Journal of Experimental Botany, Vol. 64, No. 18, pp. 5485–5496, 2013 doi:10.1093/jxb/ert314 Advance Access publication 14 October, 2013 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

Research paper How succulent leaves of avoid mesophyll conductance limitations of photosynthesis and survive drought

Brad S. Ripley1,*, Trevor Abraham1, Cornelia Klak2 and Michael D. Cramer3 1 Department of Botany, Rhodes University, 6140 Grahamstown, South Africa 2 Bolus Herbarium, Department of Biological Sciences, University of Cape Town, 7701 Rondebosch, South Africa 3 Department of Biological Sciences, University of Cape Town, 7701 Rondebosch, South Africa

* To whom correspondence should be addressed. E-mail: [email protected]

Received 23 May 2013; Revised 30 July 2013; Accepted 22 August 2013

Abstract

In several taxa, increasing leaf succulence has been associated with decreasing mesophyll conductance (gM) and an increasing dependence on Crassulacean acid metabolism (CAM). However, in succulent Aizoaceae, the photosyn- thetic tissues are adjacent to the leaf surfaces with an internal achlorophyllous hydrenchyma. It was hypothesized that this arrangement increases gM, obviating a strong dependence on CAM, while the hydrenchyma stores water and nutrients, both of which would only be sporadically available in highly episodic environments. These predictions were tested with species from the Aizoaceae with a 5-fold variation in leaf succulence. It was shown that gM values, derived from the response of photosynthesis to intercellular CO2 concentration (A:Ci), were independent of succulence, and 13 that foliar photosynthate δ C values were typical of C3, but not CAM photosynthesis. Under water stress, the degree of leaf succulence was positively correlated with an increasing ability to buffer photosynthetic capacity over several hours and to maintain light reaction integrity over several days. This was associated with decreased rates of water loss, rather than tolerance of lower leaf water contents. Additionally, the hydrenchyma contained ~26% of the leaf nitrogen content, possibly providing a nutrient reservoir. Thus the intermittent use of C3 photosynthesis interspersed with periods of no positive carbon assimilation is an alternative strategy to CAM for succulent taxa (Crassulaceae and Aizoaceae) which occur sympatrically in the Cape Floristic Region of South Africa.

Key words: Aizoaceae, Crassulaceae, Crassulacean acid metabolism, drought avoidance, leaf succulence, mesophyll conductance

Introduction Leaf succulence has evolved in diverse phylogenetic lineages et al., 2007) and leaf succulence (as opposed to stem suc- worldwide (Landrum, 2002; Ogburn and Edwards, 2010) and culence) is found in some 30 lineages within the Aizoaceae, is common in subject to frequent drought. Succulents Asphodelaceae, and Crassulaceae (Linder et al., 2010). This form a major component of the flora of the Succulent Karoo region contains some 1200 species of Aizoaceae (Goldblatt within the Greater Cape Floristic Region (GCFR; sensu Born and Manning, 2000), one clade of which has undergone very

13 Abbreviations: A, net photosynthetic rate; CAM, Crassulacean acid metabolism; Ci, intercellular CO2 concentration; δ C, stable carbon isotope ratio; Fv/Fm, poten- tial PSII photochemical efficiency; gST, stomatal conductance; gM, mesophyll conductance; Ja, rate of linear electron transport; Jmax, maximum of rate of electron transport; PPFD, photosynthetic photon flux density;R d, dark respiration; τ*, photorespiratory compensation point; Vcmax, Rubisco carboxylation; VPD, vapour pressure deficit; WC, leaf water content. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 5486 | Ripley et al. rapid and recent diversification Klak( et al., 2004). The driv- mesophyll cells, such that photosynthetic tissue is distributed ing force behind this rapid radiation is thought to be the throughout the leaf in what has been termed ‘all cell suc- innovation of several key morphological characters, includ- culence’ (von Willert et al., 1992). This anatomy is likely to ing succulent, subcylindrical to trigonous shaped leaves, in result in a reciprocal relationship between succulence and gM, response to an increasingly arid environment at the end of the and may account for the tight relationship of succulence to Miocene (Klak et al., 2004). However, the physiological role CAM in groups such as the Crassulaceae, Orchidaceae, and of leaf succulence in the tolerance of drought has not been some other families (Teeri et al., 1981; Winter et al., 1983; investigated in the Aizoaceae. Kluge et al., 1991; Nelson et al., 2005; Silvera et al., 2005; Succulent leaves have high water contents, and this, com- Nelson and Sage, 2008). In contrast, increasing succulence in bined with elastic cell walls and apoplastic polysaccharides, taxa such as the Aizoaceae is associated with an inner cortex allows leaves to avoid low leaf water potentials even after of water-storing cells, or epidermal and other peripheral cells extensive dehydration (Nobel, 1988; von Willert et al., 1992; that are achlorophyllous, and has been termed ‘storage suc- Martin, 1994; Pimienta-Barrios et al., 2002). This serves to culence’ (Ihlenfeldt, 1989). The chlorenchyma in these species buffer transpirational water loss, prolonging stomatal open- is often distributed around the periphery of the leaves and is ing and positive gas exchange, the magnitude of which corre- distinct from the water-storing hydrenchyma (Ernshaw et al., lates with the degree of leaf succulence (Nobel, 1976; Acevedo 1987). Many storage succulents do not show a strong depend- et al., 1983; Martin and Adams, 1987; Gravatt and Martin, ence on CAM and assimilate the majority of their carbon

1992; Martin, 1994). Subsequent to stomatal closure, succu- diurnally through the C3 cycle (Rundel et al., 1999; Winter and lence and the possession of relatively impervious leaf cuticles Holtum, 2002). These species may not benefit from reduced prevents cellular dehydration and allows succulent leaves to gM, which would limit their diurnal CO2 uptake. The anatomy endure long periods of drought (Hoffman et al., 2009). of storage succulence suggests that they may acquire the ben-

In addition to increasing water storage, leaf succulence efit of stored water, without becoming gM limited and may is frequently associated with the use of some form of pho- thus be less strongly dependent on CAM. This reasoning sug- tosynthetic Crassulacean acid metabolism (CAM), includ- gests no ubiquitous mechanistic link between leaf succulence ing obligate CAM, CAM-idling, CAM-cycling, and flexible and CAM per se, but rather that the co-occurrence of CAM

CAM/C3 systems (Veste et al., 2001; Herrera, 2009). Leaf and succulence should be confined only to leaves that display succulence and CAM may be mechanistically linked, or may all cell succulence (e.g. Silvera et al., 2005; Herrera, 2009). have evolved independently to optimize water use strategies This does not imply that species that develop leaf succu- in arid environments (Ogburn and Edwards, 2010). The pro- lence via a central achlorophyllous hydrenchyma are pre- posed dependence of leaf succulence on CAM arises from cluded from having CAM or being facultatively CAM. For two considerations. First, dependence on CAM is thought to example, tradescantioides can rapidly switch require the additional storage capacity for the C4 acids pro- between C3 and CAM (Herppich et al., 1996). This has been vided for by enlarged succulent cells (Gibson, 1982; Nelson demonstrated in other storage succulents via intermediate et al., 2005) associated with high vacuolar volumes (up to isotopic values (e.g. Mooney et al., 1977; Rundel et al., 1999) 98% of cell volume; Lüttge, 2004). Secondly, as leaf succu- and by more direct measures of CAM activity (Herppich lence increases, the mesophyll conductance (gM) for the dif- et al., 1996; Veste et al., 2001). CAM-idling has been shown fusion of CO2 through hydrated succulent leaf tissue to the to be important for enduring drought and, subsequent to sto- photosynthetic tissue decreases. In most C3 and C4 plants matal closure, the ability to capture and release respiratory CO2 transfer across the mesophyll is largely through gaseous CO2 allows continued light reaction activity that protects the pathways, with gM being determined by the ratio of chloro- photosynthetic apparatus from photoinhibition (Ting, 1985; plast surface to leaf area and cell wall thickness (Tomás et al., Adams et al., 1987; Griffithset al. , 1989; Herrera, 2009). Some 2013). In contrast, the mesophyll of fully hydrated succulent succulent species increase photosynthetic efficiency by CAM- leaves at least partially lacks gas spaces, and CAM may func- cycling that can save up to 10% of daily assimilated CO2 that tion to overcome gM limitations on photosynthesis (Maxwell would otherwise be lost to the atmosphere (Patel and Ting, et al., 1997; Gillon et al., 1998; Griffiths et al., 2008). CAM 1987; Herrera, 2009). These forms of CAM may capture only plants can tolerate extremely low gM values, partially due to a small proportion of assimilated carbon through PEPc, and, the capacity of phosphoenolpyruvate carboxylase (PEPc) to when this is less than a third of total CO2 fixation, do not – 13 acquire HCO3 (Maxwell et al., 1997) and because nocturnal have δ C values distinct from C3 plants (Winter and Holtum, carboxylation is limited by PEP derived from starch metabo- 2002; Herrera, 2009), and hence cannot not be identified in lism, and not by CO2 supply from the atmosphere (Nelson isotopic surveys for CAM photosynthesis. and Sage, 2008). Reduced gM is advantageous to CAM plants, A range of Aizoaceae species were used to determine the role as it limits daytime CO2 efflux from photosynthetic tissues of increasing leaf succulence in prolonging gas exchange and ensuring high intercellular CO2 concentrations during the re- maintaining photosynthetic integrity during leaf desiccation. fixation of CO2 into the C3 cycle (Nelson et al., 2005, 2008). The relationship between increasing succulence, CAM, and The extent to which increasing succulence reduces gM is likely gM was examined and the hypothesis was tested that the anat- to be dependent on leaf anatomy and how photosynthetic tis- omy of Aizoaceae succulent leaves avoids low gM and hence sue is distributed within the leaf. In some species, increasing a strong dependence on CAM. This CAM–succulence rela- succulence is via additional layers of chloroplast-containing tionship is contrasted between species from the Aizoaceae and How succulent Aizoaceae avoid conductance limitations and survive drought | 5487

Crassulaceae. The comparison was thus between plants that At the end of the experiment, leaf areas were measured by scan- possess storage succulence and those that are all cell succulent. ning cylindrical or ovoid leaves from two sides and triangular leaves from three sides. The accuracy of this method was checked by applying the same method to cylindrical, ovoid, and triangu- lar shapes of known surface area. Gas exchange parameters were Materials and methods expressed on the basis of these multisided leaf areas (in contrast to the norm of expressing only on the area of one side of planar Species selection and propagation leaves). For both the Aizoaceae and Crassulaceae, taxa were selected which Leaf or shoot turgid weights were recorded prior to the first meas- showed a range of >5-fold differences in leaf succulence. Species urement, immediately after the leaves or shoots were excised from from the Aizoaceae were selected from three of the four subfami- well-watered plants. Dry weights were determined at the end of the lies in order to cover the phylogenetic and leaf morphological diver- experiments after drying material in an oven at 60 °C to con- sity of the group. Two species were selected from the Aizooideae stant weight. Leaf succulence was calculated as (turgid weight–dry with small, planar succulent leaves where the photosynthetic tis- weight)/dry weight, and leaf water content (WC) as (wet weight–dry sue is distributed throughout the leaf, termed ‘all cell succulence’ weight)/turgid weight. (Galenia africana L. and Tetragonia fruticosa L.); one species from In a similar but independent experiment, the reduction pho- the Mesembryanthemoideae (Mesembryanthemum cordifolium L.f.) tochemical efficiency of photosystem II (PSII; Fv/Fm) over a long with planar succulent leaves and ‘all cell succulence’; nine species period of dehydration was measured. Excised leaves or shoots were from the Ruschieae (Ruschioideae), with subcylindrical to trigonous dark-adapted for 60 min using leaf clips, and chlorophyll fluores- shaped succulent, rarely planar leaves, with an inner cortex of water- cence parameters were recorded using a Walz PAM 2000 fluorometer storing cells that are achlorophyllous, termed ‘storage succulence’ (Walz, Effeltrich, Germany). After measurements, leaves of shoots [ dasyphylla (Schltr.) H.E.K.Hartmann, Carpobrotus edulis were stored on a sunlit windowsill in the laboratory and measure- (L.) L. Bolus, Delosperma echinatum Schwantes, D. tradescantioides ments were repeated routinely for a period of 40 d. Measurements L. Bolus, Drosanthemum speciosum Schwantes, Glottiphyllum were made in duplicate for each plant species. depressum N.E.Br., G. longum N.E.Br., aureus N.E.Br., For each individual replicate, the decay in A, gST, and WC over and Oscularia cedarbergensis (L. Bolus), H.E.K.Hartmann]. time (t) were fitted with the equation: Similarly, from the Crassulaceae, genetic and leaf morphological diversity within the group were selected, with most species selected a from Crassula, since it is the largest genus of Crassulaceae in South A, gST or WC = ()−ct (1) Africa [Adromischus fallax Toelken, Crassula atropurpurea (Haw.) 1+ bexp Dietr., C. fascicularis Lam., C. ovata (Mill.) Druce, C. arborescens

(Mill.) Willd., C. orbicularis L., C. pellucida L., C. sarmentosa Harv., The declines in A and gST in response to decreasing leaf WC were C. multicava Lem., C. cordata Thunb., C. sarcocaulis Eckl.&Zeyh., fitted with the same equation, but WC was substituted fort in the C. dejecta Jacq., C. ericoides Haw., Sedum pachyphyllum Rose, equation above. The decrease in Fv/Fm over time was fitted with the Tylecodon singularis (R.A.Dyer) Toelken, and T. racemosus (Harv.) equation: Toelken]. The study included mostly field-collected plants from the winter-rainfall region of the Cape and a few from the summer-rain- Fv 1 fall region of the Eastern Cape, South Africa. In addition, plants = ()c (2) were procured from nurseries, which were established as a collection Fm ab+ t of potted plants at the University of Cape Town, Biological Sciences Department Greenhouse. Plants were grown in native or nursery The parameters a, b, and c were fitted using least squared differences soils, were well watered and were supplied with Long Ashton nutri- (using the Solver add-in of Microsoft Excel). Equation 1 was used ent medium (Hewitt, 1966) modified to contain 2 mm NaNO3 (pH to derive the time required to reduce A or gST by 50%, and WC by 6.5) during pot establishment. 10%. Equation 1 was also used to calculate the WC, and the reduc- tion in turgid WC (turgid WC–WC), required to decrease A and gST by 50%. The latter measure accounts for the positive relationship Dehydration of excised shoots between succulence and WC, and shows the effect of water loss from A subsample of nine species from the family Aizoaceae (A. dasy- the initial turgid WC, and not from 100%. Equation 2 was used to phylla, M. cordifolium, C. edulis, D. speciosum, G. africana, G. depres- calculate the time required to decrease Fv/Fm by 10%. The choice of sum, L. aureus, O. cedarbergensis, and T. fructicosa) were used to 10% or 50% for calculating the decline constants of A, gST, WC, and determine the decline in photosynthetic rate (A), stomatal conduct- Fv/Fm was to ensure that estimates were not extrapolated beyond the ance (gST), and potential photochemical efficiency (Fv/Fm) during a measured data. prolonged period of shoot dehydration. Shoots, or individual leaves in the case of large-leaved species, were excised from plants and the cut ends were sealed with nail varnish. Shoots or leaves were A:Ci responses and mesophyll conductance determinations enclosed in a conifer chamber of a LI-6400 photosynthesis sys- Individual leaves of shoots were excised from the subsample tem (LI-COR Biosciences Inc., Lincoln, NE, USA). The chamber of nine Aizoaceae species and cut ends were sealed into water- was illuminated from three sides using dichroic halogen spot lights filled Eppendorf tubes using multiple wraps of Parafilm (Brand, (ECO-3000, Eurolux) such that all leaf surfaces received a phosyn- Wertheim, Germany). These were then enclosed in a conifer thetic photon flux density (PPFD) >1500 μmol m–2 s–1, leaf temper- chamber (Licor 6400-005) of a Licor 6400 photosynthesis sys- atures (estimated from energy balance using instrument software) tem with chamber conditions set as before. The response of A controlled at 25 ± 3 °C, and a vapour pressure deficit (VPD) <1.5 to intercellular CO2 concentration (Ci) was constructed accord- kPa. Gas exchange parameters were recorded at intervals during ing to Long and Bernacchi (2003) and individual replicate (n the next 10–20 h. Measurements were made in triplicate and shoots ≥3) curves fitted according to von Caemmerer (2000). Fitted or leaves were removed from the chamber between measurements, parameters were used to generate A:Ci responses with points at –2 weighed, and kept under common (~25 °C, PPFD ~100 μmol m 2 Pa intervals. gM was then determined according to the con- s–1) conditions in the laboratory. The photosynthesis system was stant J method (Warren, 2006), where the rate of linear electron ‘matched’ before each data point was recorded and after gST and transport (Ja) was calculated from generated gas exchange data photosynthesis were stable at the prevailing conditions. according to Equation 3. 5488 | Ripley et al.

  white images, and photosynthetic and achlorophyllous areas were 42 ()CAiM− /*g + τ  measured using the ‘Analyse Particles’ function in Image-J (Version JAa =+()Rd (3) CA− /*g −τ 1.44p). ()iM

Where A=photosynthetic rate, Rd=dark respiration, Ci=intercellular CO2 partial pressure, gM=mesophyll conductance, and τ*=the pho- Results torespiratory compensation point. The value of gM was obtained by iteratively changing the value between the limits of 0.01 μmol m–2 s–1 –1 –2 –1 –1 In order to retain graphical clarity, the responses of A, gST, Pa and 10 μmol m s Pa . The gM value was selected that mini- WC, and F /F to shoot excision and A:C responses are pre- mized the variance between J values determined at each C value v m i a i sented for select examples of species in the Aizoaceae. Data (2 Pa intervals, between 90 Pa and 130 Pa) and the average Ja value calculated over this same range in partial pressures. This method for the remaining species are given in the Supplementary of calculating gM has acknowledged limitations (Pons et al., 2009; Figures available at JXB online. Tholen et al., 2013), but given that the measurements were made on a range of species within a single family and that the plants were all grown under common conditions, the method retains its value for the Dehydration of excised shoots interspecific comparison made in this study. In some species it was possible to align the fibre optic of the fluo- A, gST, and WC declined over time after leaf or shoot rometer with leaves enclosed in a modified conifer chamber during excision, were non-linear, and varied between species the construction of A:Ci curves. This modification involved replac- ing the hinged ‘conifer chamber’ lid with a Perspex plate equipped (Fig. 1A–C; Supplementary Fig. S1 at JXB online). The with a port for the fibre optic probe. In these cases, simultaneous gas time required to reduce A and gST by 50% (t50%), and WC by exchange and PSII fluorescence measurements were used to confirm 10% (t10%), was calculated from these curves and showed a that ФPSII was constant when Ci ranged from 90 Pa to 130 Pa. linear response to increasing succulence (Fig. 1D–F). The

most succulent species had t50% values for A and gST that Leaf δ13C isotopic ratios and nitrogen concentration were >10-fold larger than those of the least succulent spe- cies. The t values for A were higher than those for g , Whole leaves were removed from well-watered plants for the entire 50% ST Crassulaceae and Aizoaceae species collection (n=3) at ~10:00 h. indicating that photosynthesis continued despite changes Leaf areas, and fresh and dry weights were recorded, and leaves were in gST. This is due to a curvilinear relationship between A oven-dried at 60 °C to constant weight. Dried leaves were ground and gST, particularly at high values of gST (data not shown). to a homogeneous powder using a Wiley mill with a 0.5 mm mesh Water loss was sustained for longer from more succulent (Arthur H. Thomas, CA, USA) and weighed into 8 × 5 mm tin cap- leaves, which also had the highest WC, and lost the initial sules (Elemental Microanalysis Ltd, Devon, UK). Additional leaves from well-watered plants and plants dehydrated for 1 or 2 weeks were 10% of leaf WC 10-fold more slowly than the least succu- used to make extracts of leaf carbohydrates. Fresh leaves collected at lent species. ~10:00 h were rapidly transferred into chilled 80% methanol, homog- The manner in which A and gST declined with WC enized in a mixer-mill (Retsch MM 200, Retsch, Haan, Germany), was non-linear and varied between species (Fig. 2A, B; and centrifuged in a microfuge. The supernatant was transferred Supplementary Fig. S2 at JXB online). The WC at which to ion exchange columns for the separation of the neutral fraction (mostly carbohydrate) using a modification of the method ofAtkins A and gST were decreased by 50% was strongly correlated and Canvin (1971). The column was filled with a mixed bed ion- to leaf succulence (Fig. 2C, D) and showed that the most exchange resin (Dowex Marathon MR-3, Sigma-Aldrich, St. Louis, succulent species were the least tolerant of leaf dehydra- MO, USA) with deionized water as the mobile phase. The eluted tion in comparison with the least succulent species. There neutral fraction was collected, air-dried under an N2 stream, and was no correlation between succulence and the decline in re-dissolved in 1 ml of water. Aliquots (50 μl) of this sample were placed into mass spectrometer tin capsules and oven-dried at 65 °C. turgid leaf WC required to halve A (P > 0.5) or gST (P > The neutral fraction of the methanol extraction contained no N, 0.11; Fig. 2E, F). This expression accounts for the fact that demonstrating that the carbohydrates had been effectively separated the turgid WC of leaves varied with the degree of succu- from amino compounds. lence. This shows that both A and gST were decreased by Additional leaves harvested from well-watered plants in the 50% when turgid WC was decreased by between 6% and Aizoaceae were, where possible, separated into achlorophyllous hydrenchyma and chlorophyllous mesophyll tissue using a sterile 16% (average of 11%) across all the Aizoaceae species sam- scalpel blade. Separated tissues were oven-dried, weighed, milled, pled. Only L. aureus tolerated more severe dehydration and and subject to elemental analysis to determine tissue N concentra- required a reduction in WC of 23% to halve photosynthesis tions. The stable carbon isotope ratios (δ13C) and tissue N concentra- 13 (Supplementary Fig. S2). tions for the foliar samples and the δ C values for the carbohydrate Similarly, the decline in potential PSII photochemi- samples were determined using a Thermo Flash EA 1112 series ele- mental analyser (Thermo Electron Corporation, Milan, Italy) and cal efficiency F( v/Fm) differed between species (Fig. 3A; Delta Plus XP isotope ratio mass spectrometer (Thermo Electron Supplementary Fig. S3 at JXB online). However, a much Corporation). longer dehydration period was required to affect Fv/Fm than either A or gST, and the most succulent species exhibited very little change in F /F over a period of up to 20 d. As for A Estimates of photosynthetic and achlorophyllous v m and g , however, the derived times required to reduce F /F cross-sectional areas ST v m by 10% (t ) were also positively correlated with increasing Fresh leaves taken from a subset of 10 species from the Crassulaceae 10% and 11 species from the Aizoaceae were hand sectioned with a succulence, such that the most succulent species had a t10% of razor blade and photographed with a light microscope (Nikon, 37 d, nearly 18-fold longer than the least succulent species SMZ 1500). Digital micrographs were converted to 8-bit black and (Fig. 3B). How succulent Aizoaceae avoid conductance limitations and survive drought | 5489

Fig. 1. Decline over time in (A) average photosynthesis (A), (B) stomatal conductance (gST), and (C) water content (WC) for selected examples of Aizoaceae species following leaf or shoot excision. Average time required to decrease (D) A and (E) gST by 50%, and (F) WC by 10%, for nine Aizoaceae species (n=3; mean ±SE). Linear regression lines with coefficients of determination and significance of correlations are shown. Time response data for A. dasyphylla, C. edulis, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are available in Supplementary Fig. S1 at JXB online.

Leaf δ13C isotopic ratios a small proportion to total carbon assimilation (Fig. 4B). To test if CAM could be induced by water stress, the δ13C The δ13C values for leaves harvested from the 16 well- values of carbohydrates extracted from well-watered leaves watered Crassulaceae species were positively correlated were compared with those from water-stressed plants. to leaf succulence (Fig. 4A). The range of values spanned Water stress made δ13C less negative, by on average 1.0‰ from those that would be typical of C3 to CAM plants and 1.5‰ after 7 d and 14 d, respectively (Fig. 4C). This (Winter and Holtum, 2002). A similar relationship con- small increase in δ13C is more likely to reflect the effect structed for 12 of the Aizoaceae species showed a 4-fold of decreased gST on gas exchange than a switch to CAM, weaker relationship (i.e. slope), and all values fell within the which should have a much larger effect on the δ13C of range typical of C3 plants or where CAM contributes only recently assimilated carbohydrates. 5490 | Ripley et al.

Fig. 2. (A and B) Decline in average photosynthesis (A) and stomatal conductance (gST) in response to water content (WC) for selected examples of Aizoaceae species following leaf or shoot excision. (C and D) Average WC at which A and gST were reduced by 50% for nine Aizoaceae species. (E and F) Average reduction in turgid WC required to decrease A and gST by 50% for nine Aizoaceae species (n=3; mean ±SE). Linear regression lines with coefficients of determination and significance of correlations are shown. Water content response data for A. dasyphylla, C. edulis, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are available in Supplementary Fig. S2 at JXB online.

Photosynthetic cross-sectional area and leaf N content succulent species of the Aizoaceae possess achlorophyllous hydrenchyma while the less succulent Aizoaceae species and Leaf N concentrations of neither the achlorophyllous hydren- the Crassulaceae exhibit chlorophyllous ‘all cell succulence’. chyma nor the photosynthetic mesophyll (Table 1) were cor- 2 related to succulence (all R < 0.05, data not shown). The A:C responses and mesophyll conductance N contents of the achlorophyllous hydrenchyma were high, i being on average (across species) 26 ± 6% of the total leaf N A:Ci responses for the Aizoaceae (Fig. 6A; Supplementary content, suggesting that this could be an important N store in Fig. S4 at JXB online) produced a wide range of Vcmax –2 –1 –2 –1 these leaves. The cross-sectional area of photosynthetic tissue (16–70 μmol m s ) and Jmax values (41–148 μmol m s ; was linearly correlated to succulence in both the taxonomic Supplementary Table S1 at JXB online) that were significantly groups; however, the slope of the relationship was 3-fold different between the species (Vcmax, F8,22=11.3, P < 0.0001; higher in the Crassulaceae than in the Aizoaceae (Fig. 5). The Jmax, F8,22=8.0, P < 0.0001). These parameters were not cor- difference in the slopes results from the fact that the more related to leaf succulence (all R2 >0.1; data not shown). How succulent Aizoaceae avoid conductance limitations and survive drought | 5491

Fig. 3. (A) Decline in efficiency of PSII F( v/Fm) for individual replicates of selected examples of Aizoaceae species following

leaf or shoot excision. (B) Time required to decrease Fv/Fm by 10% for nine Aizoaceae species (n=2). For each individual, replicate data are shown. Linear regression lines with coefficients

of determination and significance of correlations are shown. Fv/

Fm response data for A. dasyphylla, C. edulis, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are available in Supplementary Fig. S3 at JXB online.

The gM values derived from the CO2-saturated portions of the A:Ci responses were significantly different between species –2 –1 –1 (F8,22=4.1, P < 0.004) and ranged from 0.59 μmol m s Pa –2 –1 –1 to 1.73 μmol m s Pa , excluding the apparent outlier value 13 –2 –1 –1 Fig. 4. δ C (‰) values for well-watered leaves for species of 5.9 μmol m s Pa for C. edulis (Fig. 6B). The gM values from (A) Crassulaceae (n=16) and (B) Aizoaceae (n=12). Linear were not correlated to leaf succulence (P > 0.11). regression lines with coefficients of determination and significance of correlations are shown. (C) Response of δ13C values for select species from the Aizoaceae that were well watered or subject to Discussion water stress for 7 d or 14 d. As hypothesized, increasing succulence in the Aizoaceae was not accompanied by a decrease in mesophyll conductance succulent Kalanchoe daigremontiana (~0.5 μmol m–2 s–1 Pa–1;

(gM). The range of gM values of the Aizoaceae that were meas- Maxwell et al., 1997; Griffiths et al., 2007). The present data ured were generally higher than those reported for the CAM are, however, expressed on a total leaf area basis since several 5492 | Ripley et al.

Table 1. Leaf succulence and N concentrations (%, w/w) measured separately for achlorophyllous hydrenchyma and photosynthetic tissue (where possible) for nine species of Aizoaceae

–1 Species Succulence (g H2O g dry weight) Achlorophyllous hydrenchyma N (%) Chlorophyllous mesophyll N (%) Antimima dasyphylla 6.0 ± 0.9 – 1.97 ± 0.38 Mesembryanthemum cordifolium 17.1 ± 1.6 – 1.78 ± 0.12 Carpobrotus edulis 20.0 ± 2.3 3.83 ± 0.77 2.91 ± 0.04 Drosanthemum speciosum 14.8 ± 0.6 0.73 ± 0.06 1.15 ± 0.12 Galenia Africana 4.5 ± 0.5 – 2.98 ± 0.23 Glottiphyllum depressum 28.0 ± 3.4 0.59 ± 0.12 1.73 ± 0.14 Lampranthus aureus 18.1 ± 0.6 2.51 ± 0.15 3.95 ± 0.30 Oscularia cedarbergensis 25.5 ± 3.8 1.79 ± 0.48 2.45 ± 0.09 Tetragonia fruticosa 11.6 ± 2.5 – 1.23 ± 0.06

Values are means ±SE (n=3).

Fig. 5. Variation in cross-sectional area of chlorophyllous tissue of Crassulaceae and the Aizoaceae species (n=11 species for both) with succulence. Linear regression lines with coefficients of determination and significance of correlations are shown. leaves were cylindrical or triangular in cross-section, rather than as usual for single surfaces of planar leaves, and thus equivalent values for K. daigremontiana would be ~0.25 μmol m–2 s–1 Pa–1. The present values mostly fall withinthe range of gM values reported for C3 species (with correction to consider both leaf surfaces: ~0.38–2.18 μmol m–2 s–1 Pa–1; Flexas et al., 2007). This high gM in the Aizoaceae is remarkable consider- ing how succulent some of these leaves are, and that these succulent leaves largely lack mesophyll airspaces, which con- tribute to high gM in many C3 species. The relatively high gM Fig. 6. (A) A:Ci responses of selected examples of Aizoaceae of the Aizoaceae is, however, readily explained by the fact that species. Fitted equations for each replicate were used to anatomically the photosynthetic tissue is in a thin band adja- interpolate data to a common series of Ci values for the calculation cent to the leaf surface. Avoiding the limitations of low gM of means and standard errors. (B) Derived mesophyll conductance via anatomical modifications is not unique to the Aizoaceae, values (gM) for nine Aizoaceae species. For each species n ≥3, and and stomatal crypts play a similar role in Banksia leaves with vertical bars are standard errors. A:Ci responses for A. dasyphylla, high dry mass per area (Hassiotou et al., 2010). Additionally, D. speciosum, L. aureus, O. cedarbergensis, and T. fruticosa are limiting the water-demanding photosynthetic tissue to a thin available in Supplementary Fig. S6 at JXB online). How succulent Aizoaceae avoid conductance limitations and survive drought | 5493 layer and associating this with three-dimensional venation responses to water inputs have been demonstrated in various (Ogburn and Edwards, 2013) would maintain favourable leaf succulents including Ruschia caroli (Aizoaceae; Midgley hydraulic path lengths, whilst allowing the development of and van der Heyden, 1999), Mesembryanthemum pellitum succulent leaves. This hydraulic supply would be important (Aizoaceae), and Othonna optima (Asteraceae; Eller et al., for the uptake and recharge of photosynthetic water storage 1993). The maintenance of photochemical integrity may tissue and to service photosynthetic gM and the water loss be aided by CAM-idling, or drought-induced low-level from photosynthetic surfaces (Griffiths, 2013). CAM, which has been demonstrated in D. tradescantioides The Aizoaceae lack a strong dependence on CAM, as was (Aizoaceae; Herppich et al., 1996). CAM-idling does not evident from the δ13C isotope values, which were all more contribute to overall carbon assimilation and hence does not negative than –21‰, which is the cut-off value determined for result in discernible CAM δ13C signal (Winter and Holtum, CAM species (Winter and Holtum, 2002). The average δ13C 2002). Consequently, the importance of these mechanisms value was –29.6‰, which indicates that >60% of carbon gain may have been overlooked in many succulent species that would have occurred diurnally via the C3 cycle (Winter and have C3-like isotopic values and would not have been discern- Holtum, 2002). The fact that the δ13C values only increased ible with the present measurements. by 1.5‰ after 14 d of water stress indicates that the high δ13C The species distribution of the Aizoaceae in the GCFR values relative to those of typical CAM plants were not due overlaps that of the Crassulaceae (von Willert et al., 1990; to the well-watered conditions under which the plants were Jürgens, 1997; Goldblatt and Manning, 2002), many of generally maintained, and that CAM is not induced in these which are constitutive CAM plants (e.g. Mooney, 1977). Of species by water stress. This small shift in δ13C in response to the ~659 species of the Aizoaceae, ~524 (80%) are endemic water stress could be accounted for by changes in the Ci/Ca to the winter rainfall GCFR. In contrast, only 35 (26%) induced by stomatal closure, as in C3 species (Farquhar et al., of 134 species of Crassulaceae are endemic to this region 1989). (Goldblatt and Manning, 2002). Where the Crassulaceae are The succulent Aizoaceae leaves utilized the water stored found in the GCFR, their centre of diversity is in a region in the achlorophyllous hydrenchyma to prolong positive of higher mean annual precipitation (200–290 mm) and less gas exchange and allowed leaves to withstand long periods severe summer drought in the Cedarberg mountains and of drought. This photosynthetic buffering and the ability to surrounding valleys and the Little Karoo (Jürgens, 1997; endure drought have been demonstrated in numerous succu- Mucina et al., 2006; Rebelo et al., 2006). The centre of diver- lent species and not just those with achlorophyllous hydren- sity for the succulent Aizoaceae (subfamilies Ruschioideae chyma (e.g. Nobel, 1976; Hoffman et al., 2009). Interestingly, and Mesembranthemoideae) occurs at lower (90–164 mm) in the Aizoaceae, the more succulent species were least physi- strongly winter rainfall, in the Richtersveld and Knersvlakte ologically tolerant of lowered leaf tissue water content. The (Hartmann, 1991). It is speculated that the peripheral photo- positive correlation between succulence and the WC required synthetic tissue and achlorophyllous hydrenchyma of the suc- to halve A and gST meant that the gas exchange of the most culent Aizoaceae may be especially suited to the wet winters succulent species responded at the highest water contents. and dry summers found in this region. The greater depend- When the differences in initial turgid WC were accounted for, ence on CAM of the Crassulaceae may be associated with the it was apparent that when the initial WC was decreased by fact that these plants receive moisture and grow during the 6–16%, the photosynthetic and stomatal response were evi- hot summers. dent irrespective of the degree of leaf succulence. Hence, the The onset of the growing season in the Succulent Karoo mechanism for sustained gas exchange was not due to tol- is early May, reaching the mid-growing season in August, erance of dehydration, but the most succulent species had a with growth ceasing by early January (Wessels et al., 2011). large volume of water to buffer dehydration. Maintenance of The combination of cold and rain eliminates water stress for photosynthetic tissue water relations at the expense of water the winter and some of the spring period, during which time loss from the hydrenchyma has been demonstrated for vari- CAM would not be beneficial. Under this climate scenario, ous species (Schmidt and Kaiser, 1987; Herrera et al., 2000; the role of succulence in the Aizoaceae may be to extend the Nobel, 2006), and interspecific differences in this might growing season into the drier and warmer period of early sum- account for the range in WC loss required to produce the mer, after which the plants may become dormant during the observed gas exchange responses. hot dry period of late summer. Thus, the engagement of C3 Once positive gas exchange had ceased, the capacity for photosynthesis interspersed with periods of dormancy in net photosynthesis was maintained for long periods of drought CO2 acquisition, with or without weak CAM or CAM-idling, and was directly correlated to the degree of leaf succulence. is an alternative to constitutive CAM in these seasonally arid

Most succulent species reached t50% for A within 7 h, but t10% environments. A similar habitat differentiation based on rain- for Fv/Fm only occurred after 38 d, indicating that the energy fall has been observed for CAM plants in Madagascar. Here, provision from the photosynthetic lights reactions continued CAM plants occur in dry climatic zones or in dry micro-habi- even in the absence of net CO2 acquisition. Maintenance of tats, whereas facultative CAM physiotypes occur in less stress- photochemistry is likely to be important for energy provision ful environments (Kluge et al., 2001). Because CAM is a more to offset maintenance costs. Furthermore, a functioning pho- energetically demanding photosynthetic system than C3 (von tochemical system might enable rapid resumption of carbon Willert et al., 1992; Winter and Smith, 1996; Lüttge 2004), it assimilation after rainfall events. Such rapid gas exchange might not be ubiquitously suitable for Mediterranean climates. 5494 | Ripley et al.

CAM is particularly beneficial in hot conditions where it ena- Adams WW III, Osmond CB, Sharkey TD. 1987. Responses of two bles positive C acquisition, unlike C3 photosynthesis which CAM species to different irradiances during growth and susceptibility becomes increasingly compromised by photorespiratory CO2 to photoinhibition by high light. Plant Physiology 83, 213–218. losses at higher temperatures (Nobel, 1991). Thus, although Atkins CA, Canvin DT. 1971. Photosynthesis and CO2 evolution by both the Aizoaceae and Crassulaceae tolerate drought, the leaf discs: gas exchange, extraction, and ion-exchange fractionation seasonality of rainfall and the growing seasons may result in of 14C-labelled photosynthetic products. Canadian Journal of Botany different photosynthetic strategies being beneficial. 49, 1225–1234. While succulence is commonly associated with retaining tis- Born J, Linder HP, Desmet P. 2006. The Greater Cape floristic sue hydration, an additional limitation of seasonally arid envi- region. Journal of Biogeography 34, 147–162. ronments is the availability of soil nutrients, which require water to become soluble and accessible to plants (Cui and Caldwell, Cui M, Caldwell MM. 1997. A large ephemeral release of nitrogen 1997). This limitation is particularly pertinent in shallow soils or upon wetting of dry soil and corresponding root responses in the field. where roots are confined to rock crevices and small soil pools, Plant and Soil 191, 291–299. as is the case for many succulents in the CFR (Midgley and van Eller BM, Egli A, Flach BM-T. 1993. Influence of transpiration and der Heyden, 1999). The extension of the period over which gas photosynthetic pathway on water uptake of Cotyledon orbieulata exchange can occur during water stress can only benefit growth (CAM) and Othonna opima (C3). Botanica Helvetica 103, 207–221. if there is a simultaneous availability of nutrients for new tis- Earnshaw MJ, Carver KA, Charlton WA. 1987. Leaf anatomy, sue expansion. It is likely that succulent plants increase nutri- water relations and Crassulacean acid metabolism in the ent uptake during periods of water availability and store those chlorenchyma and colourless internal water-storage tissue of nutrients. This storage of nutrients is an additional role for the Carpobrotus edulis and Senecio mandraliscae. Planta 170, 421–432. achlorophyllous hydrenchyma in the succulent Aizoaceae and Farquhar GD, Ehleringer JR, Hubick KT. 1989. Carbon isotope explains the high N contents measured in these tissues. discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40, 503–537. Supplementary data Flexas J, Ribas-Carbó M, Diaz-Espejo A, Galmés J, Medrano H. 2008. Mesophyll conductance to CO2: current knowledge and future Supplementary data are available at JXB online. prospects. Plant, Cell and Environment 31, 602–621. Figure S1. Decline over time in average photosynthesis (A), Gibson AC. 1982. The anatomy of succulence. In: Ting IP, Gibbs M, stomatal conductance (gST), and water content (WC) for the eds. Crassulacean acid metabolism. Proceedings of the Fifth Annual indicated Aizoaceae species following leaf or shoot excision Symposium in Botany . Rockville, MD: American Society of Plant (n=3; mean ±SE). Physiologists, 1–17. Figure S2. Decline in average photosynthesis (A) and sto- Gillon JS, Borland AM, Harwood KG, Roberts A, Broadmeadow matal conductance (g ) in response to water content (WC) ST MSJ, Griffiths H. 1998. Carbon isotope discrimination in terrestrial for the indicated Aizoaceae species following leaf or shoot plants: carboxylations and decarboxylations. In: Griffiths H, ed. excision (n=3; mean ±SE). Stable isotopes: integration of biological, ecological and geochemical Figure S3. Decline in initial F /F over time for the indi- v m processes . Oxford: BIOS Scientific Publishers, 111–131. cated Aizoaceae species following leaf or shoot excision. For each species n=2 and hence individual replicate and not aver- Goldblatt P, Manning JC. 2000. Cape plants: a conspectus of the age data are presented. Cape flora of South Africa . Strelitzia 9. National Botanical Institute of South Africa, Cape Town, South Africa and Missouri Botanical Figure S4. A:Ci responses for the indicated Aizoaceae spe- cies. For each species n ≥3, and vertical bars are standard Garden, Missouri. errors. Goldblatt P, Manning JC. 2002. Plant diversity of the Cape region of southern Africa. Annals of the Missouri Botanical Gardens 89, 281–302. Gravatt DA, Martin CE. 1992. Comparative ecophysiology of five Acknowledgements species of Sedum (Crassulaceae) under well-watered and drought- Peter Bruyns (Bolus Herbarium, University of Cape Town) stressed conditions. Oecologia 92, 532–541. is thanked for supplying leaf samples of the Crassulaceae. Griffiths H. 2013. Plant venation: from succulence to succulents. Ian Newton (Department Archeometry, University of Cape Current Biology 23, R340–R341. Town) is thanked for conducting the mass spectrometer anal- Griffiths H, Cousins A, Badger M, von Caemmerer S. 2007. yses. Funding for this research was from Rhodes University Discrimination in the dark: resolving the interplay between metabolic (BSR) and the University of Cape Town (MDC). and physical constraints to PEPC activity during the CAM cycle. Plant Physiology 147, 1055–1067. Griffiths H, Ong BL, Avadhani PN, Goh CJ. 1989. Recycling of

References respiratory CO2 during Crassulacean acid metabolism: alleviation of Acevedo E, Badilla I, Nobel PS. 1983. Water relations, diurnal photoinhibition in Pyrrosia piloselloides. Planta 179, 15–122. acidity changes, and productivity of a cultivated cactus, Opuntia ficus- Griffiths, H Robe WE, Girnus J, Maxwell K. 2008. Leaf succulence indica. Plant Physiology 72, 775–780. determines the interplay between carboxylase systems and light How succulent Aizoaceae avoid conductance limitations and survive drought | 5495 use during Crassulacean acid metabolism species. Journal of Lüttge U. 2004. Ecophysiology of Crassulacean acid metabolism Experimental Botany 59, 1851–1861. (CAM). Annals of Botany 93, 629–652. Hassiotou F, Renton M, Ludwig M, Evans JR, Veneklaas EJ. Martin CE, Adams WW III. 1987. Crassulacean acid metabolism,

2010. Photosynthesis at an extreme end of the leaf trait spectrum: CO2-recycling, and tissue dessication in the Mexican epiphyte how does it relate to high leaf dry mass per area and associated Tillandsia schiedeana Steud. (Bromeliaceae). Photosynthesis Research structural parameters? Journal of Experimental Botany 61, 11, 237–244. 3015–3028. Martin CE. 1994. Physiological ecology of the Bromeliaceae. Hartmann HEK. 1991. Mesembryanthema. Contributions to the Botanical Review 60, 1–82. Bolus Herbarium 13, 75–157. Maxwell K, von Caemmerer S, Evans JR. 1997. Is a low internal

Herppich WB, Midgley G, von Willert DJ, Veste M. 1996. conductance to CO2 diffusion a consequence of succulence in plants CAM variations in the leaf-succulent Delosperma tradescantioides with Crassulacean Acid Metabolism? Australian Journal of Plant (Mesembryanthemaceae), native to southern Africa. Physiologica Physiology 24, 777–786. Plantarum 98, 85–492. Midgley GF, Van Der Heyden F. 1999. Form and function in Herrera A, Fernández MD, Taisma MA. 2000. Effects of drought on perennial plants. In: Dean WRJ, Milton SJ, eds. The Karoo: ecological CAM and water relations in plants of Peperomia carnevalii. Annals of patterns and processes . Cambridge: Cambridge University Press, Botany 86, 511–517. 91–106. Herrera A. 2009. Crassulacean acid metabolism and fitness under Mooney HA, Troughton JH, Berry JA. 1977. Carbon isotope ratio water deficit stress: if not for carbon gain, what is facultative CAM measurements of succulent plants in South Africa. Oecologia 30, good for? Annals of Botany 103, 645–653. 295–305. Hewitt EJ. 1966. Sand and water culture methods used in the Mucina L, Jürgens N, Le Roux A, Rutherford MC, Schmiedel U, study of plant nutrition , 2nd revised edn. Commonwealth Bureau Esler K, Powrie LW, Desmet PG, Milton SJ. 2006. Succulent of Horticulture and Plantation Crops, East Malling, Technical Karoo biome. In: Mucina L, Rutherford MC, eds. The vegetation of Communication No. 22. Farnham Royal, UK: Commonwealth South Africa, Lesotho and Swaziland . Strelitzia 19. South African Agriculture Bureau. National Biodiversity Institute, 221–299. Hoffman MT, Carrick PJ, Gillson L, West AG. 2009. Drought, Nelson EA, Sage RF. 2008. Functional constraints of CAM leaf climate change and vegetation response in the succulent karoo, South anatomy: tight cell packing is associated with increased CAM function Africa. South African Journal of Science 105, 54–61. across a gradient of CAM expression. Journal of Experimental Botany Ihlenfeldt H-D. 1989. Life strategies of succulent desert plants. 59, 1841–1850. Excelsa 14, 75–83. Nelson EA, Sage TL, Sage RF. 2005. Functional leaf anatomy of Jürgens N. 1986. Untersuchungen zur Ökologie sukkulenter Pflanzen plants with Crassulacean acid metabolism. Functional Plant Biology des südlichen Afrika. Mitteilungen aus dem Institut für allgemeine 32, 409–419. Botanik in Hamburg 21, 139–365. Nobel PS. 1976. Water relations and photosynthesis of a desert CAM Klak C, Reeves G, Hedderson T. 2004. Unmatched tempo of plant, Agave deserti. Plant Physiology 58, 576–582. evolution in Southern African semi-desert ice plants. Nature 427, Nobel PS. 1988. Environmental biology of agaves and cacti . 63–65. Cambridge: Cambridge University Press. Kluge M, Brulfert J, Ravelomanana D, Lipp J, Ziegler H. 1991. Nobel PS. 1991. Achievable productivities of certain CAM plants:

Crassulacean acid metabolism in Kalanchoe species collected in basis for high values compared with C3 and C4 plants. New various climatic zones of Madagascar: a survey by δ13C analysis. Phytologist 119, 183–205. Oecologia 88, 407–414. Nobel PS. 2006. Parenchyma–chlorenchyma water movement during Kluge M, Razanoelisoa B, Brulfert J. 2001. Implications of drought for the hemiepiphytic cactus Hylocereus undatus. Annals of genotypic diversity and phenotypic plasticity in the ecophysiological Botany 97, 469–474. success of CAM plants, examined by studies on the vegetation of Ogburn RM, Edwards EJ. 2010. The ecological water-use strategies Madagascar. Plant Biology 3, 214–222. of succulent plants. Advances in Botanical Research 55, 179–225. Landrum JV. 2002. Four families and 40 million years of evolution and Ogburn RM, Edwards EJ. 2013. Repeated origin of three- adaptation to xeric environments: what can stem and leaf anatomy tell dimensional leaf venation releases constraints on the evolution of us about their phylogeny? Taxon 51, 463–474. succulence in plants. Current Biology 23, 722–726. Linder HP, Johnson SD, Kuhlmann M, Matthee CA, Nyffeler R, Patel A, Ting IP. 1987. Relationship between respiration and CAM- Swartz ER. 2010. Biotic diversity in the southern African winter- cycling in Peperomia camptotricha. Plant Physiology 84, 640–642. rainfall region. Current Opinion in Environmental Sustainability 2, 109–116. Pimienta-Barrios E, Gonza´lez del Castillo-Aranda ME, Nobel PS. 2002. Ecophysiology of a wild playtopuntia exposed to prolonged Long SP, Bernacchi CJ. 2003. Gas exchange measurements, what drought. Environmental and Experimental Botany 47, 77–86. can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error. Journal of Experimental Botany 54, Pons TL, Flexas J, von Caemmerer S, Evans JR, Genty B, 2393–2401. Ribas-Carbo M, Brugnoli E. 2009. Estimating mesophyll 5496 | Ripley et al.

conductance to CO2: methodology, potential errors, and conductance to CO2 across species: quantitative limitations and recommendations. Journal of Experimental Botany 60, 2217–2234. scaling up by models. Journal of Experimental Botany 64, 2269–2281. Rebelo AG, Boucher C, Helme N, Mucina L, Rutherford MC. Veste M, Herppich WB, von Willert DJ. 2001. Variability of CAM in 2006. Fynbos biome. In: Mucina L, Rutherford MC, eds. The leaf-deciduous succulents from the Succulent Karoo (South Africa). vegetation of South Africa, Lesotho and Swaziland . Strelitzia 19. Basic Applied Ecology 2, 283–288. South African National Biodiversity Institute, Pretoria, 53–219. von Caemmerer S. 2000. Biochemical models of leaf photosynthesis. Rundel PW, Esler KJ, Cowling RM. 1999. Ecological and Canberra: CSIRO Publishing. phylogenetic patterns of carbon isotope discrimination in the winter- von Willert DJ, Eller BM, Werger MJA, Brinckmann E. 1990. rainfall flora of the Richtersveld, South Africa. Plant Ecology 142, Desert succulents and their life strategies. Vegetatio 90, 133–143. 133–148. von Willert DJ, Eller BM,Werger MJA, Brinckmann E, Ihlenfeldt Schmidt J, Kaiser W. 1987. Response of the succulent leaves H-D. 1992. Life strategies of succulents in deserts: with special of Peperomia magnoliaefolia to dehydration. Plant Physiology 83, reference to the Namib desert . Cambridge: Cambridge University 190–194. Press. Silvera KL, Santiago S, Winter K. 2005. Distribution of Warren C. 2006. Estimating the internal conductance to CO2 Crassulacean acid metabolism in orchids of Panama: evidence of movement. Functional Plant Biology 33, 431–442. selection for weak and strong modes. Functional Plant Biology 32, Wessels K, Steenkamp K, Von Maltitz G, Archibald S. 2011. 397–407. Remotely sensed vegetation phenology for describing and predicting Teeri JA, Tonsor SJ, Turner M. 1981. Leaf thickness and carbon the biomes of South Africa. Applied Vegetation Science 14, 49–66. isotope composition in the Crassulaceae. Oecologia 50, 367–369. Winter K, Holtum JAM. 2002. How closely do the δ13C values of Tholen D, Ethier G, Genty B, Pepin S, Zhu X. 2012.Variable Crassulacean acid metabolism plants reflect the proportion of CO2 mesophyll conductance revisited: theoretical background and fixed during day and night? Plant Physiology 129, 1843–1851. experimental implications. Plant, Cell and Environment 35, Winter K, Smith JAC. 1996. An introduction to Crassulacean acid 2087–2103. metabolism. Biochemical principles and ecological diversity. In: Winter Ting IP. 1985. Crassulacean acid metabolism. Annual Review of Plant K, Smith JAC, eds. Crassulacean acid metabolism: biochemistry, Physiology 36, 595–622. ecophysiology and evolution . Berlin: Springer-Verlag, 1–13. Tomás M, Flexas J, Copolovici L, Galmés J, Hallik L, Medrano Winter K, Wallace BJ, Stocker GC, Roksandic Z. 1983. H, Ribas-Carbó M, Tosens T, Vislap V, Niinemets U. 2013. Crassulacean acid metabolism in Australian vascular epiphytes and Importance of leaf anatomy in determining mesophyll diffusion some related species. Oecologia 57, 129–141.