Comparative chloroplast genomics and codon usage evolution in fourteen East Asian Aletris species

The genus Aletris (Nartheciaceae) comprises approximately 23 species with a classic East Asian–North American disjunct distribution, and the Hengduan Mountains serve as a major diversification hotspot. However, phylogenetic relationships within the genus and the genomic drivers of its diversification remain poorly resolved. We sequenced and compared chloroplast genomes for 14 East Asian Aletris species. The plastomes are highly conserved in structure (~ 154 kb) and gene order, yet exhibit pronounced sequence divergence in intergenic hotspots, providing valuable resources for phylogenetic marker development. Phylogenomic analyses robustly support Aletris monophyly with Metanarthecium as the sister lineage, resolving long-standing taxonomic uncertainty, and recover three major clades with the Japanese endemic A. foliata as the earliest-diverging lineage. Divergence-time estimation places the crown age of East Asian Aletris at approximately 26.1 Ma (late Oligocene to early Miocene), with principal diversification concentrated during the middle to late Miocene (14.5–2.2 Ma), coinciding with intensified uplift of the Qinghai–Tibetan Plateau and adjacent mountain systems. Codon usage bias is weak overall and predominantly shaped by mutational and compositional constraints, with marked functional stratification: photosynthesis-related genes exhibit significantly stronger translational optimization than ribosomal and respiratory genes. While most protein-coding genes are under strong purifying selection, rpl33 alone shows signatures of positive selection, potentially reflecting lineage-specific adaptation. This study establishes a robust, temporally calibrated chloroplast phylogenomic framework for Aletris and demonstrates that plastome evolution is characterized by strong structural conservation, mutational constraints on synonymous codon variation, and episodic adaptive signals in specific genes. These findings provide insights into the temporal assembly and evolutionary dynamics underlying diversification in this ecologically and medicinally significant East Asian lineage.