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Discrimination of Iranian honeybee populations (Apis mellifera meda) from commercial subspecies of Apis mellifera L. using morphometric and genetic methods

https://doi.org/10.1016/j.aspen.2020.04.009Get rights and content

Highlights

  • Cluster analyses differentiated the commercial subspecies from Iranian honey bees.

  • Phylogenetic tree differentiated the commercial subspecies from Iranian honey bees.

  • Phylogenetic tree confirmed the results of cluster analyses.

Abstract

The morphological characters of honeybees have an important role for discriminating honeybee subspecies. In the present research, Iranian populations of honeybee (Apis mellifera) were collected from 19 areas in Iran. The samples were collected from stationary beekeeping sites. Moreover, pictures of honeybee forewings held in the Bee Data Bank in Oberursel were compared with Iranian honeybee populations. 19 morphological characters were measured for each forewing of worker honeybee to evaluate differentiation of Iranian honeybee populations from the commercial honeybee subspecies A. m. mellifera, A. m. carnica, A. m. caucasica and A. m. ligustica. Additionally, part of the tRNAleu gene, an intergenic region and part of COII was used to confirm differentiation of the commercial subspecies from Iranian honeybee populations. Results of the cluster analyses showed that 19 morphological characters of forewings differentiated Iranian populations from the commercial subspecies. Moreover, the phylogenetic tree of part of the tRNAleu gene, an intergenic region and part of COII differentiated the commercial subspecies from Iranian honeybee populations. Results of the discriminant function analyses (DFA) indicated that the references samples of A. m. meda overlapped with Iranian populations.

Introduction

The native distribution of the honeybee (Apis mellifera) includes Europe, Africa, and the Middle East. It is commonly assumed that A. mellifera initially evolved in Asia and expanded into Europe and Africa (Cornuet and Garnery, 1991, Wallberg et al., 2014, Han et al., 2012, Ruttner, 1988, Whitfield et al., 2006). There are at least 31 A. mellifera subspecies in the world (Meixner et al., 2011, Ruttner, 1988, Chen et al., 2016, Sheppard and Meixner, 2003, Engel, 1999). A. mellifera subspecies are classified using standard morphometric, geometric morphometric and molecular techniques. The honeybee subspecies of A. mellifera L. were classified into four evolutionary lineages using standard morphometrics including C lineage (ligustica and carnica), O lineage (meda, anatoliaca, caucasica and syriaca), A lineage (jemenitica, scutellata, capennsis, lamarckii, intermissa and monticola), and M lineage (mellifera) (Ruttner, 1988). Moreover, the researchers proposed additional evolutionary lineages including lineages A (capensis, scutellata, adansonii, iberiensis and intermissa), C (meda, caucasica, ligustica, anatoliaca and carnica), M (mellifera), and Z subgroup (syriaca, lamarckii and jemenitica) based on part of COII and COI-COII intergenic region (Arias and Sheppard, 1996, Franck et al., 2000, Cridland et al., 2017, Alburaki et al., 2011). Different methods of DNA analyses such as mtDNA (Arias and Sheppard, 1996), RFLP (Martimianakis et al., 2011), and microsatellite DNA (Oleksa and Tofilski, 2014) have been used for discrimination of honeybee subspecies and populations. Honeybee subspecies can be very precisely discriminated using molecular methods (Whitfield et al., 2006), but those methods are not easily accessible to beekeepers because the molecular methods are expensive. Therefore morphometric methods have been widely used to identify honeybee subspecies (Tofilski, 2008).

Moreover, the geometric morphometric method has been applied to discriminate subspecies and populations of A. mellifera (Tofilski, 2008, Miguel et al., 2011). Traditionally, identification of the honeybee subspecies of A. mellifera L. has been based on the standard morphometric method (Ruttner, 1988, Sheppard et al., 1997, Engel, 1999, Sheppard and Meixner, 2003). Ruttner (1988) focused on seven morphological characters. Recently, some researchers applied forewing morphological characters (Uzunov et al., 2009, Tofilski, 2008, Ozbakir and Firatli, 2014, Shaibi et al., 2009, Strange et al., 2008, Dukku, 2016, Pourelmi and Fuchs, 2016).

As a consequence of gene flow between native and commercial honey bee populations, native populations have been considered to be extinct in many parts of Europe. In many countries, there is a kind of certification for local honey bees. The morphological and DNA analysis have been used to differentiate the subspecies of the native honey bee (Bouga et al., 2011, Ivanova et al., 2010). Foreign queens (A. mellifera) were imported to Iran from 1961 to 1989. Fortunately, since 1989, importation of honeybee queens has been prohibited. The introduction of Foreign queens can lead to hybridization with native populations (Zayed, 2009). Unfortunately, the beekeepers have recently imported commercial honeybee subspecies A. m. carnica, A. m. ligustica, A. m. caucasica and A. m. mellifera which has led to hybridized native Iranian populations. Therefore, the native subspecies has been seriously threatened (Dadgostar et al., 2019). The native honeybees should be preserved in stationary beekeeping sites and introduced to agricultural research centers. The researchers can monitor stationary beekeeping sites for future breeding programs because the mating process of honeybee queens can be controlled. It is important that we identify areas where the native subspecies is concentrated because useful characteristics can be transferred in future breeding programs (Bouga et al., 2011, Ebadi and Ahmadi, 2016).

Ruttner et al., (1985) studied the dispersion of A. m. meda in Iran confirming that this subspecies is native to Iran. They collected and identified the populations of A. m. meda from Iran: the subtropical coast of the Caspian Sea (Mazandaran); Northeast Iran (Mashad); West and Central, (Azerbaijan-Iranian highlands) and Southeast Iran (Kerman). Additionally they identified subspecies of A. m. meda from Southeast Anatolia (Turkey) and Iraq. The Iranian populations of honeybee have been studied using the geometric morphometric method (Gomeh et al., 2015, Dolati et al., 2013, Ozbakir et al., 2012). Researchers have studied populations of the Iranian honeybees in the south coast of the Caspian Sea, the Western and Central areas, the South and Northern areas of Iran using microsatellite markers, mtDNA (Modaber et al., 2019, Ozdil et al., 2009, Ozdil et al., 2012), allozymes (Kandemir et al., 2004) and ISSR markers (Rahimi et al., 2016). Iranian populations of honeybee have also been studied using standard morphometric methods (Mostajeran et al., 2006, Gomeh et al., 2015, Ruttner et al., 2000, Pourelmi and Fuchs, 2016, Kence et al., 2009).

The purpose of this study was to compare the efficiency of 19 standard morphometric forewing characters and part of the tRNAleu gene, an intergenic region and part of COII to differentiate Iranian honeybee populations from commercial honeybee subspecies such as A. m. carnica, A. m. ligustica, A. m. caucasica and A. m. mellifera. We aimed to investigate the trace of commercial subspecies in the native Iranian population.

Section snippets

Material and methods

The Iranian populations of honeybees (A. mellifera) were collected from stationary beekeeping sites from 19 areas in Iran. 10 colonies were selected each area. 30 honeybees were collected per colony. 25 honeybees were used in the analysis for each area. The worker honeybees were collected and killed with chloroform and preserved in 70% ethanol. The right forewings of worker honeybees (19 sampled areas) were separated and mounted using Canada Balsam (Table 1). Digital photos were taken from the

Results

The morphological characteristics passed Kolmogorov-Smirnov’s normality test (P > 0.05). The Mahalanobis distances among the 19 Iranian populations and the reference samples of A. m. meda were compared. The smallest Mahalanobis distance was observed between the Iranian population from Bandar-Abbas and the reference samples of A. m. meda (d2 = 8.84). The maximum Mahalanobis distance was seen between the Iranian population from Firuzkuh and the reference samples of A. m. meda (d2 = 18.35).

Discussion

In the present research, the researchers attempted to find stationary beekeeping sites because there is more chance to find the native subspecies of Iran, A. m. meda. The native honeybees should be preserved for future breeding programs. The stationary beekeeping sites should be remained in outside the flight distance from migratory colonies because the mating process of honeybee queens should be controlled in breeding programs (Bouga et al., 2011). In the present research, Results of the DFA

Conclusions

The results of cluster analyses using the 19 morphological forewing characters differentiated the commercial subspecies of A. m. ligustica, A. m. carnica, A. m. mellifera and A. m. caucasica from Iranian honeybee populations. Moreover in present research, the results of phylogenetic tree (using part of the tRNAleu gene, an intergenic region and part of COII) confirmed the results of cluster analyses. The phylogenetic tree differentiated commercial subspecies from the native Iranian populations.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors thank the Entomology Department, Faculty of Agriculture, University of Kurdistan, Iran, for their financial assistance. Moreover, the authors thank Professor Stefan Fuchs, who prepared the reference samples of the honeybee subspecies from the Oberursel Institute, Germany.

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    • Evaluation of the efficacy of mitochondrial ATP 6 and 8, Cyt b and 16S rDNA genes for differentiation of Iranian honeybees (Apis mellifera meda) from commercial subspecies of Apis mellifera L. and comparison with geometric morphometric method

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      Citation Excerpt :

      Researchers have examined the genetic variations of Iranian honeybees (A. m. meda) using COII and tRNAleu-COII intergenic region (Kence et al., 2009; Ozdil et al., 2009b; Rahimi, 2015; Modaber et al., 2019), ND2 (Mohammadi et al., 2018), microsatellite markers (Rahimi et al., 2016; Sahebzadeh et al., 2017) and allozymes (Kandemir et al., 2004). Moreover, Iranian honeybees have been evaluated using morphometric methods (Farshineh Adl et al., 2007; Dolati et al., 2013; Ozbakir and Firatli, 2014; Gomeh et al., 2015; Salehi and Nazemi-Rafie, 2020). The objective of the current research was assessing the efficacy of the ATP 6 and 8, Cyt b and 16S rDNA genes as barcoding candidate genes for differentiating Iranian honeybee populations from the most common commercial subspecies (A. m. carnica and A. m. ligustica).

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