HiFi-Assembled Mitogenomes of Four Pygmy Grasshoppers Reveal Mito–Nuclear Discordance in Zhengitettix transpicula and Lineage-Specific Mitochondrial Intergenic Length Variation

Mitochondrial genomes are widely used in insect taxonomy and phylogenetics, but their signals may conflict with morphology and nuclear genomic evidence because the mitochondrial genome represents a single maternally inherited locus. Here, we assembled complete mitochondrial genomes of four pygmy grasshoppers, Zhengitettix transpicula, Formosatettix sp., Gibbotettix parvipulvillus, and Bolivaritettix sp., using PacBio HiFi reads. The four mitogenomes ranged from 15,152 to 17,976 bp and contained the typical 37 mitochondrial genes. Mitochondrial phylogenies inferred by maximum likelihood and Bayesian methods were topologically identical and recovered several well-supported tetrigid relationships, including a close relationship between Formosatettix sp. and Bolivaritettix sp. However, Z. transpicula was unexpectedly placed near Macromotettixoides rather than close to other Zhengitettix representatives. In contrast, a morphology-based tree recovered Z. transpicula with Z. triangularis, and comparison with a published nuclear single-copy ortholog tree based on 1962 loci supported a non-mitochondrial placement of Zhengitettix inconsistent with the anomalous mitochondrial position of Z. transpicula. Independent assembly from the original HiFi reads, read-depth inspection, protein-coding gene checks, and nuclear-genome screening for NUMT-like sequences supported the authenticity of the assembled Z. transpicula mitogenome. These results document mito–nuclear and cyto-morphological discordance in Tetrigidae and highlight the need for integrative interpretation of mitochondrial phylogenies in taxonomically complex insect groups.