Reassessing Murinae phylogeny: integrated mitogenomic and codon usage evidence challenges the sister relationship of Micromyini and Vernayini.

backgroundThe Murinae subfamily, one of the most diverse and widely distributed rodent groups, is an important model in ecology, evolutionary biology, and biomedical research. However, deep-level phylogenetic relationships, especially the evolutionary status of key tribes, remain highly contentious. Although mitogenomes are widely used in mammalian evolutionary studies, an integrated analytical framework that combines comparative genomics, selection pressure, codon-usage dynamics, and morphological evidence is currently lacking. Here, we aim to reassess the deep phylogenetic relationships within Murinae, with a particular focus on verifying the proposed sister relationship between the tribes Micromyini and Vernayini.

resultsWe sequenced and assembled the complete mitogenomes of a single individual of Leopoldamys neilli and Micromys pygmaeus. Through a systematic comparison of mitogenomes from 117 Murinae species-representing 13 tribes and 50 genera, we identified both conserved and lineage-specific genomic features. Murinae mitogenomes were highly conserved in structure and gene order, with length variation primarily attributable to changes in the control region. Nucleotide composition was largely uniform across most lineages, though Micromyini and Vernayini displayed distinct compositional profiles, suggesting possible differences in evolutionary pressures. All protein-coding genes were under strong purifying selection, yet evolutionary rates varied nearly tenfold among genes (e.g., ATP8 versus COX1). Codon-usage bias was predominantly influenced by natural selection and correlated with phylogenetic relationships. Critically, our large‑scale phylogenomic analyses did not support the previously proposed sister‑group relationship between Micromyini and Vernayini. Instead, we recovered Vernayini as the sister group to the remaining Murinae excluding Hapalomys-a topology that received maximal statistical support (PP = 1.00, BS = 100%). This revised phylogenetic hypothesis is supported by multidimensional evidence: (i) principal component analysis of morphological traits reveals significant divergence between the two tribes; (ii) COX1‑based genetic distances (18.93 ± 0.01%) substantially exceed both the generic (12.61%) and subfamilial (18.08%) thresholds reported for Murinae; and (iii) distinct codon‑usage patterns further differentiate these tribes at the molecular level.

conclusionThis study provides the first large‑scale, integrated analysis of comparative mitogenomic data, codon usage bias, and morphological data in Murinae. We supply new genomic resources, reassess the phylogenetic framework of Murinae, definitively clarify that Micromyini and Vernayini are not sister groups, and demonstrate the power of combining genomic, compositional, and phenotypic data in systematic research. These findings offer a foundation for future phylogenomic studies while highlighting the importance of validating mitochondrial‑based hypotheses with additional data sources, such as nuclear markers or morphological traits.