Abstract
Species of the large family Orchidaceae display a spectacular array of adaptations and rapid speciations that are linked to several innovative features, including specialized pollination syndromes, colonization of epiphytic habitats, and the presence of Crassulacean acid metabolism (CAM), a water-conserving photosynthetic pathway. To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, to climatic variables and the evolution of epiphytism in tropical regions. Altitude was the most important predictor of photosynthetic pathway when all environmental variables were taken into account, with CAM being most prevalent at low altitudes. By creating integrated orchid trees to reconstruct ancestral character states, we found that C3 photosynthesis is the ancestral state and that CAM has evolved at least 10 independent times with several reversals. A large CAM radiation event within the Epidendroideae, the most species-rich epiphytic clade of any known plant group, is linked to a Tertiary species radiation that originated 65 million years ago. Our study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low-elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae.
Crassulacean acid metabolism (CAM) is a taxonomically widespread photosynthetic pathway that has evolved in plants of CO2- and water-limited environments, including tropical forest canopies with intermittent or seasonal water availability, hot semiarid regions, and some aquatic environments. The CAM pathway is characterized by the temporal separation of carbon fixation between nocturnal CO2 fixation by phosphoenolpyruvate carboxylase in the cytosol and daytime decarboxylation of organic acids to release CO2 that is then refixed by Rubisco in the chloroplast (Ting, 1985). CAM photosynthesis is found in approximately 7% of vascular plant species from 34 families (Smith and Winter, 1996; Holtum et al., 2007). About 10% of all vascular plant species are estimated to be epiphytes (Benzing, 1989), many of which exhibit CAM (Lüttge, 2004). CAM vascular epiphytes (mostly orchids and bromeliads) are an important component of the biomass and species richness of tropical forest canopies (Benzing, 1987; Lüttge, 2004; Zotz, 2004). Bromeliads, aroids, and orchids are three of the very few flowering plant lineages that were able to successfully colonize epiphytic niches (Gentry and Dodson, 1987). Yet, orchids are particularly species-rich relative to these other epiphytic groups (Gravendeel et al., 2004), making Orchidaceae a prime subject for understanding mechanisms of evolutionary radiation and diversification. About 72% of orchid species are estimated to be epiphytic (Benzing, 1989; Gravendeel et al., 2004), with the majority of these being restricted to tropical regions. Tropical forest canopies are rich in epiphytic CAM plant diversity (Benzing, 1987; Winter and Smith, 1996; Lüttge, 2004). CAM has been found in 62% and 26% of epiphytic orchid species in Australian and New Guinean rainforests, respectively (Winter et al., 1983; Earnshaw et al., 1987), 42% of orchid species in a moist lowland forest site in Panama (Zotz and Ziegler, 1997), and up to 100% of the epiphytic flora in a Mexican dry forest (Mooney et al., 1989). The abundance of CAM species in such habitats is related to limited water availability. Within a single site, the percentage of CAM epiphytes increases with canopy height, from 7% in the forest understory, to 25% at intermediate heights, to 50% in exposed canopy sites (Zotz and Ziegler, 1997). CAM species are also found in contrasting habitats such as very arid and very moist sites, which provides evidence of the biochemical flexibility of this photosynthetic adaptation (Dodd et al., 2002). Several researchers have postulated that in addition to CAM, mechanisms such as production of dust-like seeds capable of long-distance dispersal, germination associations with mycorrhizae, absorptive velamentous photosynthetic root tissue capable of rapid water uptake, and reproductive features that promote specialized pollination syndromes have contributed to the diversification of epiphytic orchids (Benzing, 1987; Gravendeel et al., 2004; Peakall, 2007; Mondragón-Palomino and Theissen, 2008). Whether CAM is linked to epiphytic diversification and species radiations throughout evolutionary time remains poorly understood.
Orchid systematics is now at an advanced stage, allowing ancestral state reconstruction and correlated evolution analysis of key adaptive traits within the context of a highly resolved phylogeny (Chase et al., 2003). We used whole leaf tissue carbon isotopic composition (δ13C) as a rapid screening method to establish whether CO2 assimilation occurs predominantly by strong CAM or C3 photosynthesis. Strong CAM and C3 photosynthetic tissues exhibit distinct δ13C values because CAM plants are enriched in 13C relative to C3 plants (Rundel et al., 1979; Winter, 1979; Winter et al., 1983; Kluge et al., 1991; Zotz and Ziegler, 1997; Crayn et al., 2001, 2004; Zotz, 2004; Silvera et al., 2005), whereas weak CAM species show overlapping δ13C values with C3 species and measurable changes in day/night titratable acidity (Silvera et al., 2005). We present data on the prevalence of CAM in the largest family of angiosperms using the regional, highly diversified, and well-described tropical orchid flora of Panama and Costa Rica (Dressler, 1993; Hammel et al., 2003) and evaluate its relationship with climate and its role in the megadiversification of epiphytes. We used available phylogenetic information to determine the distribution of photosynthetic pathways among orchid species and relate these evolutionary patterns to climate-driven habitat preferences and epiphytism. We also used phylogenetic comparative methods to demonstrate statistically the evolutionary association between epiphytism and photosynthetic pathways and to evaluate the role of CAM in species radiations in the Orchidaceae.
RESULTS
A total of 1,022 Panamanian and Costa Rican orchid species from 147 genera covering 802 sites, and a total of 1,103 species-site combinations, were analyzed. Our study covered 4% of the total number of orchids (24,910 species) as described by Chase et al. (2003) and about 68% of the total estimated number of species from Panama and Costa Rica (approximately 1,500 species) as described by Dressler (1993). Variation in whole tissue δ13C values ranged from −37.1‰ to −11.4‰, with an overall mean of −27.7‰. The isotopic values among orchid species showed a bimodal distribution, with the majority of species showing values near −28‰, indicative of the C3 photosynthetic pathway, and a smaller mode near −16‰, indicative of the CAM pathway. We found that 924 species (90%) belong to the cluster of mainly C3 photosynthesis and had δ13C values more negative than −22‰, whereas 98 species (10%) belong to the cluster of mainly CAM and had δ13C values less negative than −22‰.
A, Percentage of CAM species as a function of altitude. B, Total number of species as a function of altitude. Black bars represent C3 photosynthesis species, and red bars represent CAM species.
Relationship between δ13C and climatic variables. Each point represents a species-site combination. Black points represent C3 photosynthesis species, and red points represent CAM species.
Orchidaceae tree showing the relationship among 147 tropical genera. Tribes (in boldface) and subtribes are shown. Photosynthetic pathways based on δ13C (this study) and titratable acidity derived from Silvera et al. (2005) were mapped onto the cladogram using Mesquite version 2.5. Those lineages that show C3 photosynthesis are represented by black lines, and those with CAM are highlighted in red. The arrow indicates node 68, which shows a large contribution index to divergence correlation. These data support the multiple, independent evolutionary origins of CAM.
Orchidaceae tree showing the relationship among 53 subtribes. Black vertical lines represent five major lineages recognized as subfamilies. Presence and absence of CAM based on this study and published information derived from Silvera et al. (2005) and Smith and Winter (1996) were mapped onto the cladogram using Mesquite version 2.5. Those lineages that show CAM are depicted in red. The red area within each pie chart indicates the relative support for different ancestor states. These data support the multiple, independent evolutionary origins of CAM.
Pairwise correlation between δ13C and epiphytism (A), and divergences in δ13C and epiphytism among radiations in the tree for orchid genera (B). Epiphytism is represented as a gradient in which 0 represents terrestrial forms (no epiphytism) and 1 represents epiphytic forms. Values between 0 and 1 represent genera with species that have both growth forms. Black points represent C3 photosynthesis genera, and red points represent CAM genera.
DISCUSSION
The challenge presented by the Orchidaceae is to understand how this group evolved with the large array of adaptive characteristics that has allowed a multitude of species-rich radiations and colonization of diverse terrestrial ecosystems worldwide. Our data demonstrate patterns of CAM evolution across the Orchidaceae, including multiple independent origins of CAM, several reversals indicating the evolutionary flexibility of CAM (Figs. 3 and 4), and parallel evolution of CAM across subfamilies (Fig. 4). Our data also indicate that divergences in photosynthetic pathway and epiphytism have been consistently correlated through evolutionary time (Fig. 5), related to the prevalence of CAM epiphytic species in lower elevations and abundant species diversification of high-elevation epiphytes.
The δ13C bimodal distribution found in this study is consistent with other studies (Griffiths and Smith, 1983; Pierce et al., 2002; Holtum et al., 2004; Silvera et al., 2005; Motomura et al., 2008) showing disruptive selection on C3 photosynthesis and strong CAM δ13C values with very few intermediate δ13C values. Strong CAM is present in about 10% of orchid species studied, and the largest proportion is distributed at lower altitude. We found a weak relationship between climatic variables and the photosynthetic pathway in this study, partly because Panama and Costa Rica are concentrated in a high-precipitation region and partly because the coarse climate variables analyzed do not fully describe the microclimate of epiphytic habitats. Nonetheless, our data demonstrate that CAM has contributed to the exploitation of epiphytic habitats through mid-elevation tropical montane environments. Orchid species richness in our study, with the largest peak around 1,000 to 1,500 m of altitude, is consistent with the rule of the mid-altitudinal bulge. The mechanism creating this bulge has been explained by the interaction between temperature and water gradients caused by increasing altitude (Whittaker and Niering, 1975; Peet, 1978; Zhao et al., 2005). Higher moisture availability from elevations around 1,000 m are likely to provide a more suitable environment for epiphytic lineages (Gentry and Dodson, 1987; Cardelús et al., 2006), whereas sites above 2,000 m might have reduced canopy height with less habitat for epiphyte colonization. Increases in δ13C with increasing altitude among C3 photosynthesis groups (Fig. 2), due to the reduced photosynthetic carbon isotope discrimination at high elevation, independent of CAM (Körner et al., 1988), can contribute to the δ13C-epiphytism relationship. However, previous studies have shown that the presence of weak CAM can also contribute to a second peak of abundance within the C3 photosynthesis isotopic cluster (Winter and Holtum, 2002; Silvera et al., 2005). Evaluation of the evolutionary role of weak CAM will require extensive in situ measurements of dawn/dusk titratable acidity, an endeavor limited by the accessibility of live specimens in the field. Nevertheless, the contribution of CAM-independent factors in determining the δ13C of high-elevation epiphytes and the role of weak CAM as an evolutionary reservoir for further CAM radiations in changing environments deserve further study.
We provide evidence that C3 photosynthesis is the ancestral state in the Orchidaceae and that CAM has evolved multiple independent times (Figs. 3 and 4). There is strong evidence that evolutionary progression of photosynthesis in plants has been from C3 photosynthetic ancestors to derived weak CAM to strong CAM modes (Pilón-Smits et al., 1996; Crayn et al., 2004), paralleled by progression from terrestrial ancestors to epiphytic growth forms. Phylogenetic tree construction using phosphoenolpyruvate carboxylase sequences has shown that sequences from CAM species within the Oncidiinae clade cluster together and differ from CAM sequences of species in the Vanillineae clade, providing evidence that these paralogous genes probably arose from one or more ancient duplications, thus adding an additional line of evidence for the multiple origins of CAM (K. Silvera, L. Rodriguez, R.L. Albion, K.M. Neubig, M.W. Whitten, N.H. Williams, K. Winter, and J.C. Cushman, unpublished data). A recent study using the genus Cymbidium concluded that weak CAM was the ancestral state and that C3 and strong CAM were derived (Motomura et al., 2008). However, the results were restricted to species within one genus and limit the conclusions that can be applied at the family level. The change from terrestrial to epiphytic habitat is paralleled by a change from monopodial to sympodial growth form, a common feature in epiphytic orchids often associated with pseudobulbs, which provide tolerance to drought stress. Our study also demonstrates parallel evolution of CAM among four of the five subfamilies of orchids (Epidendroideae, Orchidoideae, Cypripedioideae, and Vanilloideae). The two largest orchid subfamilies, Orchidoideae and Epidendroideae, are considered to have diversified early in the Tertiary (Ramírez et al., 2007). The progressive aridification and declining CO2 concentrations during the Tertiary (Pearson and Palmer, 2000) are likely factors that have contributed to the large CAM radiation in Epidendroideae, which has produced the most species-rich epiphytic clades of any known plant group. CAM and epiphytism in orchids are likely to have originated about 65 million years ago. The CAM radiation events within the subfamily Epidendroideae (Fig. 4), and especially within the Neotropical subtribes Oncidiinae and Laeliinae (Fig. 3), are thus in line with paleoenvironmental conditions that favored the evolution of CAM (Monson, 1989; Ehleringer and Monson, 1993; Raven and Spicer, 1996).
Epiphytism in orchids is a pantropical phenomenon, and our study shows significant correlation between photosynthetic pathways and epiphytism (Fig. 5), indicating that throughout evolutionary time, divergence in δ13C is consistently accompanied by divergence in epiphytism and demonstrating a functional relationship between these traits. Correlated divergence between the photosynthetic pathway and epiphytism is likely an important factor contributing to the burst of speciation that occurred in diverse epiphytic orchid clades (subtribe Oncidiinae and Laeliinae; Fig. 3). These clades are species rich due to colonization of epiphytic habitats, and species within them were able to colonize wider habitat ranges compared with other plant species, due to the development of CAM as a water-conserving mode of photosynthesis, ultimately contributing to the large floristic diversity found in the Neotropics. Correlated divergence between photosynthetic pathways also indicates that epiphytic orchid species developed CAM convergently to other epiphytic or terrestrial plant species under the selectional pressure arising from the search for light in rainforest habitats. Our study shows that strong CAM species are restricted to epiphytic habitats, except for Oeceoclades maculata, a terrestrial invasive orchid species native to Africa with recent distribution in the Neotropics from Argentina to Florida (Stern, 1988; Cohen and Ackerman, 2009). The presence of strong CAM in this genus is intriguing, and whether or not the presence of CAM contributes to invasiveness in this species is unknown.
CONCLUSION
This study demonstrates several patterns of CAM evolution across the Orchidaceae, including multiple independent origins of CAM, several reversal events indicating the evolutionary flexibility of CAM, and parallel evolution of CAM across subfamilies. Divergences in the photosynthetic pathway and epiphytism have been consistently correlated through evolutionary time and are related to the prevalence of CAM epiphytic species in lower elevations and abundant species diversification of high-elevation epiphytes. Overall, our study reveals biochemical underpinnings and evolutionary interactions between CAM as a water-saving mode of photosynthesis and colonization of epiphytic habitats that have contributed to some of the most substantial plant speciations known to exist.
MATERIALS AND METHODS
Site Description
Panama and Costa Rica are equatorial tropical countries located between 7° to 11° N and 77° to 80° W. The Panamanian isthmus serves as a land bridge between North and South America and fosters a rich intermixture of plant and animal life that has migrated between the continents. The two principal mountain ranges, the Tabasará Mountains (Cordillera Central) in the west and the Cordillera de San Blas in the east, divide the country into Atlantic- and Pacific-facing slopes. Costa Rica is similarly divided into Caribbean and Pacific slopes by the Cordillera Central and the Cordillera de Talamanca. Two distinct regional seasons driven by latitudinal movement of the intertropical convergence zone produce a dry season from December to May with shorter, less intense dry seasons in sites with greater annual precipitation (Dressler, 1993). Annual precipitation is generally greater on the Caribbean coast (1,500–5,550 mm per year) than on the Pacific coast (1,140–2,290 mm per year). We present data from sites ranging in mean annual precipitation from 1,652 to 5,204 mm per year, mean annual temperatures ranging from 14.9°C to 27.6°C, mean RH ranging from 79.7% to 89.2%, and altitude from sea level to 3,290 m.
Carbon Isotope Analysis
We categorized species into C3 or CAM based on leaf δ13C values commonly observed for C3 photosynthesis plants ranging from −33‰ to −22‰ and with δ13C values typical of CAM plants ranging from −22‰ to −12‰ (Ehleringer and Osmond, 1989).
Climatic Data
We used altitude and geographic coordinates to determine climate variables for each herbarium specimen sampled for carbon isotope analysis. For each specimen, we recorded species name, herbarium code and collection number, growth form (epiphytic, terrestrial, or both), collection location, elevation, and coordinates. We used average altitude for species in which a range was given. For species with missing entries, verbal descriptions of location were converted to altitude and coordinates using the online Global Gazette Version 2.0 (http://www.fallingrain.com/world/) for Panama and Costa Rica. Coordinate information for each specimen was then used to generate mean annual temperature, mean RH, mean annual precipitation, and mean annual DTR using the Climatic Research Unit application CRU_CL_2.0 (New et al., 2002; http://www.cru.uea.ac.uk/cru/data/tmc.htm). From DTR, we calculated mean Tmin and Tmax. Simple and multiple regression analyses were used to determine relationships between climatic variables and δ13C using SAS statistics software (version 9.1.3).
Orchid Phylogeny and Species Nomenclature
We constructed two tree hypotheses for the Orchidaceae (genera and subtribes) using Phylomatic 2 (Webb et al., 2008) and Mesquite version 2.5 (Maddison and Maddison, 2008). The relationship among genera and overall tree topology followed the strict consensus tree resulting from a combined analysis of psaB + rbcL gene sequences (Cameron, 2004). The relationship among subtribes and overall topology followed the summary tree of the revised classification of Orchidaceae (Chase et al., 2003). Resulting subtribe and genera trees retained existing polytomies. The overall tree topology differs between these two trees because of different taxa and regions sampled within each publication. Because of the large size of the Orchidaceae, relationships among genera were further expanded in the genera tree using recent publications and nomenclatural changes to the Oncidiinae (Williams et al., 2001a, 2001b; Dressler and Williams, 2003), Laeliinae (Higgins, 1997; van den Berg et al., 2000; Dressler, 2002; Dressler and Higgins, 2003), Pleurothallidinae (Pridgeon and Chase, 2001; Pridgeon et al., 2001; Luer, 2004), Cranichideae (Salazar et al., 2003), and Maxillarieae (Whitten et al., 2000, 2007; Ojeda et al., 2005). Overall, our nomenclature is consistent with recent publications on nomenclatural changes presented above, the Field Guide to the Orchids of Costa Rica and Panama (Dressler, 1993), and the Royal Botanic Gardens Kew World Checklist of Monocotyledons nomenclatural database and associated authority files (http://apps.kew.org/wcsp/home.do).
Phylogenetic and Statistical Analyses
Presence and absence of CAM were traced onto the genera tree based on δ13C (this study) and titratable acidity measurements (Silvera et al., 2005). Genera were labeled as CAM if at least one species within the genus was known to conduct CAM. Character state reconstruction was performed by maximum likelihood in Mesquite version 2.5 (Maddison and Maddison, 2008), using a marginal probability reconstruction with Asymmetrical parameter Markov-k model of evolution, with an estimated forward rate of 0.1020 and backward rate of 0.2007 corresponding to gain or loss of CAM, respectively. Root state frequency in this analysis was set to be the same as equilibrium, and we specified a bias of less than 1 indicating that backward changes (loss of CAM) are more probable than forward changes. This model was chosen over parsimony or the Mk1 model (gain and loss of CAM are equally weighted) based on the prior assumption that the gain of CAM is less probable because it requires the appearance and coordination of multiple genes, whereas the loss of CAM could result from a single loss-of-function mutation. This prior assumption would likely avoid the false rejection of Dollo's law when reconstructing a character (Goldberg and Igic, 2008). Because both trees were the result of the compilation of different phylogenies, branch lengths were not available and analyses were performed with branch lengths set as equal to 1. Presence and absence of CAM based on δ13C were traced onto the orchid subtribe tree using the same reconstruction method as the orchid genera tree. Subtribes were labeled as CAM if at least one genus within the subtribe was known to conduct CAM based on the information presented in this paper and on previously published information (Smith and Winter, 1996; Silvera et al., 2005). The results of analyses using two different assumptions about the probability of each character state at the root (state frequency same as equilibrium and state frequency equal) gave nearly identical outputs for both genera and subtribe trees. The results were displayed as likelihood states reported as the proportion of total likelihood and represented as pie diagrams for the subtribe tree and as proportional to weights in each branch for the genera tree.
To analyze the relationship between the photosynthetic pathway and epiphytism, leaf δ13C was coded for each genus as the mean for all species of that genus. Epiphytism was coded for each species as 0 for terrestrial and 1 for epiphytic and for the genus as the mean for all species of that genus. For simplicity, both epiphytes and lithophytes were considered as epiphytes. Trait correlation between leaf δ13C and epiphytism was evaluated with Pearson product-moment correlation coefficients across orchid genera. The same genus-level coding was used to perform correlated divergence analysis between δ13C and epiphytism in the Analysis of Traits module in Phylocom (Felsenstein, 1985; Webb et al., 2008) using the genera tree for Orchidaceae. Branch lengths were estimated by calibrating the tree for orchid genera at a single point (first bifurcation, 76 million years ago) using the age estimate for the Orchidaceae from Ramírez et al. (2007). We then used the branch length adjustment utility in Phylocom (BLADJ) to perform age interpolations for undated nodes and produce phylogenetic distances. Node ages were treated as approximations. Analysis of Traits calculates a standardized divergence of extant taxa and handles polytomies by ranking species based on the value of the independent variable when the median is used to create two groups (Pagel, 1992). Correlated divergence analysis was performed by constructing a 0-intercept linear regression between divergence in trait 1 (δ13C) and divergence in trait 2 (epiphytism) among sister groups throughout the genera tree, so that divergence width was based on differences in traits between descendent lines (Garland et al., 1993; Garland and Díaz-Uriarte, 1999; Webb et al., 2008). We used divergence width instead of independent contrasts as a measure of absolute trait radiation because the sd (divergence width) can be used when polytomies are present in the phylogeny (Moles et al., 2005; Beaulieu et al., 2007). We also used contribution index as a measure of the amount of present-day variation within a trait that is attributed to a focal clade (Moles et al., 2005).
ACKNOWLEDGMENTS
Special thanks go to Dr. Bruce Holst (Selby Botanical Gardens); Kent Perkins, Dr. Mark Whitten, and Dr. Norris Williams (University of Florida Herbarium); Dr. Jim Solomon (Missouri Botanical Gardens Herbarium); and Mireya Correa (University of Panama Herbarium) for assisting with herbarium collections. We gratefully acknowledge Dr. Mark Whitten, Kurt Neubig, and Lorena Endara (University of Florida), the Santiago laboratory (University of California, Riverside), and two anonymous reviewers for comments to improve this paper; Dr. Todd Dawson and Dr. Stefania Mambelli (University of California, Berkeley) for assisting with isotopic analysis; Dr. Karen Schlauch (University of Nevada, Reno) for statistical advice; Dr. Doug Altshuler (University of California, Riverside) for Mesquite advice; Cristina Milsner (University of Nevada, Reno) and Michael O'Leary (University of California, Riverside) for assisting with database entry; and Vanessa Boukili (University of California, Berkeley) and Becky Albion (University of Nevada, Reno) for assistance in the laboratory.
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Author notes
This work was supported by the Environmental Protection Agency (Greater Research Opportunities Graduate Program Assistance Agreement no. MA 91685201 to K.S.), the National Science Foundation (grant nos. IOB–0543659 to J.C.C. and DEB–0706813 to L.S.S.), the National Institutes of Health (grant no. P20 RR–016464 from the Idea Network of Biomedical Research Excellence Program of the National Center for Research Resources supporting the Nevada Genomics, Proteomics, and Bioinformatics Center), the Andrew W. Mellon Foundation through the Smithsonian Tropical Research Institute (to K.W.), and the Nevada Agricultural Experiment Station (as publication no. NAES 03087114).
Corresponding author; e-mail silverak@unr.nevada.edu.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Katia Silvera (silverak@unr.nevada.edu).
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