The complete mitochondrial genome of the bumblebee, Bombus hypocrita sapporensis (Insecta: Hymenoptera: Apidae) from Hokkaido Island, Japan

Abstract

The complete is mitochondrial genome of the Japanese bumblebee Bombus hypocrita sapporensis from Hokkaido Island, Japan is analysed using next generation sequencing. The mitochondrial genome of B. h. sapporensis was observed to be a circular molecule of 15,835 bp. The average AT content in the B. h. sapporensis mitochondrial genome was 85.53%. It was predicted to contain 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes, along with one A + T-rich control region. The PCGs had ATA, ATG, or ATT as the initiation codon and were terminated by the typical stop codon TAA, except for Cytb. All the tRNA genes typically formed a cloverleaf secondary structure, except for trnE, trnF, and trnS1. The molecular phylogenetic analysis indicated that the B. h. sapporensis from Hokkaido Island population was most similar to that of the geographically isolated B. h. sapporensis from Rebun Island.

Keywords:

• Bumblebee
• Illumina sequencing
• subspecies
• Bombus hypocrita
• Hokkaido

The orange-tailed bumblebee in Far East Asian continent including North China was Bombus patagiatus, widely distributed to Russia, on the other hand, the orange-tailed bumblebee in Japan was true Bombus hypocrita (Williams, An, et al. 2012; Williams, Brown, et al. 2012). Williams, An, et al. (2012) and Williams, Brown, et al. (2012) indicated that orange-tailed bumblebee was a separate species in Japan and North China in mitochondrial DNA COI gene analyses using the DNA barcoding method. In morphological taxonomic analysis, Japanese B. hypocrita is classified into two subspecies, B. hypocrita sapporensis in Hokkaido Island, and B. h. hypocrita in Honshu, Shikoku and Kyushu Islands (Sakagami and Ishikawa 1969; Ito 1998; Ito and Munakata 1979), but the phylogenetic relationship of two B. hypocrita subspecies on Japan remains uncertain (Takahashi, Nishimoto, et al. 2016; Nishimoto et al. 2017; Takeuchi et al. 2017). Here we analysed the complete mitochondrial genome of orange-tailed bumblebee B. h. sapporensis from Hokkaido Island in Japan.

Adult workers of the B. h. sapporensis on wild flowers in the Otofuke Town (42°56′43″N 143°12′06″E), Hokkaido Islands, Japan, were collected in July 2017. The collected workers were transferred immediately to 99% ethanol for mitochondrial DNA analysis (the specimen was stored in the National Museum of Nature and Science, Japan, accession number: NSMT-I-HYM 74241). Genomic DNA isolated from males was sequenced using Illumina’s MiSeq platform (Illumina, San Diego, CA). The resultant reads were assembled and annotated using the MITOS web server (Bernt et al. 2013) and Geneious R9 (Biomatters, Auckland, New Zealand). Phylogenetic analysis was performed using the TREEFINDER (Jobb 2015) based on nucleotide sequences of the 13 protein-coding genes (PCGs).

The average AT content of the B. h. sapporensis mitochondrial genome was 85.53%. Similar to the B. hypocrita mitochondrial genomes from Honshu and Rebun Islands (Takahashi, Nishimoto, et al. 2016; Nishimoto et al. 2017), the heavy strand (H-strand) was predicted to contain nine PCGs and 13 tRNA genes, and the light strand (L-strand) was predicted to contain PCGs, nine tRNA genes, and two rDNA genes (Figure 1). The genes, ATP8 and ATP6, shared 19 nucleotides, ND4 and ND4L shared one nucleotide, and ND6 and Cytb shared 13 nucleotide. Six PCGs of the B. h. sapporensis mitochondrial genome started with ATA, ATP6, COIII, ND4, and Cytb started with ATG, and COII, ND5 and ND4L started with ATT; these starting codons have been found to be common in the other Bombus mitochondrial genomes (Cha et al. 2007; Hong et al. 2008; Du et al. 2015; Takahashi, Sasaki, et al. 2017; Zhao, Huang, et al. 2017; Zhao, Wu, et al. 2017). The stop codon in each of these PCGs was TAA, except for Cytb, which had TAG, as in other bumblebees.

Figure 1. Physical map of the mitochondrial genome of Bombus hypocrita sapporensis. Genes illustrated on the outside of the main circle are encoded on the heavy (H) strand; genes on the inside of the circle are encoded on the light (L) strand. The 13 protein-coding genes are labelled in filled arrow (green), 22 tRNA genes are labelled in triangle (red), and 16S rRNA and 12S rRNA genes are labelled in grid pattern arrow (yellow).

Molecular phylogenetic analysis was constructed by applying 13 mitochondrial PCGs with 12 closely related taxa (Figure 2). The genetic distance and mutation site of the B. hypocrita in Japan taxa mitochondrial genomes were 0.0002–0.0018 and 2–20, on the other hand that of the orange-tailed bumblebee in Asia continent and the Japanese B. hypocrita were 0.0103–0.0107 and 113–117. The B. hypocrita from three Japanese Islands were shown to be a valid subspecies from the B. hypocrita in Asia continent by complete mitochondrial DNA sequence.

Figure 2. Phylogenetic relationships (maximum likelihood) among the species of the genus Bombus (Hymenoptera) based on the mitochondrial genome nucleotide sequence of the 13 protein-coding genes. Numbers beside the nodes are percentages of 1000 bootstrap values. The Apis cerana (Takahashi et al. 2016), Apis mellifera (Nakagawa et al. 2018), and Apis dorsata (Takahashi et al. 2017) were used as outgroup. Alphanumeric in parentheses indicates the DNA database of Japan accession numbers.

Acknowledgements

We thank Dr. Takuya Kiyoshi for helping our research.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This study was supported by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry [27013B] under Ministry of Agriculture, Forestry and Fisheries of Japan; the Environment Research and Technology Development of the Ministry of Environment, Japan [Rfd-1202]; a grant from Ministry of Agriculture, Forestry and fisheries of Japan (research project for monitoring and enhancement of pollinations for crop production); JSPS KAKENHI Grant No. 17H03953.