Humans and honey bees interacted over history with consequences for distribution of the honey bee, the provider of the first sweetener or honey for humans. Honey bees are naturally distributed throughout Africa, Europe, and the Middle East. In this broad range, they have evolved into 24 subspecies, which have been grouped into three , four  or five  distinct evolutionary branches. Whitfield and colleagues  showed using SNP data that honey bees left Africa three times, first to Asia, second to Europe, and third when the African bee Apis mellifera scutellata was introduced to America in 1956. Before the introduction of the African honey bees, a subset of European subspecies and other African genotypes were introduced into the New World, first by settlers and later by beekeepers [4–6]. Since their introduction to Brazil in 1956, African bees hybridized with different European honey bees in Americas [5–13]. This was an accidental experiment, where natural hybridization between races of honey bees occurred through time. The result was the Africanized honey bees (AHB), shown to be distinct from the original African race or the European Honey Bees (EHB) in its genetics and behavioral characteristics [14–16]. On the oceanic island of Puerto Rico, AHB have been shown to have a mosaic of traits such as European-like low defensiveness behavior and African-like resistance to ectoparasites e.g. to Varroa spp. [14–16]. We dubbed these Puerto Rican bees as gAHB because of their unusual gentle behavior. AHB most likely arrived in Puerto Rico in the first half of 1990s they were reported first in 1994; , and now all feral colonies sampled are Africanized (see [14–16], 112 samples, , and this study-72 samples).
Hybridization between AHB and EHB has been previously reported in Argentina, Brazil, Perú, and Mexico [5, 6, 19, 20]. The current thinking is that when AHB arrives at a location with a resident European population, there is a large amount of hybridization, followed by elimination of European genes over time, perhaps due to selective advantage of African genes under tropical conditions . The mosaic nature of observed characteristics of gAHB may be better understood with data on population genetics [14–16], potentially providing support for different processes discussed by , extending from hybridization to founder effect to selection.
Many studies on population genetics in the Americas have been conducted [5, 6, 19–22], but in Puerto Rico, the population genetic structure of A. mellifera is unclear. Natural hybridization between EHB and AHB has been occurring over the last 17 years, and currently, based on mtDNA and morphology, Puerto Rico has a uniform geographic distribution of gAHB, both feral and domestic [14, 15]. Previous studies on the genetic structure of honey bees in Puerto Rico relied on the use of morphology, mtDNA markers, and single locus nuclear RFLP markers [14, 15]. Other approaches used worldwide to explore the population structure and hybridization zones of honey bees include morphology [13, 23] and different molecular genetic markers to study genetic variation i.e. Restriction Fragment Length Polymorphisms and Short Tandem Repeat-microsatellites, SNPs; e.g. [3, 24, 25]. To further examine the genetic structure of the Puerto Rican honey bee population, we used eight commonly selected microsatellite markers for which historical data on other Africanized and European populations exist [3, 21]. These molecular markers could help detect potential source populations for the gAHB and determine any population substructure on the island. One advantage of microsatellites is their high variability that allowed us to use individual-based measures of relatedness to study gene flow and population boundaries. In addition, the microsatellite loci could be spread across the genome, and occur at identifiable specific locations . Here we chose only eight microsatellite loci shown to be highly informative [21, 26]. More loci could be added since there are many microsatellite loci developed and published for A. mellifera (e.g. 250 reported in ).
The study of genetic structure and variation of gAHB will allow us to test two alternate hypotheses to explain the mix of AHB and EHB traits in this population: Structure and Hybridization hypotheses. Testing of these hypotheses provides a first approach to detect isolation local and global selection:
The “Structure hypothesis” is presence of different composition of European or African alleles in different subpopulations across the island of Puerto Rico. This could be due to local selection or isolation due to geographic barriers (Puerto Rico is composed of one main island and two adjacent islands and exhibit six life zones with different climate and floral phenology). In the local selection scenario, alternate European or African traits that best respond to environmental pressure under different conditions would be selected in different regions across Puerto Rico. According to this hypothesis different levels of European genes could be maintained in populations of feral bees at geographically distinct parts of the island, due to the different environmental conditions. Puerto Rico has a topography that has a strong effect on the climate, with the wetter regions on the windward, northern side of the mountains, and drier climate in the leeward rain shadow . Moreover, Puerto Rico’s six different life zones [28, 29], could provide opportunity for ecologically adapted, isolated population subdivisions. We could therefore expect European traits, for instance, on higher cooler regions of the island. In the isolation scenario, populations with high European genes in isolated regions such as surrounding islands may be observed. Structure hypothesis then predicts genetic differences among delimited geographic areas, in other words, a genetic structure for honey bee subpopulations in Puerto Rico. This will also predict a departure from Hardy-Weinberg Equilibrium (HWE) for different marker loci, indicating population substructure due to genetic drift or due to local selection on associated loci.
Alternately, the “Hybridization hypothesis” based on previous studies, states that there is only one gAHB population with large introgression of European genes, as demonstrated in one nuclear locus [14–16]. According to the hybridization hypothesis, there is no clear differentiation of subpopulations of gAHB in Puerto Rico. This could be due to a brief and effective hybridization of AHB and EHB, with a restricted flow of African genes from outside, followed by a spread of the hybrid bee across the small island. Any selective advantage for alternate AHB or EHB alleles would be present early, and spread to the whole population under a global selection scenario. In absence of a selective advantage for AHB or EHB alleles, hybridization hypothesis predicts that there will be a single, unstructured bee population with similar introgression of European genes across the sampled molecular markers. In the absence of additional Africanized bees breeding and spreading into the newly occupied territory, the sampled African alleles in Puerto Rico should not be increasing over time, but remain similar to the initial hybrid population. Alternately, if certain AHB or EHB alleles were to confer an early and fast selective advantage, hybridization hypothesis predicts that there will be different levels of introgression of EHB genes, commensurate with selective advantage at different loci, but with no current deviation from HWE. Although few known markers would be adequate to test the immediate prediction of a uniform genetic structure as per the hybridization hypothesis, early and fast hybridization would make it very unlikely to detect global selection at a resolution of 8 microsatellite markers. Future greater density marker studies, such as 1000 s of SNPs identified in SNP arrays [3, 30] or RAD-TAG sequencing with potential to generate multiples of 10000 SNPs  is more likely to provide resolution needed to determine regions of genome responding to alternate global selection pressures.