Investigating the Phylogenetic Relationships of the Iranian Honey Bee with Other Bee Breeds Worldwide Using the Cytochrome Oxidase I Region

The honey bee is an insect belonging to the class of insects and the order of diptera, found worldwide across various geographical regions (1). Based on morphological and molecular studies, 26 bee subspecies have been identified, which fall into five evolutionary lines: The Northern and Western European line (M), Central and Southern Africa (A), the Northern Mediterranean region and Eastern Europe (C), the Eastern Mediterranean, Near East, and Eastern Middle East (O), and finally the Eastern group of Ethiopia (Y) (2, 3). Among the eight subspecies of bees living in the Middle East, one subspecies, Apis mellifera meda, is found in Iran, known as the Iranian honey bee (1). In addition to Iran, this subspecies also occurs in northern Iraq, southeastern Turkey, and northern Syria (2, 3). Morphologically, it is similar to the Italian breed. In terms of biological characteristics, it is a breed with a high reproductive capacity, the ability to collect propolis, excellent wintering ability, and high aggression, all of which distinguish it from other subspecies in the region (4).

To study different populations of organisms, several methods have been widely adopted by systematic researchers, one of which is genetic diversity (5). Genetic diversity is a crucial element for genetic progress, preservation, evolution, and adaptation to changing and diverse environmental conditions (6). Phenotypic flexibility and genetic variability in traits used to identify species can lead to misidentification. However, the use of more accurate and faster systems for identifying living organisms, including molecular identifiers, can aid in the process of sample identification (7). Among molecular markers, the mitochondrial and nuclear genomes are both very useful in studying evolution and resolving relationships between different animal populations (6). The mitochondrial genome is often preferred over the nuclear genome in animals due to its multiple copies in cells, low recombination rate, haploid inheritance, and ease of extraction (8).

One of the most suitable mitochondrial genes for research using animal molecular markers is the mitochondrial cytochrome oxidase I (COI) gene (cytochrome oxidase c subunit one), which has evolved in such a way that it can distinguish even very closely related species (9). Cytochrome oxidase is the final enzyme in the mitochondrial respiratory chain, responsible for transferring electrons from cytochrome to oxygen. It is a protein complex found in the inner mitochondrial membrane, composed of 13 protein subunits, 10 of which are encoded by the nuclear genome and the remaining 3 by the mitochondrial genome (10). Cytochrome c oxidase is an enzyme present in all living organisms (11). This fact suggests that the enzyme has ancient origins, likely first arising in a primitive organism and subsequently being passed down to all organisms during evolution across generations (12).

In honey bees, genetic studies are primarily based on the analysis of both morphological (13) and molecular data (14). These two methods are currently used as powerful tools to estimate genetic diversity and determine phylogenetic relationships between different populations of bee subspecies worldwide. While morphological characteristics are useful for phylogenetic studies, they are highly sensitive to environmental factors and changing environmental conditions. In contrast, for phylogenetic studies, enzyme digestion analysis using mitochondrial DNA information provides a better and more reliable genetic marker (15). One advantage of mitochondrial DNA over morphological data is that mitochondrial DNA is more easily analyzed in the context of phylogenetic issues. Additionally, due to maternal inheritance, lack of recombination, and low efficiency of complete repair mechanisms, mtDNA evolves more rapidly than single-copy genes in nuclear DNA. Therefore, mtDNA is particularly useful for studying phylogenetic relationships and the historical changes among bee subspecies (16).

In general, the first step in achieving targeted breeding is to determine the level of genetic diversity within populations. Based on the aforementioned content, and in order to identify genetic reserves and preserve breed diversity, the present study was conducted to investigate the genetic and phylogenetic structure of native Iranian honey bees and several important honey bee species worldwide.

Previous research on animals and various genes has concluded that the cytochrome oxidase gene is a useful and reliable marker for differentiating populations (21-23). In one study (24), COI genes were used to investigate the phylogenetic characteristics of honey bee populations. The results demonstrated the suitability of the COI gene for studying and differentiating various honey bee populations in Iran. Another study conducted by previous researchers (25) in Thailand used genetic comparisons of mitochondrial genes COI and COII to differentiate populations of the small honey bee (A. florea) and the small honey bee (A. andreniformis). The results showed that these genes are effective in distinguishing the populations of these two species. The mitochondrial COI gene was also employed in research to differentiate populations of bees collected from the northwestern Himalayan region, where the results indicated that the gene region plays an important role in separating populations and subspecies of honey bees in this area (26). In another study by previous researchers (27), the COI mitochondrial gene was used to distinguish different populations of the Apis genus. The findings confirmed that the COI gene, as a molecular marker, can be effectively used to analyze the ancestral status and evolution of Apis species and has a superior ability to differentiate between bee species.

In this study, the investigation of phylogenetic relationships showed that the genetic diversity of the evaluated ecotypes does not align with their geographical diversity. Although, in most cases, the study populations were classified based on their geographical origin, it was observed that, in some instances, two populations from geographically close areas were placed into completely separate groups in the population classification based on molecular data. For example, the ecotype of Apis mellifera meda from Iran and the ecotype of Apis andreniformis from Southeast Asia were classified into separate groups in the cluster analysis, despite both populations belonging to similar climates. Such results are also evident in the cluster analysis of previous research (24), the grouping of small honey bee populations using COI and COII mitochondrial genes in earlier studies (2, 20), the grouping of different Iranian honey bee populations using the mitochondrial ND2 gene in previous research (1), the study of the genetic diversity of bee populations in Kerman province using ISSR markers (16), and the investigation of phylogenetic relationships in Iranian honeybees using both morphological and molecular markers.

To explain cases of inconsistency between groupings based on molecular data and geographical origin, it can be suggested that these groupings may reflect the unique genetic backgrounds of these populations and the presence of specific features in their genome sequences. Additionally, placing populations from geographically distant areas into the same group based on molecular data may be due to factors such as germplasm transfer, the insufficient number of markers used in genetic diversity studies, or the broad distribution of a plant species within the studied geographical area. In other words, the geographical distance between populations cannot be considered conclusive evidence of the distance or proximity of their genetic backgrounds (28-30).

Four microsatellite loci of honey bee populations in northwestern Iran have been investigated in various studies, including the study of morphometric characteristics of honey bee (Apis mellifera L.) populations in Kermanshah province (16, 28, 31), the study of morphological diversity of the small honey bee in Iran using statistical methods such as cluster analysis and minimum variance (32), and an investigation of mitochondrial genes ND1 and ND5 to explore the separation of Iranian honey bees from global subspecies (33). Additionally, the genetic diversity of bee populations in Southeast Europe has been studied using 22 microsatellite markers (34), and the genetic diversity of honey bee populations in the Tomsk region of Russia was analyzed using COI and COII genes (35). Furthermore, the genetic diversity of Argentine honey bee populations has been examined using COI and COII genes (36). All these studies have shown that there is significant genetic diversity within the species, with notable intraspecies diversity at the DNA level. According to these researchers, factors such as colony migration, migratory beekeeping, and the random mating of queens with males from different colonies play important roles in creating diversity within honey bee populations.

The substitution values obtained in this study are consistent with those reported by other researchers, who found the highest substitution rate to be in pyrimidine bases (37). Generally, the cause of these changes can be attributed to cytosine methylation (38).

The dN/dS method is one of the key indicators used to detect the process of natural selection during gene evolution. In this method, the results of nucleotide changes that lead to amino acid changes (dN) are compared to nucleotide changes that do not affect amino acids (dS) (18, 19).

Populations that have undergone expansion, or experienced a significant increase in size, or that have been subject to directional selection, typically show negative and significant D values. On the other hand, positive and significant values of D indicate the effects of genetic drift, genetic bottlenecks, or balancing selection during the evolutionary history of the population. Genetic drift leads to increased homogeneity and the loss of alleles, particularly recessive alleles. Ultimately, these factors result in reduced diversity and a decreased ability of populations to adapt to environmental changes, a process that is especially common in smaller and more homogeneous populations (18, 39).