Hybrid assembly reveals a tandem <i>trnM (tRNA-Methionine)</i> array and its origin in the <i>Vespa soror</i> mitogenome

Abstract Animal mitogenomes are often portrayed as compact and conservative, and our hybrid Illumina–Oxford Nanopore Assembly of the southern giant hornet (Vespa soror) resolved a 22 046 bp circular mitogenome with the expected gene complement and conserved order. Phylogenetic analyses of the 13 protein-coding genes recovered V. soror in its expected position within Vespa, clustering with Vespa mandarinia. Against this canonical backdrop, we have identified two large repeat systems. The control region carried six long tandem repeats, mirroring expansions in the related hornets. More strikingly, the conserved trnY–trnI–trnM–trnQ block contained a head-to-tail array of three 209 bp cassettes, each encoding a functional trnM, yielding four trnM genes in total, which is unique among Vespa. Sequence context and DNA-mode folding indicate that each cassette was a chimaera derived from the ancestral trnI–trnM–trnQ block, with a trnQ-derived fragment forming a stable hairpin. We propose that a microhomology-mediated template switch, enabled by key 8 bp microhomology (ATTTTTAT) occurring at the trnY/trnQ boundaries, seeded the first block insertion, followed by slipped-strand mispairing that expanded the tandem array. Together, these results provide a mechanism-backed example of genic repeat formation in an animal mitogenome and underscore the value of long-read sequencing in revealing hidden mitochondrial architectures.