Complete Mitochondrial Genome of Two Amathusiini Species (Lepidoideae: Nymphalidae: Satyrinae): Characterization, Comparative Analyses, and Phylogenetic Implications

<b>Background</b>: The <i>Satyrinae</i> subfamily represents a taxonomically critical group within <i>Nymphalidae</i>, 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 <i>Satyrinae</i>. Notably, <i>Amathusiini</i> exhibits a particular paucity, with only two congeneric representatives having been comprehensively sequenced to date. <b>Methods</b>: We employed high-throughput sequencing to assemble the complete mitochondrial genomes of two <i>Amathusiini</i> species, <i>Discophora sondaica</i> and <i>Aemona amathusia</i>. Our study revealed novel evolutionary insights through comparative genomics, which encompassed all available <i>Satyrinae</i> mitochondrial genomes. Additionally, we conducted phylogenetic reconstruction using maximum likelihood and Bayesian inference approaches, utilizing the most extensive dataset to date. <b>Results</b>: The closed, circular mitochondrial genomes measure 15,333 bp for <i>D. sondaica</i> and 15,423 bp for <i>A. amathusia</i>, 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 <i>atp8</i> exhibiting the highest evolutionary rate (Ka/Ks = 0.277). Phylogenetic reconstructions yielded congruent tribal-level topologies with strong nodal support: ((<i>Satyrini</i> + <i>Melanitini</i>) + (<i>Amathusiini</i> + <i>Elymniini</i>) + <i>Zetherini</i>), confirming a sister relationship between <i>Amathusiini</i> and <i>Elymniini</i>. Within <i>Satyrini</i>, five subtribes formed monophyletic groups: <i>Ypthimina</i>, <i>Erebiina</i>, <i>Maniolina</i>, <i>Satyrina</i>, and <i>Melanargiina</i>, arranged as ((<i>Ypthimina</i> + (<i>Erebiina</i> + <i>Maniolina</i>)) + (<i>Satyrina</i> + <i>Melanargiina</i>)). <i>Mycalesina</i>, <i>Lethina</i>, and <i>Parargina</i> comprised a well-supported clade (BS = 100%; PP = 1.0), though internal relationships required further resolution due to <i>Lethina</i>'s polyphyly. <b>Conclusions</b>: This study provides novel insights into mitochondrial genomic evolution within the <i>Satyrinae</i> 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.