Some problems of identification of honeybee subspecies and their solution on the example of studying the Apis mellifera in Siberia

Studies of the honeybee Apis mellifera L. are carried out by both classical morphometric and molecular methods, including whole genome sequencing. Morphometric analysis has revealed thirty subspecies of A. mellifera for four evolutionary lineages that corresponded to their geographical origin. mtDNA analysis, e.g., for the variability of COI-COII locus (the sequence between the cytochrome oxidase I and cytochrome oxidase II genes), has identified three major evolutionary lineages, A, M, and C. However, this method has a limitation associated with maternal inheritance of the mitochondrial genome. The honeybee does not have sex chromosomes, so information about inheritance in the paternal line (as well as in the maternal line) can only be obtained from the analysis of autosomal loci, such as SNP (single nucleotide polymorphism) and SSR (simple sequence repeats) markers. The present work, for the first time, evaluates the informativity of morphometric and molecular methods for the identification of A. mellifera subspecies inhabiting Siberian apiaries. We have shown that the analysis of the variability of the main parameters of the wing (cubital index, hantel index, and discoidal shift) and the mtDNA COI-COII locus accurately detect the origin of the bee colony. We also studied for the first time the genetic diversity of the A. mellifera mellifera Siberian populations for microsatellite loci. Diagnostic alleles specific for subspecies and ecotypes of the honeybees have been identified to differentiate the A. m. mellifera subspecies and its ecotypes, as well as subspecies of southern origin (Carpathian bee, Carnica). This work aimed to evaluate prospects for morphometric and molecular analysis methods in differentiation of A. mellifera subspecies reared in Siberia. Honeybees from 92 apiaries in 69 settlements located in five regions of Siberia (Tomsk and Kemerovo regions, Krasnoyarsk Territory, Altai Territory, and the Altai Republic) were studied. The first stage was the investigation of worker bees from 414 bee colonies using the morphometric method and analysis of the mtDNA COI-COII locus variability. The second stage was the identification of the A. m. mellifera colonies based on a complex of SSR markers. We examined the variability of 31 microsatellite loci. To search for unique or specific SSR markers for different honeybee subspecies, we also examined the genetic diversity of two southern subspecies, A. m. carpathica and A. m. carnica (a comparison group). Population genetic parameters (allele frequency, observed and expected heterozygosity Ho and He) were calculated using GenAlEx 6.5 software (https://biology-assets.anu.edu.au/GenAlEx/). Introgression of the genes of the evolutionary lineage C into the lineage M was assessed based on the microsatellite loci polymorphism data using the STRUCTURE 2.3.4 program (https://web.stanford.edu/group/pritchardlab/home.html). It is shown that three wing parameters, i.e., cubital index, hantel index, and discoidal shift, together with mtDNA polymorphism analysis data, are necessary and sufficient for differentiation of A. mellifera subspecies. The discoidal shift parameter is one of the first morphometric trait to deviate from the breed standard values in the honeybee hybridization. Microsatellite analysis revealed loci that differentiate both subspecies of different evolutionary lineages (M and C) and different A. m. mellifera ecotypes. Loci A043, Ap081, Ap049, AT139, A113, mrjp3 , etc. can be considered as diagnostic (subspecies-specific) loci the composition and frequency of the prevailing alleles of which differ in A. m. mellifera subspecies (lineage M) and two subspecies of southern origin ( A. m. carpathica and A. m. carnica , lineage C). In A. m. mellifera honeybees, alleles 128 bp at the A043 locus, 124 bp at the Ap081 locus, 127 bp at the Ap049 locus, 190 bp at the AT139 locus, 218 bp at the A113 locus, and 529 bp at the mrjp3 occur in high frequency (0.54-0.99). In honeybees of southern origin ( A. m. carpathica and A. m. carnica ), these alleles are rarer (0.01-0.27). The microsatellite locus A008 is the most promising molecular marker to differentiate A. m. mellifera ecotypes from Siberia, the Urals and Europe (eco-specific locus). Based on the genetic diversity of Siberian honeybees for microsatellite loci, a diagnostic panel of molecular markers has been developed to differentiate subspecies and ecotypes of honeybees belonging to the evolutionary M and C lineages ( A. m. mellifera , A. m. carpathica , and A. m. carnica ).