Research
Unified changes in cell size permit coordinated leaf evolution
Tim J. Brodribb1, Greg J. Jordan1 and Raymond J. Carpenter2
1
School of Plant Science, University of Tasmania, Hobart, Tasmania 7001, Australia; 2School of Earth and Environmental Sciences, University of Adelaide, Adelaide, SA 5005, Australia
Summary
Author for correspondence:
Tim Brodribb
Tel: + 61 362261707
Email: [email protected]
Received: 11 February 2013
Accepted: 27 March 2013
New Phytologist (2013)
doi: 10.1111/nph.12300
Key words: adaptation, cell size, genome
size, leaf thickness, stomatal density,
stomatal size, vein density.
The processes by which the functions of interdependent tissues are coordinated as lineages
diversify are poorly understood.
Here, we examine evolutionary coordination of vascular, epidermal and cortical leaf tissues
in the anatomically, ecologically and morphologically diverse woody plant family Proteaceae.
We found that, across the phylogenetic range of Proteaceae, the sizes of guard, epidermal,
palisade and xylem cells were positively correlated with each other but negatively associated
with vein and stomatal densities. The link between venation and stomata resulted in a highly
efficient match between potential maximum water loss (determined by stomatal conductance) and the leaf vascular system’s capacity to replace that water. This important linkage is
likely to be driven by stomatal size, because spatial limits in the packing of stomata onto the
leaf surface apparently constrain the maximum size and density of stomata.
We conclude that unified evolutionary changes in cell sizes of independent tissues, possibly
mediated by changes in genome size, provide a means of substantially modifying leaf function
while maintaining important functional links between leaf tissues. Our data also imply the
presence of alternative evolutionary strategies involving cellular miniaturization during
radiation into closed forest, and cell size increase in open habitats.
Introduction
Recent characterization of genes and core regulatory networks
has revolutionised our understanding of how tissues develop.
However, the development of individual tissues is only one
requirement for building complex organisms. Another, less
understood process is how the development of spatially discrete
but functionally interdependent tissues is coordinated. One possible mechanism for such coordination is colocation of primordial tissues. Thus, lymphatic and blood-carrying vessels of
mammals develop from a common embryonic vascular system,
and the xylem and phloem of plants derive from a shared
cambium. However, complex organisms also depend on the
coordinated development of many tissues with different origins
(Cavalier-Smith, 2005); for example, lung capacity, vascular volume and muscle mass are necessarily coordinated (Rubner,
1883). Similarly, developmental coordination is essential for
plants because their primordial tissues have indeterminate
growth. Thus, plants can show great plasticity in response to the
environment, but this plasticity is only effective if the diverse
tissues involved remain functionally coordinated.
One important example of coordination between discrete tissues is found between the veins and stomata in the leaves of land
plants. Branching density in the leaf vein network determines
water transport efficiency of the lamina (leaf hydraulic conductance), which is closely linked to maximum rates of photosynthesis (Brodribb et al., 2005) and transpiration (Boyce et al., 2009).
Because leaf vascular networks replace water lost by evaporation
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during the uptake of CO2 for photosynthesis (Sack & Holbrook,
2006), plants with higher rates of photosynthesis per unit leaf
area lose more water (Cowan & Farquhar, 1977) and thus
demand greater investment in leaf veins (McKown et al., 2010).
This investment comes largely as increased branching of leaf
minor veins, because a greater density of minor veins delivers
water closer to sites of evaporation in the leaf (Brodribb et al.,
2007), leading to increased transport efficiency (Sack & Frole,
2006). However, these veins are expensive to synthesize, and
plants are likely to coordinate the production of photosynthetic
and water supply tissues to maximize returns on investments in
the water transport system (Brodribb & Jordan, 2011). Furthermore, while vein density determines water supply in the leaf, the
density of stomata determines maximum rates of water loss and
photosynthesis, and thus maintaining a balance between these
traits during adaptation to the environment should be of high
functional and adaptive importance. Such coordination has been
demonstrated both within trees during plastic adaptation to light
(Murphy et al., 2012) and between species (Edwards, 2006;
Dunbar-Co et al., 2009; Zhang et al., 2012).
However, little is known about how this critically important
link between vascular and stomatal tissues is maintained. A recent
study of a tree species showed that plasticity in epidermal cell size
changed vein and stomatal density in concert during light acclimation. Hence, larger epidermal cells in the shade result in larger
leaves that have lower densities of veins and stomata than sun
leaves (Murphy et al., 2012). This coordinating role of cell size
during plastic adaptation of leaves to different evaporative and
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photosynthetic conditions of sun and shade raises the prospect
that changing cell size could also be an important mechanism for
evolutionary adaptation in plants.
A correlation between cell volume and genome size has been
long recognized as a fundamental feature of eukaryotic organisms
(Mirsky & Ris, 1951; Cavalier-Smith, 1985); however, the evolutionary significance of variation in cell size, and associated
genome size in plants and animals, has been hotly debated
(Cavalier-Smith, 1978, 2005; Petrov, 2001; Hodgson et al.,
2010). In animals, transitions in cell and genome size are implicated in several important evolutionary transitions (such as the
evolution of birds from dinosaurs; Organ et al., 2007), but in
plants the adaptive significance of cell size variation remains
obscure. Attempts to account for the enormous range in genome
and cell size in plants have recently focused on variation in stomatal size as a potentially important functional consequence of
variable cellular and nuclear volume (Beaulieu et al., 2008). Theory and observation suggest that large stomata are associated with
low rates of gas exchange as a result of limits on the packing
density of guard cells (if stomata become larger, then fewer can
fit on the leaf surface), and diminishing benefits in terms of maximum diffusive conductance of larger, deeper pores (Franks &
Beerling, 2009). Other potentially important tissues that share
size-constrained functional properties include leaf veins, which
have analogous associations between the size of cells and the density (Field & Brodribb, 2013) and conductivity (Sack & Frole,
2006; Brodribb et al., 2007) of the vascular system. Epidermal
cell size also appears to be a primary determinant of the final size
of leaves, as well as influencing the thickness of the photosynthetic mesophyll (Perez-Perez et al., 2011). Here we examine
how these interconnected systems in the leaf respond to familywide variation in cell size.
Considering the diversity of influences that cell size has on leaf
physiology, we investigate how key functional attributes of leaves
remain coordinated if cell size changes. This is of particular significance considering that cell size and genome size appear not
only to be rather labile within angiosperms, but also to exhibit
some long-term adaptive patterns (Masterson, 1994; Franks
et al., 2012b). In this study, we examine the relationship between
cell size and functional anatomy in the leaves of a morphologically and ecologically diverse sample of Proteaceae trees and
shrubs. The primary question we address in this study is whether
the cell sizes in functionally linked tissues are coordinated in such
a way as to preserve integrated function. Specifically we hypothesize that greater vein density and stomatal density should be associated with smaller cell size, leading to coordination in tissues
related to water supply and water loss in the leaf.
and it is an ecologically important group in the southern hemisphere where species range from trees in tropical rainforest to
shrubs in the arid zone. We sampled cell size and densities from
48 species and stomatal size from 417 species of Proteaceae from
all major branches of the phylogeny.
Species were categorized as being from open vegetation or
closed forest according to descriptions from regional floras.
Closed canopies are typically > 70% canopy cover, which is generally only achieved in rainforest communities. Proteaceae species
are typically canopy species, so regardless of habitat type, all
leaves were collected in the field from sun-exposed branches. In
most cases, leaves were sampled from three trees and immediately
fixed in FAA (50% ethanol, 5% (v/v) acetic acid and 3.7% (v/v)
formaldehyde). Leaves were returned to the laboratory where they
were soaked in water in preparation for anatomical sectioning.
The leaf area and mass of at least 10 leaves per species were
measured to yield leaf mass per unit area (LMA).
Stomata and vein density
Paradermal sections of leaves were made using a handheld razor
blade to remove the adaxial epidermis and palisade, exposing the
minor veins. Sections were then bleached in commercial household bleach (50 g l1 sodium hypochlorite and 13 g l1 sodium
hydroxide) until clear. Bleach was removed by washing, and sections stained in 1% toluidine blue for 30 s to colour the ligninrich veins. Finally sections were mounted in phenol glycerine jelly
and photographed with a Nikon Digital Sight DS-L1 camera
(Melville, NY, USA) mounted on a Leica DM 1000 microscope
(Nussloch, Germany) with a 910 objective. ImageJ (http://
rsbweb.nih.gov/ij/index.html) was used to measure the total
length of venation in five fields of view that were aligned midway
between the midrib and the margin. Wire frames of the veins
were drawn manually and their total length counted.
Stomatal density was measured either directly from the paradermal sections, or, where this was not possible, from stomatabearing cuticles prepared from the same leaves on which vein
density was measured. The cuticles were prepared by soaking leaf
samples in warm 10% aqueous Cr2O3 until clear, rinsing thoroughly, staining with dilute (< 0.1%) crystal violet, rinsing,
cleaning with a single-hair paintbrush (if necessary), and then
mounting on microscope slides, in phenol glycerin jelly. Stomatal
densities were measured from digital photomicrographs of the
cuticle preparation at 950 magnification using the counting tool
in ImageJ. At least three fields of view were measured from each
leaf section.
Cell sizes
Materials and Methods
The family Proteaceae was chosen for three reasons: it is a
morphologically and ecologically diverse group that has a
well-documented history of evolutionary adaptation in response
to changing climate over the Cenozoic period (Jordan et al.,
2008; Sauquet et al., 2009); it has a well-studied phylogeny, thus
allowing phylogenetically independent analysis of relationships;
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Stomatal size was determined from photographs of the prepared
cuticles described in the previous section at 940 magnification.
Guard cell length and width were measured on 20 stomata from
each stomata-bearing surface of the leaves.
Sizes of epidermal cells were measured from paradermal sections of leaves. Length (maximum dimension) and breadth
(width perpendicular to the length) were measured on a total of
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at least 20 epidermal cells from three leaves. Epidermal cell size
was then estimated as the square root of the product of length
and breadth. The square root was used to ensure that this trait
showed the same dimensionality as the other linear dimensions.
In one species, Hollandaea sayeriana, epidermal cells could not be
reliably differentiated from light microscopy or scanning electron
microscopy of paradermal preparations (see, e.g., figs 127 and
128 in Carpenter, 1994). For this species, the size was estimated
from mean epidermal cell width in cross-section, using a regression of this dimension against epidermal cell size for the other 47
species.
Palisade and minor vein conduit width were measured in
cross-sections prepared from the same leaves as all other anatomical data. Squares of c. 5 mm2 were cut from the region between
the midrib and margin and sectioned on a freezing microtome.
Sections were stained with toluidine blue and mounted in phenol
glycerine jelly. From these sections, we measured leaf thickness
and palisade width by taking the maximum cell width from the
20 widest cells per section.
Conduit width was measured in minor veins, identified in the
cross-sections by their lack of bundle sheath cells. These minor
veins generally contained 10–20 conduits for which lumen
width was measured. Three to five minor veins were measured
per section.
Maximum stomatal conductance
To examine the balance between water supply determined by
vein density, and the demand for water produced by stomatal size
and density, we calculated the theoretical maximum leaf conductance (Franks & Farquhar, 2001) to water vapour based on the
measured stomatal anatomy using the following equation:
d
a
pffiffiffiffiffiffiffiffi
gmax ¼ D v
l þ p2 a=p
Phylogenetically independent correlations
To assess the strength of association between pairs of traits, Pearson product–moment correlation coefficients and phylogenetically adjusted correlation coefficients were calculated. Because
stomatal density and vein density were expected to show curvilinear associations with other dimensions, all of the correlations
involving these two traits were on a log-log basis. The other correlations were all linear-linear. Pairwise scatter plots confirmed
the linear nature of all these relationships. The phylogenetically
adjusted correlations were performed using phylogenetically
independent contrasts generated using the ‘ape’ package of R
(Paradis et al., 2001), based on the phylogeny described earlier
but cut down to the 48 species for which the full complement of
traits were available.
Eqn 1
where gmax is the maximum leaf stomatal conductance to water
vapour (mmol m2 s1); d is the diffusivity of water in air (m2
s1); v is the molar volume of air (m3 mol1); D is the stomatal
density (stomata m2); a is the maximum pore area (m2); and l is
the pore depth (m).
The mean guard cell width was substituted for pore depth
based on the assumption that guard cells were approximately
circular in cross-section. Maximum pore area was calculated from
the guard cell length, assuming that guard cells opened into a circular aperture.
Phylogenetic analysis guard cell length
The phylogenetic distribution of guard cell length was investigated using a phylogeny of 143 species of Proteaceae and six
species of its sister clade, Platanus, as an outgroup (Supporting
Information, Fig. S1). The phylogeny was created by concatenating the phylogenies of Sauquet et al. (2009), Mast & Givnish
(2002) and Mast et al. (2008) with a few additional species added
by assuming the monophyly of individual genera. In each of these
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latter cases, the genera are well defined and accepted. The 149
species were selected to summarize data from 417 of the c. 1700
species of Proteaceae (including all accepted genera) and nine of
the 10 species of Platanaceae. Clades in which stomatal density
varied by < 15% among species were represented by a single, typical species; in other cases 90th and 10th percentile species were
included. The evolution of stomatal length was reconstructed
using parsimony with a squared cost assumption, implemented
in Mesquite 2.75 (Maddison & Maddison, 2011). Guard cell
lengths were allocated to classes based on a log-linear relationship
rounded to the nearest lm.
To test for any association between guard cell length and habitat, we used a phylogenetically adjusted one-way ANOVA comparing open vegetation species and closed forest species. This was
using a phylogenetic generalized linear model based on this phylogeny, assuming branch lengths of 1, and implemented in the
‘ape’ package of R (Paradis et al., 2001).
Results
Interspecies variation
In our sample of 48 species of Proteaceae, we found enormous
interspecific diversity in all aspects of leaf morphology and anatomy (Fig. 1). Physical attributes of leaves such as lamina size varied by up to three orders of magnitude (mean leaf sizes ranging
from < 1 to > 500 cm2 in the largest species), and leaf thickness
by more than fourfold (Table 1). Very large differences between
species were observed in the densities of minor veins (ranging
from 2.3 to 13.2 mm mm2) and stomata (from 44 to
520 mm2). Cell size also varied substantially between species,
with a fourfold range in the widths of epidermal and palisade
cells and the length of stomatal guard cells (Table 1).
Stomata and veins
The densities of stomata (Dstomata) and minor veins (Dvein)
were very strongly linearly correlated among all species with an
almost proportional relationship encompassing all species
(Dstomata = 23.95 Dvein + 25; P < 0.0001; Fig. 2). The species
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Fig. 1 Representative images from two
sampled Proteaceae species with contrasting
leaf anatomy. Images show, from left to
right, stomatal size and density; vein density;
and cross-sections illustrating epidermal and
palisade cell sizes. All images are at the same
magnification to highlight the very large
differences in the sizes of cells and densities
of veins and stomata.
with the lowest Dstomata (44 stomata mm2) also had the lowest
vein density (2.3 mm mm2), while the highest Dstomata (521
stomata mm2) was found in a rainforest species with a high vein
density of 12.8 mm mm2. The density of veins showed no relationship with leaf area, and the density of stomata showed a weak
positive relationship with leaf area; however, both densities
showed strong and highly significant negative correlations with
leaf thickness (Table 2).
among species conformed to the predicted linear relationship
between the capacity to supply water (proportional to Dvein) and
the capacity of leaves to lose water (proportional to maximum
stomatal conductance). Species with stomata on both leaf surfaces
were not expected to fall on the same relationship (see the Materials and methods section) and, indeed, we found a nonsignificant
slope in these 11 species (Fig. 5).
Phylogenetic patterns in stomatal size
Cell size
The sizes of stomatal, epidermal, xylem and palisade cells were all
interrelated. The most important associations were found between
epidermal cell width and both the guard cell length (Table 2,
Fig. 3a) and xylem lumen diameter in minor veins (Table 2), all of
which were related by very strong linear correlations among species.
Palisade cell width was also significantly correlated with these other
cell types, but these correlations were not as strong (Table 2).
Cell sizes were significantly linked to key functional traits,
Dvein, Dstomata and the thickness of leaves, but not leaf area
(Table 2). The strongest association was between the size of stomatal guard cells and Dstomata, with diminishing guard cell length
in species with higher Dstomata (Lguardcell = 255Dstomata0.43;
r2 = 0.82). Confirming that this relationship is probably driven
by spatial constraints, the Dstomata in amphistomatic species fitted
the same regression against stomatal size as hypostomatic species
only when just stomata on the lower leaf surface were considered
(Fig. 4). Other cell sizes, particularly xylem and epidermal cell
size, were also strongly correlated with Dstomata (Table 2). Similar
to Dstomata, we found that Dvein was strongly correlated with cell
size, with highly significant correlations (P < 0.001) among species between Dvein and all cell types (Table 2). High vein density
was strongly associated with small cell size in stomata, epidermis,
palisade and xylem cells across all species.
Functional coordination
Maximum predicted stomatal conductances to water vapour were
found to be strongly correlated with Dvein (Fig. 5). This pattern
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Stomatal size data spanning the full generic diversity of Proteacae
demonstrated different patterns in different clades (Fig. 6).
Moderately large stomata (31–54 lm) were found almost
throughout the large, and mainly open-habitat, subfamily Proteoideae, with the only major variation being the appearance of
very large stomata within the recently evolved genus, Protea (also
of open habitats), and smaller stomata in a few genera. By
contrast, the subfamily Grevilleoideae, in which most of the
phylogenetic diversity is in rainforest, mostly has small stomata
(< 31 lm), particularly in rainforest genera. Finally, the mainly
open-habitat subfamily Persoonioideae shows markedly large
stomata (> 60 lm). The ancestral state reconstruction suggests
the independent evolution of both very large stomata (in Persoonioideae, Protea, Agastachys and Strangea) and small stomata (in
several clades within Grevilleoideae and within a few genera of
Proteoideae) (Fig. S1). The emergent pattern of small stomata in
rainforest clades and large stomata in open-habitat clades is
confirmed by the presence of a phylogenetically independent
association between habitat type and stomatal size, with clades of
open environments being very significantly more likely
(P < 0.001) to have large stomata than closed forest clades.
Discussion
As leaf anatomy and morphology evolve and adapt in response to
environmental change, there is the potential for functionally interdependent tissues to change independently of one another. Without some coordinating mechanism, this would lead to inefficiency
or plant death. Our results show that coordinated changes in cell
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Conospermum
longifolium
Isopogon fletcheri
Leucadendron
pubescens
Leucospermum
cordifolium
Persoonia
lanceolata
Persoonia muelleri
Protea cynaroides
Protea nitida
Protea repens
Synaphea
petiolaris
Synaphea
spinulosa
Agastachys
odorata
Alloxylon
pinnatum
Athertonia
diversifolia
Austromuellera
trinervia
Bankia grandis
Bellendena
montana
Brabejum
stellatifolium
Buckinghamia
celsissima
Cardwellia
sublimis
Carnarvonia
araliifolia
Catalepidia
heyana
Cenarrhenes
nitida
Darlingia
ferruginea
Eidothea
hardeniana
Species
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0.393
0.724
0.313
0.176
0.196
0.345
0.638
0.336
0.289
0.222
0.362
0.307
Amphistomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
0.25
0.714
0.587
0.515
0.525
0.396
Amphistomatic
Amphistomatic
Amphistomatic
Amphistomatic
Amphistomatic
Hypostomatic
0.405
Amphistomatic
0.244
0.61
Amphistomatic
Hypostomatic
0.594
0.561
Amphistomatic
Amphistomatic
0.629
0.59
Amphistomatic
Hypostomatic
Lamina
thickness
(mm)
Stomatal
distribution
Table 1 Characteristics of species used in this study
28.1
31.7
27.8
20.1
40.8
21.0
19.0
21.0
22.0
31.8
16.7
18.8
31.5
39.7
46.1
64.4
63.1
31.9
30.6
50.4
92.4
46.2
47.0
39.1
32.9
Epidermal
cell size
(lm)
20.9
13.5
30.4
24.1
22.2
17.2
14.8
25.5
18.2
32.8
14.7
22.1
20.7
40.4
22.7
48.2
32.0
22.3
21.3
21.5
25.3
28.9
29.0
20.9
23.1
Palisade
cell width
(lm)
29
38.6
33.5
24.2
39
25.3
23.3
27.7
31.9
42.7
27
20.8
39.3
60
46
67.7
71.5
38.9
61
45.9
79.7
40.5
41.3
41.7
25.2
Guard cell
length
(lm)
5.78
5.70
3.91
3.50
4.70
4.10
3.65
8.00
4.50
4.56
3.38
4.43
7.42
5.38
4.49
6.43
6.77
8.55
6.25
8.94
8.86
11.23
5.32
5.64
4.97
Diameter of
vessel lumens
(lm)
8.28
10.80
4.67
7.58
6.33
9.12
8.51
10.64
11.94
4.97
12.80
7.13
4.83
3.40
5.68
2.29
4.20
6.60
9.52
5.91
4.03
4.62
2.84
4.82
7.95
Vein
density
(mm mm2)
204
234
226
198
137
130
156
185
220
229
150
94
140
273
244
269
210
288
230
169
134
255
215
198
174
Leaf mass
per unit
area (g m2)
129
177
210
340
145
248
312
279
170
128
521
267
98
46
73 (67)
27 (27)
33 (3)
141 (124)
60 (56)
48 (47)
27 (23)
103 (99)
109 (90)
130 (99)
150 (143)
Stomatal
density
(mm2)
19.7
99.4
7.5
43.4
400
86.2
39.2
18.3
72.6
1.2
429
660
9.8
3.8
24.6
1.9
44.5
32.1
5.0
14.5
5.2
8.3
11.3
0.93
8.7
Leaf
area
(cm2)
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0.27
0.612
0.62
0.611
0.45
0.452
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
0.359
0.221
Hypostomatic
Hypostomatic
0.218
Hypostomatic
0.269
0.244
0.338
0.325
0.291
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
0.398
Hypostomatic
0.641
0.27
Hypostomatic
Hypostomatic
0.246
0.349
0.338
0.241
Hypostomatic
Hypostomatic
Hypostomatic
Hypostomatic
0.329
0.254
Hypostomatic
Hypostomatic
Lamina
thickness
(mm)
Stomatal
distribution
25.0
31.6
27.6
22.4
68.0
16.9
16.5
20.8
85.7
20.8
24.6
17.3
34.0
19.8
31.1
17.3
24.1
39.6
16.3
27.7
21.8
22.0
29.9
Epidermal
cell size
(lm)
18.2
23.7
20.7
16.8
27.8
16.8
22.0
20.8
30.7
14.5
18.4
18.6
19.7
20.7
21.8
19.1
39.9
41.7
13.3
23.0
17.2
18.7
18.9
Palisade
cell width
(lm)
33.8
33.7
27.8
31.3
59.6
37.3
26.6
50.3
62.7
20.5
32
23.1
41.1
25.6
28
24.4
26.5
27.4
26.3
32.3
26.1
30
30.7
Guard cell
length
(lm)
3.61
4.33
4.67
5.89
11.01
6.88
5.00
4.37
8.75
4.41
3.89
4.28
4.13
3.98
4.20
3.97
5.91
4.42
4.19
4.04
4.87
4.80
5.07
Diameter of
vessel lumens
(lm)
11.44
6.66
4.43
6.34
3.52
3.77
5.51
5.30
3.18
5.88
6.63
13.21
10.39
11.46
4.83
12.79
5.21
6.25
12.44
6.18
7.62
8.13
6.88
Vein
density
(mm mm2)
184
92
226
202
191
225
381
300
111
89
83
147
261
109
244
114
202
151
110
124
163
114
137
Leaf mass
per unit
area (g m2)
227
137
129
196
45
131
305
79
43
250
196
444
125
359
161
303
189
106
326
129
246
231
161
Stomatal
density
(mm2)
Stomatal distribution is either amphi- or hypostomatic. Stomatal densities are for the abaxial leaf surface, and adaxial densities are given in parentheses for amphistomatic species.
Embothrium
coccineum
Floydia praealta
Gevuina avellana
Grevillea hilliana
Helicia
australasica
Hicksbeachia
pilosa
Hollandaea
sayeriana
Lambertia inermis
Lasjia whelanii
Lomatia tinctoria
Megahertzia
amplexicaulis
Musgravea
heterophylla
Neorites
kevedianus
Opisthiolepis
heterophylla
Orites diversifolius
Orites milliganii
Placospermum
coriaceum
Roupala
pseudocordata
Sphalmium
racemosum
Stenocarpus
sinuatus
Telopea
truncata
Triunia
montana
Xylomelum
pyriforme
Species
Table 1 (Continued)
31.7
30.5
4.9
155
13.9
56
2.9
2.6
249
435
368
259
0.78
19.1
10.1
150
123
560
58.8
28.8
67.9
28.2
17.8
Leaf
area
(cm2)
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Research 7
Fig. 2 Correlation between the mean density of stomata on both leaf
surfaces and the mean density of veins in the leaves of 48 species of
Proteaceae trees and shrubs. Despite an enormous range in leaf size, shape
and habitat, a highly significant linear correlation (r2 = 0.45; P < 0.0001)
between the densities of water supply tissue and stomatal pores was
observed. See Table 1 for species list.
size can function as a linkage between the vascular system and epidermis during adaptive variation in leaves of Proteaceae. Over an
extreme range of leaf size, morphology and habitat, our sample of
48 species of shrubs and trees showed strongly correlated changes
in the sizes of distinct cell types (stomatal, epidermal, xylem and
palisade cells) in the leaf. This resulted in a unified relationship
between the density of stomata on the leaf surface and the density
of vein branching in the lamina. As these tissues are responsible for
regulating interlocked processes of water delivery and water loss in
leaves, modification of cell size therefore provides a rapid way for
plants to adapt and evolve to the prevailing conditions without
compromising the coordination of component tissues necessary
for the whole leaf to function effectively (Fig. 7).
Variation in the sizes of cells and genomes appears to be closely
related across biological kingdoms (Cavalier-Smith, 2005), but
the selective processes driving this variation has been the subject
of considerable controversy. Based upon a linear correlation
(P < 0.01; data not shown) between the stomatal size data presented here for Proteaceae and genome size published by Stace
et al. (1998), we can say that Proteaceae provide an exemplary
demonstration of this so called ‘C-size paradox’. A commonly
cited explanation for cell and genome size variation suggests that
different cell sizes may be suited to different ecological strategies,
and in particular whether a species is the product of r- or K-type
Table 2 Correlations among pairs of traits
Guard cell
length
Guard cell length
Stomatal density
Vein density
Epidermal cell width
Xylem lumen diameter
Palisade cell width
Lamina thickness
Leaf mass per unit area
Leaf area
0.89 ***
0.62 ***
0.79 ***
0.44 **
0.40 *
0.58 ***
0.35 *
0.34 *
Stomatal
density
Vein
density
Epidermal
cell width
Xylem lumen
diameter
Palisade cell
width
Lamina
thickness
Leaf mass per
unit area
0.91 ***
0.59 ***
0.71 ***
0.84 ***
0.88 ***
0.65 ***
0.56 ***
0.63 ***
0.49 ***
0.64 ***
0.47 ***
0.58 ***
0.65 ***
0.51 ***
0.28 ns
0.56 ***
0.58 ***
0.68 ***
0.43 **
0.35 *
0.58 ***
0.27 ns
0.29 *
0.29 *
0.04 ns
0.18 ns
0.28 ns
0.62 ***
0.72 ***
0.86 ***
0.52 ***
0.58 ***
0.53 ***
0.30 *
0.38**
0.72 ***
0.42 *
0.69 ***
0.65 ***
0.35 *
0.38 **
0.53 ***
0.57 ***
0.41 **
0.09 ns
0.07 ns
0.30 *
0.36 *
0.31 *
0.21 ns
0.59 ***
0.30 *
0.12 ns
0.65 ***
0.54 ***
0.57 ***
Leaf area
0.30 *
0.41 **
0.34 *
0.13 ns
0.24 ns
0.05 ns
0.44 **
0.48 ***
Numbers above the diagonal are unadjusted Pearson correlation coefficients, numbers below the diagonal are phylogenetically adjusted coefficients.
***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.005.
(a)
(b)
(c)
Fig. 3 All species showed coordinated changes in species mean cell sizes from stomatal, epidermal, vein and palisade tissues. Linear regressions were highly
significant in all cases regardless of whether data were phylogenetically adjusted (Table 2) or unadjusted (shown here). See Table 1 for species list.
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Fig. 4 Spatial limits to stomatal packing produce a general relationship
between the size and density of stomata on the lower leaf surface (black
circles) in all Proteaceae. Larger stomatal size leads to lower density,
following a predictable relationship; stomatal length = 289.1(stomatal
density)0.43. Species with stomata on both leaf surfaces (amphistomy;
green circles) were able to achieve higher total stomatal densities (the sum
of both leaf surfaces) by avoiding the strict spatial limits associated with
confining stomata to the lower leaf surface. See Table 1 for species list.
Fig. 5 Coordination in cell size leads to a strong correlation between vein
density and maximum stomatal conductance. In all species with stomata
confined to the lower surface (black circles), the density of veins providing
water supply to the leaf (a proxy for the water transport efficiency of the
leaf; Brodribb et al., 2007) was linearly related to the maximum potential
rate of water loss, modelled from the stomatal anatomy and density.
Leaves with stomata distributed on both surfaces (green circles) did not
conform to the general relationship found in typical hypostomatic species
and were able to produce higher maximum stomatal conductances from
the same venous supply.
selection (Cavalier-Smith, 1978). Owing to an apparent negative
correlation between cell size and rates of cell division (Van’t Hof
& Sparrow, 1963), large cells/genomes are proposed to result
from K-selection, yielding slow-growing species, while small cells
are suggested as being associated with r-selected species, with
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rapid turnover. This idea has received some support from studies
in vertebrates (Vinogradov, 1995), and has been tested in seed
plants on the premise that K-selected species are typically those
with high LMA and hence that LMA should be positively correlated with genome size. Although early studies provided some
support for this hypothesis (Knight et al., 2005), a larger study
found nonsignificant associations within a phylogenetically independent framework (Beaulieu et al., 2007), and similarly within
the Proteaceae we found no correlations between cell size and
LMA (Table 2).
Rather than expecting cell size to affect leaf economics in a predictable way, we argue here that by coordinating cell sizes in the
leaf, plants are free to adapt the water transport and photosynthetic gas-exchange properties of leaves while still maintaining an
optimal balance between water transport and water loss tissues.
Coordination in stomatal and hydraulic physiology provides
important functional advantages in terms of maintaining optimal
investment and safety (Brodribb, 2009) and this pressure may be
the adaptive driver that has led to cell size-dependent coordination between xylem and stomatal tissues in the leaf. Along with
other studies showing correlations between cell size and the density of stomata and veins (Murphy et al., 2012; Zhang et al.,
2012), the data here from such a diverse family as the Proteaceae
provide strong evidence of the primacy of the stomata–vein linkage. Size correlations between other leaf cells, such as epidermal
cells and palisade cells (Fig. 3), may have functional significance
as well, but it is also possible that the correlated size of these cells
is merely a consequence of a common genetic control of cell sizes,
perhaps via genome size.
Other studies have indicated the importance of cell size in regulating the density of stomata on the leaf, because constraints on
packing density mean that fewer large stomata can fit on the leaf
than smaller stomata (Beaulieu et al., 2008; Franks & Beerling,
2009). Our data show that this relationship is particularly strong
among Proteaceae (Fig. 3, Table 2). Owing to the connection
between size and density in stomata, a critical implication of
reduced stomatal cell size is a dramatic increase in the amount of
water and CO2 that can be exchanged over the leaf epidermis,
because stomatal density is allowed to increase and pore depth is
reduced. The net result of this is that a leaf epidermis built from
smaller cells will support a much higher capacity for gas
exchange, in terms of both photosynthetic CO2 and transpirational water vapour. However, without a parallel increase in the
density of minor veins in the leaf, the increased capacity for photosynthesis cannot be realized because stomata will not be sufficiently irrigated to open fully (Brodribb, 2009). Hence the
discovery here, that vein density is also sensitive to cell size in
such a way as to change in proportion with stomatal density, is
highly significant because the result is a match between water
supply and water loss from the leaf. This match is most clearly
demonstrated by the proportionality between modelled maximum stomatal conductance and vein density across all species
(Fig. 5), indicating that the hydraulic efficiency of the vein network (as determined by vein density; Brodribb et al., 2007)
remains in proportion to the water-loss capacity of the leaf. Interestingly, we found that vein density was most strongly correlated
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Fig. 6 Stomatal length and habitat type
(open vegetation or closed forest, or both)
across the full phylogenetic range of
Proteaceae, with the evolution of stomatal
length reconstructed using parsimony. A
phylogenetically adjusted one-way ANOVA
showed significantly greater stomatal length
in open vegetation species than in closed
forest species (P < 0.001). Persoonioideae has
been abbreviated to Pers. See Supporting
Information Fig. S1 for individual species
labels.
with the size of stomatal and epidermal cells, and whilst these
cells are the key determinants of stomatal density, they are separated from the xylem and mesophyll cells that make up the vein
network and surrounding tissue.
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One pathway to evolving enhanced photosynthetic rate in
leaves can be easily visualized as a process of selection for small
cell size, leading to high densities of stomata and veins in leaves
(Fig. 7). Such an evolutionary trajectory appears to have been
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Fig. 7 Schematic representation of the linkages between variation in cell
size and species adaptation, explaining why cell size should be coordinated
and why variation in cell size is a useful adaptive tool. As long as mutations
in cell size produce parallel changes in the size of all cell lines in the leaf,
the critical link between water supply and transpiration/photosynthesis will
be maintained. Selection can then act upon the functional outputs of cell
size variation in the leaf, which are photosynthetic performance and leaf
thickness, both of which are highly adaptive under different conditions.
The selective advantage of high rates of photosynthesis is relatively
straightforward, while leaf thickness is involved in a suite of associated
functional characters, such as leaf mass per unit area (LMA), stomatal
positioning and leaf water content (see the Discussion section).
significant during the evolution of the Proteaceae. Thus, one
major clade (the Grevilleoideae) is dominated by taxa with small
stomata and high vein densities. This pattern makes ecological
sense because cellular miniaturization in this group is associated
with species that occupy tropical rainforests, where rapid gas
exchange and growth tend to be selected for (K€orner, 1994).
Interestingly, however, several clades, including two large and
ecologically successful groups (Persoonioideae and Protea, each
with c. 100 species), have very large stomata. These cases are of
particular interest because they imply that apparently less productive, large cell size must also confer selective advantages to plants
under some conditions. In the Proteaceae, very large stomata
occur almost exclusively in clades of open habitats (Fig. 6), and
almost exclusively in species with thick and mostly amphistomous leaves. This association also fits the evidence that selection
under high light conditions favours thick leaves and amphistomy
(Smith et al., 1998; Cooper & Cass, 2003). Amphistomous
leaves are highly efficient in terms of photosynthesis, water use
and hydraulic supply (Mott et al., 1982), and the ability to spread
stomata more sparsely on both leaf surfaces will greatly ameliorate stomatal crowding constraints, probably allowing stomatal
size to increase. It is uncertain whether the primary selective force
in open conditions is for thick leaves and hence large cells, ultimately allowing amphistomy to develop, or whether amphistomy
is selected for initially, leading to a relaxation in spatial constraints in the leaf and allowing cell size and leaf thickness to
increase.
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Evidence suggests that amphistomy is strongly associated
with high light environments and selected against in shady
environments (Carpenter, 1994; Smith et al., 1998). While
amphistomy provides for a more efficient use of water (Mott
& O’Leary, 1984), mesophyll tissue (Parkhurst, 1978) and
vein tissue (because veins can deliver water simultaneously to
both surfaces of the leaf), these benefits require substantial illumination of both leaf surfaces, and under low light the significant internal self-shading makes this a highly inefficient
architecture. Alternatively, the more common configuration of
stomata confined to the lower leaf surface seems to provide
plants with a more efficient light-harvesting configuration
under shadier conditions. However, the restriction of stomata
to the lower leaf surface requires that stomata become densely
concentrated and hence vein density must be very high to sustain rapid water transport to this highly evaporative surface.
The combination of hypostomy and high rates of gas exchange
would therefore produce substantial pressure for simultaneous
miniaturization of both stomata and veins (Field & Brodribb,
2013). However, as long as the size of these cells declines in
concert, leaves will remain balanced in terms of hydraulic supply and evaporative load. Our data for Proteaceae indicate that
this cell size-dependent coordination does operate, and we suggest that this is possibly mediated by the size of the species
genome.
Recently, it has been suggested that changes in stomatal and
genome size in land plants may be a response to Phanerozoic
megacycles of CO2 in the atmosphere, with periods of low
CO2 favouring smaller stomata (Franks et al., 2012a). The
common link between stomatal and vein density, demonstrated
here through cell size, provides an important connection
between the evolutionary patterns described for stomatal density and similar patterns reported for vein density (Boyce et al.,
2009). Studies of leaf veins across the phylogeny of vascular
plants show that, although vein densities range from < 1 to
> 25 mm mm2, only angiosperms are able to produce leaves
with vein densities in the upper part of this range (> 6 mm
mm2). This unique capacity in angiosperms has been discussed as an important factor contributing to their success over
competing plant groups (Brodribb & Feild, 2010), and, similar
to the stomatal trend, the rise in vein density requires a
decrease in cell size (Field & Brodribb, 2013) and is likely to
respond to changes in atmospheric CO2 (Brodribb & Feild,
2010). It seems reasonable to suggest that the transition from
ferns and gymnosperms characterized by low densities of veins
and stomata to angiosperms with high densities of veins and
stomata might be mediated by a decrease in genome size, a
proposition supported by the fact that ferns and gymnosperms
tend to have large genome sizes relative to angiosperms (Leitch
et al., 2005). It remains unresolved, however, whether changing
genome size is a driver or a product of macroevolutionary
changes in vascular plants. In the context of this question, it is
relevant to note that the evolution of high vein density in angiosperms actually occurred after the divergence of early angiosperm lineages, and that the ancestral state for angiosperm vein
density is low, despite the fact that the early angiosperms are
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thought to have small genomes (Masterson, 1994) and therefore potentially small cells. A focus on the evolutionary trajectories of genome size and cell size at the base of the angiosperms
will provide important insights into the drivers of this critical
evolutionary event, and whether a linkage among genome size,
cell size and water use is universal among vascular plant species.
References
Beaulieu JM, Leitch IJ, Knight CA. 2007. Genome size evolution in relation
to leaf strategy and metabolic rates revisited. Annals of Botany 99:
495–505.
Beaulieu JM, Leitch IJ, Patel S, Pendharkar A, Knight CA. 2008. Genome size is
a strong predictor of cell size and stomatal density in angiosperms. New
Phytologist 179: 975–986.
Boyce CK, Brodribb TJ, Feild TS, Zwieniecki MA. 2009. Angiosperm leaf vein
evolution was physiologically and environmentally transformative. Proceedings
of the Royal Society of London Series B 276: 1771–1776.
Brodribb T, Feild T, Jordan G. 2007. Leaf maximum photosynthetic rate and
venation are linked by hydraulics. Plant Physiology 144: 1890–1898.
Brodribb TJ. 2009. Xylem hydraulic physiology: the functional backbone of
terrestrial plant productivity. Plant Science 177: 245–251.
Brodribb TJ, Feild TS. 2010. Leaf hydraulic evolution led a surge in leaf
photosynthetic capacity during early angiosperm diversification. Ecology Letters
13: 175–183.
Brodribb TJ, Holbrook NM, Zwieniecki MA, Palma B. 2005. Leaf hydraulic
capacity in ferns, conifers and angiosperms: impacts on photosynthetic
maxima. New Phytologist 165: 839–846.
Brodribb TJ, Jordan GJ. 2011. Water supply and demand remain balanced
during leaf acclimation of Nothofagus cunninghamii trees. New Phytologist 192:
437–448.
Carpenter RJ. 1994. Cuticular morphology and aspects of the ecology and fossil
history of North Queensland rainforest Proteaceae. Botanical Journal of the
Linnean Society 116: 249–303.
Cavalier-Smith T. 1978. Nuclear volume control by nucleoskeletal DNA,
selection for cell-volume and cell-growth rate, and solution of DNA C-value
paradox. Journal of Cell Science 34: 247–278.
Cavalier-Smith T. 1985. Cell volume and the evolution of genome size. Chichester,
UK: Wiley.
Cavalier-Smith T. 2005. Economy, speed and size matter: evolutionary forces
driving nuclear genome miniaturization and expansion. Annals of Botany 95:
147–175.
Cooper RL, Cass DD. 2003. A comparative epidermis study of the Athabasca
sand dune willows (Salix; Salicaceae) and their putative progenitors. Canadian
Journal of Botany-Revue Canadienne De Botanique 81: 749–754.
Cowan IR, Farquhar GD. 1977. Stomatal function in relation to leaf metabolism
and environment. Symposium of the Society for Experimental Biology 31:
471–505.
Dunbar-Co S, Sporck MJ, Sack L. 2009. Leaf trait diversification and design in
seven rare taxa of the Hawaiian Plantago radiation. International Journal of
Plant Sciences 170: 61–75.
Edwards EJ. 2006. Correlated evolution of stem and leaf hydraulic traits in
Pereskia (Cactaceae). New Phytologist 172: 479–789.
Field TS, Brodribb TJ. 2013. Hydraulic tuning of vein cell microstructure in the
evolution of angiosperm venation networks. New Phytologist, doi: 10.1111/
nph.12311.
Franks PJ, Beerling DJ. 2009. Maximum leaf conductance driven by CO2 effects
on stomatal size and density over geologic time. Proceedings of the National
Academy of Sciences, USA 106: 10343–10347.
Franks PJ, Farquhar GD. 2001. The effect of exogenous abscisic acid on stomatal
development, stomatal mechanics, and leaf gas exchange in Tradescantia
virginiana. Plant Physiology 125: 935–942.
Franks PJ, Freckleton RP, Beaulieu JM, Leitch IJ, Beerling DJ. 2012a.
Megacycles of atmospheric carbon dioxide concentration correlate with fossil
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
Research 11
plant genome size. Philosophical Transactions of the Royal Society B-Biological
Sciences 367: 556–564.
Franks PJ, Leitch IJ, Ruszala EM, Hetherington AM, Beerling DJ. 2012b.
Physiological framework for adaptation of stomata to CO2 from glacial to
future concentrations. Philosophical Transactions of the Royal Society B-Biological
Sciences 367: 537–546.
Hodgson JG, Sharafi M, Jalili A, Diaz S, Montserrat-Marti G, Palmer C,
Cerabolini B, Pierce S, Hamzehee B, Asri Y et al. 2010. Stomatal vs. genome
size in angiosperms: the somatic tail wagging the genomic dog? Annals of
Botany 105: 573–584.
Jordan GJ, Weston PH, Carpenter RJ, Dillon RA, Brodribb TJ. 2008. The
evolutionary relations of sunken covered, and encrypted stomata to dry habitats
in proteaceae. American Journal of Botany 95: 521–530.
Knight CA, Molinari NA, Petrov DA. 2005. The large genome constraint
hypothesis: evolution, ecology and phenotype. Annals of Botany 95:
177–190.
K€orner C. 1994. Leaf diffusive conductances in the major vegetation types of the
globe. In: Schulze ED, Caldwell MM, eds. Ecophysiology of photosynthesis.
Berlin, Germany: Springer, 463–490.
Leitch IJ, Soltis DE, Soltis PS, Bennett MD. 2005. Evolution of DNA amounts
across land plants (Embryophyta). Annals of Botany 95: 207–217.
Maddison WP, Maddison DR. 2011. Mesquite: a modular system for
evolutionary analysis. Version 2.75. URL [WWW document] http://
mesquiteproject.org [accessed on 22 November 2012].
Mast AR, Givnish TJ. 2002. Historical biogeography and the origin of stomatal
distributions in Banksia and Dryandra (Proteaceae) based on their cpDNA
phylogeny. American Journal of Botany 89: 1311–1323.
Mast AR, Willis CL, Jones EH, Downs KM, Weston PH. 2008. A smaller
Macadamia from a more vagile tribe: inference of phylogenetic
relationships, divergence times, and diaspore evolution in Macadamia and
relatives (tribe Macadamieae; Proteaceae). American Journal of Botany 95:
843–870.
Masterson J. 1994. Stomatal size in fossil plants - evidence for polyploidy in
majority of angiosperms. Science 264: 421–424.
McKown AD, Cochard H, Sack L. 2010. Decoding leaf hydraulics with a
spatially explicit model: principles of venation architecture and implications for
its evolution. American Naturalist 175: 447–460.
Mirsky AE, Ris H. 1951. The DNA content of animal cells and its evolutionary
significance. Journal of General Physiology 34: 451–462.
Mott KA, Gibson AC, O’Leary JW. 1982. The adaptive significance of
amphistomatic leaves. Plant, Cell & Environment 5: 455–460.
Mott KA, O’Leary JW. 1984. Stomatal behavior and CO2 exchange
characteristics in amphistomatous leaves. Plant Physiology 74: 47–51.
Murphy MRC, Jordan GJ, Brodribb TJ. 2012. Differential leaf expansion can
enable hydraulic acclimation to sun and shade. Plant, Cell & Environment 35:
1407–1418.
Organ CL, Shedlock AM, Meade A, Pagel M, Edwards SV. 2007. Origin of
avian genome size and structure in non-avian dinosaurs. Nature 446:
180–184.
Paradis E, Bolker B, Claude J, Cuong HS, Desper R, Durand B, Dutheil J,
Gascuel O, Heibl C, Lawson D et al. 2001. Package ‘ape’. URL [WWW
document] http://ape.mpl.ird.fr/ [accessed on 22 August 2012].
Parkhurst DF. 1978. The adaptive significance of stomatal occurrenceon one or
both surfaces of leaves. Journal of Ecology 66: 367–383.
Perez-Perez JM, Rubio-Diaz S, Dhondt S, Hernandez-Romero D, SanchezSoriano J, Beemster GTS, Ponce MR, Micol JL. 2011. Whole organ, venation
and epidermal cell morphological variations are correlated in the leaves of
Arabidopsis mutants. Plant, Cell & Environment 34: 2200–2211.
Petrov DA. 2001. Evolution of genome size: new approaches to an old problem.
Trends in Genetics 17: 23–28.
€
Rubner M. 1883. Uber
den einfluss der k€orpergr€osse auf stoff- und kraftwechsel.
Zeitschrift fur Biologie 19: 536–562.
Sack L, Frole K. 2006. Leaf structural diversity is related to hydraulic capacity in
tropical rainforest trees. Ecology 87: 483–491.
Sack L, Holbrook NM. 2006. Leaf Hydraulics. Annual Review of Plant Physiology
and Molecular Biology 57: 361–381.
New Phytologist (2013)
www.newphytologist.com
New
Phytologist
12 Research
Sauquet H, Weston PH, Anderson CL, Barker NP, Cantrill DJ, Mast AR,
Savolainen V. 2009. Contrasted patterns of hyperdiversification in
Mediterranean hotspots. Proceedings of the National Academy of Sciences, USA
106: 221–225.
Smith WK, Bell DT, Shepherd KA. 1998. Associations between leaf structure,
orientation, and sunlight exposure in five Western Australian communities.
American Journal of Botany 85: 56–63.
Stace HM, Douglas AW, Sampson JF. 1998. Did ‘paleo-polyploidy’ really occur
in proteaceae? Australian Systematic Botany 11: 613–629.
Van’t Hof J, Sparrow AH. 1963. A relationship between DNA content, nuclear
volume, and minimum mitotic cycle time. Proceedings of the National Academy
of Sciences, USA 49: 897–902.
Vinogradov AE. 1995. Nucleotypic effect in homeotherms: body-mass-corrected
basal metabolic rate of mammals is related to genome size. Evolution 496:
1249–1259.
Zhang S-B, Guan Z-J, Sun M, Zhang J-J, Cao K-F, Hu H. 2012. Evolutionary
association of stomatal traits with leaf vein density in Paphiopedilum,
Orchidaceae. PLoS ONE 7: e40080.
New Phytologist (2013)
www.newphytologist.com
Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Phylogeny of Proteaceae (and outgroup, Platanaceae),
with evolutionary reconstruction of guard cell length.
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
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