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Relationships between leaf succulence and Crassulacean acid metabolism in the genus Sansevieria (Asparagaceae)
Flora 261 (2019) 151489
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Relationships between leaf succulence and Crassulacean acid metabolism in the genus Sansevieria (Asparagaceae)
T
Craig E. Martina,b, Werner B. Herppichb,*, Yvonne Roscherc, Michael Burkartc a
Department of Ecology & Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045, USA Abteilung Technik im Gartenbau, Leibniz-Institute für Agrartechnik und Bioökonomie, Max-Eyth-Allee 100, 14469 Potsdam, Germany c Institut für Biochemie und Biologie, Universität Potsdam, Maulbeerallee 3, 14469 Potsdam, Germany b
ARTICLE INFO
ABSTRACT
Edited by: Hermann Heilmeier
Relationships between different measures of succulence and Crassulacean acid metabolism (CAM; defined here as nocturnal increases in tissue acidity) were investigated in leaves of ten species of Sansevieriaunder greenhouse conditions. CAM was found in seven of the ten species investigated, and CAM correlated negatively with leaf thickness and leaf hydrenchyma/chlorenchyma ratio. Similarly, CAM correlated negatively with leaf water content, but only when expressed on a fresh mass basis. CAM was not correlated with “mesophyll succulence”, but weakly with leaf chlorophyll concentration. These results indicate that CAM is associated more with “all-cell succulence” and not with the amount of leaf hydrenchyma in the genus Sansevieria. The findings of this study emphasize the importance of defining the nature of “leaf succulence” in studies of photosynthetic pathways and leaf morphology. Evidence is also provided that CAM and succulence arose multiple times in the genus Sansevieria.
Keywords: Anatomy CAM Chlorenchyma Chlorophyll Hydrenchyma Morphology Phylogeny
1. Introduction Plants growing in arid regions often display a wide variety of adaptive features that serve to avoid or minimize the effects of drought stress (Ehleringer, 1985; von Willert et al., 1992; Veste et al., 2001). These features include tissue water storage, deep and/or widespread root systems, leaf morphologies that reduce high temperatures, sunken stomata at low densities, and water-conservative photosynthetic pathways, especially Crassulacean acid metabolism (CAM), one of three photosynthetic pathways found in plants (Edwards and Walker, 1983; Taiz and Zeiger, 1998; Schulze et al., 2002). A common morphoanatomical feature often associated with plants having CAM photosynthesis is succulence (Kluge and Ting, 1978; Gibson, 1982; Osmond, 1978; Winter, 1985; Males, 2017), which typically occurs in one of two forms: distinctive achlorophyllous waterstorage tissue (parenchyma) termed “hydrenchyma”, or simply very large, mostly chlorophyllous cells comprising the bulk of a photosynthetic organ, e.g., leaf or stem. The latter form has been referred to as “all-cell succulence” (Ihlenfeldt, 1985; von Willert et al., 1992; Ogburn and Edwards, 2010, 2012, 2013; Males, 2017; Ho et al., 2018). In both cases, the large cells harbor large vacuoles that occupy up to, and often greater than, 90% of the cell volume (Kluge and Ting, 1978; Steudle et al., 1980; Smith and Heuer, 1981). The function of these
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large cells and associated vacuoles in both types of succulence is obvious when considering the arid environments occupied by such succulent plants, i.e., water storage and supply during times of low ambient water availability (MacDougal and Spalding, 1910; Smith et al., 1987; Nowak and Martin, 1997; Chiang et al., 2013; Ho et al., 2018). Furthermore, CAM conserves water in these plants by limiting stomatal opening and, hence, water loss, to the nighttime when the atmospheric evaporative demand is lower than during the warmer and less humid days (Kluge and Ting, 1978; Osmond, 1978; Winter, 1985; Lüttge, 1987). As Rubisco is inoperative at night, CO2 assimilation is catalyzed by PEP carboxylase during CAM, which results in the production of malic acid. The latter is stored in the large vacuoles of the photosynthetic cells throughout the nighttime period of CO2 fixation. Thus, it has been proposed that there is a causal correlation between the degree of leaf (or stem) succulence and the occurrence of the CAM photosynthetic pathway (Winter et al., 1983; Larcher, 2003; Heyduk et al., 2016; Males, 2017, 2018). The latter correlation, however, is complicated by the occurrence of both CAM and C3 plants having distinct layers of non-photosynthetic hydrenchyma tissue, age and size of the photosynthetic organ, and potential problems of low mesophyll conductance to CO2 in succulent tissues, the latter being a result of minimal amounts of intercellular air space (Smith and Heuer, 1981; Ripley et al., 2013).
Corresponding author. E-mail address: [email protected] (W.B. Herppich).
https://doi.org/10.1016/j.flora.2019.151489 Received 12 April 2019; Received in revised form 22 October 2019; Accepted 23 October 2019 Available online 25 October 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.
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Table 1 The ten species of Sansevieria used in this study, species authority, ranges of leaf length, width, thickness, general shape of the leaf, and general geographic distribution. Information taken from Newton (2004); Budweg and Mansfeld (2015); African Plant Database (2019) and current study. Species
Species Authority
Leaf Length, cm
Leaf Width, cm
Leaf Thickness, mm
Hydrenchyma Thickness, mm
Leaf Transverse Section Shape
Species Distribution
ballyi (ba) cylindrica (cy)
L. E. Newton Bojer ex Hook.
0.6–1.2 2.5–4
cylindrical with groove cylindrical
central East Africa southern Africa
Schweinf. ex Baker
28–48
cylindrical with groove
central East Africa
fischeri (fi) gracilis (gr)
Baker (Marais) N. E. Br.
2–4.5 0.7
6–12 25–40 20–30 30–50 30–45 20–45 7 6–10
3–8 23–38
ehrenbergii (eh)
6–12 75–150 60–150 75–180
18–43 0.5
central East Africa central East Africa
parva (pa) raffillii (ra)
N. E. Br. N. E. Br.
0.8–3 5–13
2–3 6–8
none only near midrib
cylindrical cylindrical with partial groove slightly concave concave
senegambica (se) suffruticosa (su)
Baker N. E. Br.
3–6.5 1.3–2
none 11–18
volkensii (vo)
Gürke
2–5 13–20 13–19 13–20 13–19
45–245 15–90 23–80 20–45 60–105 68–152 30–70 15–60 45–100 45–122
2.5–3.5
1.3–2
11–18
A considerable number of studies have shown strong correlations between tissue succulence and the presence of CAM in several groups of plants, including species of Crassulaceae (Teeri et al., 1981), epiphytic orchids and ferns (Winter et al., 1983), and species in the grape and pepper families (Virzo De Santo et al., 1983). Partly as a result of these studies and partly based on first principles, several workers have argued that succulence is an evolutionary prerequisite for the historical development of CAM (see above). In contrast, Martin et al. (2009) found poor correlations between leaf thickness and degree (not presence) of CAM in two tropical CAM species in the Asclepiadaceae. Furthermore, Virzo De Santo et al. (1983) argued that any correlation between CAM and succulence may simply reflect two independent adaptations to arid environments (von Willert et al., 1992). One problem with such correlative studies is the definition of “succulence” used for these studies. For example, it may be expected that succulence–CAM correlations may be causally linked in plants with all-cell succulence, because all parenchyma cells in the photosynthetic tissue engage in CAM and, as a result, require very large vacuoles, whereas such a correlation appears to be less likely in CAM plants with well-defined hydrenchyma/chlorenchyma divisions of parenchyma tissue, given that the hydrenchyma cells, although contributing to tissue succulence, are not involved in CAM photosynthesis. Furthermore, many C3 halophytes have highly succulent photosynthetic tissues, yet this succulence appears to be primarily an adaptation that dilutes high concentrations of potentially toxic inorganic solutes (Turner and Kramer, 1980; Leuschner and Ellenberg, 2018). As a result of the above problems linking CAM and succulence, numerous measures of succulence have been attempted in such correlations (e.g., von Willert et al., 1992; Males, 2017, 2018; Ho et al., 2018). Most such measures relate photosynthetic tissue water content to leaf mass or structure (e.g., thickness or amount of hydrenchyma). One measure which often successfully correlates CAM and succulence was proposed by Kluge and Ting (1978) and relates tissue water content to tissue chlorophyll content and was termed “mesophyll succulence”. Other confounding factors might obfuscate reported correlations – or the lack thereof – between the CAM photosynthetic pathway and succulence of the photosynthetic organ. Such factors include the use of different measures of succulence, as indicated above, the use of widely divergent taxa, comparison of different organ types, as well as comparing plants under different environmental conditions. The monocot genus Sansevieria (Asparagaceae), containing around 70 species, is distributed in Africa and Asia, with a center of species diversity in East Africa (Brown, 1915; Newton, 2001; Webb and Newton, 2017; van Kleinwee, 2018). All species of this genus are characterized by a clear subdivision of the mesophyll into
slightly concave cylindrical with partial groove cylindrical with groove
central East Africa central East Africa central West Africa central East Africa central East Africa
chlorenchyma and a unique type of central colorless water-storage tissue (Koller and Rost, 1988a). Besides scattered vascular and fiber bundles, the latter comprises two different cell types (Koller and Rost, 1988a, 1988b; van Kleinwee, 2018). Water is effectively stored in clusters of large non-living thin-walled water-storage cells (Alfani et al., 1989), which are embedded in and interconnected by a network of small living cells, the so-called network cells (Koller and Rost, 1988a). While this general arrangement seems to be similar among all species of this genus, the relative contribution of both cell types varies (Koller and Rost, 1988a). Different Sansevieria species exhibit different leaf forms (cylindrical vs. “flat” concave), different degrees of xeromorphism and different types of succulence, e.g., hydrenchyma vs. all-cell succulence, and the degrees of succulence vary greatly among the different species (Alfani et al., 1989). For example, S. parva lacks a distinct layer of foliar hydrenchyma, while that of S. cylindrica is often 1–2 cm thick (Brown, 1915; Budweg and Mansfeld, 2015). Given the widely scattered results of attempts to correlate photosynthetic pathway (specifically, CAM vs. non-CAM) with “succulence”, the goal of the current study was to further investigate this correlation by comparing different measures of succulence and photosynthetic pathway (C3 or CAM) in leaves of different species of the genus Sansevieria largely differing in anatomical/morphological features and covering the full variability of leaf morphology in the genus but grown under identical conditions in the greenhouses of the University of Potsdam. This allows us to allows testing the hypothesis that succulence per se is an essential prerequisite for the occurrence and the expression of CAM, without the limits of family-specific or environmental effects on both features. This analysis also helps to evaluate whether differences in the chlorenchyma to hydrenchyma ratio in the different species of the genus Sansevieria might influence metabolic capabilities such as CAM. 2. Materials and methods 2.1. Plant material Whole plants and cuttings of most accessions examined were originally collected in Africa (Table 1) from the 1960s to the 1980s, then grown in several greenhouses in Germany, until they were transferred to the greenhouses of the University of Potsdam mainly as potted plants in 2015–2016. Thus, plants used for this study were at least, and often much more than, 20 years old. Plant sample sizes (usually 4–5; 2 for two species) varied according to availability of plants. Leaves for morphological and succulence analyses were excised from the plants and taken to the laboratory during May and June 2016, while leaves for 2
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physiological measurements were collected in July 2016. Air temperatures in the greenhouses at these two times ranged from approximately 15 to 25 °C, depending on time of day and position of sun and clouds. Relative humidity in the greenhouses was relatively constant at approximately 70%. Daytime Photosynthetic Photon Flux Densities were highly variable throughout the day, with maximum values of 1600 μmol m−2 s-1. Plants were watered up to watered up field capacity once weekly throughout the year, but with longer intervals during winter, and fertilized once monthly from May to September. Leaves collected for morphological and succulence analyses were immediately placed in a refrigerator (10 °C), and those used for physiological measurements were frozen at −12 °C upon arrival in the adjacent laboratory. The latter leaves were excised from the plants in the greenhouse at 16:30 h and at 07:30 h the next day (same plants) in order to determine overnight accumulation of malic acid. One leaf per plant was used for each type of measurement.
645 nm and 662 nm. After recovery from the centrifuge tube, the pellet was dried at 85 °C then weighed until no further mass loss. Chlorophyll a and b concentrations were calculated according to Lichtenthaler and Wellburn (1983). 2.5. Statistics Mean morning and evening titratable acidities were compared with the paired t-test for each species. In three cases (S. ballyi, S. gracilis, and S. senegambica), this comparison did not meet the assumptions for parametric tests, so the non-parametric Wilcoxon Signed Rank test was used (Sokal and Rohlf, 2012). Mean (for each species) overnight accumulations (AM-PM) of leaf acidities were compared with species means of the leaf morphologies, succulence, and chlorophyll measures using linear regressions. All statistical analyses were performed using the SigmaPlot 12.5 (SystatSoftware, Inc., San Jose, CA, USA) software package.
2.2. Morphological and succulence determinations
3. Results
Freehand sections (approximately 0.3 mm thick) were cut from the middle of mature leaves, then photographed with a dnt (StratfordUpon-Avon, UK) DigiMicro digital camera mounted on a light microscope (Fig. S1, right column). Because most of the leaves were large, multiple images were taken, then assembled for analysis using ImageJ (NIH, Bethesda, MD, USA) v1.50i software to determine the proportion of the leaf cross-section as achlorophyllous hydrenchyma and as green chlorenchyma. Leaf succulence was quantified via six different ways, using the middle (lengthwise and laterally) of fully developed, mature leaves (c.f. Table 1; Fig. S1):
Several key leaf morpho-anatomical variables varied largely in the ten species of Sansevieria selected for this study (Table 1; Fig. 1, S1). For example, (mid-) leaf thickness varied over ten-fold, from 0.2 cm in S. parva to > 2 cm in S. cylindrica (Fig. 2), while the leaf fresh mass-to-dry mass ratio varied almost three-fold, from near 8 in S. volkensii to 20 in S. cylindrica (Fig. 3). In addition, the proportion of leaf hydrenchyma, i.e., the area of hydrenchyma occupying the cross-section of the middle of a leaf, expressed relative to the area of the chlorenchymatous tissue (“chlorenchyma”) in these ten species of Sansevieria varied greatly, from zero or near zero in S. senegambica and S. parva to over 2 in S. cylindrica (Fig. 4). Relationships among these morpho-anatomical parameters were highly variable. For example, those species with the thickest leaves also exhibited the greatest amount of hydrenchyma (compare Figs. 2 and 4). In fact, leaf thickness accounted for nearly 99% of the variability in the hydrenchyma/chlorenchyma ratio of these ten species of Sansevieria (P < 0.001; regression not shown). On the other hand, there was no significant relationship between leaf thickness and leaf fresh mass-todry mass ratio (compare Figs. 2 and 3; R2 = 0.11, P = 0.38; regression not shown). Given the similarity of the following two measures, the leaf fresh mass:dry mass ratio correlated well with leaf water content on a dry mass basis (compare Figs. 3 and 5; R2 = 0.65, P < 0.01; regression not shown), but not when water content is expressed on a fresh mass basis (compare Figs. 3 and 6; R2 = 0.00, P = 0.89; regression not shown). Foliar chlorophyll concentrations were also highly variable in the ten species of Sansevieria examined (Fig. 8). In addition, the degree of CAM, measured as the increase in leaf tissue acidity overnight, varied nearly seven-fold, from a minimum near 180 μmol g−1 in S. fischeri to over 1300 μmol g−1 in S. senegambica (Table 2; Figs. 2–8). Seven of the ten species of Sansevieria expressed CAM (Table 2). Mean morning and evening acidities were not significantly different in S. ballyi, S. gracilis, and S. senegambica. The degree of CAM was strongly inversely correlated with leaf thickness in the ten species of Sansevieria. In fact, leaf thickness accounted for over 60% of the variation in the nocturnal accumulation of acidity, with species having thinner leaves showing greater amounts of CAM (Fig. 2). Furthermore, given the strong positive correlation between leaf thickness and leaf hydrenchyma-to-chlorenchyma ratio, the degree of CAM was also highly negatively correlated with the latter ratio, i.e., thicker leaves had relatively more hydrenchyma and lower amounts of CAM (Fig. 4). Likewise, thinner leaves had greater proportional amounts of chlorenchyma, and these species exhibited greater degrees of CAM. On the other hand, species having leaves with greater amounts of water on a fresh mass basis did not have more hydrenchyma, as explained above and, as a result, there appears to be more CAM in leaves with higher
1) leaf thickness, measured with a standard, hand-held cm ruler; 2) leaf hydrenchyma/chlorenchyma cross-sectional area ratio: from the ImageJ images as above; 3) fresh mass/dry mass ratio (FM/DM), measured from 1 cm2 sections; 4) water content, fresh mass basis (g H2O g−1 FM), measured from 1 cm2 sections; 5) water content, dry mass basis (g H2O g−1 DM), measured from 1 cm2 sections; and 6) mesophyll succulence (Kluge and Ting, 1978) (g H2O mg−1 Chl), measured from 1 cm2 sections. 2.3. Titratable acidity After thawing, approximately 1 cm2 of tissue was excised from the center of the leaf, weighed, then placed in a 2-ml plastic reaction tube, whereupon the leaf tissue was obliterated with a glass rod. After two days at −30 °C, then thawing, all liquid (minus cell debris) was pipetted into a new tube, a 250 μl aliquot of the leaf liquid was filled up to 40 ml with demineralized water and then titrated to pH 8.2 using 0.01 N NaOH and a T50 M auto-titrator with a Rondo 20-sample changer (Mettler Toledo, Gießen, Germany). The remaining cell debris was dried at 85 °C and then weighed until no further mass loss. 2.4. Chlorophyll concentration After freezing, then thawing, another 1 cm2 of tissue was excised from the center of the leaf, weighed, then sliced into smaller pieces, collected in a 50 ml centrifuge tube and 10 ml of 100% acetone added. After one day at 10 °C, the leaf pieces were blended in acetone with an Ultra Turrax (Atkinson, NH, USA) t18 blender. An aliquot of the acetone/leaf mixture was then transferred into a 2 ml reaction tube, and centrifuged for two min at 14,500 rpm with an Eppendorf (Hamburg, Germany) MiniSpin Plus micro-centrifuge, after which the absorption of the supernatant was measured with a Specord 210 Plus spectrophotometer (Analytik Jena AG, Jena, Germany) at wavelengths of 3
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Fig. 1. Cross sections of mature leaf section of all Sansevieria species investigated. Hydrenchyma is indicated as light grey area, while the dark grey-hatched area denotes the extension of chlorenchyma. Dots and small circles represent vascular bundles. Bars indicate 5 mm.
fresh mass-based water content, but this relation is not significant (Fig. 6). The degree of CAM was also not significantly correlated with leaf water content when expressed on a dry mass basis (Fig. 5), nor with leaf fresh mass:dry mass ratio (Fig. 3). When leaf water content is expressed on a chlorophyll mass basis, a measure referred to as “mesophyll succulence,” the degree of CAM is inversely, but not significantly, correlated with the latter (Fig. 7). Overall, it is clear that plants with leaves having more water in nonphotosynthetic tissue exhibited lesser degrees of CAM. The degree of CAM appears to be positively correlated with the amount of chlorophyll in the leaves of the ten species of Sansevieria in this study (Fig. 8).
4. Discussion The results of this study serve to emphasize two major conclusions. First, the CAM photosynthetic pathway and succulent leaves are widespread in the genus Sansevieria (Virzo De Santo et al., 1981–1982); and, second, when considering various measures of succulence, the most important predictor of CAM vs. non-CAM is the amount of chlorophyllous tissue (chlorenchyma) in the leaf, especially in plants having a clear separation of hydrenchyma and chlorenchyma tissue in the photosynthetic organs. In such leaves, “succulence” primarily reflects the amount of water in the achlorophyllous, non-CAM-performing tissue in the leaf; thus, any correlation between overall leaf succulence 4
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Fig. 2. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf thickness at the center of the leaf for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 4. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf hydrenchyma:chlorenchyma ratio for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 3. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf fresh mass:dry mass ratio for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 5. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf water content on a dry mass basis for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
and CAM is negative. When such an anatomical distinction is lacking, e.g., when organs are “all-cell succulent”, then water content of the entire tissue should correlate well with the CAM photosynthetic pathway both at the species (Herppich et al., 1998) and at the genus level (Herppich and Herppich, 1996). The genus Sansevieria is well known for its wide variety of leaf forms and shapes, ranging from flat and lanceolate to cylindrical leaves (Newton, 2001; Schwerdtfeger, 2009). All species, however, show the distinct internal parenchyma made from both dead water storage cells and living network cells. This, in particular, makes Sansevieria an excellent tool for comparing the potential interactions between leaf succulence and CAM. In addition, the species chosen cover all sections and subsections proposed by Pfennig (1977); Jankalski (2009) and Mansfeld (2013), which are largely congruent. They also cover 7 out of 16 species groups proposed by Jankalski (2015). Regarding phylogenetic studies,
the selected species cover all 4 main clades in Lu and Morden (2014), both main clades in Baldwin and Webb (2016), 2 out of 4 main clades in Takawira-Nyenya et al. (2018), and 5 out of 7 main clades in van Kleinwee (2018). Nevertheless, the phylogenetic trees proposed in these studies, although of different resolution and quality, are largely incongruent, and none of them represents a consolidated state of phylogenetic relationships within Sansevieria. Hence, no further analysis of this topic is included in the presented considerations. Overall, species of Sansevieria with thick leaves have proportionally large amounts of hydrenchyma, which does not engage in CAM photosynthesis. Such leaves have a high water content on a fresh mass, but not a dry mass, basis. The latter reflects the fact that thick leaves have a relatively large dry mass; thus, they have a low FM:DM ratio. Although the “mesophyll succulence” index proposed by Kluge and Ting (1978) was intended to provide a physiological basis of leaf water 5
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Fig. 6. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf water content on a fresh mass basis for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 7. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf mesophyll succulence for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Table 2 Morning (AM), evening (PM), and delta (AM-PM) titratable acidities for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling). Values are means ( ± SE) of 4–5 plants per species, except N = 2 for S. ballyi and S. gracilis. Morning (AM) means are significantly different from PM means (* indicates P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001), except forS. ballyi,S. gracilis, and S. senegambica (ns; P = 0.48, 0.35, and 0.13, respectively). Species
AM acidity, μmol g−1DM
PM acidity, μmol g−1DM
Δ acidity, μmol g−1DM
ballyi cylindrica ehrenbergii fischeri gracilis parva raffillii senegambica suffruticosa volkensii
1520.8 ± 875.4 1630.8 ± 121.6 1310.1 ± 219.8 676.0 ± 71.0 1333.8 ± 236.9 2325.3 ± 130.0 2238.9 ± 348.9 2466.2 ± 482.9 1490.0 ± 180.0 1074.8 ± 135.9
875.4 ± 73.6 1017.9 ± 52.0 903.6 ± 130.0 494.3 ± 64.6 919.0 ± 16.2 1047.5 ± 130.5 1278.8 ± 351.7 1162.4 ± 174.8 921.1 ± 131.9 480.3 ± 91.5
645.4 ± 12.8 ns 612.9 ± 99.7** 406.5 ± 107.1* 181.7 ± 20.3*** 414.8 ± 253.1 ns 1277.8 ± 101.3*** 960.1 ± 91.5*** 1303.8 ± 358.2 ns 568.8 ± 131.4** 594.5 ± 76.6***
Fig. 8. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf chlorophyll concentration (DM basis) for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
content that might predict the photosynthetic pathway of a plant, this index was a poor indicator of the degree of CAM in the current study of the genus Sansevieria. The reason for this, as indicated above, is related to the amount of hydrenchyma in the leaf. Kluge and Ting (1978) were well aware of this limitation, as they suggested that the water content value in the mesophyll succulence index should be limited to the water content of the photosynthetic chlorenchyma tissue and not to the whole leaf, if possible. Thus, the findings of the current study strongly support this suggested limitation of this succulence measure. Strictly speaking, the unique type of water-storage tissue could not be important for CAM because dead water-storage cells should not be involved in the storage of malic acid. In fact, Alfani et al. (1989) assumed that it exclusively functions as a fast, “metabolically low-cost” water storage system that could lose considerable amounts of water without damages of living cells during dehydration. On the other hand, Koller and Rost (1988a) reported that the relative area occupied by the water storage tissue was larger in more xeromorphic physiognomy, i.e. thicker cuticle and higher fiber content. The latter species also contain a larger number of dead water storage compared to living network cells. All the species in this study are naturally found in arid regions
throughout eastern Africa (some species of Sansevieria are native to less arid regions of Southeast Asia). Both CAM and leaf succulence should prove beneficial by conserving and reducing water loss in such arid regions (von Willert et al., 1992). Furthermore, neither CAM nor succulence was limited to certain phylogenetic clades as also suggested by van Kleinwee (2018). Thus, it appears that both CAM and leaf succulence evolved multiple times in the genus Sansevieria; alternatively, it might have evolved at the beginning of the evolution of the genus. The latter seems to be plausible for succulence due to the fact that all species of Sansevieria show the strict separation in internal genus-specific water storage tissue and surrounding chlorenchyma. Nevertheless, similar findings of multiple origins of CAM have been reported in other taxonomic groups, including the Bromeliaceae (Crayn et al., 2004), Aeonium in the Crassulaceae (Mort et al., 2007), and in the family Orchidaceae 6
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(Silvera et al., 2010). Perhaps more important, similar findings about multiple origins of both succulence and CAM have been found in the Agavoideae (Heyduk et al., 2016), a subfamily of the Asparagaceae, in which Sanseveria is also found.
(Asparagaceae). Molec. Phylogenet. Evol. 105, 102–113. Ho, C.-L., Chiang, J.-M., Lin, T.-C., Martin, C.E., 2018. First report of C4/CAM-cycling photosynthetic pathway in a succulent grass, Spinifex littoreus (Brum. f.) Merr., in coastal regions of Taiwan. Flora 254, 194–202. Ihlenfeldt, H.-D., 1985. Lebensformen und Überlebensstrategien bei Sukkulenten. Plant Biol. 98, 409–423. Jankalski, S., 2009. The Sansevieria inflorescence and new sections proposed. Sansevieria 19, 8–10. Jankalski, S., 2015. Infrageneric species groups in Sansevieria. Sansevieria 33, 15–19. Kluge, M., Ting, I.P., 1978. Crassulacean Acid Metabolism. Analysis of an Ecological Adaptation. Springer-Verlag, Berlin. Koller, A.L., Rost, T.L., 1988a. Leaf anatomy in Sansevieria (Agavaceae). Am. J. Bot. 75, 615–633. Koller, A.L., Rost, T.L., 1988b. Structural analysis of water-storage tissue in leaves of Sansevieria (Agavaceae). Bot. Gaz. 149, 260–274. Larcher, W., 2003. Physiological Plant Ecology. Ecophysiology and Stress Physiology of Functional Groups, 4th ed. Springer-Verlag, Berlin. Leuschner, C., Ellenberg, H., 2018. Salt marshes and inland saline habitats. Vegetation Ecology of Central Europe, vol. 2. Ecology of Central European Non-Forest Vegetation: Coastal to Alpine, Natural to Man-Made Habitats. Springer International Publishing, AG, Cham, pp. 3–62. https://doi.org/10.1007/978-3-319-43048-5. Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592. Lu, P.-L., Morden, C.W., 2014. Phylogenetic relationships among Dracaenoid genera (Asparagaceae: Nolinoideae) inferred from chloroplast DNA loci. Syst. Bot. 39, 90–104. Lüttge, U., 1987. Carbon dioxide and water demand: Crassulacean acid metabolism (CAM), a versatile ecological adaptation exemplifying the need for integration in ecophysiological work. New Phytol. 106, 593–629. MacDougal, D.T., Spalding, E.S., 1910. The water-balance of succulent plants. Carn. Inst. Wash. Publ. No. 141, 1–77. Males, J., 2017. Secrets of succulence. J. Exp. Bot. 68, 2121–2134. Males, J., 2018. Concerted anatomical change associated with CAM in the Bromeliaceae. Funct. Plant Biol. 45, 681–695. Mansfeld, P.A., 2013. Neugliederung der Gattung Sansevieria (Asparagaceae). Kakt. and. Sukk. 64, 35–38. Martin, C.E., Hsu, R.C.-C., Lin, T.-C., 2009. The relationship between CAM and leaf succulence in two epiphytic vines, Hoya carnosa and Dischidia formosana (Asclepiadaceae), in a subtropical rainforest in northeastern Taiwan. Photosynthetica 47, 445–450. Mort, M.E., Solits sic, D.E., Soltis, P.S., Santos-Guerra, A., Francisco-Ortega, J., 2007. Physiological evolution and association between physiology and growth form in Aeonium(Crassulaceae). Taxon 56, 453–464. Newton, L.E., 2001. Sansevieria. In: Eggli, U. (Ed.), Illustrated Handbook of Succulent Plants. Monocotyledons, pp. 261–272. Newton, L.E., 2004. Sansevieria ballyi– a name at last for a long-cultivated plant. Brit. Cact. Succ. J. 22, 10–13. Nowak, E.J., Martin, C.E., 1997. Physiological and anatomical responses to water deficits in the CAM epiphyte Tillandsia ionantha (Bromeliaceae). Int. J. Plant Sci. 158, 818–826. Ogburn, R.M., Edwards, E.J., 2010. The ecological water-use strategies of succulent plants. Adv. Bot. Res. 55, 179–225. Ogburn, R.M., Edwards, E.J., 2012. Quantifying succulence: a rapid, physiologically meaningful metric of plant water storage. Plant Cell Environ. 35, 1533–1542. Ogburn, R.M., Edwards, E.J., 2013. Repeated origin of three-dimensional leaf venation releases constraints on the evolution of succulence in plants. Curr. Biol. 23, 722–726. Osmond, C.B., 1978. Crassulacean acid metabolism: a curiosity in context. Annu. Rev. Plant Physiol. 29, 379–414. Pfennig, H., 1977. Rasenbildend bis baumartig: die Sansevierien. Gartenpraxis 1977, 506–511. Ripley, B.S., Abraham, T., Klak, C., Cramer, M.D., 2013. How succulent leaves of Aizoaceae avoid mesophyll conductance limitations of photosynthesis and survive drought. J. Exp. Bot. 64, 5485–5496. Schulze, E.-D., Beck, E., Müller-Hohenstein, K., 2002. Plant Ecology. Springer-Verlag, Berlin. Schwerdtfeger, M., 2009. Die Faszination der Sansevierien. Kakt. and. Sukk. 60, 239–246. Silvera, K., Santiago, L.S., Cushman, J.C., Winter, K., 2010. The incidence of Crassulacean Acid Metabolism in Orchidaceae derived from carbon isotope ratios: a checklist of the flora of Panama and Costa Rica. Bot. J. Linn. Soc. 163, 194–222. Smith, J.A.C., Heuer, S., 1981. Determination of the volume of intercellular spaces in leaves and some values for CAM plants. Ann. Bot. 48, 915–917. Smith, J.A.C., Schulte, P.J., Nobel, P.S., 1987. Water flow and water storage in Agave deserti: Osmotic implications of Crassulacean acid metabolism. Plant Cell Environ. 10, 639–648. Sokal, R.R., Rohlf, F.J., 2012. Biometry. The Principles and Practice of Statistics in Biological Research, 4th ed. WH Freeman & Co, New York. Steudle, E., Smith, J.A.C., Lüttge, U., 1980. Water-relation parameters of individual mesophyll cells of the Crassulacean acid metabolism plant Kalanchoe daigremontiana. Plant Physiol. 66, 1155–1163. Taiz, L., Zeiger, E., 1998. Plant Physiology, 2nd ed. Sinauer Associates, MA, USA. Takawira-Nyenya, R., Mucina, L., Cardinal-McTeague, W.M., Thiele, K.R., 2018. Sansevieria (Asparagaceae, Nolinoideae) is a herbaceous clade within Dracaena: inference from non-coding plastid and nuclear DNA sequence data. Phytotaxa 376, 254–276. Teeri, J.A., Tonsor, S.J., Turner, M., 1981. Leaf thickness and carbon isotope composition
5. Conclusions These results complement current understanding of the relationship between the requirement of large vacuoles in photosynthetic cells, hence large cells, for the nocturnal storage of malic acid during CO2 assimilation in Crassulacean acid metabolism. As a result, the amount of water stored in achlorophyllous tissue is not directly related to the biochemistry and physiology of CAM. It should be emphasized that water storage in achlorophyllous tissue also constitutes an excellent adaptation to arid environments, as does CAM, but the results of the current study emphasize that the positive correlation between tissue succulence and CAM is applicable primarily to the photosynthetic tissue (chlorenchyma). Declarations of Competing Interest None Acknowledgments We heartily thank Ms. G. Wegner and Ms. C. Rolleczek at the ATB for assistance with the acidity measurements. In addition, we thank Jil Roßberg for the perfect drawings of the cross sections and Christiane Benthin for taking care of the plants as well as Doris Pfennig, Michael Schwerdtfeger and Uwe Scharf for providing plants. C.E.M. gratefully acknowledges DAAD and AvH for funding during his stays at the ATB. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.flora.2019.151489. References African Plant Database, 2019. Conservatoire Et Jardin Botaniques De La Ville De Genève. http://www.ville-ge.ch/musinfo/bd/cjb/africa/recherche.php. Alfani, A., Ligrone, R., Fioretto, A., Virzo De Santo, A., 1989. Histochemistry, ultrastructure and possible significance of dead parenchyma cells with specialized walls in the leaf and rhizome of Sansevieria. Plant Cell Environ. 12, 249–259. Baldwin, A.S., Webb, R.H., 2016. The genus Sansevieria: An introduction to molecular (DNA) analysis and preliminary insights to intrageneric relationships. Sansevieria 34, 14–26. Brown, N.E., 1915. Sansevieria. A monograph of all known species. Bull. Misc. Inform. Kew 1915, 1–81. Budweg, H.G., Mansfeld, P.A., 2015. Sansevieria Online. Sonderheft 2015. (Accessed 12 April 2019). http://www.sansevieria-online.de/pdf/so_sonderheft_2015.pdf. Chiang, J.-H., Lin, T.-C., Luo, Y.-C., Chang, C.-T., Cheng, J.-Y., Martin, C.E., 2013. Relationships among rainfall, leaf hydrenchyma, and Crassulacean acid metabolism in Pyrrosia lanceolata(L.) Fraw. (Polypodiaceae) in central Taiwan. Flora 208, 343–350. Crayn, D.M., Winter, K., Smith, J.A.C., 2004. Multiple origins of Crassulacean acid metabolism and the epiphytic habitat in the Neotropical family Bromeliaceae. Proc. Nat. Acad. Sci. USA 101, 3703–3708. Edwards, G., Walker, D.A., 1983. C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis. Blackwell Science Publ., Oxford. Ehleringer, J., 1985. Annuals and perennials of warm deserts. In: Chabot, B.F., Mooney, H.A. (Eds.), Physiological Ecology of North American Plant Communities. Chapman & Hall, N.Y, pp. 162–180. Gibson, A.C., 1982. The anatomy of succulence. In: Ting, I.P., Gibbs, M. (Eds.), Crassulacean Acid Metabolism. Amer. Soc. Plant Physiol., Rockville, MD. pp. 1–17. Herppich, W.B., Herppich, M., 1996. Ecophysiological investigations on plants of the genus Plectranthus(Fam. Lamiaceae) native to Yemen and southern Africa. Flora 191, 401–408. Herppich, W.B., Herppich, M., von Willert, D.J., 1998. Ecophysiological investigations on plants of the genus Plectranthus(Lamiaceae). Influence of environment and leaf age on CAM, gas exchange and leaf water relations in Plectranthus marrubioides Benth. Flora 193, 99–109. Heyduk, K., McKain, M.R., Lalani, F., Leebens-Mack, J., 2016. Evolution of a CAM anatomy predates the origins of Crassulacean metabolism in Agavoideae
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C.E. Martin, et al. in the Crassulaceae. Oecologia 50, 367–369. Turner, N.C., Kramer, P.J. (Eds.), 1980. Adaptation of Plants to Water and High Temperature Stress. John Wiley and Sons, New York. van Kleinwee, I., 2018. In the Name of Sansevieria (Asparagaceae [sic]). An Integrative Study on Identification and Classification of the Sansevieria Diversity. Master of Science thesis. Ghent University, Ghent, Belgium. Veste, M., Herppich, W.B., von Willert, D.J., 2001. Variability of CAM in leaf-deciduous succulents from the Succulent Karoo (South Africa). Basic Appl. Ecol. 2, 283–288. Virzo De Santo, A., Fioretto, A., Alfani, A., 1982. The Adaptive Significance of Growth Form, Leaf Morphology, and CAM in the Genus Sansevieria. Virzo De Santo, A., Alfani, A., Russo, G., Fioretto, A., 1983. Relationship between CAM and succulence in some species of Vitaceae and Piperaceae. Bot. Gaz. 144, 342–346.
von Willert, D.J., Eller, B.M., Werger, M.J.A., Brinckmann, E., Ihlenfeldt, H.-D., 1992. Life Strategies of Succulents in Deserts: With Special Reference to the Namib Desert. Cambridge Univ. Press, Cambridge. Webb, R.H., Newton, L.E., 2017. Biogeography of the genus Sansevieria: Should plants without a known type locality be considered valid species? Sansevieria 36, 15–21. Winter, K., 1985. Crassulacean acid metabolism. In: Barber, J., Baker, N.R. (Eds.), Photosynthetic Mechanisms and the Environment. Elsevier Science Publ. BV, Amsterdam, pp. 329–387. Winter, K., Wallace, B.J., Stocker, G.C., Roksandic, Z., 1983. Crassulacean acid metabolism in Australian vascular epiphytes and some related species. Oecologia 57, 129–141.
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Relationships between leaf succulence and Crassulacean acid metabolism in the genus Sansevieria (Asparagaceae)
T
Craig E. Martina,b, Werner B. Herppichb,*, Yvonne Roscherc, Michael Burkartc a
Department of Ecology & Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045, USA Abteilung Technik im Gartenbau, Leibniz-Institute für Agrartechnik und Bioökonomie, Max-Eyth-Allee 100, 14469 Potsdam, Germany c Institut für Biochemie und Biologie, Universität Potsdam, Maulbeerallee 3, 14469 Potsdam, Germany b
ARTICLE INFO
ABSTRACT
Edited by: Hermann Heilmeier
Relationships between different measures of succulence and Crassulacean acid metabolism (CAM; defined here as nocturnal increases in tissue acidity) were investigated in leaves of ten species of Sansevieriaunder greenhouse conditions. CAM was found in seven of the ten species investigated, and CAM correlated negatively with leaf thickness and leaf hydrenchyma/chlorenchyma ratio. Similarly, CAM correlated negatively with leaf water content, but only when expressed on a fresh mass basis. CAM was not correlated with “mesophyll succulence”, but weakly with leaf chlorophyll concentration. These results indicate that CAM is associated more with “all-cell succulence” and not with the amount of leaf hydrenchyma in the genus Sansevieria. The findings of this study emphasize the importance of defining the nature of “leaf succulence” in studies of photosynthetic pathways and leaf morphology. Evidence is also provided that CAM and succulence arose multiple times in the genus Sansevieria.
Keywords: Anatomy CAM Chlorenchyma Chlorophyll Hydrenchyma Morphology Phylogeny
1. Introduction Plants growing in arid regions often display a wide variety of adaptive features that serve to avoid or minimize the effects of drought stress (Ehleringer, 1985; von Willert et al., 1992; Veste et al., 2001). These features include tissue water storage, deep and/or widespread root systems, leaf morphologies that reduce high temperatures, sunken stomata at low densities, and water-conservative photosynthetic pathways, especially Crassulacean acid metabolism (CAM), one of three photosynthetic pathways found in plants (Edwards and Walker, 1983; Taiz and Zeiger, 1998; Schulze et al., 2002). A common morphoanatomical feature often associated with plants having CAM photosynthesis is succulence (Kluge and Ting, 1978; Gibson, 1982; Osmond, 1978; Winter, 1985; Males, 2017), which typically occurs in one of two forms: distinctive achlorophyllous waterstorage tissue (parenchyma) termed “hydrenchyma”, or simply very large, mostly chlorophyllous cells comprising the bulk of a photosynthetic organ, e.g., leaf or stem. The latter form has been referred to as “all-cell succulence” (Ihlenfeldt, 1985; von Willert et al., 1992; Ogburn and Edwards, 2010, 2012, 2013; Males, 2017; Ho et al., 2018). In both cases, the large cells harbor large vacuoles that occupy up to, and often greater than, 90% of the cell volume (Kluge and Ting, 1978; Steudle et al., 1980; Smith and Heuer, 1981). The function of these
⁎
large cells and associated vacuoles in both types of succulence is obvious when considering the arid environments occupied by such succulent plants, i.e., water storage and supply during times of low ambient water availability (MacDougal and Spalding, 1910; Smith et al., 1987; Nowak and Martin, 1997; Chiang et al., 2013; Ho et al., 2018). Furthermore, CAM conserves water in these plants by limiting stomatal opening and, hence, water loss, to the nighttime when the atmospheric evaporative demand is lower than during the warmer and less humid days (Kluge and Ting, 1978; Osmond, 1978; Winter, 1985; Lüttge, 1987). As Rubisco is inoperative at night, CO2 assimilation is catalyzed by PEP carboxylase during CAM, which results in the production of malic acid. The latter is stored in the large vacuoles of the photosynthetic cells throughout the nighttime period of CO2 fixation. Thus, it has been proposed that there is a causal correlation between the degree of leaf (or stem) succulence and the occurrence of the CAM photosynthetic pathway (Winter et al., 1983; Larcher, 2003; Heyduk et al., 2016; Males, 2017, 2018). The latter correlation, however, is complicated by the occurrence of both CAM and C3 plants having distinct layers of non-photosynthetic hydrenchyma tissue, age and size of the photosynthetic organ, and potential problems of low mesophyll conductance to CO2 in succulent tissues, the latter being a result of minimal amounts of intercellular air space (Smith and Heuer, 1981; Ripley et al., 2013).
Corresponding author. E-mail address: [email protected] (W.B. Herppich).
https://doi.org/10.1016/j.flora.2019.151489 Received 12 April 2019; Received in revised form 22 October 2019; Accepted 23 October 2019 Available online 25 October 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.
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Table 1 The ten species of Sansevieria used in this study, species authority, ranges of leaf length, width, thickness, general shape of the leaf, and general geographic distribution. Information taken from Newton (2004); Budweg and Mansfeld (2015); African Plant Database (2019) and current study. Species
Species Authority
Leaf Length, cm
Leaf Width, cm
Leaf Thickness, mm
Hydrenchyma Thickness, mm
Leaf Transverse Section Shape
Species Distribution
ballyi (ba) cylindrica (cy)
L. E. Newton Bojer ex Hook.
0.6–1.2 2.5–4
cylindrical with groove cylindrical
central East Africa southern Africa
Schweinf. ex Baker
28–48
cylindrical with groove
central East Africa
fischeri (fi) gracilis (gr)
Baker (Marais) N. E. Br.
2–4.5 0.7
6–12 25–40 20–30 30–50 30–45 20–45 7 6–10
3–8 23–38
ehrenbergii (eh)
6–12 75–150 60–150 75–180
18–43 0.5
central East Africa central East Africa
parva (pa) raffillii (ra)
N. E. Br. N. E. Br.
0.8–3 5–13
2–3 6–8
none only near midrib
cylindrical cylindrical with partial groove slightly concave concave
senegambica (se) suffruticosa (su)
Baker N. E. Br.
3–6.5 1.3–2
none 11–18
volkensii (vo)
Gürke
2–5 13–20 13–19 13–20 13–19
45–245 15–90 23–80 20–45 60–105 68–152 30–70 15–60 45–100 45–122
2.5–3.5
1.3–2
11–18
A considerable number of studies have shown strong correlations between tissue succulence and the presence of CAM in several groups of plants, including species of Crassulaceae (Teeri et al., 1981), epiphytic orchids and ferns (Winter et al., 1983), and species in the grape and pepper families (Virzo De Santo et al., 1983). Partly as a result of these studies and partly based on first principles, several workers have argued that succulence is an evolutionary prerequisite for the historical development of CAM (see above). In contrast, Martin et al. (2009) found poor correlations between leaf thickness and degree (not presence) of CAM in two tropical CAM species in the Asclepiadaceae. Furthermore, Virzo De Santo et al. (1983) argued that any correlation between CAM and succulence may simply reflect two independent adaptations to arid environments (von Willert et al., 1992). One problem with such correlative studies is the definition of “succulence” used for these studies. For example, it may be expected that succulence–CAM correlations may be causally linked in plants with all-cell succulence, because all parenchyma cells in the photosynthetic tissue engage in CAM and, as a result, require very large vacuoles, whereas such a correlation appears to be less likely in CAM plants with well-defined hydrenchyma/chlorenchyma divisions of parenchyma tissue, given that the hydrenchyma cells, although contributing to tissue succulence, are not involved in CAM photosynthesis. Furthermore, many C3 halophytes have highly succulent photosynthetic tissues, yet this succulence appears to be primarily an adaptation that dilutes high concentrations of potentially toxic inorganic solutes (Turner and Kramer, 1980; Leuschner and Ellenberg, 2018). As a result of the above problems linking CAM and succulence, numerous measures of succulence have been attempted in such correlations (e.g., von Willert et al., 1992; Males, 2017, 2018; Ho et al., 2018). Most such measures relate photosynthetic tissue water content to leaf mass or structure (e.g., thickness or amount of hydrenchyma). One measure which often successfully correlates CAM and succulence was proposed by Kluge and Ting (1978) and relates tissue water content to tissue chlorophyll content and was termed “mesophyll succulence”. Other confounding factors might obfuscate reported correlations – or the lack thereof – between the CAM photosynthetic pathway and succulence of the photosynthetic organ. Such factors include the use of different measures of succulence, as indicated above, the use of widely divergent taxa, comparison of different organ types, as well as comparing plants under different environmental conditions. The monocot genus Sansevieria (Asparagaceae), containing around 70 species, is distributed in Africa and Asia, with a center of species diversity in East Africa (Brown, 1915; Newton, 2001; Webb and Newton, 2017; van Kleinwee, 2018). All species of this genus are characterized by a clear subdivision of the mesophyll into
slightly concave cylindrical with partial groove cylindrical with groove
central East Africa central East Africa central West Africa central East Africa central East Africa
chlorenchyma and a unique type of central colorless water-storage tissue (Koller and Rost, 1988a). Besides scattered vascular and fiber bundles, the latter comprises two different cell types (Koller and Rost, 1988a, 1988b; van Kleinwee, 2018). Water is effectively stored in clusters of large non-living thin-walled water-storage cells (Alfani et al., 1989), which are embedded in and interconnected by a network of small living cells, the so-called network cells (Koller and Rost, 1988a). While this general arrangement seems to be similar among all species of this genus, the relative contribution of both cell types varies (Koller and Rost, 1988a). Different Sansevieria species exhibit different leaf forms (cylindrical vs. “flat” concave), different degrees of xeromorphism and different types of succulence, e.g., hydrenchyma vs. all-cell succulence, and the degrees of succulence vary greatly among the different species (Alfani et al., 1989). For example, S. parva lacks a distinct layer of foliar hydrenchyma, while that of S. cylindrica is often 1–2 cm thick (Brown, 1915; Budweg and Mansfeld, 2015). Given the widely scattered results of attempts to correlate photosynthetic pathway (specifically, CAM vs. non-CAM) with “succulence”, the goal of the current study was to further investigate this correlation by comparing different measures of succulence and photosynthetic pathway (C3 or CAM) in leaves of different species of the genus Sansevieria largely differing in anatomical/morphological features and covering the full variability of leaf morphology in the genus but grown under identical conditions in the greenhouses of the University of Potsdam. This allows us to allows testing the hypothesis that succulence per se is an essential prerequisite for the occurrence and the expression of CAM, without the limits of family-specific or environmental effects on both features. This analysis also helps to evaluate whether differences in the chlorenchyma to hydrenchyma ratio in the different species of the genus Sansevieria might influence metabolic capabilities such as CAM. 2. Materials and methods 2.1. Plant material Whole plants and cuttings of most accessions examined were originally collected in Africa (Table 1) from the 1960s to the 1980s, then grown in several greenhouses in Germany, until they were transferred to the greenhouses of the University of Potsdam mainly as potted plants in 2015–2016. Thus, plants used for this study were at least, and often much more than, 20 years old. Plant sample sizes (usually 4–5; 2 for two species) varied according to availability of plants. Leaves for morphological and succulence analyses were excised from the plants and taken to the laboratory during May and June 2016, while leaves for 2
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physiological measurements were collected in July 2016. Air temperatures in the greenhouses at these two times ranged from approximately 15 to 25 °C, depending on time of day and position of sun and clouds. Relative humidity in the greenhouses was relatively constant at approximately 70%. Daytime Photosynthetic Photon Flux Densities were highly variable throughout the day, with maximum values of 1600 μmol m−2 s-1. Plants were watered up to watered up field capacity once weekly throughout the year, but with longer intervals during winter, and fertilized once monthly from May to September. Leaves collected for morphological and succulence analyses were immediately placed in a refrigerator (10 °C), and those used for physiological measurements were frozen at −12 °C upon arrival in the adjacent laboratory. The latter leaves were excised from the plants in the greenhouse at 16:30 h and at 07:30 h the next day (same plants) in order to determine overnight accumulation of malic acid. One leaf per plant was used for each type of measurement.
645 nm and 662 nm. After recovery from the centrifuge tube, the pellet was dried at 85 °C then weighed until no further mass loss. Chlorophyll a and b concentrations were calculated according to Lichtenthaler and Wellburn (1983). 2.5. Statistics Mean morning and evening titratable acidities were compared with the paired t-test for each species. In three cases (S. ballyi, S. gracilis, and S. senegambica), this comparison did not meet the assumptions for parametric tests, so the non-parametric Wilcoxon Signed Rank test was used (Sokal and Rohlf, 2012). Mean (for each species) overnight accumulations (AM-PM) of leaf acidities were compared with species means of the leaf morphologies, succulence, and chlorophyll measures using linear regressions. All statistical analyses were performed using the SigmaPlot 12.5 (SystatSoftware, Inc., San Jose, CA, USA) software package.
2.2. Morphological and succulence determinations
3. Results
Freehand sections (approximately 0.3 mm thick) were cut from the middle of mature leaves, then photographed with a dnt (StratfordUpon-Avon, UK) DigiMicro digital camera mounted on a light microscope (Fig. S1, right column). Because most of the leaves were large, multiple images were taken, then assembled for analysis using ImageJ (NIH, Bethesda, MD, USA) v1.50i software to determine the proportion of the leaf cross-section as achlorophyllous hydrenchyma and as green chlorenchyma. Leaf succulence was quantified via six different ways, using the middle (lengthwise and laterally) of fully developed, mature leaves (c.f. Table 1; Fig. S1):
Several key leaf morpho-anatomical variables varied largely in the ten species of Sansevieria selected for this study (Table 1; Fig. 1, S1). For example, (mid-) leaf thickness varied over ten-fold, from 0.2 cm in S. parva to > 2 cm in S. cylindrica (Fig. 2), while the leaf fresh mass-to-dry mass ratio varied almost three-fold, from near 8 in S. volkensii to 20 in S. cylindrica (Fig. 3). In addition, the proportion of leaf hydrenchyma, i.e., the area of hydrenchyma occupying the cross-section of the middle of a leaf, expressed relative to the area of the chlorenchymatous tissue (“chlorenchyma”) in these ten species of Sansevieria varied greatly, from zero or near zero in S. senegambica and S. parva to over 2 in S. cylindrica (Fig. 4). Relationships among these morpho-anatomical parameters were highly variable. For example, those species with the thickest leaves also exhibited the greatest amount of hydrenchyma (compare Figs. 2 and 4). In fact, leaf thickness accounted for nearly 99% of the variability in the hydrenchyma/chlorenchyma ratio of these ten species of Sansevieria (P < 0.001; regression not shown). On the other hand, there was no significant relationship between leaf thickness and leaf fresh mass-todry mass ratio (compare Figs. 2 and 3; R2 = 0.11, P = 0.38; regression not shown). Given the similarity of the following two measures, the leaf fresh mass:dry mass ratio correlated well with leaf water content on a dry mass basis (compare Figs. 3 and 5; R2 = 0.65, P < 0.01; regression not shown), but not when water content is expressed on a fresh mass basis (compare Figs. 3 and 6; R2 = 0.00, P = 0.89; regression not shown). Foliar chlorophyll concentrations were also highly variable in the ten species of Sansevieria examined (Fig. 8). In addition, the degree of CAM, measured as the increase in leaf tissue acidity overnight, varied nearly seven-fold, from a minimum near 180 μmol g−1 in S. fischeri to over 1300 μmol g−1 in S. senegambica (Table 2; Figs. 2–8). Seven of the ten species of Sansevieria expressed CAM (Table 2). Mean morning and evening acidities were not significantly different in S. ballyi, S. gracilis, and S. senegambica. The degree of CAM was strongly inversely correlated with leaf thickness in the ten species of Sansevieria. In fact, leaf thickness accounted for over 60% of the variation in the nocturnal accumulation of acidity, with species having thinner leaves showing greater amounts of CAM (Fig. 2). Furthermore, given the strong positive correlation between leaf thickness and leaf hydrenchyma-to-chlorenchyma ratio, the degree of CAM was also highly negatively correlated with the latter ratio, i.e., thicker leaves had relatively more hydrenchyma and lower amounts of CAM (Fig. 4). Likewise, thinner leaves had greater proportional amounts of chlorenchyma, and these species exhibited greater degrees of CAM. On the other hand, species having leaves with greater amounts of water on a fresh mass basis did not have more hydrenchyma, as explained above and, as a result, there appears to be more CAM in leaves with higher
1) leaf thickness, measured with a standard, hand-held cm ruler; 2) leaf hydrenchyma/chlorenchyma cross-sectional area ratio: from the ImageJ images as above; 3) fresh mass/dry mass ratio (FM/DM), measured from 1 cm2 sections; 4) water content, fresh mass basis (g H2O g−1 FM), measured from 1 cm2 sections; 5) water content, dry mass basis (g H2O g−1 DM), measured from 1 cm2 sections; and 6) mesophyll succulence (Kluge and Ting, 1978) (g H2O mg−1 Chl), measured from 1 cm2 sections. 2.3. Titratable acidity After thawing, approximately 1 cm2 of tissue was excised from the center of the leaf, weighed, then placed in a 2-ml plastic reaction tube, whereupon the leaf tissue was obliterated with a glass rod. After two days at −30 °C, then thawing, all liquid (minus cell debris) was pipetted into a new tube, a 250 μl aliquot of the leaf liquid was filled up to 40 ml with demineralized water and then titrated to pH 8.2 using 0.01 N NaOH and a T50 M auto-titrator with a Rondo 20-sample changer (Mettler Toledo, Gießen, Germany). The remaining cell debris was dried at 85 °C and then weighed until no further mass loss. 2.4. Chlorophyll concentration After freezing, then thawing, another 1 cm2 of tissue was excised from the center of the leaf, weighed, then sliced into smaller pieces, collected in a 50 ml centrifuge tube and 10 ml of 100% acetone added. After one day at 10 °C, the leaf pieces were blended in acetone with an Ultra Turrax (Atkinson, NH, USA) t18 blender. An aliquot of the acetone/leaf mixture was then transferred into a 2 ml reaction tube, and centrifuged for two min at 14,500 rpm with an Eppendorf (Hamburg, Germany) MiniSpin Plus micro-centrifuge, after which the absorption of the supernatant was measured with a Specord 210 Plus spectrophotometer (Analytik Jena AG, Jena, Germany) at wavelengths of 3
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Fig. 1. Cross sections of mature leaf section of all Sansevieria species investigated. Hydrenchyma is indicated as light grey area, while the dark grey-hatched area denotes the extension of chlorenchyma. Dots and small circles represent vascular bundles. Bars indicate 5 mm.
fresh mass-based water content, but this relation is not significant (Fig. 6). The degree of CAM was also not significantly correlated with leaf water content when expressed on a dry mass basis (Fig. 5), nor with leaf fresh mass:dry mass ratio (Fig. 3). When leaf water content is expressed on a chlorophyll mass basis, a measure referred to as “mesophyll succulence,” the degree of CAM is inversely, but not significantly, correlated with the latter (Fig. 7). Overall, it is clear that plants with leaves having more water in nonphotosynthetic tissue exhibited lesser degrees of CAM. The degree of CAM appears to be positively correlated with the amount of chlorophyll in the leaves of the ten species of Sansevieria in this study (Fig. 8).
4. Discussion The results of this study serve to emphasize two major conclusions. First, the CAM photosynthetic pathway and succulent leaves are widespread in the genus Sansevieria (Virzo De Santo et al., 1981–1982); and, second, when considering various measures of succulence, the most important predictor of CAM vs. non-CAM is the amount of chlorophyllous tissue (chlorenchyma) in the leaf, especially in plants having a clear separation of hydrenchyma and chlorenchyma tissue in the photosynthetic organs. In such leaves, “succulence” primarily reflects the amount of water in the achlorophyllous, non-CAM-performing tissue in the leaf; thus, any correlation between overall leaf succulence 4
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Fig. 2. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf thickness at the center of the leaf for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 4. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf hydrenchyma:chlorenchyma ratio for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 3. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf fresh mass:dry mass ratio for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 5. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf water content on a dry mass basis for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
and CAM is negative. When such an anatomical distinction is lacking, e.g., when organs are “all-cell succulent”, then water content of the entire tissue should correlate well with the CAM photosynthetic pathway both at the species (Herppich et al., 1998) and at the genus level (Herppich and Herppich, 1996). The genus Sansevieria is well known for its wide variety of leaf forms and shapes, ranging from flat and lanceolate to cylindrical leaves (Newton, 2001; Schwerdtfeger, 2009). All species, however, show the distinct internal parenchyma made from both dead water storage cells and living network cells. This, in particular, makes Sansevieria an excellent tool for comparing the potential interactions between leaf succulence and CAM. In addition, the species chosen cover all sections and subsections proposed by Pfennig (1977); Jankalski (2009) and Mansfeld (2013), which are largely congruent. They also cover 7 out of 16 species groups proposed by Jankalski (2015). Regarding phylogenetic studies,
the selected species cover all 4 main clades in Lu and Morden (2014), both main clades in Baldwin and Webb (2016), 2 out of 4 main clades in Takawira-Nyenya et al. (2018), and 5 out of 7 main clades in van Kleinwee (2018). Nevertheless, the phylogenetic trees proposed in these studies, although of different resolution and quality, are largely incongruent, and none of them represents a consolidated state of phylogenetic relationships within Sansevieria. Hence, no further analysis of this topic is included in the presented considerations. Overall, species of Sansevieria with thick leaves have proportionally large amounts of hydrenchyma, which does not engage in CAM photosynthesis. Such leaves have a high water content on a fresh mass, but not a dry mass, basis. The latter reflects the fact that thick leaves have a relatively large dry mass; thus, they have a low FM:DM ratio. Although the “mesophyll succulence” index proposed by Kluge and Ting (1978) was intended to provide a physiological basis of leaf water 5
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Fig. 6. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf water content on a fresh mass basis for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Fig. 7. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf mesophyll succulence for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
Table 2 Morning (AM), evening (PM), and delta (AM-PM) titratable acidities for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling). Values are means ( ± SE) of 4–5 plants per species, except N = 2 for S. ballyi and S. gracilis. Morning (AM) means are significantly different from PM means (* indicates P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001), except forS. ballyi,S. gracilis, and S. senegambica (ns; P = 0.48, 0.35, and 0.13, respectively). Species
AM acidity, μmol g−1DM
PM acidity, μmol g−1DM
Δ acidity, μmol g−1DM
ballyi cylindrica ehrenbergii fischeri gracilis parva raffillii senegambica suffruticosa volkensii
1520.8 ± 875.4 1630.8 ± 121.6 1310.1 ± 219.8 676.0 ± 71.0 1333.8 ± 236.9 2325.3 ± 130.0 2238.9 ± 348.9 2466.2 ± 482.9 1490.0 ± 180.0 1074.8 ± 135.9
875.4 ± 73.6 1017.9 ± 52.0 903.6 ± 130.0 494.3 ± 64.6 919.0 ± 16.2 1047.5 ± 130.5 1278.8 ± 351.7 1162.4 ± 174.8 921.1 ± 131.9 480.3 ± 91.5
645.4 ± 12.8 ns 612.9 ± 99.7** 406.5 ± 107.1* 181.7 ± 20.3*** 414.8 ± 253.1 ns 1277.8 ± 101.3*** 960.1 ± 91.5*** 1303.8 ± 358.2 ns 568.8 ± 131.4** 594.5 ± 76.6***
Fig. 8. The relationship between the degree of CAM (defined as leaf acidity in the morning - leaf acidity in the evening AM-PM, with acidity expressed on a dry mass (DM) basis) and leaf chlorophyll concentration (DM basis) for ten species of Sansevieria in the greenhouses of the University of Potsdam (see Materials and Methods for environmental conditions during leaf sampling; see Table 1 for species names). The correlation coefficient (R) and significance (P) of the correlation are given in the figure. Data points are means of 4–5 plants per species of Sansevieria, except N = 2 for S. ballyi and S. gracilis.
content that might predict the photosynthetic pathway of a plant, this index was a poor indicator of the degree of CAM in the current study of the genus Sansevieria. The reason for this, as indicated above, is related to the amount of hydrenchyma in the leaf. Kluge and Ting (1978) were well aware of this limitation, as they suggested that the water content value in the mesophyll succulence index should be limited to the water content of the photosynthetic chlorenchyma tissue and not to the whole leaf, if possible. Thus, the findings of the current study strongly support this suggested limitation of this succulence measure. Strictly speaking, the unique type of water-storage tissue could not be important for CAM because dead water-storage cells should not be involved in the storage of malic acid. In fact, Alfani et al. (1989) assumed that it exclusively functions as a fast, “metabolically low-cost” water storage system that could lose considerable amounts of water without damages of living cells during dehydration. On the other hand, Koller and Rost (1988a) reported that the relative area occupied by the water storage tissue was larger in more xeromorphic physiognomy, i.e. thicker cuticle and higher fiber content. The latter species also contain a larger number of dead water storage compared to living network cells. All the species in this study are naturally found in arid regions
throughout eastern Africa (some species of Sansevieria are native to less arid regions of Southeast Asia). Both CAM and leaf succulence should prove beneficial by conserving and reducing water loss in such arid regions (von Willert et al., 1992). Furthermore, neither CAM nor succulence was limited to certain phylogenetic clades as also suggested by van Kleinwee (2018). Thus, it appears that both CAM and leaf succulence evolved multiple times in the genus Sansevieria; alternatively, it might have evolved at the beginning of the evolution of the genus. The latter seems to be plausible for succulence due to the fact that all species of Sansevieria show the strict separation in internal genus-specific water storage tissue and surrounding chlorenchyma. Nevertheless, similar findings of multiple origins of CAM have been reported in other taxonomic groups, including the Bromeliaceae (Crayn et al., 2004), Aeonium in the Crassulaceae (Mort et al., 2007), and in the family Orchidaceae 6
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(Silvera et al., 2010). Perhaps more important, similar findings about multiple origins of both succulence and CAM have been found in the Agavoideae (Heyduk et al., 2016), a subfamily of the Asparagaceae, in which Sanseveria is also found.
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5. Conclusions These results complement current understanding of the relationship between the requirement of large vacuoles in photosynthetic cells, hence large cells, for the nocturnal storage of malic acid during CO2 assimilation in Crassulacean acid metabolism. As a result, the amount of water stored in achlorophyllous tissue is not directly related to the biochemistry and physiology of CAM. It should be emphasized that water storage in achlorophyllous tissue also constitutes an excellent adaptation to arid environments, as does CAM, but the results of the current study emphasize that the positive correlation between tissue succulence and CAM is applicable primarily to the photosynthetic tissue (chlorenchyma). Declarations of Competing Interest None Acknowledgments We heartily thank Ms. G. Wegner and Ms. C. Rolleczek at the ATB for assistance with the acidity measurements. In addition, we thank Jil Roßberg for the perfect drawings of the cross sections and Christiane Benthin for taking care of the plants as well as Doris Pfennig, Michael Schwerdtfeger and Uwe Scharf for providing plants. C.E.M. gratefully acknowledges DAAD and AvH for funding during his stays at the ATB. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.flora.2019.151489. References African Plant Database, 2019. Conservatoire Et Jardin Botaniques De La Ville De Genève. http://www.ville-ge.ch/musinfo/bd/cjb/africa/recherche.php. Alfani, A., Ligrone, R., Fioretto, A., Virzo De Santo, A., 1989. Histochemistry, ultrastructure and possible significance of dead parenchyma cells with specialized walls in the leaf and rhizome of Sansevieria. Plant Cell Environ. 12, 249–259. Baldwin, A.S., Webb, R.H., 2016. The genus Sansevieria: An introduction to molecular (DNA) analysis and preliminary insights to intrageneric relationships. Sansevieria 34, 14–26. Brown, N.E., 1915. Sansevieria. A monograph of all known species. Bull. Misc. Inform. Kew 1915, 1–81. Budweg, H.G., Mansfeld, P.A., 2015. Sansevieria Online. Sonderheft 2015. (Accessed 12 April 2019). http://www.sansevieria-online.de/pdf/so_sonderheft_2015.pdf. Chiang, J.-H., Lin, T.-C., Luo, Y.-C., Chang, C.-T., Cheng, J.-Y., Martin, C.E., 2013. Relationships among rainfall, leaf hydrenchyma, and Crassulacean acid metabolism in Pyrrosia lanceolata(L.) Fraw. (Polypodiaceae) in central Taiwan. Flora 208, 343–350. Crayn, D.M., Winter, K., Smith, J.A.C., 2004. Multiple origins of Crassulacean acid metabolism and the epiphytic habitat in the Neotropical family Bromeliaceae. Proc. Nat. Acad. Sci. USA 101, 3703–3708. Edwards, G., Walker, D.A., 1983. C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis. Blackwell Science Publ., Oxford. Ehleringer, J., 1985. Annuals and perennials of warm deserts. In: Chabot, B.F., Mooney, H.A. (Eds.), Physiological Ecology of North American Plant Communities. Chapman & Hall, N.Y, pp. 162–180. Gibson, A.C., 1982. The anatomy of succulence. In: Ting, I.P., Gibbs, M. (Eds.), Crassulacean Acid Metabolism. Amer. Soc. Plant Physiol., Rockville, MD. pp. 1–17. Herppich, W.B., Herppich, M., 1996. Ecophysiological investigations on plants of the genus Plectranthus(Fam. Lamiaceae) native to Yemen and southern Africa. Flora 191, 401–408. Herppich, W.B., Herppich, M., von Willert, D.J., 1998. Ecophysiological investigations on plants of the genus Plectranthus(Lamiaceae). Influence of environment and leaf age on CAM, gas exchange and leaf water relations in Plectranthus marrubioides Benth. Flora 193, 99–109. Heyduk, K., McKain, M.R., Lalani, F., Leebens-Mack, J., 2016. Evolution of a CAM anatomy predates the origins of Crassulacean metabolism in Agavoideae
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von Willert, D.J., Eller, B.M., Werger, M.J.A., Brinckmann, E., Ihlenfeldt, H.-D., 1992. Life Strategies of Succulents in Deserts: With Special Reference to the Namib Desert. Cambridge Univ. Press, Cambridge. Webb, R.H., Newton, L.E., 2017. Biogeography of the genus Sansevieria: Should plants without a known type locality be considered valid species? Sansevieria 36, 15–21. Winter, K., 1985. Crassulacean acid metabolism. In: Barber, J., Baker, N.R. (Eds.), Photosynthetic Mechanisms and the Environment. Elsevier Science Publ. BV, Amsterdam, pp. 329–387. Winter, K., Wallace, B.J., Stocker, G.C., Roksandic, Z., 1983. Crassulacean acid metabolism in Australian vascular epiphytes and some related species. Oecologia 57, 129–141.
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