Acta Oecologica 37 (2011) 381e385
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Acta Oecologica
journal homepage: www.elsevier.com/locate/actoec
Original article
Density and length of stomatal and epidermal cells in "living fossil"
trees grown under elevated CO2 and a polar light regime
R. Ogaya a, *, L. Llorens b,1, J. Peñuelas a
a
b
Global Ecology Unit CREAF-CEAB-CSIC, CREAF (Center for Ecological Research and Forestry Applications), Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 November 2010
Accepted 27 April 2011
Available online 14 May 2011
During the Cretaceous and early Tertiary, when the climate was warm and the atmospheric CO2
concentration ([CO2]) was at least double that of the present-day, polar forests populated high latitude
landmasses. We investigated the density and length of stomata and other epidermal cells of two
deciduous and three evergreen "living fossil" tree species representative of these ancient forests. These
tree species were grown in a simulated Cretaceous high latitude environment at either ambient
(400 ppmv) or elevated (800 ppmv) [CO2] during four years. After 4 years growing at elevated [CO2], the
leaf stomatal density and index (percentage of leaf epidermal cells that are stomata) of these plants were
similar to those of their counterparts growing at ambient [CO2]. While the CO2 enrichment only modified
the stomatal pore length in two of the five studied species, it increased significantly the overall length of
the epidermal cells of all the species, reducing their density. These results revealed that leaf epidermal
cells of these "living fossil" species were more sensitive than stomata to an experimental doubling of
atmospheric CO2 concentration.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Ancient species
Climate change
CO2
Photosynthesis
Polar forests
Stomatal density
1. Introduction
Polar regions were populated by extensive forests during the
Cretaceous and early Tertiary (Kumagai et al., 1995; Cantrill and
Poole, 2005), when atmospheric CO2 concentration ([CO2]) was at
least double than that of the present day (Crowley and Berner,
2001) and climate was warm (Spicer and Chapman, 1990;
Kumagai et al., 1995). The high latitude light environment was
characterized by summers of continuous, low to moderate, irradiance followed by several months of darkness or extremely low
irradiance during winter season. Therefore, these ancient high
latitude forests would have experienced a CO2-rich atmosphere
interacting with extreme seasonal variations in daylight.
The exchange of CO2 and water vapour between a leaf and the
atmosphere is principally controlled by stomatal density (number
of stomata per unit of leaf area) and their mean aperture. Stomatal
density is known to be affected by environmental variables such as
light and atmospheric [CO2] (Bergmann, 2004; Casson and Gray,
2008). On one hand, a genotypic decrease in stomatal density has
* Corresponding author. Tel.: þ34 935814036; fax: þ34 935814151.
E-mail address: [email protected] (R. Ogaya).
1
Present address: Department of Environmental Sciences, Faculty of Sciences,
University of Girona, Campus Montilivi, E-17071 Girona, Spain.
1146-609X/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.actao.2011.04.010
been observed induced by shading conditions (Schoch et al., 1980;
Lake et al., 2001) and an increase in response to high irradiance
(Thomas et al., 2003). On the other hand, the observation of
stomatal density and index (percentage of leaf epidermal cells that
are stomata) on herbarium leaves revealed a decrease in these
variables in response to atmospheric [CO2] increases during the last
centuries (Woodward, 1987; Peñuelas and Matamala, 1990; Van
Hoof et al., 2006). In accordance, a similar phenotypic response
has been observed in plants living under different [CO2]
(Woodward et al., 2002; Driscoll et al., 2006; Casson and Gray,
2008; Sekiya and Yano, 2008). Although both density and index
respond to CO2, stomatal index is rather insensitive to changes in
soil moisture supply, atmospheric humidity and temperature
(Beerling, 1999) making it a more suitable indicator of palaeo-CO2
changes.
However, it has been demonstrated that the phenotypic
response of the stomatal index to the atmospheric [CO2] is
nonlinear (Radoglou and Jarvis, 1990; Royer, 2001; Marchi et al.,
2004; Haworth et al., 2010). Indeed, previous studies have found
that in contemporary plants the stomatal index repeatedly shows
a lower sensitivity to atmospheric CO2 levels above 340 ppm in the
short (Beerling and Royer, 2002; Casson and Gray, 2008; Riikonen
et al., 2008) and in the long term (Jones et al., 1995; Bettarini
et al., 1998), which constrains the estimation of higher-thanpresent palaeo-CO2 levels on the basis of this index.
382
R. Ogaya et al. / Acta Oecologica 37 (2011) 381e385
Another possible consequence of an atmospheric CO2-enrichment might be an increase in the length of the stomatal aperture
(length between the junctions of the guard cells at each end of the
stomata), or pore size (Wagner et al., 1996), which might contribute
to counteract a reduced stomatal density in response to high CO2
levels. Indeed, a negative correlation between stomatal size and
density has recently been shown (Franks et al., 2009; Franks and
Beerling, 2009). Also recently, Miller-Rushing et al. (2009) reported an increase in stomatal length and a decrease in stomatal
density as the [CO2] increased during the lifespan of some tree
species, although no changes took place in their intrinsic leaf water
use efficiency during this time.
Therefore, the aim of the present study was to elucidate how the
stomatal index and the stomatal and epidermal cell density and
size of tree species growing in ancient high latitude forests might
have been modified by a CO2-rich atmosphere. We determined the
stomatal and other epidermal cell densities, and the stomatal pore
and epidermal cell length in five "living fossil" tree species after
four years growth in a simulated Cretaceous high latitude environment. All of the species belong to genera known as components
of the Late Cretaceous high latitude forests.
Plants were grown in a silica sandevermiculiteepeat medium
(13:5:2), watered twice daily via an automated drip irrigator, and
fertilized with Rorison’s nutrient solution. Leaf growth started in
April and continued through September in all species. Measurements were performed during the fourth year of plant exposure to
the treatments. In August, stomata and other epidermal cell length
and density were determined from one leaf of two plants per
species, chamber and CO2 treatment.
2.3. Stomata and other epidermal cell measurements
For microscopic observations, plant leaves were bleached in
a 30% sodium hypochlorite solution during 24 h to remove the
mesophyll. The abaxial surface of bleached leaves (one leaf per
plant) was observed in a microscope (Olympus CH-2, Olympus
Optical Co., Ltd., Tokyo, Japan), and the total number of stomata and
other epidermal cells were counted in three fields of view per leaf
(0.065 mm2 per field of view) (Fig. 1). Stomatal index was estimated
as [number of stomata/(number of stomata þ number of epidermal
cells)]*100. The length of three stomatal pores (length between the
junctions of the guard cells at each end of the stomata) and six
epidermal cells per field of view was also measured.
2. Material and methods
2.1. Experimental design
We simulated an ancient polar environment using three replicate growth rooms, each one divided into two isolated chambers,
one with ambient [CO2] (400 ppm) and the other with elevated
[CO2] (800 ppm). The elevated CO2 treatment corresponded to
a conservative estimate for the Late Cretaceous (Royer et al., 2001).
A daytime photon flux of 300e400 mmol m2 s1 was provided
using sodium lamps, with cool water being pumped through a glass
jacket surrounding the bulb to minimize radiant heat flux
(Sunbeam Hydrostar; Avon Gro-Lite Systems, Bristol, UK). For the
first three years, the plants experienced a photoperiod of 69 N
with day length being adjusted weekly (Beerling and Osborne,
2002). For the fourth year, the photoperiod was changed in the
same way, i.e. weekly to simulate 75 N of latitude. During this year,
the continuous light period started at the beginning of May and
ended at mid-August. Plants in both CO2 treatments were always
submitted to the same photoperiod. Temperature within the
growth rooms ranged from 10 C in winter to 25 C in summer, and
was warmed by 5 C compared with the outside air (see Llorens
et al., 2009a for details) in order to match mean temperatures
estimated for high latitudes of the Cretaceous (Markwick, 1998).
Relative humidity within the growth rooms was maintained above
75% by using an automated misting system. Further details about
the experimental design are provided elsewhere (Beerling and
Osborne, 2002; Osborne and Beerling, 2003).
2.4. Statistical analyses
Effects of [CO2] and species on the stomatal density and index,
and on the stomatal pore and epidermal cell lengths were assessed
by means of two-way analyses of variance (ANOVA). Post-hoc
analyses were performed to test differences between species.
Stomatal index values (i) were transformed to sin1 i1/2 to meet the
normality assumptions of the ANOVA. All analyses were performed
with the Statview software package (Abacus concepts Inc., Cary,
North Carolina, USA).
2.2. Plant material
One-year old saplings of five species, three deciduous and two
evergreens, of "living fossil" trees were grown in our growth rooms
for four years. These species were: Taxodium distichum (L.) Rich
(deciduous taxodioid), Metasequoia glyptostroboides Hu Cheng
(deciduous taxodioid), Sequoia sempervirens (D. Don) Endl. (evergreen taxodioid), Araucaria araucana (Molina) K. Koch (evergreen
conifer), and Nothofagus cunninghamii (Hook.) Oerst. (evergreen
angiosperm). These taxa have long fossil records at the generic level
(>65 Myr), and their ancestors were dominant elements in Cretaceous and Paleogene polar forests (Spicer and Chapman, 1990;
Kumagai et al., 1995; Cantrill and Poole, 2005).
Fig. 1. Detail of a microscope observation of stomata and epidermal cells in a Sequoia
sempervirens leaf abaxial surface.
R. Ogaya et al. / Acta Oecologica 37 (2011) 381e385
3. Results
Stomatal density ranged from 63 stomata mm2 in A. araucana to
502 stomata mm2 in N. cunninghamii. N. cunninghamii was also the
species with the largest number of epidermal cells per mm2,
whereas the other species showed similar numbers, close to 1000
cells mm2 (Fig. 2). The deciduous gymnosperm species (T. distichum and M. glyptostroboides) had larger stomatal density
(P < 0.05) and index (P < 0.01) than evergreen gymnosperm species
(S. sempervirens and A. araucana). The evergreen angiosperm
383
N. cunninghamii showed intermediate stomatal index values
between deciduous and evergreen gymnosperm species (Fig. 2).
CO2 enrichment did not significantly affect the leaf stomatal
density or index (Fig. 2), but plants growing at elevated [CO2]
showed a lower overall number (Fig. 2) and a higher length (Fig. 3)
of epidermal cells compared to those growing at ambient [CO2]
(P < 0.01 in both cases). The effect of CO2 enrichment on the
stomatal pore length was not the same for all the species (Fig. 3).
Indeed, elevated [CO2] significantly increased and decreased the
stomatal pore length of N. cunninghamii and T. distichum respectively, while it did not significantly affect the stomatal pore length
of the other three species.
4. Discussion
The studied species did not show any phenotypical change in
their stomatal density or index in response to a doubling of
atmospheric CO2 concentration (Fig. 2). Royer et al. (2001) documented a similar lack of response in one of the species studied here,
M. glyptostroboides, after studying historical collections of leaves of
this species developed during the anthropogenically driven CO2
increase of the past 145 years, as well as saplings of this species
grown in CO2-controlled greenhouses. Several studies have reported phenotypic or genotypic decreases in stomatal density and/or
index in response to a rise in atmospheric (Woodward, 1987;
Woodward et al., 2002; Sekiya and Yano, 2008), although other
studies have shown small effects (Radoglou and Jarvis, 1990; Ryle
and Stanley, 1992) or even increases (Rowland-Bamford et al.,
Fig. 2. Mean values of leaf abaxial stomatal density, epidermal cell density and
stomatal index for plants growing at ambient (400 ppm) and elevated (800 ppm) CO2
concentrations. A.a., N.c., M.g., T.d., and S.s. correspond to Araucaria araucana, Nothofagus cunninghamii, Metasequoia glyptostroboides, Taxodium distichum, and Sequoia
sempervirens, respectively. Vertical bars indicate the standard errors of the mean.
Fig. 3. Mean values of the length of leaf abaxial stomatal pores and epidermal cells for
plants growing at ambient (400 ppm) and elevated (800 ppm) CO2 concentrations. A.a.,
N.c., M.g., T.d., and S.s. correspond to Araucaria araucana, Nothofagus cunninghamii,
Metasequoia glyptostroboides, Taxodium distichum, and Sequoia sempervirens, respectively. Vertical bars show the standard errors of the mean. Significant differences
within each species are indicated as: *P < 0.05 and **P < 0.01.
384
R. Ogaya et al. / Acta Oecologica 37 (2011) 381e385
1990; Royer, 2001; Lawson et al., 2002; Marchi et al., 2004). In fact,
experiments conducted under above present-day values of atmospheric [CO2] often show little or no change in stomatal density and
index (Estiarte et al., 1994; Ceulemans et al., 1995; Poole et al.,
2000; Medlyn et al., 2001; Reid et al., 2003; Herrick et al., 2004;
Tricker et al., 2005; Van Hoof et al., 2006; Buckley, 2008), suggesting a saturation of the response close to present-day levels. Our
results support these conclusions since we did not find a significant
effect of the CO2-enrichment on the stomatal density or index of the
5 studied species after 4 years growth under a doubling of [CO2]. On
the other hand, our measurements were performed on saplings,
and their response to CO2-enrichment is not necessarily the same
than that of adult plants.
Our study also shows that there was not a general response of the
stomatal pore length of these species to the CO2 enrichment (Fig. 3). In
previous studies with these plants in this experimental system, larger
leaf photosynthetic rates and instantaneous water use efficiency
values were found under the CO2-enriched atmosphere (Llorens et al.,
2009a, b; Royer et al., 2005; Osborne and Beerling, 2003). Therefore,
although the studied species were able to make a profit of high
atmospheric [CO2] (Llorens et al., 2009a, b; Royer et al., 2005; Osborne
and Beerling, 2003), we did not observe any phenotypic adaptation at
the level of stomatal morphology. Nevertheless, plants growing under
elevated [CO2] showed less but longer epidermal cells than those
growing at ambient [CO2], in agreement with previous studies (Taylor
et al., 1994; Kürschner et al., 1998; Poole et al., 2000; Uprety et al.,
2002; Driscoll et al., 2006). A decrease in epidermal cell density
along with an increase in the size of the epidermal cells in response to
elevated [CO2] has been related to an increased osmotic potential
associated with their higher saccharide content which causes the
cells to absorb more water and thus enlarge (DeLucia et al., 1985).
Evidences from other studies suggest that this carbon-induced
growth effect on leaf cell expansion occurs because of changes in
the biophysical properties of the cell wall, particularly enhanced
wall extensibility (Ferris and Taylor, 1994; Taylor et al., 1994;
Ranasinghe and Taylor, 1996; Ferris et al., 2002). However, there is
no evidence of a long term response, or evolutionary response, of this
epidermal cell elongation under high [CO2] concentrations.
To conclude, our results show that epidermal cells of the studied
"living fossil" species were more sensitive than stomata to an
increase of atmospheric [CO2] within the range experienced during
the Cretaceous (Crowley and Berner, 2001) when these plant genera
populated the high latitude forests (Kumagai et al., 1995; Cantrill and
Poole, 2005). Saplings often respond differently to CO2 compared to
adult trees, but the results of this study show that at least for the
studied saplings, the stomatal index (calculated from the number of
stomata and other epidermal cells) reflects the effects of high
atmospheric [CO2] on stomatal frequency better than stomatal
density, when the other epidermal cells are not taken into account.
Acknowledgments
The authors thank Steve Ellin for technical assistance and David J.
Beerling for allowing us to participate in the experiment. This study
was supported by a European Union Marie Curie postdoctoral
fellowship to L.L. (contract N EVK2eCTe2002e50022), by the
Spanish Government CGL2006-4025/BOS, CGL2007-64583/BOS and
Consolider-Ingenio MONTES CSD2008-00040 projects, the Catalan
government SGR2009-458 project, and the Spanish National
Research Council CSIC-PIF08-006-3 project.
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