Short communication Phylogeny of Calvin cycle enzymes supports Plantae monophyly Adrian Reyes-Prieto, Debashish Bhattacharya * University of Iowa, Department of Biological Sciences and the Roy J. Carver Center for Comparative Genomics, 446 Biology Building, Iowa City, IA 52242, USA Received 10 November 2006; revised 5 February 2007; accepted 13 February 2007 Available online 4 March 2007 1. Introduction Photosynthesis is a critical biochemical process on our planet providing food for most life. The common ancestor of plants and their algal sisters gained photosynthesis through the engulfment and retention of a cyanobacterial primary endosymbiont that evolved into a photosynthetic organelle, the plastid (Bhattacharya et al., 2004). In photosynthetic eukaryotes, the essential series of reactions that capture the products of photosynthetic light reactions (ATP and NADPH 2 ) to fix CO 2 (Fig. 1), known as the Calvin cycle (CC; Calvin and Benson, 1948), takes place in the plastid stroma. The eukaryotic CC involves 11 different enzymes (Table 1) that are nuclear encoded and plastid targeted to express their function, with the excep- tion of ribulose-1,5-bisphosphate carboxylase (RuBisCO) subunits (large and small) that remain plastid encoded in red and glaucophyte algae. In green algae (and land plants) the RuBisCO large subunit is encoded in the plastid gen- ome but the small subunit is nuclear encoded. Photosyn- thetic eukaryotes also contain cytosolic enzymes involved in glycolysis and gluceoneogenesis that catalyze reactions similar to those in the CC and were present in eukaryotes before plastid origin (Martin and Schnarrenberger, 1997). Molecular phylogenetic analyses suggest that land plants (Martin and Schnarrenberger, 1997) and red algae acquired at least a subset of the CC enzymes via intracellular (endo- symbiotic) gene transfer (EGT) from the captured cyano- bacterium prior to the divergence of green and red algae (Matsuzaki et al., 2004). However, it is well known that some CC enzymes in land plants and red algae have a non-cyanobacterial origin (Martin and Schnarrenberger, 1997; Matsuzaki et al., 2004). A likely explanation is that these pre-existing host enzymes took over the role of the original cyanobacterial proteins that have been lost over evolutionary time. It is unknown whether these putative gene replacements were an ancient feature of eukaryotic CC evolution or whether gene recruitments-replacements occurred more recently in different photosynthetic lineages. To gain a clearer picture of early CC enzyme evolution it is critical to analyze genome data from the three major groups that presumably diversified from the first photosyn- thetic eukaryote: the red algae, the green algae (including land plants), and the glaucophyte algae. These taxa, referred to as the Plantae (Cavalier-Smith, 1981) or Archaeplastida (Adl et al., 2005), are postulated to share a single origin. Phylogenetic analyses using multi-gene nuclear and plastid data strongly support Plantae mono- phyly (e.g., Rodriguez-Ezpeleta et al., 2005), however some single- and multi-gene analyses do not recover this clade (e.g., Nozaki et al., 2003; Stiller and Harrell, 2005). Phylogenetic analysis of enzymes involved in conserved, ancient metabolic processes is a potentially valuable source of information for elucidating the evolutionary history of major eukaryotic groups. Here, we use this comparative approach to elucidate the phylogeny of CC enzymes with molecular data from all three primary photosynthetic lin- eages that share the cyanobacterial endosymbiont; i.e., including expressed sequence tag (EST) data generated in our lab from the glaucophyte alga Cyanophora paradoxa (Reyes-Prieto et al., 2006). 2. Materials and methods 2.1. cDNA library construction Total RNA from a culture of Cyanophora paradoxa Pringsheim strain (CCMP329) was extracted with Trizol (GibcoBRL) and the mRNA purified using the Oligotex 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.02.026 * Corresponding author. Fax: +1 319 335 1069. E-mail address: debashi-bhattacharya@uiowa.edu (D. Bhattacharya). www.elsevier.com/locate/ympev Molecular Phylogenetics and Evolution 45 (2007) 384–391