Other studies in this region have revealed a consistent detected (Hall 1990; Muralidharan & Hall 1990; McMichael increase in the level of Africanization of managed colonies & Hall 1996; Suazo et al. 1998). over time (Quezada-Euán et al. 1996; Quezada-Euán & Various hypotheses for the apparent asymmetry between Medina 1998). African- and European-derived mitotypes mitochondrial and nuclear markers have been proposed. are now associated with a diverse array of morphotypes. In First, there may be selective advantages of Africanized contrast, however, some degree of gene flow was initially bees over European bees in the tropics (Page 1989; Hall detected from domestic European colonies to Africanized 1991, 1992; Rinderer et al. 1993) or large differences in feral populations, but by 1994 feral populations were essen- population densities (Page 1989; Rinderer et al. 1991; Hall tially Africanized (Quezada-Euán et al. 1996). 1992). Second, fitness differences between European and In this study, we analysed a collection obtained from African mitochondria have been postulated (Harrison & the Yucatan in 1985 and reanalysed the 1989 collection of Hall 1993). Finally, it is possible that there have been inap- Rinderer et al. (1991) using a DraI mtDNA restriction frag- propriate assumptions about the fixation of mitochondrial ment length polymorphism (RFLP) test (Garnery et al. 1993). DNA (mtDNA) markers in Old World populations of We report baseline haplotype frequencies for popula- honeybees (e.g. Schiff et al. 1994; Sheppard et al. 1999). tions obtained from throughout the putative origins of A hybrid zone between Africanized and European Yucatecan bees. We then compare these data sets with a populations is present in Argentina that corresponds to a contemporary (1998) collection of Yucatecan bees obtained change in climatic conditions (Sheppard et al. 1991). African- from identical transects to the 1989 study to ascertain the derived bees were found in the tropical north of the extent of maternal gene flow before, during and after the country where Africanized bees could be expected to have arrival of Africanized bees. a selective advantage. European-derived bees were found in the temperate south. In a transition zone between these Materials and methods two habitats, both European and African mitochondrial haplotypes (mitotypes) were associated with a range of Sampling design morphometric and allozyme phenotypes, indicating that hybridization was occurring. In May 1998, we retraced the 10 transects through the In 1989 the neotropical Yucatan peninsula in Mexico Yucatan and Quintana Roo states of the Yucatan peninsula was chosen as an area of particular interest for the study of chosen by Rinderer et al. (1991) in 1989. Collections of adult the hybridization between Africanized and European bees worker bees and drones were made at four to 10 apiaries (Rinderer et al. 1991). In this region, in contrast to other