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Saxifragales is a 15-family order of early-divergent Eudicots with a rich morphological diversity and an ancient rapid radiation. Codon usage bias (CUB) analyses have emerged as an essential tool for understanding the evolutionary dynamics in genes. Thus far, the codon utilization patterns had only been reported in four separate genera within Saxifragales. This study provides a comprehensive assessment of the codon manipulation based on 50 plastid genes, covering 11 constituent families at a larger sampling scale. Our results first showed a high preference for AT bases and AT-ending codons. We then used effective number of codons (ENC) to assess a range of codon bias levels in the plastid genes. We also detected high-informative intrafamilial differences of ENC in three families. Subsequently, parity rule 2 (PR2) plot analyses revealed both family-unique and order-shared bias patterns. Most importantly, the ENC plots and neutrality analyses collectively supported the dominant roles of selection in the CUB of Saxifragales plastid genes. Notably, the phylogenetic affinities inferred by both ML and BI methods were consistent with each other, and they all comprised two primary clades and four subclades. These findings significantly enhance our understanding of the evolutionary processes of the Saxifrage order, and could potentially inspire more CUB analyses at higher taxonomic levels. [ABSTRACT FROM AUTHOR]

• This study assembled and annotated four new plastomes from the Telephium clade. • Monophyletic Telephium clade further formed Hylotelephium and Rhodiola subclade. • No positive selection gene was observed but 19 genes had positive selection sites. • Codon usage was biased, shaped by natural selection, mutations and other factors. • 34 taxon-specific codon aversion motifs were identified from 49 plastid genes. The Telephium clade of the Crassulaceae family contains many medicinal, ornamental, and ecologically restorative plants. However, the phylogenetic relationships within the clade remain debated, and comprehensive analyses of codon usage and selection pressure in Telephium plastomes are limited. In this study, we assembled and annotated four plastomes and performed extensive analyses. The plastomes exhibited a typical quadripartite structure and high conservation. The lengths ranged from 151,357 bp to 151,641 bp with 134 genes identified. The GC content was the highest within IR, followed by LSC, and lowest in the SSC region. Meanwhile, a unique inversion was observed within the LSC region of Meterostachys sikokianus. Polymorphisms analysis revealed minimum nucleotide diversity in the IR regions, with over ten highly polymorphic regions identified. Phylogenetically, two subclades formed within the monophyletic Telephium clade, with Umbilicus as the sister group to the remaining Hylotelephium subclade members. Notably, no significant positive selection was found among the 79 plastid genes, which showed varying evolutionary patterns. However, 19 genes contained codons under positive selection. The specific functions of these sites require further investigation. Synonymous codon usage was biased and conserved across the tested plastomes, shaped by natural selection, mutations and other factors of varying influence. We also identified 34 taxon-specific codon aversion motifs from 49 plastid genes. Our plastomic analyses elucidate phylogenetic relationships and evolutionary patterns in this medicinal clade, providing a foundation for further research on these ecologically and pharmaceutically important plants. [ABSTRACT FROM AUTHOR]