Characterization of the Complete Mitochondrial Genome of Eight Diurnal Hawkmoths (Lepidoptera: Sphingidae): New Insights into the Origin and Evolution of Diurnalism in Sphingids

SIMPLE SUMMARY: We newly sequenced the mitochondrial genomes of 22 species from three subfamilies in the family Sphingidae. The mitogenomes of six diurnal species were newly sequenced, assembled, and annotated in this study (Hemaris radians, Macroglossum bombylans, Macroglossum fritzei, Macroglossum pyrrhosticta, Neogurelca himachala, and Sataspes xylocoparis) and, together with those of two previously reported diurnal hawkmoths (Cephonodes hylas and Macroglossum stellatarum), were analyzed in terms of sequence length, nucleotide composition, relative synonymous codon usage, non-synonymous/synonymous substitution ratio, gene spacing, and repeat sequences. The phylogenetic relationships among the subfamilies of Sphingidae were analyzed to explore the origin, divergence time, and evolutionary status of diurnal sphingids. In addition, we performed divergence time estimation and reconstruction of ancestral characters. Diurnal behavior of hawkmoths first originated 29.19 million years ago (Mya) and may have been influenced by the combination of herbaceous flourishing, which occurred 26–28 Mya, the uplift of the Tibetan Plateau, and the large-scale evolution of bats in the Oligocene to Pre-Miocene. Moreover, diurnalism in hawkmoths had multiple independent origins. ABSTRACT: In this study, the mitochondrial genomes of 22 species from three subfamilies in the Sphingidae were sequenced, assembled, and annotated. Eight diurnal hawkmoths were included, of which six were newly sequenced (Hemaris radians, Macroglossum bombylans, M. fritzei, M. pyrrhosticta, Neogurelca himachala, and Sataspes xylocoparis) and two were previously published (Cephonodes hylas and Macroglossum stellatarum). The mitochondrial genomes of these eight diurnal hawkmoths were comparatively analyzed in terms of sequence length, nucleotide composition, relative synonymous codon usage, non-synonymous/synonymous substitution ratio, gene spacing, and repeat sequences. The mitogenomes of the eight species, ranging in length from 15,201 to 15,461 bp, encode the complete set of 37 genes usually found in animal mitogenomes. The base composition of the mitochondrial genomes showed A+T bias. The most commonly used codons were UUA (Leu), AUU (Ile), UUU (Phe), AUA (Met), and AAU (Asn), whereas GCG (Ala) and CCG (Pro) were rarely used. A phylogenetic tree of Sphingidae was constructed based on both maximum likelihood and Bayesian methods. We verified the monophyly of the four current subfamilies of Sphingidae, all of which had high support. In addition, we performed divergence time estimation and ancestral character reconstruction analyses. Diurnal behavior in hawkmoths originated 29.19 million years ago (Mya). It may have been influenced by the combination of herbaceous flourishing, which occurred 26–28 Mya, the uplift of the Tibetan Plateau, and the large-scale evolution of bats in the Oligocene to Pre-Miocene. Moreover, diurnalism in hawkmoths had multiple independent origins in Sphingidae.