Diversity of the honeybee Apis mellifera L. in Tomsk region according to morphometric and molecular genetic markers

In Siberia, the honeybee was introduced about 230 years ago. It was the darkcolored forest bee Apis mellifera mellifera L., that was cultivated in Siberia as the most adapted to the harsh climatic conditions of the region. At the end of the last century, bees of the southern breeds, mainly Apis mellifera carpathica subspecies (a derivative of A. m. carnica subspecies), were actively imported to Siberia. Introgressive bee hybridization leads to the reduction of the range of native subspecies and the formation of hybrids, modifies the genetic pool of local honeybee populations leading to the loss of their genetic identity. Russia, including Siberia, still has unique abilities to preserve the aboriginal populations of the honeybee. For Siberia, such a unique subspecies is the dark-colored forest bee A. m. mellifera, which is considered endangered in Europe. At present, the knowledge of honeybee subspecies living in Siberia, including Tomsk region, is insufficient; data on the genetic diversity of honeybees are fragmentary. In this regard, the aim of this work was to identify the biological diversity of the A. mellifera honeybee living in Tomsk region using morphometric and molecular genetic markers. A total of 337 bee colonies obtained from 65 apiaries of Tomsk region were investigated using mtDNA analysis (variability of the COI-COII locus) and morphometric method (analysis of wing parameters: cubital and hantel indexes, discoidal shift) (See Fig. 1). The genetic diversity of honeybees was studied using 9 microsatellite loci (A008, AC117, A043, A113, A024, Ap243, Ap049, H110, SV185); a total of 106 bee colonies and 893 individuals were investigated. According to the analysis of variability of the COI-COII mtDNA locus, 62.9% of bee colonies were of A. m. mellifera origin on maternal line, 29.1% of bee colonies were of the origin from the southern subspecies and 8.0% were from mixed colonies. Three variants of the mtDNA COI-COII locus were registered: PQQ, PQQQ (characteristic of A. m. mellifera) and Q (characteristic of subspecies of the southern origin) (See Fig. 2). According to a morphometric study, about 56% of the studied bee colonies conformed to the A. m. mellifera standard according to the majority of morphometric parameters, but for some individual characteristics (mainly the indicator “discoidal shift”), a deviation from the values adopted for this subspecies was recorded. About 24% of the studied bee colonies are more consistent with the A. m. carpathica standard, but also have some signs characteristic of A. m. mellifera (hybrids based on the A. m. carpatica subspecies). Finally, a comparative analysis of the variability of morphometric parameters and variability of the COI-COII mtDNA locus allowed us to identify bee colonies (the so-called “inverted colonies”), which corresponded to the A. m. mellifera standard according to morphometric parameters, but had the Q variant of mtDNA (colony origin from the southern bee subspecies on the maternal line) or, on the contrary, the colonies were the A. m. carpathica subspecies according to morphometric parameters, while mtDNA was specific for A. m. mellifera (See Table 1). Consequently, the study of honeybees in Tomsk region using a comprehensive approach, including morphometric and mtDNA analysis, showed that most bee colonies are represented by hybrid forms both on the basis of A. m. mellifera subspecies and on the basis of A. m. carpathica; hybrids based on A. m. mellifera prevail (See Fig. 3). No large areas were found with a genetically homogeneous array of bees, originating from A. m. mellifera subspecies. At the same time, the apiaries, where A. m. mellifera bees are preserved and bred, were identified in some districts of Tomsk region (Molchanovsky, Chainsky, Zyryansky, Teguldetsky, Tomsky) (See Fig. 3). In order to characterize the genetic diversity of honeybees in Tomsk region, as well as to assess the process of bee hybridization, we analyzed the variability of nine microsatellite loci in honeybees (See Table 2). A comparative analysis of the variability of the studied microsatellite loci in purebred bees (A. m. mellifera and A. m. carpathica) showed differences in the spectrum and/or frequency of alleles between subspecies for most loci. In addition, for some loci (A008, A043, A113, A024, and Ap049), the predominant alleles were recorded (the frequency of their registration was more than 0.40), and the spectrum of these alleles differed in bees of different evolutionary lineages (M and C) (See Table 2). Evaluation of genetic diversity on heterozygosity of most of the studied loci revealed similar results for two bee subspecies, namely lower values of the observed heterozygosity compared with the expected heterozygosity (See Table 2). The revealed differences between honeybees of A. m. mellifera and A. m. carpathica subspecies on the variability of the studied loci were used to assess the genetic diversity of hybrid bees obtained from apiaries of Tomsk region. We have established that both in hybrids based on A. m. mellifera subspecies (variants PQQ and PQQQ of the COI-COII mtDNA locus) and in hybrids based on subspecies of the southern origin (variant Q of the COI-COII locus) the nuclear genome is more consistent with the A. m. mellifera genome in the spectrum and/or frequency of alleles of the studied DNA markers. Using the Principal Coordinate Analysis (PCoA) method (See Fig. 4 and 5), we showed that the genetic diversity of honeybees living in apiaries of Tomsk region is not determined by a single indicator (geographical localization and isolation of apiaries, the bee breed (origin), the level of gene introgression), but by the complex effect of the above factors, whose importance to a certain extent depends on human activity. Thus, the study of genetic diversity, which is determined by numerous factors, as well as evaluation of the level of introgression between aboriginal and adventive subspecies of honey bees are important to establish the effects of hybridization and to preserve the gene pool of local bee subspecies. To preserve and restore the unique gene pool of A. m. mellifera, a Coordinating Council on the problems of selection, rational use and protection of A. m. mellifera gene pool was created in 2019 in Russia. Two researchers from “Apis” Scientific and Production Center, Tomsk State University are its members. The success of measures to preserve aboriginal bee ecotypes will primarily depend on detecting and restoring the unique surviving populations, creating bee nurseries and reserves, as well as on studying the current state of various honeybee populations to understand genetic processess going on in them. The paper contains 5 Figures, 2 Tables and 46 References.

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