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

Abstract

Background/Objectives:Scutiger ningshanensis (Fang, 1985) is an endemic Chinese amphibian species within the genus Scutiger (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 Scutiger, this study aims to (1) characterize the complete mitogenome of S. ningshanensis, (2) analyze its molecular evolution, and (3) clarify its phylogenetic position and divergence history within Megophryidae. Methods: 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. Results: The mitogenome of S. ningshanensis 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 Scutiger, forming a sister clade to Oreolalax and Leptobrachium, and that S. ningshanensis and S. liubanensis 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 S. ningshanensis diverging~32.67 MYA (Oligocene). Conclusions: This study provides the first comprehensive mitogenomic characterization of S. ningshanensis, 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.

Keywords: Ka/Ks; Scutiger ningshanensis; divergence time; mitochondrial genome; phylogenetic analysis.

Figure 1

Complete mitogenome circles map of S. ningshanensis. Purple represents tRNA, green represents rRNA, black areas represent GC content; in addition, the map also shows the GC-skew value.

Figure 2

Amino acid content of PCGs in S. ningshanensis.

Figure 3

Relative synonymous codon usage (RSCU) in the mitogenome of S. ningshanensis. (The codon is represented by the X-axis, and the RSCU value is represented by the Y-axis).

Figure 4

Putative secondary structure of transfer RNA (tRNA) genes of mitogenome of S. ningshanensis.

Figure 5

The prognostic map of 12S rRNA secondary structures in S. ningshanensis.

Figure 6

The prognostic map of 16S rRNA secondary structures in S. ningshanensis.

Figure 7

Phylogenetic relationships within Megophryidae derived from ML method based on 13 PCGs; the numbers on the nodes represent the support values of these nodes, the first number represents the bootstrap value of the ML tree, and the second number represents the posterior probability from the BI tree. Species grouped under the same color on the right side of the tree belong to the same taxonomic unit. The species S. ningshanensis used in this study is indicated by the Latin name with an asterisk.

Figure 8

The divergence time of Megophryidae with 95% highest posterior probability density. Numbers nearby nodes refer to divergence times. The “*” represents the current study.

Figure 9

The Ka/Ks values among the S. ningshanensis species. Note: PV083741, KU127230, NC_030627, NC_088425, and NC_056343 represent S. ningshanensis (this study), O. major, O. lichuanensis, L. alpina, and O. schmidti, respectively.