The stomatal response to vapor pressure deficit drives the apparent temperature response of photosynthesis in tropical forests.

As temperature rises, net carbon uptake in tropical forests decreases, but the underlying mechanisms are not well understood. High temperatures can limit photosynthesis directly, for example by reducing biochemical capacity, or indirectly through rising vapor pressure deficit (VPD) causing stomatal closure. To explore the independent effects of temperature and VPD on photosynthesis we analyzed photosynthesis data from the upper canopies of two tropical forests in Panama with Generalized Additive Models. Stomatal conductance and photosynthesis consistently decreased with increasing VPD, and statistically accounting for VPD increased the optimum temperature of photosynthesis (Topt) of trees from a VPD-confounded apparent Topt of c. 30-31°C to a VPD-independent Topt of c. 33-36°C, while for lianas no VPD-independent Topt was reached within the measured temperature range. Trees and lianas exhibited similar temperature and VPD responses in both forests, despite 1500 mm difference in mean annual rainfall. Over ecologically relevant temperature ranges, photosynthesis in tropical forests is largely limited by indirect effects of warming, through changes in VPD, not by direct warming effects of photosynthetic biochemistry. Failing to account for VPD when determining Topt misattributes the underlying causal mechanism and thereby hinders the advancement of mechanistic understanding of global warming effects on tropical forest carbon dynamics.

A medida que aumenta la temperatura, disminuye la absorción neta de carbono en los bosques tropicales, sin embargo, aún no se conocen bien los mecanismos que la subyacen. Las altas temperaturas pueden limitar la fotosíntesis directamente, por ejemplo, reduciendo la eficiencia de los procesos bioquímicos, pero también de forma indirecta a través del aumento del déficit de presión de vapor (DPV) que resulta en el cierre estomático. Para explorar los efectos independientes de la temperatura y el DPV en la fotosíntesis, analizamos datos de la absorción neta de carbono del dosel de dos bosques tropicales en Panamá utilizando modelos aditivos generalizados. La conductancia estomática y la fotosíntesis disminuyó consistentemente con el aumento de DPV, y considerando el DPV en modelas estadísticas, la temperatura óptima de la fotosíntesis (Topt) aumentó, de un Topt aparente influida por la DVP de c. 30­31°C a un Topt independiente del DPV de c. 33­36°C. Los árboles y las lianas mostraron respuestas similares a la temperatura y a la DVP en ambos bosques, a pesar de la diferencia de 1500 mm en la precipitación media anual. La fotosíntesis en los bosques tropicales está limitada en gran medida por los efectos indirectos del aumento de la temperatura, a través de cambios en el DPV y no por los efectos directos en los procesos bioquímicos. Si no se tiene en cuenta el DPV al determinar el Topt, se atribuye erróneamente el mecanismo causal subyacente y, por lo tanto, se obstaculiza el avance en la comprensión de los efectos del calentamiento global en la dinámica del carbono.

Evidence for phylogenetic signal and correlated evolution in plant-water relation traits.

Evolutionary relationships are likely to play a significant role in shaping plant physiological and structural traits observed in contemporary taxa. We review research on phylogenetic signal and correlated evolution in plant-water relation traits, which play important roles in allowing plants to acquire, use, and conserve water. We found more evidence for a phylogenetic signal in structural traits (e.g. stomatal length and stomatal density) than in physiological traits (e.g. stomatal conductance and water potential at turgor loss). Although water potential at turgor loss is the most-studied plant-water relation trait in an evolutionary context, it is the only trait consistently found to not have a phylogenetic signal. Correlated evolution was common among traits related to water movement efficiency and hydraulic safety in both leaves and stems. We conclude that evidence for phylogenetic signal varies depending on: the methodology used for its determination, that is, model-based approaches to determine phylogenetic signal such as Blomberg's K or Pagel's λ vs statistical approaches such as ANOVAs with taxonomic classification as a factor; on the number of taxa studied (size of the phylogeny); and the setting in which plants grow (field vs common garden). More explicitly and consistently considering the role of evolutionary relationships in shaping plant ecophysiology could improve our understanding of how traits compare among species, how traits are coordinated with one another, and how traits vary with the environment.

Plant water use theory should incorporate hypotheses about extreme environments, population ecology, and community ecology.

Plant water use theory has largely been developed within a plant-performance paradigm that conceptualizes water use in terms of value for carbon gain and that sits within a neoclassical economic framework. This theory works very well in many contexts but does not consider other values of water to plants that could impact their fitness. Here, we survey a range of alternative hypotheses for drivers of water use and stomatal regulation. These hypotheses are organized around relevance to extreme environments, population ecology, and community ecology. Most of these hypotheses are not yet empirically tested and some are controversial (e.g. requiring more agency and behavior than is commonly believed possible for plants). Some hypotheses, especially those focused around using water to avoid thermal stress, using water to promote reproduction instead of growth, and using water to hoard it, may be useful to incorporate into theory or to implement in Earth System Models.

Dissecting succulence: Crassulacean acid metabolism and hydraulic capacitance are independent adaptations in Clusia leaves.

Succulence is found across the world as an adaptation to water-limited niches. The fleshy organs of succulent plants develop via enlarged photosynthetic chlorenchyma and/or achlorophyllous water storage hydrenchyma cells. The precise mechanism by which anatomical traits contribute to drought tolerance is unclear, as the effect of succulence is multifaceted. Large cells are believed to provide space for nocturnal storage of malic acid fixed by crassulacean acid metabolism (CAM), whilst also buffering water potentials by elevating hydraulic capacitance (CFT ). The effect of CAM and elevated CFT on growth and water conservation have not been compared, despite the assumption that these adaptations often occur together. We assessed the relationship between succulent anatomical adaptations, CAM, and CFT , across the genus Clusia. We also simulated the effects of CAM and CFT on growth and water conservation during drought using the Photo3 model. Within Clusia leaves, CAM and CFT are independent traits: CAM requires large palisade chlorenchyma cells, whereas hydrenchyma tissue governs interspecific differences in CFT . In addition, our model suggests that CAM supersedes CFT as a means to maximise CO2 assimilation and minimise transpiration during drought. Our study challenges the assumption that CAM and CFT are mutually dependent traits within succulent leaves.

Brief reflections on 50 years as a plant ecophysiologist.

SCOPE: This paper is a short biographical sketch of my life as a plant ecophysiologist in which serendipity and outstanding collaborators have been key allies.

Forty years of research into crassulacean acid metabolism in the genus Clusia: anatomy, ecophysiology and evolution.

Clusia is the only genus containing dicotyledonous trees with a capacity to perform crassulacean acid metabolism (CAM). Since the discovery of CAM in Clusia 40 years ago, several studies have highlighted the extraordinary plasticity and diversity of life forms, morphology and photosynthetic physiology of this genus. In this review, we revisit aspects of CAM photosynthesis in Clusia and hypothesize about the timing, the environmental conditions and potential anatomical characteristics that led to the evolution of CAM in the group. We discuss the role of physiological plasticity in influencing species distribution and ecological amplitude in the group. We also explore patterns of allometry of leaf anatomical traits and their correlations with CAM activity. Finally, we identify opportunities for further research on CAM in Clusia, such as the role of elevated nocturnal accumulation of citric acid, and gene expression in C3-CAM intermediate phenotypes.

Leaf vein density correlates with crassulacean acid metabolism, but not hydraulic capacitance, in the genus Clusia.

BACKGROUND AND AIMS: Many succulent species are characterized by the presence of Crassulacean acid metabolism (CAM) and/or elevated bulk hydraulic capacitance (CFT). Both CAM and elevated CFT substantially reduce the rate at which water moves through transpiring leaves. However, little is known about how these physiological adaptations are coordinated with leaf vascular architecture. METHODS: The genus Clusia contains species spanning the entire C3-CAM continuum, and also is known to have >5-fold interspecific variation in CFT. We used this highly diverse genus to explore how interspecific variation in leaf vein density is coordinated with CAM and CFT. KEY RESULTS: We found that constitutive CAM phenotypes were associated with lower vein length per leaf area (VLA) and vein termini density (VTD), compared to C3 or facultative CAM species. However, when vein densities were standardized by leaf thickness, this value was higher in CAM than C3 species, which is probably an adaptation to overcome apoplastic hydraulic resistance in deep chlorenchyma tissue. In contrast, CFT did not correlate with any xylem anatomical trait measured, suggesting CAM has a greater impact on leaf transpiration rates than CFT. CONCLUSIONS: Our findings strongly suggest that CAM photosynthesis is coordinated with leaf vein densities. The link between CAM and vascular anatomy will be important to consider when attempting to bioengineer CAM into C3 crops.

The CAM lineages of planet Earth.

BACKGROUND AND SCOPE: The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages. RESULTS AND CONCLUSIONS: We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.

CAM photosynthesis: the acid test.

There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesis - or key elements associated with it, such as increased water-use efficiency - into C3 plants. Resolving how CAM photosynthesis evolved from the ancestral C3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C3 and CAM.

Evolution of crassulacean acid metabolism (CAM) as an escape from ecological niche conservatism in Malagasy Bulbophyllum (Orchidaceae).

Despite growing evidence that niche shifts are more common in flowering plants than previously thought, little is known of whether such shifts are promoted by changes in photosynthetic pathways. Here we combine the most complete phylogeny for epiphytic Malagasy Bulbophyllum orchids (c. 210 spp.) with climatic niche and carbon isotope ratios to infer the group's spatial-temporal history, and the role of strongly expressed crassulacean acid metabolism (CAM) in facilitating niche shifts and diversification. We find that most extant species still retain niche (Central Highland) and photosynthesis (C3 ) states as present in the single mid-Miocene (c. 12.70 million yr ago (Ma)) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c. 7.36 Ma) invasion of species from the sub-humid highland first into the island's humid eastern coastal, and then into the seasonally dry 'Northwest Sambirano' rainforests, yet without significant effect on diversification rates. These findings indicate that CAM in tropical epiphytes may be selectively advantageous even in high rainfall habitats, rather than presenting a mere adaptation to dry environments or epiphytism per se. Overall, our study qualifies CAM as an evolutionary 'gateway' trait that considerably widened the spatial-ecological amplitude of Madagascar's most species-rich orchid genus.

Large differences in leaf cuticle conductance and its temperature response among 24 tropical tree species from across a rainfall gradient.

More frequent droughts and rising temperatures pose serious threats to tropical forests. When stomata are closed under dry and hot conditions, plants lose water through leaf cuticles, but little is known about cuticle conductance (gmin ) of tropical trees, how it varies among species and environments, and how it is affected by temperature. We determined gmin in relation to temperature for 24 tropical tree species across a steep rainfall gradient in Panama, by recording leaf drying curves at different temperatures in the laboratory. In contrast with our hypotheses, gmin did not differ systematically across the rainfall gradient; species differences did not reflect phylogenetic patterns; and in most species gmin did not significantly increase between 25 and 50°C. gmin was higher in deciduous than in evergreen species, in species with leaf trichomes than in species without, in sun leaves than in shade leaves, and tended to decrease with increasing leaf mass per area across species. There was no relationship between stomatal and cuticle conductance. Large species differences in gmin and its temperature response suggest that more frequent hot droughts may lead to differential survival among tropical tree species, regardless of species' position on the rainfall gradient.

Photosynthetic plasticity of a tropical tree species, Tabebuia rosea, in response to elevated temperature and [CO2 ].

Atmospheric and climate change will expose tropical forests to conditions they have not experienced in millions of years. To better understand the consequences of this change, we studied photosynthetic acclimation of the neotropical tree species Tabebuia rosea to combined 4°C warming and twice-ambient (800 ppm) CO2 . We measured temperature responses of the maximum rates of ribulose 1,5-bisphosphate carboxylation (VCMax ), photosynthetic electron transport (JMax ), net photosynthesis (PNet ), and stomatal conductance (gs ), and fitted the data using a probabilistic Bayesian approach. To evaluate short-term acclimation plants were then switched between treatment and control conditions and re-measured after 1-2 weeks. Consistent with acclimation, the optimum temperatures (TOpt ) for VCMax , JMax and PNet were 1-5°C higher in treatment than in control plants, while photosynthetic capacity (VCMax , JMax , and PNet at TOpt ) was 8-25% lower. Likewise, moving control plants to treatment conditions moderately increased temperature optima and decreased photosynthetic capacity. Stomatal density and sensitivity to leaf-to-air vapour pressure deficit were not affected by growth conditions, and treatment plants did not exhibit stronger stomatal limitations. Collectively, these results illustrate the strong photosynthetic plasticity of this tropical tree species as even fully developed leaves of saplings transferred to extreme conditions partially acclimated.

Leaf heat tolerance of 147 tropical forest species varies with elevation and leaf functional traits, but not with phylogeny.

Exceeding thermal thresholds causes irreversible damage and ultimately loss of leaves. The lowland tropics are among the warmest forested biomes, but little is known about heat tolerance of tropical forest plants. We surveyed leaf heat tolerance of sun-exposed leaves from 147 tropical lowland and pre-montane forest species by determining the temperatures at which potential photosystem II efficiency based on chlorophyll a fluorescence started to decrease (TCrit ) and had decreased by 50% (T50 ). TCrit averaged 46.7°C (5th-95th percentile: 43.5°C-49.7°C) and T50 averaged 49.9°C (47.8°C-52.5°C). Heat tolerance partially adjusted to site temperature; TCrit and T50 decreased with elevation by 0.40°C and 0.26°C per 100 m, respectively, while mean annual temperature decreased by 0.63°C per 100 m. The phylogenetic signal in heat tolerance was weak, suggesting that heat tolerance is more strongly controlled by environment than by evolutionary legacies. TCrit increased with the estimated thermal time constant of the leaves, indicating that species with thermally buffered leaves maintain higher heat tolerance. Among lowland species, T50 increased with leaf mass per area, suggesting that in species with structurally more costly leaves the risk of leaf loss during hot spells is reduced. These results provide insight in variation in heat tolerance at local and regional scales.

Photosynthetic quantum efficiency in south-eastern Amazonian trees may be already affected by climate change.

Tropical forests are experiencing unprecedented high-temperature conditions due to climate change that could limit their photosynthetic functions. We studied the high-temperature sensitivity of photosynthesis in a rainforest site in southern Amazonia, where some of the highest temperatures and most rapid warming in the Tropics have been recorded. The quantum yield (Fv /Fm ) of photosystem II was measured in seven dominant tree species using leaf discs exposed to varying levels of heat stress. T50 was calculated as the temperature at which Fv /Fm was half the maximum value. T5 is defined as the breakpoint temperature, at which Fv /Fm decline was initiated. Leaf thermotolerance in the rapidly warming southern Amazonia was the highest recorded for forest tree species globally. T50 and T5 varied between species, with one mid-storey species, Amaioua guianensis, exhibiting particularly high T50 and T5 values. While the T50 values of the species sampled were several degrees above the maximum air temperatures experienced in southern Amazonia, the T5 values of several species are now exceeded under present-day maximum air temperatures.

Safety and acute efficacy of cryoballoon ablation for atrial fibrillation at community hospitals.

AIMS: Pulmonary vein isolation (PVI) using cryoballoon ablation (CBA) is an established procedure for treating symptomatic paroxysmal and persistent atrial fibrillation (AF). The safety and efficacy of PVI performed at community hospitals are unknown. We aimed to determine the safety and acute efficacy of PVI using CBA performed at community hospitals with limited annual case numbers. METHODS AND RESULTS: This registry study included 1004 consecutive patients who had PVI performed for symptomatic paroxysmal (n = 563) or persistent AF (n = 441) from January 2019 to September 2020 at 20 hospitals. Each hospital performed fewer than 100 CBA-PVI procedures/year according to local standards. Procedural data, efficacy, and complication rates were determined. The mean number of CBA procedures performed/year at each centre was 59 ± 25. The average procedure time was 90.1 ± 31.6 min and the average fluoroscopy time was 19.2 ± 11.4 min. Isolation of all pulmonary veins was documented in 97.9% of patients. The most frequent reason for not achieving complete isolation was development of phrenic nerve palsy. No hospital deaths were observed. Two patients (0.2%) suffered a clinical stroke. Pericardial effusion occurred in six patients (0.6%), two of whom (0.2%) required pericardial drainage. Vascular complications occurred in 24 patients (2.4%), two of whom (0.2%) required vascular surgery. Phrenic nerve palsy occurred in 48 patients (4.8%) and persisted up to hospital discharge in six patients (0.6%). CONCLUSION: Pulmonary vein isolation procedures for paroxysmal or persistent AF using CBA can be performed at community hospitals with high acute efficacy and low complication rates.

Directional change in leaf dry matter δ 13C during leaf development is widespread in C3 plants.

BACKGROUND AND AIMS: The stable carbon isotope ratio of leaf dry matter (δ 13Cp) is generally a reliable recorder of intrinsic water-use efficiency in C3 plants. Here, we investigated a previously reported pattern of developmental change in leaf δ 13Cp during leaf expansion, whereby emerging leaves are initially 13C-enriched compared to mature leaves on the same plant, with their δ 13Cp decreasing during leaf expansion until they eventually take on the δ 13Cp of other mature leaves. METHODS: We compiled data to test whether the difference between mature and young leaf δ 13Cp differs between temperate and tropical species, or between deciduous and evergreen species. We also tested whether the developmental change in δ 13Cp is indicative of a concomitant change in intrinsic water-use efficiency. To gain further insight, we made online measurements of 13C discrimination (∆ 13C) in young and mature leaves. KEY RESULTS: We found that the δ 13Cp difference between mature and young leaves was significantly larger for deciduous than for evergreen species (-2.1 ‰ vs. -1.4 ‰, respectively). Counter to expectation based on the change in δ 13Cp, intrinsic water-use efficiency did not decrease between young and mature leaves; rather, it did the opposite. The ratio of intercellular to ambient CO2 concentrations (ci/ca) was significantly higher in young than in mature leaves (0.86 vs. 0.72, respectively), corresponding to lower intrinsic water-use efficiency. Accordingly, instantaneous ∆ 13C was also higher in young than in mature leaves. Elevated ci/ca and ∆ 13C in young leaves resulted from a combination of low photosynthetic capacity and high day respiration rates. CONCLUSION: The decline in leaf δ 13Cp during leaf expansion appears to reflect the addition of the expanding leaf's own 13C-depleted photosynthetic carbon to that imported from outside the leaf as the leaf develops. This mixing of carbon sources results in an unusual case of isotopic deception: less negative δ 13Cp in young leaves belies their low intrinsic water-use efficiency.

Photosynthetic heat tolerance of shade and sun leaves of three tropical tree species.

Previous studies of heat tolerance of tropical trees have focused on canopy leaves exposed to full sunlight and high temperatures. However, in lowland tropical forests with leaf area indices of 5-6, the vast majority of leaves experience varying degrees of shade and a reduced heat load compared to sun leaves. Here we tested whether heat tolerance is lower in shade than in sun leaves. For three tropical tree species, Calophyllum inophyllum, Inga spectabilis, and Ormosia macrocalyx, disks of fully developed shade and sun leaves were subjected to 15-min heat treatments, followed by measurement of chlorophyll a fluorescence after 48 h of recovery. In two of the three species, the temperature causing a 50% decrease of the fluorescence ratio Fv/Fm (T50) was significantly lower (by ~ 1.0 °C) in shade than in sun leaves, indicating a moderately decreased heat tolerance of shade leaves. In shade leaves of these two species, the rise in initial fluorescence, F0, also occurred at lower temperatures. In the third species, there was no shade-sun difference in T50. In situ measurements of photosynthetic CO2 assimilation showed that the optimum temperature for photosynthesis tended to be lower in shade leaves, although differences were not significant. At supra-optimal temperatures, photosynthesis was largely constrained by stomatal conductance, and the high-temperature CO2 compensation point, TMax, occurred at considerably lower temperatures than T50. Our study demonstrates that the temperature response of shade leaves of tropical trees differs only marginally from that of sun leaves, both in terms of heat tolerance and photosynthetic performance.

Ecophysiology of constitutive and facultative CAM photosynthesis.

In plants exhibiting crassulacean acid metabolism (CAM), CAM photosynthesis almost always occurs together with C3 photosynthesis, and occasionally with C4 photosynthesis. Depending on species, ontogeny, and environment, CAM input to total carbon gain can vary from values of <1% to 100%. The wide range of CAM phenotypes between and within species is a fascinating example of functional diversity and plasticity, but poses a significant challenge when attempting to define CAM. CO2 gas exchange experiments designed for this review illustrate key patterns of CAM expression and highlight distinguishing features of constitutive and facultative CAM. Furthermore, they help to address frequently recurring questions on CAM terminology. The functional and evolutionary significance of contrasting CAM phenotypes and of intermediate states between extremes is discussed. Results from a study on nocturnal malate accumulation in 50 species of Aizoaceae exposed to drought and salinity stress suggest that facultative CAM is more widespread amongst vascular plants than previously thought.

Operating at the very low end of the crassulacean acid metabolism spectrum: Sesuvium portulacastrum (Aizoaceae).

Demonstration of crassulacean acid metabolism (CAM) in species with low usage of this system relative to C3-photosynthetic CO2 assimilation can be challenging experimentally but provides crucial information on the early steps of CAM evolution. Here, weakly expressed CAM was detected in the well-known pantropical coastal, leaf-succulent herb Sesuvium portulacastrum, demonstrating that CAM is present in the Sesuvioideae, the only sub-family of the Aizoaceae in which it had not yet been shown conclusively. In outdoor plots in Panama, leaves and stems of S. portulacastrum consistently exhibited a small degree of nocturnal acidification which, in leaves, increased during the dry season. In potted plants, nocturnal acidification was mainly facultative, as levels of acidification increased in a reversible manner following the imposition of short-term water-stress. In drought-stressed plants, nocturnal net CO2 exchange approached the CO2-compensation point, consistent with low rates of CO2 dark fixation sufficient to eliminate respiratory carbon loss. Detection of low-level CAM in S. portulacastrum adds to the growing number of species that cannot be considered C3 plants sensu stricto, although they obtain CO2 principally via the C3 pathway. Knowledge about the presence/absence of low-level CAM is critical when assessing trajectories of CAM evolution in lineages. The genus Sesuvium is of particular interest because it also contains C4 species.

Facultative crassulacean acid metabolism in a C3-C4 intermediate.

The Portulacaceae enable the study of the evolutionary relationship between C4 and crassulacean acid metabolism (CAM) photosynthesis. Shoots of well-watered plants of the C3-C4 intermediate species Portulaca cryptopetala Speg. exhibit net uptake of CO2 solely during the light. CO2 fixation is primarily via the C3 pathway as indicated by a strong stimulation of CO2 uptake when shoots were provided with air containing 2% O2. When plants were subjected to water stress, daytime CO2 uptake was reduced and CAM-type net CO2 uptake in the dark occurred. This was accompanied by nocturnal accumulation of acid in both leaves and stems, also a defining characteristic of CAM. Following rewatering, net CO2 uptake in the dark ceased in shoots, as did nocturnal acidification of the leaves and stems. With this unequivocal demonstration of stress-related reversible, i.e. facultative, induction of CAM, P. cryptopetala becomes the first C3-C4 intermediate species reported to exhibit CAM. Portulaca molokiniensis Hobdy, a C4 species, also exhibited CAM only when subjected to water stress. Facultative CAM has now been demonstrated in all investigated species of Portulaca, which are well sampled from across the phylogeny. This strongly suggests that in Portulaca, a lineage in which species engage predominately in C4 photosynthesis, facultative CAM is ancestral to C4. In a broader context, it has now been demonstrated that CAM can co-exist in leaves that exhibit any of the other types of photosynthesis known in terrestrial plants: C3, C4 and C3-C4 intermediate.