Genetic Relationships Between Commercially Produced and Natural Populations of Bombus Terrestris Dalmatinus in Terms of Mitochondrial COI and CytB

Bombus species are adapted to different climatic and environmental conditions, having a wide range of habitats from sea level up to 5800 m. Bombus terrestris (Linnaeus, 1758) shows abilities for high migration, early colony development, to benefit from a large number of flowers and to withstand low temperatures (Goka et al., 2001; Goulson et al., 2008). This species is one of the most important pollinators of both natural and cultivated plants (Michener, 2000) and constitutes about 90% of commercially produced colonies (Velthuis & van Doorn, 2006). It is now used for pollination in over fifty-seven countries including Turkey and has not been found naturally in sixteen of them. Bombus terrestris dalmatinus Dalla Torre is one of the most suitable subspecies for commercial breeding because of its high adaptability and reproductive performance under captive conditions (Velthuis & van Doorn, 2006).

The many benefits of commercially produced Bombus colonies including increased yield and quality of fruit in undergrowth cultivation and reduced use of hormones and chemical drugs have increased demand globally since the 1980s (Ono, 1998; Gürel et al., 1999; Goulson & Hanley, 2004; Hingston, 2006; Velthuis & van Doorn, 2006; Inoue et al., 2008; Williams & Osborne, 2009). However, bees emerging from commercial colonies are reported to cause problems (Goulson & Hughes, 2015) such as competition with local Bombus species for nesting and ecotypes (Goulson, 2003; Inoue & Yokoyama, 2010; Aizen, 2018) because it has escaped from greenhouses (Seabra et al., 2019) and colonized the environment (Kraus et al., 2011). Trillo et al. (2019) reported the presence of managed bumblebees escaping from greenhouses and feeding on the same flowering plants with the native ones, especially in winter. Thus, there has likely been genetic contamination due to hybridization (Rhymer & Simberloff, 1996; Ings et al., 2006; Kanbe et al., 2008; Goulson, 2010). In addition, this species, which is highly invasive (Hingston et al., 2002), is thought to cause the spread of exotic diseases (Whitehorn et al., 2013). Due to such reasons, natural bumblebee populations are thought to be negatively affected and even at risk of extinction in some places (Goulson, 2003; Cejas et al., 2018; Tsuchida et al., 2019). However, there is a lack of knowledge about the current state of most populations (Chandler et al., 2019). Genetic studies on the detection of introgression between commercial and native Bombus populations are still scarce (Kraus et al., 2011; Seabra et al., 2019; Cejas et al., 2020).

Each year, approximately two million commercially produced Bombus colonies are estimated to be shipped globally (Lecocq et al., 2016), and almost 300,000 colonies are used per year, especially in tomato pollination, in Turkey alone (Cilavdaroglu & Gurel, 2020). In greenhouses in Turkey there are no measures in place to prevent the escape of bumblebees into the natural habitat. Furthermore, as is also usually practiced in Portugal, hive boxes at the end of their useful life are left outside of greenhouses with bees still in residence (Seabra et al., 2019). In many species, mitochondrial DNA (mtDNA) is used for identifying the inter and intra specific differences due to high evolution ratio compared to nuclear DNA and to non-recombining and maternal inheritance (Rubinoff & Holland, 2005). In this context, animal mtDNA is a reliable and valid marker for population genetics and phylogenetic studies. Although the B. terrestris subspecies differ morphologically, reliable molecular tools are needed because the features are insufficient in some subtype and ecotype level definitions. mtDNA is widely used in phylogenetic studies, in particular, COI and CytB sequencing data accumulated for bumblebee species are used to reveal genetic relationships between bumblebee populations (Pedersen, 1996; Widmer & Schmid-Hempel, 1999; Yoon et al., 2003; Murray et al., 2008; Kim, 2009; Tokoro, 2010; Williams et al., 2012; Han et al., 2019).

The genetic structure of commercial colonies taken from companies in Antalya and their possible genetic effects on local genotypes are not fully known. Therefore, the genetic diversity of B. t. dalmatinus populations in habitats may be at risk of being lost without being identified. The information obtained about the genetic basis and the population structure of the geographical variation will serve as an essential resource for the use and safeguarding of bumblebees, but for now molecular genetic studies on bumblebees are limited in Turkey. In order to assess this concern, this study was carried with the use of COI and CytB haplotypes to evaluate whether there has been a hybridization between commercially reared and indigenous populations of B. t. dalmatinus in Antalya.

Both COI and CytB have a common haplotype (H1) in all populations, except in the Bayatbademler and Termessos populations, suggesting that the studied populations belong to the same subspecies, B. t. dalmatinus. Ricardo et al. (2020) noticed that despite a high intraindividual variation, most individuals of the B. morio populations in Brazilian territory had one haplotype predominant. On the other hand, there were some haplotypes that only occurred in specific groups, either company, greenhouse regions and natural fields in current study. The commercially produced individuals and the individuals collected from around the greenhouses carry more common haplotypes than the individuals from distant natural areas (Tab. 2 and Tab. 3). Seabra et al. (2019) supports this result reporting a relatively common haplotype in commercial individuals and in wild individuals collected nearby the greenhouses where the commercial hives are used. The presence of jointly shared haplotypes indicates an affinity between commercially produced individuals and individuals collected from the greenhouse environment. This result suggests that individuals can go leave greenhouses and survive around greenhouses in the natural environment. Candidate hybrids were detected in the wild, as well as putatively escaped commercial bumblebees, some being potentially fertile males (Seabra et al., 2019).

In another study, commercial individuals escaping from greenhouses were shown to hybridize with individuals in the vicinity. In addition, if natural bees are far enough from greenhouse areas, they show the ability to preserve their original genetic structure (Kraus et al., 2011). Cejas et al. (2018) showed 16s haplotype as evidence of integration between B. terrestris subspecies. As seen in Tables 2 and 3, similar haplotype numbers (COI=16 and CytB=20) were determined in this present study. The similarity of the number of haplotypes supports that we can use it as evidence to demonstrate integration among sub-populations in the Antalya region. Pedersen (1996) identified the nucleotide sequence of the fragment of 532 bp in the mitochondrial COI gene of bumblebees and revealed the phylogenetic relationships of eleven Bombus species. Tokoro et al. (2010) studied the mitochondrial COI (1048 bp) gene and identified fifteen haplotypes in order to determine the risk of the genetic breakdown in local Japanese bumblebees and the outsourced gene flow of samples collected from Japan, China and Korea. When the literature is examined, the number of haplotypes calculated in this study is notably higher than those found in other studies. This situation is due to Turkey's very different geographical and climatic conditions, the intersection of Europe, Asia and Africa in Turkey and the high genetic diversity of Turkey's B. terrestris populations.

AMOVA analysis revealed that genetic variation within populations is greater than among populations. One reason for this situation may be hybridization between populations. Individuals carrying a genomic structure from one population to another may have increased intra-population variation due to hybridization, while reducing genetic variation between populations. The B. t. dalmatinus species in Antalya may be genomically a large population and the differences between sub-populations are still low. Genetic similarities and differences were calculated through the pairwise F_ST values for mtDNA COI and CytB gene fragments of twelve populations. According to these results, individuals gathered from around the greenhouses were genetically closer to commercially produced individuals than natural ones. When Tab. 4 and Tab. 5, which show the results of AMOVA, were examined, the remarkable results were that the intra-genetic variations among the commercial populations and among the populations from the near the greenhouses were lower than the natural populations far from greenhouse areas. Understandably commercial populations produced thorugh culturing and the populations from the near the greenhouses showed a more homogeneous genetic structure. Moreira et al. (2015) investigated the genetic structure in twenty-two natural and two commercial B. terrestris L. populations using eight microsatellites and two mitochondrial genes (COI and CytB). The barrier formed by the Irish Sea and the dominant south winds is thought to prevent gene flow between Ireland and Britain, and also heterozygosity in Ireland and Isle of Man was reported to be lower than in the European continent and commercial populations. The data suggest that B. terrestris individuals in the west of Ireland, where the use of managed bumblebees is rare, associated with populations from the European mainland in terms of COI. Estoup et al. (1996) stated that B. terrestris populations in the European continent have a relatively homogeneous genetic structure due to the low overall geographical barriers. Based on this idea, we think that individuals who do not encounter any barrier in greenhouses without precautions easily affect the genetics of natural bees.

Although few studies have been conducted in Turkey, up to fifty different Bombus species have been identified. Based on world-species distribution, Turkey is understood to be an important genetic resource in terms of bumblebees (Özbek, 1997; Barkan & Aytekin, 2013; Meydan et al., 2016), but it is not clear whether there is a hybridization between natural populations and commercial ones. Thus, the genetic diversity of Turkey's local B. t. dalmatinus populations must be determined and utilized in protection strategies as the source of genes. The data revealed by this completed study will be useful for this purpose.

The Antalya province is where B. t. dalmatinus is widely found in natural habitats, and many sub-species are also encountered (Barkan & Aytekin, 2013), which is where very intensive greenhouse activities are conducted. Intensive use of commercial B. t. dalmatinus colonies are thought to increase the numbers of escaped bumblebees from greenhouses, but the spread of a large number of commercially produced individuals leads to increased competition with natural populations for nesting and feeding, spread of diseases and uncontrolled crossbreeding with local genotypes (Dafni, 1998; Goka et al., 2001; Whitehorn et al., 2013; Aizen, 2018). Furthermore, the introduction of foreign Bombus terrestris (Linnaeus) has resulted in a decline in native bumblebee populations in such countries as Japan, Chile and Argentina (Cejas et al., 2018).

Although it is difficult to prove that commercially produced bees and wild bees are hybridized, bees fleeing from greenhouses can compete with natural bees in terms of nest and nutrient sharing. The invasive potential of commercially produced bumblebees has been clearly identified in several studies. Ings et al. (2006) used a paired design to compare the nectar-foraging performance and reproductive outputs of commercial and native colonies growing under identical field conditions, and they found that the commercial colonies have high reproductive success, superior foraging ability and large colony size. In a study conducted in Turkey, Gösterit (2017) determined that the colonies founded by commercial queens produced more than twice the number of gynes (82.11±9.32) than colonies founded by native queens (32.85±3.99).

Pirounakis et al. (1998) reported that the CytB gene could be successfully used to identify geographical subspecies of bumblebees. In another study using the CytB gene, the genetic structure of B. terrestris bees on two islands in the African Gulf was found to be heterogeneous (Widmer et al., 1998). Koulianos and Hempel (2000) investigated the genetic differences and kinship relationships using mitochondrial CytB and COI genes in nineteen bombus species collected from sixteen regions in Europe and three areas in North and South America. Morath (2007) used the mtDNA CytB gene to determine genetic variation in Bombus impatiens. In addition, the genetic structure of seven mainland and island Asian populations of Bombus ignitus was investigated with the use of nine microsatellite markers, and the sequences of part of the mitochondrial CytB gene were determined. The results of this study, carried out with the samples collected from most of the companies in Antalya and the areas where the bumblebees are concentrated, overlap with many of the previous studies and summarize the genetic relationships among the populations in the Antalya region.

Although the investigation of heteroplasmy was not the subject of this study we surprisingly encountered individuals with double peaks, which were too obvious to be considered as artifacts in sequencing of both directions. (Sup. Fig. 1). As mentioned in previous similar studies (Wernick et al., 2016; Williams et al., 2019; Pizzirani et al., 2020; Ricardo et al., 2020; Tikochinski et al., 2020) double peaks on sequence traces with both alleles from one individual were identified as mitochondrial heteroplasmy and a single individual with two haplotypes. At least one heteroplasmy was detected in 93 of 157 individuals for COI and at least 105 of 170 individuals for CytB in the current study. Ricardo et al. (2020) reported that heteroplasmy was detected in individuals from all the ten sampled locations with an average of six heteroplasmic haplotypes per individual. Moreover, they found that some of these heteroplasmic haplotypes are shared between individuals from different locations. This situation regarding heteroplasmy in this study suggested that new studies are needed to prove it. Similarly, after the initial definition of heteroplasmy for Bombus morio (Francoso et al., 2016) further investigation was necessary (Ricardo et al., 2020). The bee samples from near the greenhouses have been determined to have high similarity with the commercially produced individuals. These results suggest that commercial colonies escaped from greenhouses and can cause genetic hybridization with local populations around greenhouses. However, the natural populations collected from different native habitats far away from greenhouses constitute different branches of phylogenetic tree from the commercial. This shows that they can still retain their genetic constructs, but uncontrolled and imprudent use of commercial colonies may soon be a serious threat to the original genetic stock of B. t. dalmatinus. In such areas as Antalya where hundreds of thousands of commercial colonies are produced every year and used in greenhouses, very serious measures must be taken.