PEPC Gene Family Evolution Across Arecaceae: Complete Five-Subfamily Evidence for a Two-Lock Model of Irreversible C4 Exclusion
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Background
C4 photosynthesis has evolved more than sixty times across flowering plants, but never in palms. Over 2,500 palm species have spent more than 100 million years in high-light, water-stressed habitats where C4 physiology would be advantageous, yet none have taken this path. Phosphoenolpyruvate carboxylase (PEPC) is the enzyme that gates entry into the C4 pathway. In every C4 grass, a commelinid-specific paralog called PEPC1 drives the CO₂-concentrating mechanism that defines the syndrome. Until this study, one of the five palm subfamilies — Ceroxyloideae — had never been examined for PEPC gene content.
Results
We present a complete PEPC census across 13 palm species spanning all five subfamilies (Calamoideae, Nypoideae, Coryphoideae, Ceroxyloideae, Arecoideae). Ceroxyloideae was characterized for the first time, using deep tBLASTn screening of ten million whole-genome shotgun reads. PEPC1 is absent from every palm species examined. Copy numbers are tightly constrained to 5–7 per genome, in stark contrast with the 6–25 copies found in grasses. A phylogenetic analysis of 337 PEPC sequences places all 55 palm loci at basal positions relative to the PEPC1 clade, consistent with the palm lineage having diverged approximately 120 million years ago — roughly 15 million years before the PEPC1 duplication that occurred within the commelinids. Birth-death modeling reveals long-term gene family stasis in palms. Codon substitution analyses yield a genome-wide ω of 0.0582, indicating pervasive purifying selection and no signature of C4-specific adaptation. Coconut PEPC promoters uniformly lack the mesophyll-specific MESP-1/GT-1/DOF regulatory module that is required for C4-type gene expression.
Conclusions
We propose a Two-Lock Model of irreversible C4 exclusion in palms. The first is a Gene Lock: PEPC1 is phylogenetically absent from all five subfamilies. The second is a Regulatory Lock: the ancestral promoter architecture of palm PEPC genes lacks the cis-elements needed for mesophyll-specific expression. Together, these two locks — one phylogenetic, one regulatory — explain why C4 photosynthesis has never arisen in one of the largest C4-absent plant families. The model provides a testable framework for investigating other lineages that have remained closed to the C4 path.
