JHR 96: 495-506 (2023) yee Se, JOURNALOR See teetenscsiett doi: 10.3897/jhr.96. 102569 RESEARCH ARTICLE ) Hymenopter a % https://jhr.pensoft.net Thelnternaionl Society of Hymenoptariss, RESEARCH Genetic variation and phylogenetic relationships of commercial populations of Bombus ignitus (Hymenoptera, Apidae) with wild populations in Eastern Asia Saeed Mohamadzade Namin', Jiaxing Huang’, Jiandong An’, Chuleui Jung'” | Agricultural Science and Technology Institute, Andong National University, Andong, Republic of Korea 2. Key Laboratory for Insect-Pollinator Biology of the Ministry of Agriculture, Institute of Apiculture, Chinese Academy of Agricultural Sciences, Beijing 100093, China 3 Department of Plant Medicals, Andong National Univer- sity, Andong, Republic of Korea Corresponding author: Chuleui Jung (cjung@andong.ac.kr) Academic editor: Jack Neff | Received 23 February 2023 | Accepted 7 June 2023 | Published 21 June 2023 https://zoobank. org/C549C40C-DE48-425B-96 1 F-02F03 18228B0 Citation: Mohamadzade Namin S, Huang J, An J, Jung C (2023) Genetic variation and phylogenetic relationships of commercial populations of Bombus ignitus (Hymenoptera, Apidae) with wild populations in Eastern Asia. Journal of Hymenoptera Research 96: 495-506. https://doi.org/10.3897/jhr.96.102569 Abstract The bumblebee, Bombus ignitus (Hymenoptera, Apidae), plays a vital role in pollination in Northeast Asia, including Korea, China, Japan, and Far East Russia. Understanding the genetic makeup of the population can aid in its conservation. This study explores the DNA barcode region of cytochrome C oxidase subunit I (COD) of B. ignitus in commercial populations from Korea and Japan. The results reveal low intraspecific genetic diversity among commercially reared populations, with a maximum sequence divergence of 0.3%. Analysis of a 458-bp region of the COI gene, including 384 previously reported sequences, identified 20 haplotypes with the highest sequence divergence of 2.01% in East Asia. Commercial populations show a genetic similarity primarily with the Japanese population. Cross-mating with native populations could result in competition and genetic contamination, leading to reduced fitness and sensitivity to future envi- ronmental conditions. Morphological similarities make monitoring of such effects challenging. This study provides a basis for further research on population studies, conservation, and commercialization of local populations of B. ignitus for better pollination services while minimizing risks of reducing genetic diversity and increasing competition between native and introduced populations. Keywords Bombus ignitus, Bumblebee, COI, wild populations, commercial population Copyright Saeed Mohamadzade Namin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 496 Saeed Mohamadzade Namin et al. / Journal of Hymenoptera Research 96: 495-506 (2023) Introduction Bumblebees, play a critical role in pollinating many agricultural crops (Grixti et al. 2009; Hou et al. 2009). Bombus ignitus is an important pollinator species throughout its distributional range, including Korea (Yoon et al. 1999), China (An et al. 2010; Zhang et al. 2018) and Japan (Asada and Ono 2000) where it is used commercially in pollination of greenhouse plants (Cooley and Vallejo-Marin 2021). It plays a signifi- cant role in the pollination of various crops such as tomatoes, strawberries, and blue- berries (Toni et al. 2020). The decline of B. ignitus populations could have adverse ef- fects on agricultural production and ecosystem health in Korea, highlighting the need for conservation measures. However, B. ignitus populations in Korea are threatened by various factors, including habitat loss, fragmentation, and pesticide exposure (Carvell et al. 2006; Wu et al., 2010). Furthermore, the introduction of non-native bumblebees such as B. terrestris for pollination purposes has further exacerbated the decline of the species through competition for food, spread of diseases, and hybridization with native populations (Dimou et al. 2008; Whittington et al. 2004; Ings et al. 2005; Kanbe et al. 2008; Tsuchida et al. 2019; Keum et al. 2020). These negative impacts have led some countries to ban the introduction of foreign bumblebee species and promote the use of local pollinators instead (Velthuis and Doorn 2006). Maintaining genetic diversity in local populations is essential for survival and fit- ness in different environmental conditions, such as climate changes and food avail- ability (Kawecki and Ebert 2004). Inbreeding can occur in populations with lower genetic diversity, leading to inbreeding depression and reduced fitness, including lower reproductive rates, slower growth rates, and less flexibility in adapting to different envi- ronmental conditions (Zayed and Packer 2005). Failure to adapt could ultimately lead to extinction. Therefore, knowledge of genetic diversity and geographic relationships of B. ignitus is crucial for long-term conservation, artificial selection, and the develop- ment of effective conservation strategies (Lee et al. 2006; Goulson et al. 2008; Zayed 2009; Whitehorn et al. 2009; Goka 2010; Habel et al. 2014). Mitochondrial DNA is inherited maternally and characterized by a relatively fast mutation rate. It exhibits high genetic variation between related species and low in- traspecific variation (Moritz et al. 1987). Among mitochondrial genes, Cytochrome oxidase subunit I (COI) has emerged as the most interesting and widely used gene due to its lower mutation rates and high incidence of nucleotide substitution at the third codon position compared to other protein-coding genes (Yi et al. 2002; McClellan 2000). Prior studies have demonstrated that Japanese populations of B. ignitus form a distinct clade that is genetically divergent from Korean and Chinese populations, using COI and microsatellites (Shao et al. 2004; Tokoro et al. 2010; Oh et al. 2013). Additionally, Han et al. (2018) used DNA barcoding part of COI to determine that the original stocks of commercially bred B. ignitus in Belgium were from Japan. ‘This study aims to assess the genetic diversity of commercial populations of B. ignitus in Korea and Japan and explore the phylogenetic relationships between these populations and wild East Asian populations of B. ignitus using both newly collected samples and previously deposited sequences in GenBank. Understanding the genetic diversity and Genetic variation of commercial Bombus ignitus 497 relationships between populations, is helpful to develop strategies for conservation, artificial selection and improving the fitness of B. ignitus populations in the face of environmental changes. Materials and methods Sample collection In this study 35 worker bees (one individual from each colony) were collected from three different commercial populations of B. ignitus in South Korea and Japan. All populations were acquired from Biobest (Belgium) between 2017 and 2018, as pre- sented in Table 1. Voucher specimens were stored at -20 °C in 100% ethanol until DNA extraction. All analyzed samples are stored in ethanol and deposited in the insect collection of Andong National University, South Korea. DNA extraction, primer, PCR, and sequencing Total DNA was extracted from the hind leg of each of the 35 samples using the DNeasy Blood and Tissue kit (Qiagen, Germany). We used Polymerase Chain Reac- tion (PCR) to amplify a 658-bp region of the COI gene that corresponds to the “DNA Barcode” region (Herbert et al. 2003). The universal primer set LCO-1490 (5’-GGT- CAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGT- GACCAAAAAATCA-3’) (Flomer et al. 1994) was used in the PCR reaction, and Ac- cuPower PCR PreMix (Bioneer, Daejeon, Korea) was used as the PCR master mix. Thermocycler conditions consisted of initial denaturation step for 5 minutes at 95 °C, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 52 °C for 30 seconds, and extension at 72 °C for 30 seconds, and a final extension step for 5 minutes at 72 °C. Sequencing was performed commercially by BIONICS (Seoul, South Korea). All sequences were generated in both directions by Sanger sequencing. Sequence analysis and genetic diversity estimates The consensus sequence was assembled from forward and reverse sequences using BI- OEDIT v7.0.5.2 (Hall 1999) and all sequences were aligned in MEGA7 (Kumar et al. 2016) using ClustalW. COI sequences of this study have been archived on the GenBank under accession numbers MN022949-MN022985 and MN423343— MN423360 (Table 1, Suppl. material 1: appendix S1). The within-locality diversity estimates in terms of haplotype diversity (H), mean number of pairwise differences (MPD), and nucleotide diversity (x) which reflect ge- netic diversity within each locality were analyzed for commercially reared populations of B. ignitus based on 658 bp of COI sequences using DNAsp v5 (Librado and Rozas 2009). Because commercial populations are isolated from each other and even from wild populations, we did not calculate the pairwise fixation indices and migration rates. 498 Saeed Mohamadzade Namin et al. / Journal of Hymenoptera Research 96: 495-506 (2023) Table |. Sampling information of the B. ignitus and GenBank accession numbers. Locality No. of samples Accession No. Commercial — Korea A* (BIKOA1-—BIKOA15) 15 MN022949-58, MN022981-85 Commercial — Korea B* (BIKOB1—BIKOB10) 10 MN022959-68 Commercial — Japan** (BIJAO1—BIJA10) 10 MN022969-76, MW080642-3 * Commercial stocks imported from a Belgian company and kept in Korea. ** Commercial stocks imported from a Belgian company and kept in Japan. Network construction In order to evaluate the phylogenetic relationship between commercial and wild pop- ulations of B. ignitus, all previously reported B. ignitus COI sequences in the nucleo- tide database of the National Centre for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov) were also included in our analysis. Only sequences that over- lapped part of the gene by 458 bp were selected for analysis. To construct the most re- liable haplotype network, all frequencies of the sequences in Tokoro et al. (2010) were included (Suppl. material 1: appendix S2). The final dataset included 419 sequences, consisting of 35 from the present study and 384 from GenBank (Suppl. material 1: appendix S1). Since B. terrestris is closely related to B. ignitus (Cameron et al., 2007), this species was used as an outgroup to root the tree. The evolutionary distances between haplo- types were calculated using Kimura's 2-parameter model (Kimura 1980) implemented in MEGA7 (Kumar et al. 2016). Bayesian analyses were conducted in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2011), using the HKY+G mutation model which were se- lected by jModeltest 2.1.10 (Darriba et al. 2012). The analyses were performed for 5 x 10° generations, with four chains each. Trees were sampled every 500 generations with 25% burn-in. A haplotype network was constructed using median-joining method (Bandelt et al. 1999) in Network software version 5.0.0.1 to infer the relationships among haplotypes and their geographical distribution. Results Sequence analysis and genetic diversity estimates The Commercial-Japan population was found to have no genetic diversity, as all ten in- dividuals possessed the same haplotype (H9). Although most individuals of Commer- cial-Korea A and Commercial-Korea B possessed H9 haplotype, two more haplotypes occurred. In terms of x and HD, all three localities displayed low estimates and the highest nucleotide diversity (x = 0.00058) and haplotype diversity (HD = 0.362) were found in Commercial-Korea A population and the estimates of Commercial-Korea B (x = 0.00031) was as low as nearly half of that obtained from Commercial-Korea A (Table 2). Genetic variation of commercial Bombus ignitus 499 Table 2. Within locality diversity estimates in commercially reared populations of B. ignitus based on 658-bp partial COI gene. Locality N NH HF HD NP MPD ™ Japan 10 1 H9;.1.0 - - - - KoreaA 15 So E15; 00067; H9, 0:87 111.0013 0.362 2 0.3809 0.00058 Korea B 10 2 FD, 0,95: 1155001 0.2 1 0.2 0.00031 — Zero estimates were obtained either by one haplotype or by in individual from corresponding locality; N: number of sampled individuals; NH: Number of haplotypes; HF: Haplotype frequency; HD: Haplotype diversity; NP: Number of polymorphic sites; MPD: Mean number of pairwise differences; x: Nucleotide diversity Genetic variation based on 458-bp of COI gene A total of 20 haplotypes was obtained from 419 studied sequences (Suppl. material 1: appendix S3). The most divergent haplotypes (H18 versus H2) differed by 9 substitu- tions (2.01%) across the 458-bp sequence region (Suppl. material 1: appendix S3). The highest within country genetic distances were found in China (8 substitutions) followed by 4 substitutions in Korean and Japanese and 2 within Belgian commercial haplotypes. Sequence alignments revealed 17 variable sites; of these, 13 substitutions (76.5%) were transitions (AG, C<>T), whereas four (23.5%) were transversions (three T H15 Figure |. Median joining haplotype network of B. ignitus based on 458-bp partial COI gene. Each circle represents one haplotype and the size is proportional to its frequency among the studied sequences. Small black dashes represent mutational steps (Belgium = commercial population). commercial purposes. (Hedrick and Kalinowski 2000; Cole 2003). The dominant haplotypes in Korea and China are H1 and H4, which differ in one nucleotide substi- tution, and other Korean haplotypes are derived from these two haplotypes (Fig. 1). These two haplotypes are also the most frequent haplotypes in China. The most wide- spread haplotypes with highest frequency are likely the oldest ones and appear in the center of haplotype network while recently derived haplotypes distributed in the re- stricted localities forming a star-shape phylogeny (Watterson and Guess 1977). Thus Haplotype 1 is the most widespread haplotype in Korea and China and Haplotype 9 is the oldest and most widespread haplotype in Japan and Belgium commercially reared populations (Fig. 1) which suggests that the commercial populations have close relat- edness to Japanese wild populations. Han et al. (2018) also revealed that the Japanese sequence from Honshu made one cluster with Belgian sequences. We found that haplotype 5 is shared between Belgian (commercial) and Korean populations and it is phylogenetically closely related to Korean haplotypes. Among the three commercial populations studied here, the Commercial-Korea A population showed the highest haplotype and nucleotide diversity (Table 2). Since high genetic diversity is known to play a crucial role in enabling populations to adapt to new environmental conditions (Suaraz and Tsutsui 2008; Handley et al. 2011), this population appears to be particularly well-suited for utilization in variable environments for the purpose of pollinating agricultural products. However, it is important to consider the potential risks associated with widespread introduction of alien populations of Bombus ignitus Genetic variation of commercial Bombus ignitus 501 H4 Kor, Chi H5 Kor, Bel an H6 Kor H16 Kor H17 Kor H1 Kor, Chi H7 Kor = H18 Kor H19 Chi H20 Chi Hi5 Jap H14 Jap H13 Jap H12 Jap H11 Bel H10 Jap H9 Jap, Bel 91 H2 Chi i H3 Chi H8 Jap Bombus terrestris (KJ837593) 0.02 Figure 2. Bayesian inference of 458-bp partial COI gene of the haplotypes of B. ignitus. Numbers repre- sent posterior probability. Bel: Belgium (commercial), Chi: China, Jap: Japan, Kor: Korea. originated from Japan, due to the potential risk of their escape from greenhouses. ‘These include the loss of genetic variation within native populations, decrease in adaptation rate, and disruption of population structure of local populations. Additionally, releasing alien populations can result in competition for food and the spread of diseases and para- sites, which may reduce the size of local populations (Dimou et al. 2008). Hybridiza- tion between alien and native populations is also a major concern, as it can alter genetic diversity, viability, and productivity of the populations. Furthermore, this phenomenon can reduce individual fitness, negatively impacting the ability of populations to adapt to future environmental changes (Rhymer and Simberloff 1996; Laikre et al. 2010). The importation of the European bumblebee, B. terrestris, to Japan as a pollinator for tomato production has resulted in negative impacts on native bumblebee popu- lations. The species is currently widespread in Hokkaido and has interfered repro- 502 Saeed Mohamadzade Namin et al. / Journal of Hymenoptera Research 96: 495-506 (2023) ductively with native bumblebees, leading to declines in populations of B. Aypocrita (Inoue et al. 2008; Kondo et al. 2009). As a result of these risks, Williams et al. (2012) proposed preventing the movement of B. patagiatus from China into Japan and B. hypocrita from Japan into China for pollination services. At the population level, the ongoing release of introduced bees could increase the dominance of the alien popula- tion in relation to local bumblebee communities. Additionally, due to the morphologi- cal similarities of the female castes of B. ignitus and B. ardens (Mohamadzade Namin et al., 2021) and similarities of alien populations of B. ignitus with native ones, moni- toring the adverse effects of the introduction on local populations can be challenging. Therefore, it is essential to carefully weigh the potential risks and benefits of introduc- ing alien bumblebees for pollination services, and to implement measures to minimize negative impacts on native bumblebee populations. While steps are taken to minimize the impact of insecticide spraying on bumblebees, releasing alien populations can have significant negative ecological and genetic consequences. ‘Thus, it is important to thor- oughly evaluate the potential risks and benefits before introducing alien populations and to take measures to minimize negative impacts on native populations. 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PNAS 102: 10742-10746. https://doi. org/10.1073/pnas.0502271102 Zhang H, Zhou Z, Huang J, Yuan X, Ding G, An J (2018) Queen traits and colony size of four bumblebee species of China. Insectes Sociaux 65: 537-547. https://doi.org/10.1007/ s00040-018-0639-2 Supplementary material | Information for COI sequences of Bombus ignitus from this study and NCBI- Genbank database Authors: Saeed Mohamadzade Namin, Jiaxing Huang, Jiandong An, Chuleui Jung Data type: phylogenetic (.docx file) Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.96.102569.suppl1