Abstract
<title>Abstract</title> <p>Synonymous variation in plastid genes may record both lineage history and local sequence-context constraints. We integrated phylogenetic codon-use analysis with exact-codon conditional randomization across 18 Platycerium species. Eighty-four reference coding features were reconstructed from a published whole-plastome alignment and audited; the primary matrix comprised 44 editing-free, intact genes and 792 species–gene sequences. Relative synonymous codon usage (RSCU) distance increased with plastome-tree distance, and an RSCU phenogram recovered the three recognized geographic lineages, indicating that codon profiles retain phylogenetic structure despite narrow ranges of genomic GC content and effective number of codons. Gene-level RSCU divergence decreased with coding-sequence length, emphasizing the need to distinguish short-gene sampling effects from evolutionary differentiation. For each coding sequence, 9,999 randomizations retained protein sequence, the exact number of every codon, nucleotide composition, and within-codon dinucleotides while changing synonymous-codon placement. Codon-boundary CpG was lower than its conditional expectation, with negative median effects in all species and most genes and persistence under reconstruction and length sensitivities. TpA showed a pooled deficit but greater gene heterogeneity. Analysis of all 16 boundary dinucleotides placed CpG within a broader redistribution of neighbouring codon contexts. Together, the results connect lineage-associated synonymous divergence with a conserved codon-order tendency and support an evolutionary model in which shared plastid composition, ancestry, gene-level constraints, and local sequence context jointly shape codon organization.</p>