Phylogenetic analysis of the complete mitochondrial genome of the Japanese peacock butterfly Aglais io geisha (Stichel 1907) (Insecta: Lepidoptera: Nymphalidae)

Abstract The peacock butterfly Aglais io (Linnaeus, 1758) (Nymphalidae: Nymphalinae: Nymphalini) is a colorful and charismatic flagship butterfly species whose range spans from the British Isles and Europe through temperate Asia and the Far East. In Europe, it has been used as a model species for studying the effects of GMO maize pollen on caterpillar growth and survivorship. The Japanese subspecies, Aglais io geisha (Stichel 1907), is not as well studied as its European counterpart. Genome skimming by Illumina sequencing allowed the assembly of a complete circular mitochondrial genome (mitogenome) of 15,252 bp from A. io geisha consisting of 80.6% AT nucleotides, 13 protein-coding genes, 22 tRNAs, two rRNAs, and a control region in the gene order typical of butterfly species. Aglais io geisha COX1 gene features an atypical start codon (CGA) while COX1, COX2, CYTB, ND1, ND3, ND4, and ND5 display incomplete stop codons finished by the addition of 3’ A residues to the mRNA. Bayesian phylogenetic reconstruction places A. io geisha within a clade with European A. io mitogenomes in the tribe Nymphalini, which is consistent with previous phylogenetic hypotheses.

The peacock butterfly Aglais io (Linnaeus, 1758) (Nymphalidae: Nymphalinae: Nymphalini) is an indicator species for studying the effects of GMO maize pollen on non-targeted insects in Europe (Arpaia et al. 2018;Leclerc et al. 2018). The natural range of A. io includes the British Isles, Europe, temperate Asia, and the Far East, but has recently expanded its range into North America (Nazari et al. 2018).
Aglais io is a colorful bivoltine species producing two broods of offspring, with one that flies in summer and one that over-winters as adults (Arpaia et al. 2018;Leclerc et al. 2018). The adults feed on nectar-bearing plants, while the caterpillars feed on members of the nettle family Urticaceae which is the route by which GMO maize pollen is ingested (Leclerc et al. 2018). Adult females lay eggs in large pyramidal clusters on hops plants (Humulus lupulus) to protect the innermost layer of eggs from parasitism by flies (Tachnidae) and wasps (Ichneumonidae) (Hondo et al. 1995;Audusseau et al. 2021). Adults have been observed to fake death upon wings being pinched together, with antennae becoming immobile and legs stiffening against the body (Loxdale 2017). Adults also have sound-producing eye-spots on their wings to deter predators including bats and birds (Møhl and Miller 1976;Vallin et al. 2005;Loxdale 2017). Less is known about the focus of the current study, the Japanese peacock butterfly, subspecies A. io geisha (Stichel 1907), than its European counterpart A. io.
Here we report the complete mitochondrial genome (mitogenome) sequence of A. io geisha from specimen Ai2015.2, collected in Saitama, Japan (GPS 35.90807 N,139.65657E) in July 2015 that has been pinned, spread, and deposited in the Wallis Roughley Museum of Entomology, University of Manitoba (http://www.wallisroughley.ca/, Jason Gibbs, Jason.Gibbs@umanitoba.ca) voucher WRME0507739.
The A. io geisha circular 15,252 bp mitogenome assembly was composed of 2700 paired reads with nucleotide composition: 40.1% A, 11.9% C, 7.5% G, and 40.5% T. The gene composition and order in A. io geisha is typical of the arrangement found in most butterfly mitogenomes (Park et al. 2016). The A. io geisha protein-coding gene start codons include ATG (ATP6, COX2, COX3, CYTB, ND1, ND4), ATT (ND2, ND3, ND5), ATC, (ND6), CGA, an atypical COX1 start codon that is also found in the COX1 gene of many other insects (Liao et al. 2010). Additionally, ATP8 and ND4L have ATA start codons that are infrequently used in insect mitochondria but are frequently used in other animal groups (Okimoto et al. 1990;Han et al. 2016;Alexiuk et al. 2020b). The mitogenome contains five protein-coding genes (COX1, COX2, CYTB, ND3, ND5) with single-nucleotide (T--) stop codons, and two protein-coding genes (ND1, ND4) with twonucleotide (TA-) stop codons completed by post-transcriptional addition of 3 0 A residues. All structures of the tRNAs were verified using ARWEN v.1.2 (Laslett and Canback 2008) and have typical cloverleaf secondary structures with the exception for trnS (AGN) where the dihydrouridine arm is replaced by a loop, whereas the control region and mitochondrial rRNAs are typical for Lepidoptera (McCullagh and Marcus 2015).

Acknowledgments
Thanks to Genome Quebec for assistance with library preparation and sequencing.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov] (https://www.ncbi.nlm.nih.gov/) under the accession nos. MZ322948 and MZ322949. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA733565, SRX11064013, and SAMN19415664 respectively.