Abstract: Chenopodiaceae s.s. (Amaranthaceae s.l.) contains the largest number of C₄ species among eudicots. Despite this, plastome evolution within this family has been investigated in only a few species. Here, we analyzed 119 plastomes from 115 species, including 78 newly sequenced plastomes, representing all subfamilies and most C₄ lineages of Chenopodiaceae s.s.. Plastome structural variants, rearrangements, and codon usage bias were compared across subfamilies and photosynthetic types. Multiple phylogenetic approaches were employed to reconstruct the evolutionary relationships within Chenopodiaceae s.s., and Bayesian divergence time estimation was performed. Various Mk models for discrete character evolution were tested to investigate the evolution of C₄ photosynthesis, and stochastic character mapping simulations were used to reconstruct shifts in photosynthetic pathways through time. Several plastome structural variants and rearrangements were identified, but associations with photosynthetic types were observed only in the subfamily Suaedoideae. Codon usage bias analysis revealed significant bias exclusively in C₄ species, suggesting enhanced translational efficiency and accuracy as an adaptation to environmental conditions. We inferred multiple independent origins of the C₄ pathway, with the oldest lineages—Bienertia (Suaedoideae) and Caroxyleae (Salsoloideae)—dating to approximately 34 and 32 million years ago (Ma), respectively, during the Oligocene. A marked increase in the number of C₄ lineages occurred between 20 and 15 Ma. Declining atmospheric CO₂ concentrations, combined with genetic, ecological, and environmental factors, likely promoted the expansion of C₄ photosynthesis until recently. Finally, we identified five new hypervariable regions that will be valuable for phylogenetic and DNA barcoding applications in Chenopodiaceae s.s..

Key words: Chloroplast genome, Chenopodiaceae s.s., C₄ photosynthesis, IR/SC boundary, Codon bias, Evolution