We have constructed a composite phylogeny of 92 green plant taxa with representatives from all major clades to examine the level and degree of codon usage for the gene rbcL and to search for potential links between primary and secondary levels of organization. rbcL encodes the large subunit of RUBISCO, an important enzyme in photosynthesis. Such functional importance predicts that changes in primary genetic substructure, and perhaps secondary structure and enzymatic efficiency should be infrequently tolerated. Specifically, this conservation predicts that changes in codon bias and usage should occur infrequently, if at all. We have tested this claim and found it to be largely false. Instead the degree of codon bias among green plant lineages shifted in correspondence with phylogeny. One notable finding was a major transition to decreased codon bias at the boundary between spore plants and seed plants. The codon bias cannot be explained by overall genome composition bias, and must have other explanations, related to selection. Codon preference also changed among green plant lineages, but these changes were most often homoplasious. However, the shifts in preference were predominantly to G or C ending codons, indicating possible biochemical canalization. To search for mechanisms that may explain these changes, we examined the relationship between the degree of bias and rates of Kn/Ks substitutions in rbcL. Lower Kn/Ks ratios were correlated with higher codon bias, indicating tracking of preferred codons within amino acid families. These data support the hypothesis that codon bias and usage are under selective pressure. In general, our study identifies how comparative analyses of genetic organization may help clarify the mechanisms linking genes, proteins, and patterns of microevolution. Our study also indicates that usage of nucleotide sequence alone for phylogenetic reconstruction may be overly simple; consideration of how the selective environment may effect phylogenetic results is critical.

Key words: Chloroplast, Codon Bias, Green Plants, rbcL