Evolution along the crassulacean acid metabolism continuum Katia Silvera A , Kurt M. Neubig B , W. Mark Whitten B , Norris H. Williams B , Klaus Winter C and John C. Cushman A,D A Department of Biochemistry and Molecular Biology, MS200, University of Nevada, Reno, NV 89557-0200, USA. B Florida Museum of Natural History, University of Florida, Gainesville, FL 32611-7800, USA. C Smithsonian Tropical Research Institute, PO Box 0843-03092, Balboa, Ancón, Republic of Panama. D Corresponding author. Email: jcushman@unr.edu This paper is part of an ongoing series: ‘The Evolution of Plant Functions’. Abstract. Crassulacean acid metabolism (CAM) is a specialised mode of photosynthesis that improves atmospheric CO 2 assimilation in water-limited terrestrial and epiphytic habitats and in CO 2 -limited aquatic environments. In contrast with C 3 and C 4 plants, CAM plants take up CO 2 from the atmosphere partially or predominantly at night. CAM is taxonomically widespread among vascular plants and is present in many succulent species that occupy semiarid regions, as well as in tropical epiphytes and in some aquatic macrophytes. This water-conserving photosynthetic pathway has evolved multiple times and is found in close to 6% of vascular plant species from at least 35 families. Although many aspects of CAM molecular biology, biochemistry and ecophysiology are well understood, relatively little is known about the evolutionary origins of CAM. This review focuses on five main topics: (1) the permutations and plasticity of CAM, (2) the requirements for CAM evolution, (3) the drivers of CAM evolution, (4) the prevalence and taxonomic distribution of CAM among vascular plants with emphasis on the Orchidaceae and (5) the molecular underpinnings of CAM evolution including circadian clock regulation of gene expression. Additional keywords: phosphoenolpyruvate carboxylase, photosynthesis, d 13 C. Introduction Crassulacean acid metabolism (CAM) is one of three modes of photosynthetic assimilation of atmospheric CO 2 , along with C 3 and C 4 photosynthesis. The net result of CAM is an improvement in water use efficiency (WUE; CO 2 fixed per unit water lost) generally 6-fold higher than for C 3 plants and 3-fold higher than for C 4 plants under comparable conditions (Nobel 1996). Thus, CAM is an important ecophysiological metabolic adaptation that permits plants to occupy semiarid habitats and habitats with intermittent or seasonal water availability (Winter and Smith 1996; Cushman 2001). In this review, we examine the permutations and plasticity of CAM in the context of evolution, discuss the metabolic and genetic requirements for CAM – including leaf succulence – and consider the likely drivers for the evolution of CAM. We next review the current surveys of the taxonomic distribution of CAM species and the several survey methods used to estimate the prevalence of CAM. We then discuss the molecular evolution of CAM, including its origins, describe molecular markers used to study the evolutionary progression of gene family changes and to analyse circadian clock control. Finally, we address future directions for research. Phases of CAM The physiological and biochemical temporal sequence of events that constitute CAM have been described in detail as being separable into four discrete phases (Osmond 1978; Winter 1985; Lüttge 1987; Griffiths 1988). Phase I is typically characterised by nocturnal stomatal opening, CO 2 uptake and fixation by phosphoenolpyruvate carboxylase (PEPC) in the cytosol and the formation of C 4 organic acids (usually malic acid), which are stored in the vacuole (Fig. 1). The rate of nocturnal CO 2 assimilation is governed by mesophyll processes, such as regulation of carbohydrate storage reserves (Cushman et al. 2008a) or vacuolar storage capacity, rather than stomatal conductance (Winter 1985; Winter et al. 1985). Depending on the CAM species, a variety of storage carbohydrates (e.g. starch, glucans, soluble hexoses) might be catabolised to produce phosphoenolpyruvate (PEP), the substrate for carboxylation (Christopher and Holtum 1996, 1998; Holtum et al. 2005). Phase I reflects the fundamental adaptation of CAM that results in reduced transpiration and improved water economy due to lower night-time evapotranspirational demands and associated water losses (Griffiths 1988). Phase II describes the transition from PEPC to ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO)-mediated carboxylation during the early light period leading to carbohydrate production. During this phase, CO 2 is derived from both organic acid decarboxylation and direct uptake from the atmosphere. Phase III encompasses the period of major efflux of organic acids from the vacuole and their subsequent decarboxylation (Fig. 1). This decarboxylation can lead to CSIRO PUBLISHING Review www.publish.csiro.au/journals/fpb Functional Plant Biology, 2010, 37, 995–1010 Ó CSIRO 2010 10.1071/FP10084 1445-4408/10/110995