Crassulacean Acid Metabolism (CAM) is a water-conserving mode of photosynthesis present in 7% of vascular plant species worldwide. In tropical orchids, the CAM pathway is found in up to 50% of species. To better understand the role of CAM in species radiations and the molecular mechanisms of CAM evolution in orchids, we performed carbon stable isotopic composition of leaf samples from 1,103 species native to Panama and Costa Rica, and character state reconstruction and phylogenetic trait analysis of CAM and epiphytism. The multiple independent evolutionary origins of CAM in orchids suggest that evolution from C₃ to weak and strong CAM may involve relatively few genetic changes. To test this corollary, we performed an analysis of the phosphoenolpyruvate carboxylase (PEPC) gene family structure and mRNA relative abundance, using closely-related species exhibiting C₃, weak CAM and strong CAM.

Results/Conclusions

When ancestral state reconstruction of CAM is overlain onto a phylogeny of orchids, the distribution of photosynthetic pathways shows that C₃ photosynthesis is the ancestral state and that CAM has evolved  independently several times within the Orchidaceae. Using phylogenetic trait analysis, we found that divergences in photosynthetic pathway and epiphytism are consistently correlated through evolutionary time and are related to the prevalence of CAM epiphytes in lower elevations and abundant species diversification of high elevation epiphytes. Using PEPC analysis, we found that at least five isoforms are present in orchids, with one putative CAM-specific PEPC isogene with discrete amino acid changes identified in CAM species based on cDNA clone sampling. Validation of the isotopic analysis and the molecular genetic analysis of PEPC gene family using 24-hour gas exchange showed that weak CAM species exhibit limited degree of nocturnal CO₂ uptake when compared to strong CAM species. This study demonstrates several patterns of CAM evolution in orchids, thus improving our understanding of the functional significance and evolutionary origins of CAM.