Mitochondrial Genome of Scutiger ningshanensis (Anura, Megophryidae, Scutiger): Insights into the Characteristics of the Mitogenome and the Phylogenetic Relationships of Megophryidae Species

<b>Background/Objectives:</b><i>Scutiger ningshanensis</i> (Fang, 1985) is an endemic Chinese amphibian species within the genus <i>Scutiger</i> (Megophryidae). Despite its ecological significance, its mitochondrial genome architecture and evolutionary relationships remain poorly understood. Given the high structural variability in Megophryidae mitogenomes and unresolved phylogenetic patterns in <i>Scutiger</i>, this study aims to (1) characterize the complete mitogenome of <i>S. ningshanensis</i>, (2) analyze its molecular evolution, and (3) clarify its phylogenetic position and divergence history within Megophryidae. <b>Methods:</b> The complete mitochondrial genome was sequenced and annotated, followed by analyses of nucleotide composition, codon usage bias, and selection pressures (Ka/Ks ratios). Secondary structures of rRNAs and tRNAs were predicted, and phylogenetic relationships were reconstructed using maximum likelihood and Bayesian methods. Divergence times were estimated using molecular clock analysis. <b>Results:</b> The mitogenome of <i>S. ningshanensis</i> is 17,282 bp long, encoding 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and a control region, with a notable AT bias (61.05%) with nucleotide compositions of T (32.51%), C (24.64%), G (14.3%), and A (28.54%). All tRNAs exhibited cloverleaf structures except trnS1, which lacked a DHU stem. Phylogenetic analysis confirmed the monophyly of <i>Scutiger</i>, forming a sister clade to <i>Oreolalax</i> and <i>Leptobrachium</i>, and that <i>S. ningshanensis</i> and <i>S. liubanensis</i> are sister species with a close evolutionary relationship. Positive selection was detected in Atp8 (Ka/Ks > 1), suggesting adaptation to plateau environments, while other PCGs underwent purifying selection (Ka/Ks < 1). Divergence time estimation placed the origin of Megophryidae at~47.97 MYA (Eocene), with <i>S. ningshanensis</i> diverging~32.67 MYA (Oligocene). <b>Conclusions:</b> This study provides the first comprehensive mitogenomic characterization of <i>S. ningshanensis</i>, revealing its evolutionary adaptations and phylogenetic placement. The findings enhance our understanding of Megophryidae's diversification and offer a genomic foundation for future taxonomic and conservation studies.