The Complete Mitochondrial Genomes of Two Amathusiini Species, Discophora sondaica and Aemona amathusia, and Phylogenomic Analysis of the Satyrinae

Background: The Satyrinae subfamily represents a taxonomically critical group within Nymphalidae, characterized by its remarkable species diversity. Despite its evolutionary significance, the phylogenetic relationships among tribal and subtribal lineages remain poorly resolved. Although mitochondrial genomes have become crucial molecular markers in Lepidoptera phylogenetics, their potential remains underutilized in the systematics of Satyrinae. Notably, Amathusiini exhibits a particular paucity, with only two congeneric representatives having been comprehensively sequenced to date. Methods: To address this gap, we employed high-throughput sequencing to assemble the complete mitochondrial genomes of two Amathusiini species, Discophora sondaica and Aemona amathusia. Our study revealed novel evolutionary insights through comparative genomics, which encompassed all available Satyrinae mitochondrial genomes. Additionally, we conducted phylogenetic reconstruction using maximum likelihood and Bayesian inference approaches, utilizing the most extensive dataset to date. Results: The closed circular mitochondrial genomes measure 15,333 bp for D. sondaica and 15,423 bp for A. amathusia, maintaining the ancestral lepidopteran architecture: 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and an AT-rich control region. Comparative analyses of 71 mitochondrial genomes revealed strong evolutionary conservation across multiple parameters: nucleotide composition (AT content range: 77.9% - 81.8%), codon usage bias (ENC = 30.83 - 37.55), tRNA secondary structures, and control region organization. All PCGs showed purifying selection signals (Ka/Ks < 1.0), with atp8 exhibiting the highest evolutionary rate (Ka/Ks = 0.277). Phylogenetic reconstructions yielded congruent tribal-level topologies with strong nodal support: (((Satyrini + Melanitini) + (Amathusiini + Elymniini) + Zetherini), confirming a sister relationship between Amathusiini and Elymniini. Within Satyrini, five subtribes formed monophyletic groups: Ypthimina, Erebiina, Maniolina, Satyrina, and Melanargiina, arranged as (((Ypthimina + (Erebiina + Maniolina)) + (Satyrina + Melanargiina)). Mycalesina, Lethina, and Parargina comprised a well-supported clade (BS = 100%; PP = 1.0), though internal relationships required further resolution due to Lethina's polyphyly. Conclusions: This study provides novel insights into mitochondrial genomic evolution within the Satyrinae subfamily, while elucidating the efficacy of mitogenomic data for resolving deep phylogenetic relationships within this ecologically significant subfamily. Our findings establish critical genome baselines for further systematic research and underscore essential pathways for refining subtribal-level taxonomy through integrative molecular approaches.