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Plant, Cell and Environment (2014)

doi: 10.1111/pce.12331

Original Article

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants Matt Stata1, Tammy L. Sage1, Troy D. Rennie1, Roxana Khoshravesh1, Stefanie Sultmanis1, Yannay Khaikin1, Martha Ludwig2 & Rowan F. Sage1 1

Department of Ecology and Evolutionary Biology, The University of Toronto, Toronto, ON, Canada M5S 3B2 and 2School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6009, Australia

ABSTRACT The evolution of C4 photosynthesis from C3 ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol.These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C3, C3–C4 intermediate and C4 species from 12 lineages of C4 evolution. All C3 species had more chloroplasts per M cell area than their C4 relatives in high-light growth conditions. C3 species also had higher chloroplast coverage of the M cell periphery than C4 species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C4 lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C4 species than their C3 relatives. C3–C4 intermediate species generally exhibited similar patterns as their C3 relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C4 function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C4 M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C4 photosynthesis. Key-words: C3–C4 intermediate; C4 evolution; C4 photosynthesis; Kranz anatomy; mesophyll conductance.

INTRODUCTION C4 photosynthesis is a CO2 concentrating mechanism where phosphoenolpyruvate carboxylase (PEPC) first assimilates atmospheric CO2 in the cytosol of the leaf mesophyll (M) cells. In addition to a series of biochemical modifications to create the C4 metabolic cycle, the leaf structure of C4 plants has been modified from that of their C3 ancestors to create a radial arrangement of M and enlarged bundle sheath (BS) cells commonly termed Kranz anatomy (Dengler & Nelson 1999). Mesophyll cell numbers are generally reduced in C4 leaves relative to those of closely related C3 species, such that each photosynthetically active M cell either touches a BS cell Correspondence: R. F. Sage. Fax: +1 416 978 5878; e-mail: r.sage@ utoronto.ca © 2014 John Wiley & Sons Ltd

or is no more than one cell away from a BS cell (Dengler & Nelson 1999; Edwards & Voznesenskaya 2011). Chloroplast numbers and size are increased in the BS cells, giving the C4 leaf a distinctly different appearance than a typical C3 leaf (Black & Mollenhauer 1971; Brown & Hattersley 1989; Voznesenskaya et al. 2007; Muhaidat et al. 2011; Sage et al. 2011a). Whereas C3 leaves can have colourless veins that are often visible to the naked eye, C4 leaves have dark green veins, and instead of the C3 pattern of dark green M cells between clear veins, the C4 leaf has light green M tissue (Dengler & Nelson 1999; Yamada et al. 2009; Maai et al. 2011). The reason for the light green M tissue of C4 leaves has never been systematically examined, although it has been noted that C4 M cells often have fewer chloroplasts than C4 BS cells (Black & Mollenhauer 1971; Liu & Dengler 1994; Dengler & Nelson 1999; Maai et al. 2011). Although chloroplast numbers in C4 BS cells are increased over those of their closest C3 relatives (Brown & Hattersley 1989), it is uncertain whether chloroplast number in M cells is similar in C3 and C4 relatives or is reduced in the C4 species, reflecting a decline in M chloroplast investment during the C4 evolutionary process. Numerous functional changes could affect the number of M chloroplasts in C4 relative to C3 plants. Firstly, the chloroplast of C4 M cells is no longer engaged in carboxylation, so this function should not be a driver for M chloroplast investment. Secondly, in C3 species, the chloroplast stroma is the site of carboxylation, and close positioning of many chloroplasts against the cell wall enhances mesophyll conductance for CO2 diffusion (Syvertsen et al. 1995; Evans & Loreto 2000; Tholen et al. 2008; Evans et al. 2009). In C4 species, by contrast, the cytosol is the initial site of carbon fixation and large numbers of chloroplasts could interfere with diffusion into this compartment. These considerations, coupled with the lighter green appearance of the C4 M tissue, lead us to hypothesize that the chloroplast investment in C4 M cells is reduced relative to their C3 relatives. Differences in M chloroplast number and position would have important implications for understanding the function of M cells in both the C3 and C4 context and the evolutionary changes needed to form a C4 leaf. Moreover, changes in M chloroplast number would need to be considered in ongoing programs to engineer the C4 pathway into C3 crops to ensure efficient C4 function in 1

2 M. Stata et al. the M tissue (von Caemmerer et al. 2012; http://c4rice.irri .org). C4 photosynthesis is one of the most convergent evolutionary events in the biosphere, with over 68 independent origins (Sage et al. 2011a, 2012; Christin et al. 2013). This high number of distinct origins provides an opportunity to examine the adaptive significance of the many traits in C4 plants, using a comparative approach where multiple lineages serve as independent evolutionary replicates (Ackerly 1999; Christin et al. 2013). If a trait is an absolute requirement for C4 function, it should be present in all C4 lineages. For example, the initial PEP carboxylation step is common to all C4 plants and considered essential for C4 photosynthesis (Kellogg 1999; Leegood 2002). Alternatively, a trait may be specific to certain anatomical or biochemical subtypes, and thus essential for a subset of C4 groups. As a third possibility, a trait could be characteristic of a given phylogenetic lineage rather than a feature unique to the C4 pathway. For example, greater drought tolerance of certain C4 grasses is proposed to be a common phylogenetic feature inherited from C3 ancestors, rather than a C4-specific characteristic (Edwards & Smith 2010). To evaluate these possibilities, it is necessary to examine many distinct lineages of C4 evolution, treating each one as an independent replicate. Over the past decade, we have assembled a large, living collection of closely related C3 and C4 species from a dozen independent evolutionary lineages of the C4 pathway. The acquisition of these species was guided by molecular phylogenies to identify close C3 relatives of the C4 clades, and in some cases, to identify species that branch in intermediate positions between related C3 and C4 species. Here, we utilize this collection to examine changes in M cell properties from C3 to related C4 sister species in 11 clades that contain 12 independent lineages of the C4 pathway and six independent lineages of C3–C4 intermediate photosynthesis (Supporting Information Table S1). C3–C4 intermediate species have recently been termed ‘C2’ species, in recognition that they gain fitness via C2 photosynthesis, which is a carbonconcentrating mechanism that enhances CO2 around BS Rubisco using a photorespiratory glycine shuttle (Muhaidat et al. 2011; Sage et al. 2012). C2 refers to the two carbons in the glycine molecules that transport CO2 into the BS, following the logic of C4 photosynthesis where a four-carbon molecule moves CO2 into the BS. ‘C2 plant’ is preferred to ‘C3–C4 intermediate plant’ because the name C2 photosynthesis refers to a photosynthetic trait without presuming evolutionary intermediacy. Many C2 species are not C3–C4 intermediates as they lack C4 relatives, and it is important to recognize the C2 condition as an adaptive trait in its own right. One of our C2 species, Mollugo verticillata, occurs on a distinct phylogenetic branch from that containing the C4 Mollugo species (Christin et al. 2011), indicating it is not a true C3–C4 intermediate. In the present study, we treat each of the 12 distinct C4 lineages and six C2 lineages as evolutionary replicates, allowing us for the first time to generate a comprehensive picture of the change in chloroplast number, location and size of M cells during the C4 evolutionary process.

MATERIALS AND METHODS Plant material and growth conditions We measured chloroplast number, size and location in the M cells of 35 species from 11 phylogenetic clades (Supporting Information Table S1). The clades are from nine distinct angiosperm families, representing five orders [Caryophyllales (six clades), Poales (two clades) and Boraginales, Brassicales, Malpighiales (one clade each)]. In the Nyctaginaceae, two distinct lineages of C4 origin are represented (Allionia and Boerhavia; Douglas & Manos 2007; Sage et al. 2011a), giving 12 independent C4 lineages in the 11 clades studied. Six lineages of C2 origin were also represented in five clades. One clade (Molluginaceae) contained two distinct C2 lineages – one that was sister to the C4 clade (M. nudicaulis) and one (M. verticillata) that was on a separate branch of the Mollugo phylogeny than the C4 clade (Christin et al. 2011). Ten of the C4 lineages include species characterized as being predominantly NADP-malic enzyme (NADP-ME) subtypes, whereas two are NAD-malic enzyme subtypes (NAD-ME; Supporting Information Table S1). No PEP carboxykinase subtypes are included in the study because we could not obtain a close C3 relative. We also sampled three additional C4 grass species and one sedge species for images only (Zea mays, Saccharum officinarum, Echinochloa glabrescens and the sedge Eleocharis baldwinii). These images are included to broaden the monocot sample but were not quantified as we lacked closely related C3 and C4 sister species in these groups. All species except those in the Neurachninae were grown in a greenhouse at the University of Toronto between April and August at approximately 28/23 °C (day/night) temperature, and sampled from June to early August, when day length and mean light intensity were the greatest.The Neurachninae species were grown in a naturally illuminated glasshouse with mean temperatures of 27/14 °C at the University of Western Australia, Perth, Western Australia, and sampled in September 2011. In both locations, plants frequently experienced direct sunshine with light intensity exceeding 1600 μmol m−2 s−1 for at least 4 h per day. Plants at the University of Toronto were grown in 4–20 L pots of a sandy loam potting soil, and were watered and fertilized regularly to avoid drought and nutrient deficiency. The Neurachninae species were grown in 4 L pots of native soil from near the collection sites and were regularly watered to prevent drought yet avoid waterlogging. Sampling of all plants occurred between 0900 h and noon after leaves had been exposed to direct sun (photon flux density >1200 μmol photons m−2 s−1) for at least 30 min.

Imaging procedures Numerous methods are used to estimate organelle numbers, size and distribution in leaves. These methods include the use of (1) light microscopy and transmission electron microscopy (TEM) to analyse planar sections, which are thin sections in two dimensions (Oguchi et al. 2005; McKown & Dengler 2007; Muhaidat et al. 2011; Grace et al. 2013); (2) light microscopy to examine single cells (Osteryoung et al. 1998); and (3) © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

C3 versus C4 mesophyll chloroplasts confocal microscopy in combination with two- or threedimension morphometric analysis to examine whole cells (Park et al. 2009; Kubinova et al. 2014; Rosnow et al. 2014). For the questions addressed here, where relative differences in chloroplast number, size and position are being assessed, we used TEM and light microscopy of single-cell isolates to assess whether C4 plants exhibit altered chloroplast properties. TEM provides the high-resolution imaging capability necessary for measuring certain chloroplast parameters that cannot be accurately obtained with confocal or light microscopy while imaging of single-cell isolates allows measurements of chloroplast numbers per cell as would the more time consuming and expensive confocal microscopy. For all imaging, leaf tissue was cut from the most recent, fully expanded, mature leaves at a point halfway between the base and tip of a leaf, and midway between the midrib and leaf margin as described previously (Sage & Sage 2009). Chloroplast movement within mesophyll cells can occur in response to rapid changes in light intensity (Maai et al. 2011; Terashima et al. 2011). Thus, all leaves were removed from plants while in the greenhouse, immediately immersed and sectioned into 2 mm2 pieces in a 2% glutaraldehyde fixative. Leaf sections were then transferred in a fixative-filled pipette into fixative-filled vials. Vials were completely filled with fixative to prevent air bubbles from forming when capped. This procedure ensures rapid penetration of fixative into tissues as indicated by leaf samples sinking within minutes (O’Brien & McCully 1981). After fixation, samples for anatomical and ultrastructural analysis were prepared for TEM (Sage & Williams 1995) and single-celled isolation (Osteryoung et al. 1998). TEM images from transverse leaf sections (cross sections) were used to quantify chloroplast size and number in planar sections, chloroplast coverage of the cell periphery (including the relative coverage of the periphery opposite the intercellular air spaces, IAS), the fraction of the outer chloroplast edge that was appressed against the cell periphery, and thickness of M cell walls as described in Supporting Information Fig. S1. Quantification of cells in transverse sections was conducted for all species in the study except maize, sorghum, Eleocharis, Echinochloa, Heliotropium calcicola and Boerhavia coccinea. To minimize the potential for artefacts associated with plane of section, we also used TEM to examine paradermal leaf sections from eight species representing four lineages. All parameters quantified in the transverse sections were also measured in cells of the paradermal sections, except for M cell wall thickness and the fraction of the outer chloroplast envelope in contact with the M cell periphery. Fourteen C3 and C4 species from seven lineages were also sampled for single-cell isolations. Single-cell isolates enabled us to use a light microscope to focus through the cell to count chloroplast numbers per cell, and quantify the projected chloroplast area as an index of chloroplast size. In the single-cell isolates, both adaxial and abaxial (spongy) mesophyll cells were measured. Quantifications were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and a Wacom Cintiq graphics tablet (Wacom Technology Corporation, Vancouver, WA, USA). © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

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Experimental design and statistical analysis With the exception of Euphorbia and the Neurachninae plants, each species for the TEM measurements were sampled in triplicate, where one leaf sample was obtained from each of three separate plants. For the single-cell isolate measurements, seven species were sampled in triplicate, except for the M. cerviana cells that were sampled from two plants, and the Cleome gynandra sections that were obtained from three leaves on one plant; untimely mortality reduced the sample size of these two species. Due to poor germination, we substituted H. calcicola in place of its sister species H. tenellum, and B. coccinea in place of B. burbidgeana, for the single-cell isolate work. For the transverse sections imaged by TEM, 9–10 adaxial M cells were randomly sampled from one plant for quantification. Fifteen adaxial M cells per plant were randomly sampled per paradermal section. For single-cell isolation, five adaxial M cells and five abaxial M cells per plant were sampled. For each plant, parameter values determined from each cell were averaged to provide distinct mean values for transverse sections, paradermal sections, adaxial single cells and abaxial single cells. Two statistical approaches were employed for the data from transverse sections. For the first, we averaged all plant means of a common photosynthetic type from a given lineage, to give one mean value per photosynthetic pathway per lineage. In most cases, there was only one species mean per pathway type within a given lineage, derived from the three replicate individuals. In the case of each Euphorbia species, the mean was derived from one plant. In C3 Alternanthera and C3 Neurachninae, there were two and three species, respectively, that contributed to the mean of the photosynthetic type for that lineage. In the Aizoaceae, two C4 species (Cypselea humifusa and Trianthema portulacastrum) contributed to the C4 mean. By pooling the samples to provide one mean per pathway per lineage, we were able to eliminate effects of variable samples sizes between the lineages. We then tested differences between photosynthetic pathway types using a one-way analysis of variance (anova) with a Student–Newman–Keuls post hoc test (when equal variance and normality tests passed) or a Dunn’s post hoc test on ranks when either equal variance or normality assumptions failed (using SigmaPlot; Systat Software, San Jose, CA, USA). The second approach treated the C3 and C4 measures within a lineage as a before and after measurement (before and after C4 evolution) and evaluated differences with a paired t-test using SigmaPlot, where each lineage was a replicate. For paradermal sections that had equal sample size per species, data were analysed using a one-way anova with plant mean as the unit of replication; for single-cell isolates, C3 and C4 differences were tested with a paired t-test using species as the unit of replication.

RESULTS In each of the three perspectives examined, the C3 species had greater numbers of chloroplasts per planar area of M cells than the corresponding C4 species of the same clade

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Figure 1. Transmission electron micrographs of transverse sections from adaxial mesophyll cells of C3, C2 and C4 species of the Boraginaceae genus Heliotropium (panels a–c) and Poaceae genus Neurachne (panels d–f). C, chloroplast; HeCo, H. convolvulaceum; HeTn, H. tenellum; HeTx, H. texanum; IAS, intercellular air space; NeAl, N. alopecuroides; NeMi, N. minor; NeMu, N. munroi; V, vacuole. Scale bars = 10 μm. See Supporting Information Figs S2–S4 for images from the additional C3, C4 and C2 species examined in this study.

(transverse sections – Figs 1 & 2a, and Supporting Information Figs S2–S4; paradermal sections – Fig. 3, and Supporting Information Fig. S5a; single-cell isolates – Fig. 4 and Supporting Information Fig. S6a). The relative differences between chloroplast properties of C3 and C4 M cells were similar in the three perspectives. In cells imaged from transverse and paradermal sections, the mean chloroplast number per M cell area in the C4 species was 38–47% of the C3 value, respectively (Tables 1 & 2). In the single-cell isolates, the number of chloroplasts per C4 M cell was about 42% of the C3 M cells in both the adaxial and abaxial regions of the leaf (Table 2). Variation in chloroplast number per planar area was apparent between clades, such that C4 species in some clades had more chloroplasts per M cell area than C3 species in other clades (Fig. 2a). The C4 Euphorbia, for example, had twice as many chloroplasts per M cell area than C3 species in the Amaranthaceae and Nyctaginaceae; however, the C4 Euphorbia species had 60% of the chloroplast numbers per M cell area as the C3 Euphorbia plants (Fig. 2a). Differences in M chloroplast numbers between the C3 and C4 lineages were apparent in both the grass and eudicot lineages, and the NADP-ME subtypes and NAD-ME subtypes (Fig. 2a,

Supporting Information Figs S5a & S6a). In Atriplex, an NAD-ME line, the differences in chloroplast numbers between C3 and C4 M cells were not large in transverse section, but were in the single-cell isolates (Fig. 2a, Supporting Information Fig. S6a). A C4 sedge and the C4 grasses maize, sugar cane and Echinochloa also have relatively few chloroplasts in the M cells (Supporting Information Fig. S7). Mean chloroplast numbers per planar cell area were statistically similar between the C2 and C3 species, although in two clades (Heliotropium and Neurachninae), the C2 species had fewer chloroplasts per M cell area than their respective C3 relative within the clade (Table 1; Fig. 1, and Supporting Information Fig. S2). The greater numbers of chloroplasts per planar cell area in the C3 and C2 species allowed them to maintain high coverage of the M cell perimeter (Tables 1 & 2; Fig. 2c, Supporting Information Fig. S5b; see also images in Figs 1, 3 & 4). Chloroplasts in the C3 species covered 60–90% of the entire cell perimeter in transverse and paradermal sections, for an average cover of 73% in the transverse sections and 89% in the paradermal sections (Fig. 2c, Supporting Information Fig. S5b; Tables 1 & 2). By contrast, chloroplasts of the C4 species covered 36–41% of the entire cell perimeter on © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

C3 versus C4 mesophyll chloroplasts

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Figure 2. Quantified properties of chloroplasts in adaxial mesophyll cells of C3, C2 and C4 species in 12 lineages where C4 photosynthesis has evolved. Note: two lineages are present in the Nyctaginaceae and the chloroplast coverage values do not incorporate variation in chloroplast contact with the cell periphery. All parameters were measured from cells in tranverse sections. (a) Chloroplast number per planar cell area; (b) the % of the outer chloroplast length that contacts the cell perimeter; (c) the % of the entire cell perimeter that is covered by chloroplasts; (d) the % of cell perimeter facing the intercellular air spaces (IAS) that is covered by chloroplasts. See Supporting Information Fig. S1 for description of the measurements and Supporting Information Table S1 for species information and samples size. Mean ± SE for each species, where C3 species are represented by white columns, C2 species by grey columns and C4 species by black columns. Clades and species are as follows: from left to right: Aerva: Ae. lanata (C3), Ae. persica (C4); Alternanthera: Alt. brasiliensis (C3), Alt. sessilis (C3), Alt. tenella (C2), Alt. caracasana (C4); Euphorbia: Eu. angusta (C3), Eu. acuta (C2), Eu. mesembryanthoides (C4); Heliotropium: H. tenellum (C3), H. convolvulaceum (C2), H. texanum (C4); Mollugo: M. pentaphylla (C3), M. nudicaulis (C2), M. verticillata (C2), M. cerviana (C4); Neurachninae: Thyridolepis mitchelliana (C3), Neurachne alopecuroidea (C3), N. lanigera (C3), N. minor (C2), N. munroi (C4); Dica./Set: Dichanthelium oligosanthes (C3), Setaria viridis (C4); Nyctaginaceae: Mirabilis jalapa (C3), Allionia incarnata (C4), Boerhavia burbidgeana (C4); Aizoaceae: Sesuvium verrucosum (C3), Cypselea humifusa (C4); Trianthema portulacastrum (C4); Cleome: Cl. viscosa (C3), Cl. gynandra (C4); Atriplex: At. prostrata (C3), At. rosea (C4). All species are NADP-malic enzyme species except for the Cleome and Atriplex species, which are NAD-malic enzyme types.

average (Fig. 2c, Supporting Information Fig. S5b; Tables 1 & 2). In the C3 and C2 plants, most chloroplasts occurred along the portion of the cell perimeter immediately adjacent to the IAS, such that the coverage of the IAS perimeter averaged © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

89% in the C3 species and 85% in the C2 species in transverse section (Table 1; Fig. 2d). The C3 chloroplast coverage of the IAS perimeter averaged 94% in the paradermal views (Table 2). In the C4 species, only 40–43% of the IAS

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Figure 3. Transmission electron micrographs of paradermal sections from adaxial mesophyll cells of C3 (a, c, e) and C4 species (b, d, f) of the Alternanthera (a, b), Heliotropium (c, d) and Dichanthelium/Setaria lineages (e, f). AlCa, Alt. caracasana; AlSe, Alt. sessilis; C, chloroplast; DiOl, D. oligosanthes; HeTn, H. tenellum; HeTx, H. texanum; IAS, intercellular air space; SeVi, Se. viridis; V, vacuole. Bars = 5 μm. See Supporting Information Fig. S5 for quantified data from the paradermal sections for chloroplast number, position and size.

Parameter

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C2 species

Chloroplast number per planar cell area, μm−2 × 1000 Planar area per chloroplast, μm2

17.4 ± 2.7a

18.8 ± 8.2ab

8.1 ± 1.6b (47%*)

13.2 ± 1.2a

11.9 ± 2.1a

21.4 ± 3.1a

17.2 ± 4.3a

17.9 ± 2.1a (136%, P = 0.06) 12.2 ± 1.4b (57%*)

72.8 ± 2.9a

67.7 ± 6.9a

36.3 ± 2.1b (50%*)

89.1 ± 1.8a

84.8 ± 6.1a

40.4 ± 2.4b (45%*)

74.1 ± 4.2a

74.0 ± 4.3a

41.2 ± 7.3b (56%*)

160 ± 30a

140 ± 30a

Chloroplast planar area per planar cell area, % Chloroplast coverage of the entire cell perimeter, % Chloroplast coverage of the cell perimeter adjacent to IAS, % % of the outer chloroplast length contacting the cell periphery Cell wall thickness, nm

C4 species (% of C3)

Table 1. Properties of the mesophyll cell chloroplasts and walls in C3, C2 and C4 plants from six lineages of C2 evolution, and 12 lineages of C4 evolution (see Supporting Information Table S1 for species)

160 ± 30a

Values were obtained from transmission electron microscopy images of transverse sections of leaves. Area measurements are from planar views through leaf sections. Means ± SE, where data from all species within a lineage were pooled to give one observation per photosynthetic type per lineage, for a total of 11 C3 observations, 6 C2 observations and 12 C4 observations. Mean differences were tested using one-way analysis of variance with a Student–Newman–Keuls test, or if normality failed, a Dunn’s post hoc test on ranks. Statistical groups are shown by letters at P < 0.05. The ‘*’ indicates C4 means within a lineage differed from C3 means at P < 0.05 using a paired t-test, where values within a lineage were considered before and after C4 evolution measurements. IAS, intercellular air space. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

C3 versus C4 mesophyll chloroplasts

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Figure 4. Light micrographs of isolated mesophyll cells from C3 (a, c, e) and C4 (b, d, f) species of Atriplex (a, b), Heliotropium (c, d) and Dichanthelium/Setaria (e, f). AtPr, At. prostrata; AtRo, At. rosea; C, chloroplast; DiOl, D. oligosanthes; HeCa, H. calcicola; HeTx, H. texanum; SeVi, Se. viridis. Bars = 10 μm. See Supporting Information Fig. S6 for quantified data from the single-cell isolates for chloroplast numbers and size.

perimeter was covered by chloroplasts in the transverse and paradermal views (Tables 1 & 2). Chloroplasts are largely appressed against the M cell periphery in C3 species (Evans & Loreto 2000; Sage & Sage 2009; Busch et al. 2013), but whether they remain so in C2 and C4 M cells is uncertain. We therefore examined the degree to which chloroplasts are appressed against the M cell periphery in a planar section. A chloroplast edge was considered appressed if a ribbon of cytosol was either not apparent, or was less than 0.1 μm across. If the cytosol between the plasmalemma and chloroplast membrane was clearly greater than the 0.1 μm across, that part of the chloroplast was considered to be recessed from the cell periphery (Fig. 5). In the C3 species examined here, we observed that 50–90% of the outer chloroplast membrane was appressed against the M cell periphery (74% on average across all C3 lineages; Table 1, Fig. 2b). The pattern in the C2 species was the same as the C3 plants. By contrast, in all but two C4 lineages (Neurachne munroi and the Nyctaginaceae Allionia incarnata), we observed a reduced proportion (20– 40%) of the outer chloroplast membrane pressed against the M cell periphery (Fig. 2b). Chloroplasts were still © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

arrayed near the cell periphery, but unlike the C3 pattern, a distinct lane of cytosol was observed between the plasmalemma and much of the outer chloroplast membrane in the C4 species (Fig. 5). In numerous lineages, the C4 species had larger chloroplasts on average in planar sections than the corresponding C3 species (Fig. 6, Supporting Information Figs S5c & S6b), which contributed to a marginally significant enhancement of mean chloroplast area in the C4 relative to the C3 plants (Tables 1 & 2). The increased chloroplast size in certain C4 taxa partially compensated for their reduced chloroplast numbers, such that the M cell area covered by C4 chloroplasts was 57% of the mean value observed for the C3 species when measured in transverse section (Table 1). In the four clades where the C4 species had markedly larger chloroplasts (Mollugo, Nyctaginaceae-Boerhavia, Aizoaceae, Atriplex; Fig. 6a), the total chloroplast area per cell area was similar to their C3 counterparts (Fig. 6b). The thickness of M cell walls facing the IAS has been suggested to differ between C3 and C4 species as a result of evolutionary selection for differences in mesophyll conductance (Evans & Loreto 2000). We observed no difference in

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Table 2. Properties of the mesophyll Values from paradermal sections chloroplasts of C3 and C4 species determined (% of C3) from paradermal sections of adaxial mesophyll Parameter C3 species C4 species 20.9 ± 1.3a 8.0 ± 0.7b (38%) Chloroplast number per planar cell area, cells, or from single-cell isolates of adaxial μm−2 × 1000 mesophyll and abaxial mesophyll cells Planar area per chloroplast, μm2 15.5 ± 1.4a 22.4 ± 2.1b (145%) Chloroplast planar area per planar cell area, % 30.8 ± 2.7a 16.5 ± 1.3b (54%) Chloroplast coverage of the entire cell perimeter, % 88.8 ± 1.5a 40.5 ± 3.1b (46%) Chloroplast coverage of the cell perimeter 93.8 ± 1.7a 43.3 ± 3.3b (46%) adjacent to IAS, % Values from single-cell isolates Adaxial cells Chloroplast number per cell 46.8 ± 2.5a 19.7 ± 2.6b (42%) 28.6 ± 1.7a 49.5 ± 8.0b (173%) Projected area per chloroplast, μm2 Abaxial cells Chloroplast number per cell 48.4 ± 2.4a 21.4 ± 3.7b (43%) 25.6 ± 1.2a 45.8 ± 10.9a (184%, Projected area per chloroplast, μm2 P = 0.08) Mean ± SE. Values from paradermal sections are from four lineages with one C3 and C4 species per lineage. Values from single-cell isolates are from seven lineages with one C3 and C4 species per lineage. Means differences for paradermal data were tested using a one-way analysis of variance at P < 0.05, with a plant as the unit of replication (n = 12); for single-cell isolates, differences were tested using a paired t-test with species as the replicate (n = 7 for adaxial cells and 5 for spongy cells). Statistical groups are shown by letters at P < 0.05. IAS, intercellular air space.

M cell wall thickness facing the IAS between the C3 and C4 species in the large majority of lineages (Fig. 7), and no difference in average M wall thickness calculated from C3, C4 and C2 species values (Table 1).

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Figure 5. Transmission electron micrographs showing the placement of the mesophyll chloroplasts in relation to the cell periphery in Mollugo (a, b) and Heliotropium (c, d) species. C, chloroplast; CYT, cytoplasm; HeTn, H. tenellum; HeTx, H. texanum; IAS, intercellular air space; MI, mitochondrion; MoCe, Mollugo cerviana; MoPe, Mollugo pentaphylla; V, vacuole. Bars = 5 μm.

DISCUSSION In all C4 lineages examined, there was a decline in chloroplast numbers in the M cells of C4 compared with C3 species and a substantial reduction in the degree to which the chloroplasts cover the M cell periphery, particularly in the cell region facing the intercellular air spaces. In most C4 species, the chloroplasts of the M cells were partially recessed away from the cell periphery, creating noticeable lanes of cytoplasm between the chloroplast envelope and plasmalemma. These patterns were observed across a broad range of angiosperm orders, including monocots and eudicots, and the two principal subtypes of C4 photosynthesis – NADP-malic enzyme and NAD-malic enzyme. Our results are specific to the high-light conditions the plants were grown under and it is unknown whether the C3 to C4 differences also occur in shadegrown plants. In a survey of the literature, we generally observed that C4 species have relatively few chloroplasts in the M cells, whereas (typically unrelated) C3 species have many (e.g. Black & Mollenhauer 1971; Frederick & Newcomb 1971; Laetch 1971; Ueno et al. 1988; Evans & Loreto 2000; Wakayama et al. 2006). Images in the literature from PEP carboxykinase (PCK) type species indicate that M cells of this C4 subtype also have relatively few chloroplasts and peripheral coverage (Frederick & Newcomb 1971; Morgan & Brown 1979; Voznesenskaya et al. 2007). For example, M cells of the PCK species Chloris gayana averaged 1.6 chloroplasts per planar cell section, less than 20% of the values determined for wheat and oat (Frederick & Newcomb 1971). Single-celled C4 plants also exhibit reduced chloroplast investment. In both known lineages of single-celled C4 species (Bienertia cycloptera and Suaeda aralocaspica), chloroplast density and coverage of the M cell periphery is reduced where PEPCase is © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

(a)

C3 species C2 species C4 species

40

48

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40

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NAD-ME

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Aizo. Cleome Atriplex

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0

Neurach.

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Euphorbia Heliotrop. Mollugo

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Aerva Altern.

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Euphorbia Heliotrop. Mollugo

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Aerva Altern.

Area per chloroplast, µm–2

48

9

Chloroplast area per mesophyll cell area, %

C3 versus C4 mesophyll chloroplasts

Figure 6. Planar area per chloroplast (a) and the proportion of the adaxial mesophyll cell area covered by chloroplast planar area (b) of C3, C2 and C4 species from 12 lineages where C4 photosynthesis evolved. Means ± SE. Values were compiled from planar area determinations of transverse sections of adaxial mesophyll cells. See Supporting Information Table S1 for sample size and the Fig. 2 legend for full generic and species names corresponding to each column. C3 species are represented by white columns, C2 species by grey columns and C4 species by black columns.

active and Rubisco is not (Voznesenskaya et al. 2005; Chuong et al. 2006; Rosnow et al. 2014). By themselves, these earlier studies provide circumstantial support for the hypothesis that reduced M chloroplast investment is a fundamental feature of C4 photosynthesis. Because they do not present phylogenetically informed comparisons of multiple C3 to C4 lineages, the ability of these studies to evaluate evolutionary hypotheses is limited. We have overcome this limitation by examining closely related C3 and C4 species from a dozen independent lineages of C4 evolution. In combination with the literature observations, our results allow us to conclude that fewer M cell chloroplasts and reduced chloroplast coverage of the M cell periphery is a general feature of the C4 pathway and can therefore be regarded as an essential adaptation for efficient C4 function. We propose two categories of evolutionary drivers to explain why natural selection favoured fewer, more widely spaced M chloroplasts during C4 evolution and hypothesize that they acted in concert to strengthen selection upon M chloroplast properties. The first category of drivers reflects the location of carboxylation and CO2 diffusion to the carboxylation site. An important limitation on C3 photosynthesis is the diffusive conductance from the intercellular air spaces to the site of carboxylation in the chloroplast stroma (gm; Evans et al. 2009). Limitations in gm reduce stromal CO2 relative to intercellular CO2 values by © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

40–110 μmol mol−1 in most plants at moderate temperatures (Evans & Loreto 2000; Scafaro et al. 2011; Evans & von Caemmerer 2013). In C3 leaves, gm is enhanced by pressing high numbers of chloroplasts against the M cell periphery (Sharkey et al. 1991; Evans & Loreto 2000). This minimizes the liquid phase resistance caused by the cytoplasm and increases the surface area of chloroplasts directly opposite the CO2 source in the intercellular air space (Syvertsen et al. 1995; Tholen et al. 2008; Evans et al. 2009). In transgenic tobacco, for example, overexpression of phytochrome caused chloroplasts to bow away from the cell periphery, resulting in a 25% reduction in gm and a 42% reduction in net CO2 assimilation rate at current CO2 (Sharkey et al. 1991). In C4 plants, Rubisco is absent from the M cell chloroplasts, and the site of fixation is the cytosol where PEPC resides (Evans & von Caemmerer 1996). PEPC is abundant in the cytoplasm lining the cell periphery, including the thin layer of cytoplasm where chloroplasts are recessed away from the plasmalemma (Ueno 1996, 1998, 2001; Ueno & Sentoku 2006; Voznesenskaya et al. 2006). If a C4 leaf exhibited the C3 pattern of many chloroplasts appressed against the M cell periphery, the high number of chloroplasts could restrict CO2 access to the cytosol. By reducing chloroplast number and partially backing the chloroplasts away from the cell perimeter, lanes of cytoplasm are opened up for direct diffusive access of CO2 to the site of carboxylation in the C4 M cell.

M. Stata et al.

C3 species C2 species C4 species

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200

Atriplex

Cleome

Aizo.

Nyctag.

Dica./Set.

Neurach.

Mollugo

Heliotrop.

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Altern.

100 Aerva

Mesophyll wall thickness, nm

10

Figure 7. The thickness of adaxial mesophyll cell walls facing the intercellular air spaces in C3, C2 and C4 species from 11 lineages of C4 photosynthesis. Means ± SE. See Supporting Information Table S1 for sample size and the Fig. 2 legend for full generic and species names corresponding to each column. C3 species are represented by white columns, C2 by grey columns and C4 by black columns.

High chloroplast coverage of the C3 M cell periphery also enhances the trapping and refixation of photorespired CO2 in these cells, which can boost carbon assimilation over 10% (Sage & Sage 2009; Tholen et al. 2012; Busch et al. 2013). Without M Rubisco, there is no photorespiration in C4 M cells and CO2 trapping by M chloroplasts cannot happen. Given these considerations, we propose that the downregulation of Rubisco expression in the M cells relaxed selection pressure favouring chloroplast coverage of the M cell periphery of C4 plants, whereas PEPC enhancement increased selection for greater cytosol exposure to the plasmalemma. As shown by studies with Flaveria, reduction of M Rubisco expression and up-regulation of PEPC activity occurred late in the evolution of C4 photosynthesis, during the formation of C4-like plants (Monson & Rawsthorne 2000; Sage et al. 2012). We therefore hypothesize that the reduction in M chloroplast numbers also occurred late in the evolutionary process, which is supported by our observation that most C2 species have C3 patterns of M chloroplast number, size and distribution. With the exception of H. convolvulaceum and N. minor, all of the C2 species had similar chloroplast numbers as their C3 relatives and all except N. minor exhibited C3-like values of chloroplast coverage of the M cell periphery. All C2 species also have C3 patterns of chloroplast appression against the plasmalemma. Because the initial site of CO2 fixation in C2 plants is Rubisco in the M chloroplasts (von Caemmerer 1989; Monson & Rawsthorne 2000), the

diffusive constraints for C2 species would be the same as for C3 species, and thus require the same chloroplast number and distribution. The advantage of C2 photosynthesis relative to C3 photosynthesis is the ability of a glycine shuttle to move Rubisco oxygenation products into the BS mitochondria, where glycine decarboxylase is located and the release of photorespired CO2 occurs (Rawsthorne 1992). This photorespired CO2 is refixed with high efficiency in the BS but not the M tissue, and thus should not influence chloroplast numbers in the M cells. A second driver affecting chloroplast properties of C4 M cells may arise in response to changes in the energetic demands on M cell chloroplasts during C4 evolution. In a C3 M cell, the energy requirements for photosynthesis and photorespiration are met by the resident chloroplasts. By contrast, in C4 photosynthesis, a significant portion of the photosynthetic energy production is assumed by the BS chloroplasts (Kanai & Edwards 1999; Evans et al. 2007; Furbank 2011; Bellasio & Griffiths 2014). If the M chloroplasts in a C4 leaf were as dense as they are in a C3 leaf, they would form a tube-like parasol that would substantially shade the BS chloroplasts (Dengler & Nelson 1999; Ueno & Sentoku 2006; Evans et al. 2007), which in turn could create imbalances between M and BS energy production.An advantage of fewer M chloroplasts in a C4 leaf would therefore be reduced shading of BS chloroplasts. In addition, the transfer of some energy production to the C4 BS should reduce the overall energetic demand on the M cell chloroplasts relative to a C3 plant, allowing for reduced investment in M chloroplasts. The magnitude of this effect is difficult to gauge, however, due to energy sharing between M and BS chloroplasts (Furbank 2011; Bellasio & Griffiths 2014). C4 M cells support the entire ATP demands of the C4 cycle, which is estimated to be 40% of the ATP requirement of C4 photosynthesis (von Caemmerer 2000; Ubierna et al. 2011, 2013). In addition, they can also support the C3 metabolic cycle by exporting reducing power to the BS as malate, and reducing PGA imported from the BS (Leegood 2000; Furbank 2011; Bellasio & Griffiths 2014). In NADP-ME species, most of the reducing power needed for carbon reduction is supplied by M chloroplasts due to the depletion of PSII in BS chloroplasts; however, the degree of this contribution varies between species and growth conditions (Furbank 2011; Bellasio & Griffiths 2014). In NAD-ME species, the BS chloroplasts generate much of the reducing power for carbon fixation (Kanai & Edwards 1999); hence, it follows that NAD-ME species may invest in fewer M chloroplasts than NADP-ME species. This hypothesis is not supported, however, because chloroplast size and number in M cells do not clearly differ between the NADP-ME and NAD-ME lineages. NAD-ME plants also export PGA from the BS to M chloroplasts for reduction (Furbank et al. 2000) and this sharing of the workload may explain in part the similarity of M chloroplast size and numbers between NADP-ME and NAD-ME species. The gm of C4 M tissue is presumed to be about twice that of C3 M tissue, although direct measurements of gm by isotope discrimination and fluorescence methods are not © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

C3 versus C4 mesophyll chloroplasts possible in C4 plants (Evans & von Caemmerer 1996; Evans & Loreto 2000; Yin et al. 2011). The higher gm estimates have led to speculation that C4 M cell walls are thinner than those of C3 plants (Evans & von Caemmerer 1996; Evans & Loreto 2000). Our results show no difference in M wall thickness between related C3, C2 and C4 species. Mesophyll wall thickness varied fivefold between lineages, but within lineages, all of the species had similar values. Previous differences in wall thickness reported for C3 and C4 species were between unrelated species (Evans & Loreto 2000), and hence may be due to lineage differences and not photosynthetic pathway differences. We conclude that wall thickness cannot explain differences in gm between C3 and C4 species.

CONCLUSION In this study, we show that evolutionary specialization occurs in the C4 M cells in a manner that is consistent with optimizing gm and M versus BS energetics, for efficient C4 photosynthesis. Instead of the optimal C3 pattern where many, often smaller chloroplasts cover most of the plasma membrane, C4 plants have fewer chloroplasts with large amounts of cytoplasm adjacent to the cell periphery. As well, chloroplast numbers increase in the BS cells during the C3 to C4 transition (Black & Mollenhauer 1971; Brown & Hattersley 1989; Muhaidat et al. 2011) in contrast to the decline in the M cells reported here. This demonstrates separate developmental trajectories in the two cell types with respect to chloroplast size, numbers and position. Understanding the molecular control over these distinct features will be essential for ongoing efforts to establish a functional C4 pathway in C3 species such as rice (von Caemmerer et al. 2012; http://c4rice.irri.org). For example, in their attempt to increase chloroplast numbers in the BS tissue, C4 engineers will need to identify the cell-specific control over M and BS chloroplast properties to avoid enhancing chloroplast numbers in all leaf tissues. The data presented here will also guide the improvement of theoretical models of C4 photosynthesis (Yin et al. 2011; von Caemmerer 2013; Ubierna et al. 2013; Bellasio & Griffiths 2014). The knowledge that C4 M cells have reduced chloroplast investment will help modellers better parameterize gm and the energy distribution between the chloroplasts of C4 M and BS cells. Finally, the results here demonstrate the depth of convergence associated with complex trait evolution. All the C4 lineages showed similar changes in M chloroplast numbers and coverage of the M cell periphery, exemplifying the high potential for evolution to repeatedly fine tune cellular mechanics to allow for efficient C4 function.

ACKNOWLEDGMENTS We thank Dan Johnson and Phil Cutter for sample fixation. We also thank Drs O. Björkman (Palo Alto, CA, USA), S. Covshoff (Cambridge University, UK); Professors E. Kellogg (University of Missouri at St. Louis), D. Longstreth (Louisiana State University, Baton Rouge, LA, USA); A. M. Powell (Sol Ross State University, Alpine, TX, USA); A. © 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

11

Raghavendra (University of Hyderabad, India), Dr C. Root (Reno, NV, USA) and A. Tyler and T. Huxman (University of Arizona University, Tuscon, AZ, USA) for providing seeds and plant material.This research was supported by Discovery grants # 154273-2012 to R.F.S. and 155258-2008 to T.L.S. from the Natural Science and Engineering Research Council (NSERC) of Canada, and a Canadian International Development Agency (CIDA) GCIAR-Canada linkage fund grant to T.L.S. and R.F.S.

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Received 27 November 2013; received in revised form 3 March 2014; accepted for publication 17 March 2014

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. A diagram showing the measurements used to parameterize chloroplast properties in the mesophyll cells examined in this study. The insert shows the measurements

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for wall thickness and the % contact of the outer chloroplast length with the cell periphery. Note that chloroplast coverage estimates do not incorporate variation in chloroplast contact with the mesophyll cell periphery. Figure S2. Transmission electron micrographs of adaxial mesophyll cells from C3 (a, d, g), C2 (b, e, h) and C4 (c, f, i) species of Alternanthera (a–c), Euphorbia (d–f) and Mollugo (g–i). Abbreviations: AlSe, Alt. sessilis; AlTe, Alt. tenella; AlCa, Alt. caracasana; EuAn, Eu. angusta; EuAc, Eu. acuta; EuMe, Eu. mesembryanthoides; MoPe, M. pentaphylla; MoNu, M. nudicaulis; MoCe, M. cerviana; C, chloroplast; IAS, intercellular air space; V, vacuole. Bars = 10 μm. Figure S3. Transmission electron micrographs of adaxial mesophyll cells from C3 (a, c, e, g) and C4 (b, d, f, h) species of Aerva (a, b), Atriplex (c, d), Cleome (e, f), and Dichanthelium/ Setaria (g, h). Abbreviations: AeLa, Ae. lanata; AePe, Ae. persica; AtPr, At. prostrata: AtRo, At. rosea; ClVi, Cl. viscosa; ClGy, Cl. gynandra; DiOl, D. oligosanthes; SeVi, Se. viridis; C, chloroplast; IAS, intercellular air space; V, vacuole. Bars = 10 μm. Figure S4. Adaxial mesophyll cells of the C3 (a, b) and C4 (b,c,e,f) species of the Sesuviodeae (a,b,c) and Nyctaginaceae (d, e, f) clades of C4 evolution. Abbreviations: SeVe, Sesuvium verrucosum; CyHu, Cypselea humifusa; TrPo, Trianthema portulacastrum; MiJa, Mirabilis jalapa; Bobu, Boerhavia burbidgeana; Alin, Allionia incarnata; C, chloroplast; IAS, intercellular air space; V, vacuole. Bars = 10 μm. Note: Boerhavia and Allionia are in distinct C4 lineages while Cypselea and Trianthema are of the same lineage. Figure S5. Quantified properties of chloroplasts in adaxial mesophyll cells of C3 and C4 species in four clades where C4 photosynthesis has evolved. All parameters were measured from leaf paradermal sections. (a) Chloroplast number per planar cell area; (b) the % of the cell perimeter that is covered by chloroplasts; (c) the area per chloroplast in planar section and (d) the % of the planar cell area in a paradermal section that is covered by chloroplast planar area. Mean ± SE of a single species, n = 3. C3 species are represented by white columns and C4 species by black columns. Clades and species are as follows, from left to right: Alternanthera: Alt. sessilis (C3), Alt. caracasana (C4); Heliotropium: H. tenellum (C3), H. texanum (C4); Dica./Set: Dichanthelium oligosanthes (C3) and Setaria viridis (C4); Cleome: Cl. viscosa (C3), Cl. gynandra (C4). Figure S6. Quantified properties of chloroplasts from single isolated mesophyll cells of C3 and C4 species from seven clades where C4 photosynthesis has evolved. (a) Chloroplast number per cell and (b) the projected area per chloroplast. Mean ± SE of a single species (n = 3 except for Cleome gynandra where n = 1 and Mollugo cerviana where n = 2). Data are from the adaxial region of the leaf. C3 species are represented by white columns and C4 species by black columns. Clades and species are as follows, from left to right: Alternanthera: Alt. sessilis (C3), Alt. caracasana (C4); Heliotropium: H. calcicola (C3), H. texanum (C4); Mollugo: M. pentaphylla (C3), M. cerviana (C4); Nyctaginaceae: Mirabilis jalapa (C3), Boerhavia coccinea (C4); Dica./Set:

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Dichanthelium oligosanthes (C3) and Setaria viridis (C4); Cleome: Cl. viscosa (C3), Cl. gynandra (C4); Atriplex: At. prostrata (C3), At. rosea (C4). Figure S7. Transmission electron micrographs of leaf M cells of the C4 grasses Zea mays (panel a), Saccharum officinarum (b), Echinochloa glabrescens (c) and the C4 sedge Eleocharis baldwinii (d). C, chloroplast; IAS, intercellular air space; V, vacuole. Bars = 10 μm. Table S1. Lineages and species used in this study. Sample size refers to the number of individual plants sampled. Letters after lineage number indicate independently derived

lineages of C2 photosynthesis in the Neurachnineae. References after source location indicate prior publication of the species; these may have more detailed collection information. The phylogeny reference for a lineage is given in parenthesis in the first line of a lineage listing. NP indicates NADP-malic enzyme type of C4 photosynthesis, whereas N indicates the NAD-malic enzyme type (Sage et al. 2011a). Orders are indicated after the family name, where Bo is Boraginales, Br is Brassicales, Ca is Caryophyllales, Ma is Malpighiales and Po is Poales. Taxonomy follows Tropicos (http://www.tropicos.org).

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