Tree Physiology 31, 997–1006
doi:10.1093/treephys/tpr087
Research paper: Part of a special issue on canopy processes
in a changing climate
Leaf photosynthesis, respiration and stomatal conductance
in six Eucalyptus species native to mesic and xeric environments
growing in a common garden
James D. Lewis1,2,5, Nathan G. Phillips1,3, Barry A. Logan1,4, Carolyn R. Hricko4 and David T. Tissue1
1Hawkesbury Institute for the Environment, University of Western Sydney, Richmond, NSW 2753, Australia; 2Department of Biological Sciences, Louis Calder Center—Biological
Field Station, Fordham University, PO Box 887, Armonk, NY 10504, USA; 3Department of Geography and Environment, Boston University, 675 Commonwealth Avenue, Boston,
MA 02215, USA; 4Biology Department, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA; 5Corresponding author ([email protected])
Received May 3, 2011; accepted August 4, 2011; handling editor Michael Ryan
Trees adapted to mesic and xeric habits may differ in a suite of physiological responses that affect leaf-level carbon balance,
including the relationship between photosynthesis (A) and respiration at night (Rn). Understanding the factors that regulate
physiological function in mesic and xeric species is critical for predicting changes in growth and distribution under changing
climates. In this study, we examined the relationship between A and Rn, and leaf traits that may regulate A and Rn, in six
Eucalyptus species native to mesic or xeric ecosystems, during two 24-h cycles in a common garden under high soil moisture.
Peak A and Rn generally were higher in xeric compared with mesic species. Across species, A and Rn covaried, correlated with
leaf mass per area, leaf N per unit area and daytime soluble sugar accumulation. A also covaried with gs, which accounted for
93% of the variation in A within species. These results suggest that A and Rn in these six Eucalyptus species were linked
through leaf N and carbohydrates. Further, the relationship between A and Rn across species suggests that differences in this
relationship between mesic and xeric Eucalyptus species in their native habitats may be largely driven by environmental factors rather than inter-specific genetic variation.
Keywords: diel gas exchange, Eucalyptus, nitrogen, nocturnal, photosynthesis, respiration, source:sink balance, stomatal
conductance.
Introduction
Water availability regulates plant carbon dynamics in many
­terrestrial ecosystems (Webb et al. 1983, Weltzin et al. 2003).
Trees exhibit a suite of physiological responses to water availability, which affect leaf-level carbon balance (Niinemets 2001,
Merchant et al. 2006, Mitchell et al. 2008). For example, the
relationship between instantaneous net photosynthetic rates
(A) and respiration rates (R) has been observed to differ
between species from mesic and xeric habitats (Wright et al.
2006, Cai et al. 2010). These phenotypic differences may
reflect both genetic and environmental effects (Abrams 1994,
Warren et al. 2006, Tjoelker et al. 2008), but few studies have
examined whether species native to xeric and mesic habitats
differ in the relationship between A and R under common-­
garden conditions (Wright et al. 2006), where genetic effects
can be isolated from environmental factors. Understanding the
factors that regulate species differences in the relationship
between A and R is critical for predicting environmental effects
on leaf-level carbon balance, as carbon uptake (A) and loss (R)
drive leaf-level carbon balance.
A key factor driving differences between mesic and xeric
species in the relationship between A and R is leaf mass per
area (LMA). Xeric species often have higher LMA compared
with mesic species (Abrams et al. 1994, Niinemets 2001,
Warren et al. 2006, Mitchell et al. 2008), and increasing LMA
is closely related to increased A (Field and Mooney 1986,
© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
998 Lewis et al.
Evans 1989, Reich et al. 1998, Wright et al. 2006) and R in
many species (Wright et al. 2006). Increasing LMA may
increase A and R through increased leaf nitrogen (N) or carbohydrates (Dijkstra and Lambers 1989, Tissue et al. 2002). A
and R are broadly correlated with leaf N, which is an indicator
of enzyme concentrations because it is a major component of
proteins (Reich et al. 1998, Burton et al. 2002, Wright et al.
2006). A and R may also be linked due to the role of carbohydrates as substrates for respiration (Azcon-Bieto and Osmond
1983, Anekonda et al. 1999, Amthor 2000, Turnbull et al.
2004, Whitehead et al. 2004, Tjoelker et al. 2008).
Stomatal conductance (gs) is another key factor that may
vary between mesic and xeric species, thereby affecting the
relationship between A and R (Wright et al. 2006). Stomatal
conductance often regulates A, particularly when water is
­limiting, and a positive relationship between A and gs has been
widely observed across a broad range of species and environmental conditions (Farquhar and Sharkey 1982). At a larger
scale, water stress limits primary production of most terrestrial
ecosystems (Webb et al. 1983), suggesting a close relationship between carbon dynamics and water use. However, mesic
and xeric species may differ in the relationship between A and
gs. For example, xeric species may exhibit more conservative
diurnal patterns of gs (Austin et al. 2009, Zeppel et al. 2010),
leading to differences in diurnal patterns of A. As a result, gs
may affect the relationship between A and R through ­differential
effects on A in mesic and xeric species.
Further, assessing differences between mesic and xeric
­species in the relationship between A and R requires consideration of dark respiration at night (Rn), which may play a significant role in total daily leaf-level carbon balance (Shapiro et al.
2004). The relationship between A and R is often assessed by
comparing A with R measured on darkened leaves during the
day, but daytime R may not reflect Rn (Shapiro et al. 2004,
Wright et al. 2006, Ayub et al. 2011). Accordingly, understanding physiological traits that may differentiate mesic and xeric
species requires an understanding of the relationship between
A and Rn, and associated leaf characteristics, including leaf N,
carbohydrate status and gs.
Understanding the factors that regulate differences between
mesic and xeric species is critical for predicting changes in
growth and distribution of plants under changing climates
(Weltzin et al. 2003, Carter and White 2009). However, relatively little is known about physiological differences between
mesic and xeric species in many genera. For example, water
supply is a key factor driving Eucalyptus species distributions,
yet comparatively little research has been done on the leaflevel factors that improve drought tolerance in this genus in
xeric habitats (Ngugi et al. 2004, Merchant et al. 2006,
Merchant et al. 2007, Warren et al. 2011). Further, research on
plant responses to water a­vailability frequently focuses on
responses to water limitation, while less is known about
Tree Physiology Volume 31, 2011
­ ifferences between mesic and xeric species under high soil
d
moisture conditions. In this study, we examined relationships
among diel patterns of carbon exchange, leaf chemistry and gs
under high soil moisture conditions. Specifically, we addressed
the following questions: (i) Does the relationship between A
and Rn differ between mesic and xeric Eucalyptus species
under high soil moisture conditions in a common garden? (ii)
Do species differences in A and Rn reflect differences in LMA,
leaf [N] and leaf carbohydrate accumulation? (iii) Do species
differences in A reflect differences in gs?
To address these questions, we compared diel leaf-level
­carbon exchange, chemistry and gs of six Eucalyptus species,
including three mesic species (Eucalyptus argophloia,
Eucalyptus dunnii and Eucalyptus globulus), two xeric woodland
species (Eucalyptus sideroxylon and Eucalyptus tereticornis)
and a phreatophyte (Eucalyptus camaldulensis) (Phillips et al.
2010), grown in a common garden under high soil moisture.
The use of six species native to a wide range of habitats
allowed us to test our questions across a broad range of
­physiological capacities. In contrast to most previous studies,
which could not separate inter-specific genetic variation and
habitat-based differences, the use of a common garden
allowed us to identify genetically based differences by
minimizing the confounding effects of phenotypic variation
­
when comparing plants growing in different habitats.
Materials and methods
Site description
The study was conducted in a common garden at the Sefton
Plantation (33 °36′ 20.3′S, 150 °44′ 11.5′E) at the Hawkesbury
Forest Experiment site, a climate change research facility on the
University of Western Sydney campus in Richmond, NSW,
Australia (Sefton 2003, Phillips et al. 2010). The forest plantation was established in April 2000 (Sefton 2003); trees were 7
years old during the study period. Twenty individuals of each of
the six Eucalyptus species (E. argophloia Blakely, E. camaldulensis Dehnh., E. dunnii Maiden, E. globulus Labill. subsp. maidenii
(F. Muell) Kirkpatr., E. sideroxylon Cunn. ex Woolls subsp. sideroxylon and E. tereticornis Smith) were initially planted in adjacent
10 m × 16 m plots. Seeds were obtained from Ensis (Australian
Tree Seed Centre, ACT, Australia).
The site has been fully described in Phillips et al. (2010).
Following are the key characteristics to note about it for this
study. Soil in the Sefton Plantation is classified as a Blackendon
sand extending to 0.9 m depth, underlain by a clay hardpan.
Mean annual rainfall is 801 mm. However, cumulative rainfall
between 1 November 2007 and 31 July 2008 was 19% more
than the long-term average (Australian Government Bureau of
Meteorology, Richmond—UWS Hawkesbury Station; www.
bom.gov.au). An important implication of the unusually wet
Photosynthesis and respiration covary in Eucalyptus 999
period was that soil moisture was high and unlikely to generate
large or persistent plant water stress during this study.
Relationships between A, Rn and gs
Leaf-level carbon dynamics and gs of sunlit, unshaded, fully
expanded, upper crown leaves of the six Eucalyptus species
were measured during two 24-h periods (2 and 11 January
2008). Both days were generally cloudless, and air ­temperature,
vapour pressure deficit and photosynthetically active radiation
(Q, μmol m−2 s−1) were similar on both days. The crowns of three
to four trees of the six Eucalyptus species, E. argophloia (four
trees), E. camaldulensis (three), E. dunnii (four), E. grandis (four),
E. sideroxylon (three) and E. tereticornis (three), were accessed
using aluminium scaffolding with work platforms. From each
tree, two leaves were chosen for repeated measurements of net
carbon dioxide exchange (A and Rn) and gs. Gas exchange measurements were conducted about every 3 h using LI-6400
Portable Photosynthesis Systems (LICOR Inc., Lincoln, NE, USA),
commencing at 07:00 Australian Eastern Daylight Time (AEDT)
and ending at 04:00 AEDT the following morning. The same two
leaves were used for measurement every 3 h and during both
dates, except on the rare occasions when leaf damage necessitated using a new leaf. During each measurement period, cuvette
block temperatures, leaf-to-air vapour pressure deficits (D) and
Q were maintained under prevailing ambient conditions for the
site. Accordingly, across species, all leaves were measured
under the same temperature, D, Q and [CO2] conditions at a
given sampling period. The reference [CO2] was set to
400 µmol mol−1. Q was regulated using the built-in blue and red
light-emitting diode source mounted above the leaf cuvette
(LI-6400-02B) and the cuvette air flow rate was set to
500 µmol s−1 during daytime. For measurements at night of Rn,
the cuvette air flow rate was reduced to 300 µmol s−1. Readings
were recorded after leaves reached stable values for at least
5 min. For each leaf, we recorded three to five readings at a
given sampling period; presented values and statistical analyses
were based on the average of these readings. In addition, spot
measurements were ­periodically made on adjacent leaves and
we found that repeated measurements on the same leaves did
not affect measured values.
Leaf chemistry
Leaf samples for nitrogen and carbohydrate analyses were
­collected three times (07:00, 19:00 and 04:00) during both
24-h measurement periods and immediately placed into liquid
nitrogen. Leaf tissues were subsequently oven-dried at 80 °C
for 48 h and then dried leaves were milled to a fine powder
using a ball mill. Leaf [N] was determined using a CHN analyser
(LECO TruSpec, LECO Corporation, MI, USA). Leaf soluble sugars and starch were determined colorimetrically using the
phenol–­sulphuric acid technique of Tissue and Wright (1995);
total non-structural carbohydrate (TNC) was calculated as the
sum of soluble sugars and starch. Daytime carbohydrate accumulation was calculated as the difference between carbohydrate concentrations in the early morning (07:00) and at dusk
(19:00) and nocturnal carbohydrate loss was calculated as the
difference between carbohydrate c­oncentrations at dusk
(19:00) and pre-dawn (04:00).
Statistical analyses
This study examined the relationships between A, Rn, leaf
chemistry and gs across six Eucalyptus species. Statistical tests
were conducted using R version 2.13.0 (R Development Core
Team 2009). Repeated-measures analysis of variance was
used to test for the effect of species and measurement date on
diel patterns of gas exchange and leaf chemistry. Results indicated that most variables did not differ between dates, and
generally there were no interactions between species and
dates; therefore, we only present effects of species and time of
day. Regression analyses were used to examine relationships
between A, Rn, leaf chemistry and gs within and across species.
Normal probability plots and plots of residuals versus predicted
values indicated that assumptions of normality and homogeneity of variances were met for all variables and no transformations were necessary. For significant effects, Tukey’s honest
significant difference test was used for pairwise comparisons
of means. In all analyses, test results were considered significant if P ≤ 0.05.
Results
Relationships between A, Rn and gs
To examine if relationships between A and Rn were conserved
across species, we measured daytime and nocturnal patterns
of carbon dynamics in six species of Eucalyptus. Diel carbon
dynamics (Figure 1) and gs (Figure 2) varied among species
(Table 1). Light-saturated A and gs at 10:00 were highest in E.
camaldulensis, E. sideroxylon and E. tereticornis (the phreatophyte and the two xeric species, respectively) and lowest in E
argophloia, E. dunnii and E. grandis (the three mesic species).
The highest values for A were observed at 10:00 in E. dunnii, E.
grandis and E. tereticornis, while A was similar at 10:00 and
13:00 in E argophloia, E. camaldulensis and E. sideroxylon.
Across the nocturnal measurement periods (22:00–04:00), Rn
values were highest in E. grandis, E. sideroxylon and E. tereticornis, and were lowest in E. argophloia and E. dunnii (Figure 1).
Nocturnal gs values increased between 22:00 and 04:00 in E.
grandis, but did not vary over time in the other five species.
To examine relationships among A, Rn and gs, we compared
A at 10:00 with Rn at 22:00 and gs at 10:00. A at 10:00 was
representative of maximum observed A, while Rn at 22:00 was
representative of Rn across the night, as Rn did not vary across
measurement periods. Across species, Rn increased with
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1000 Lewis et al.
Figure 1. ​Mean (±SE) net photosynthetic and respiration rates during the day and night (hatched zones), respectively, for six Eucalyptus species
averaged across two 24-h measurement cycles. Note the differences in the y-axis scales for daytime and nocturnal rates. Xeric species (E. sideroxylon,
E. tereticornis) and the phreatophyte (E. camaldulensis) shown in solid symbols, mesic species (E. argophloia, E. dunnii and E. globulus) shown with
open symbols. n = 3–4 for each species during each measurement period.
increasing A (P < 0.001); A accounted for 44% of the variation
in Rn (Figure 3). A increased with gs at 10:00 (P < 0.001). The
relationship between A and gs at 10:00 varied among species
(P = 0.008), with gs and species combined accounting for
93% of the variation in A. The response of A to gs was similar
in all species except E. camaldulensis, which had a lower A for
a given gs than the other species. Nonetheless, E. camaldulensis had the highest A at 10:00 among all six species because it
had the highest gs at 10:00.
Leaf chemistry
Leaf N per unit area differed among species (Tables 1 and
2), but did not vary over the day. Leaf N per unit area was
Tree Physiology Volume 31, 2011
­highest in E. sideroxylon and E. camaldulensis, and lowest in
E. dunnii. Leaf soluble sugar, starch and TNC per unit area
increased over the day, decreased over the night and differed among species. Leaf TNC concentrations per unit area
at sunset (19:00) were lowest in E. sideroxylon, and highest
in E. camaldulensis and E. dunnii. The magnitude of the
increases in leaf ­soluble sugars, starch and TNC over the
course of the day, as well as the magnitude of the decreases
over the course of the night, was highest in E. sideroxylon
and E. camaldulensis, and lowest in E. dunnii. Species with
the highest leaf N had the highest LMA (Table 2). However,
there was no clear relationship between leaf carbohydrates
and LMA across species.
Photosynthesis and respiration covary in Eucalyptus 1001
Figure 2. ​Mean (±SE) stomatal conductance (gs) during the day and night (hatched zones), respectively, averaged across two 24-h measurement
cycles. Note the differences in the y-axis scales for daytime and nocturnal measurements. Species are differentiated by discrete symbols as in
Figure 1. n = 3–4 for each species during each measurement period.
Table 1. ​Summary of repeated-measures ANOVA results for the effect of species (S) over time (T) and between measurement days (D) on the
indicated variables.
Variable
S
T
D
S × T
S × D
T × D
S × T × D
A
gs
Leaf [N]
Leaf soluble sugar
concentration
Leaf starch concentration
Leaf TNC concentration
***
***
*
*
***
***
ns
*
ns
ns
ns
***
**
***
ns
ns
ns
ns
ns
ns
ns
ns
ns
***
ns
ns
ns
ns
***
***
**
***
***
ns
ns
ns
*
ns
ns
*
*
ns
ns, not significant.
*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001
Relationships between leaf physiology and chemistry
Higher light-saturated A at 10:00 was associated with
increased daytime accumulation of leaf soluble sugar (Figure
4; P = 0.024) and with a marginal increase in starch accumulation (Figure 4; P = 0.068). A at 10:00 increased with leaf N
per unit area (Figure 4; P < 0.001). Rn increased with leaf N
per unit area and daytime accumulation of soluble sugars
(Figure 4; P < 0.001 in both cases). Further, increasing Rn
was associated with greater nocturnal depletion of leaf soluble sugars (P = 0.002). Rn did not vary with daytime accumulation of starch (Figure 4; P = 0.900) or nocturnal depletion of
starch (P = 0.935).
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1002 Lewis et al.
relationship between A and Rn, despite greater A and Rn in the
xeric species in this study. Rather, our results suggest that
inter-specific variation in diurnal patterns of gs, which accounted
for 93% of the variation in A, plays a greater role in regulating
differences in leaf-level carbon balance among these species,
even under high soil moisture conditions.
At least two factors may account for our observed general
relationship between A and Rn across species, in contrast to
previous research. Higher R at a given A in xeric compared
with mesic species may be due to physiological responses to
drier soils (Wright et al. 2006). In our study, we measured
plants grown under common-garden conditions that enabled
us to compare species under similar environmental conditions.
Hence, the consistent relationship between A and Rn across
our species suggests that physiological differences between
mesic and xeric species observed in other studies may be
­primarily driven by environmental factors, such as soil moisture
(Warren et al. 2006). Also, in our study, respiration was
­measured at night, rather than on dark-adapted leaves during
the daytime. Rn may differ from daytime R because Rn reflects
the coupled respiratory biochemistry and lower air temperatures observed at night, while darkened leaves in the daytime
reflect potentially uncoupled respiratory biochemistry and
higher air temperatures than commonly observed at night
(Shapiro et al. 2004, Wright et al. 2006).
Relationships among respiration, photosynthesis and
leaf chemistry
Figure 3. ​Relationships between net photosynthetic rates at 10:00
(A), respiration rates at 22:00 (Rn ) (a), and stomatal conductance at
10:00 (gs, 10:00) (b) for six Eucalyptus species. Species are differentiated by discrete symbols as in Figure 1. Regression analyses incorporated species as an independent variable, to account for potential
differences among species. The slope (m) and coefficient of determination (r 2) are shown for each regression.
Discussion
Peak midday A and nocturnal respiration (Rn) generally were
higher in xeric species compared with mesic species. A
accounted for 44% of the variation in Rn across species,
­suggesting that A and Rn share a common relationship across
the six Eucalyptus species. The shared relationship across
­species native to mesic and xeric habitats differs from a previous study in which the relationship between A and Rn differed
between mesic and xeric species (Wright et al. 2006).
However, the shared relationship across species is consistent
with that of Warren et al. (2006), who observed that leaf traits
of 29 Eucalyptus species generally did not differ between
mesic and xeric species. These results suggest that adaptations in Eucalyptus to xeric habitats do not involve shifts in the
Tree Physiology Volume 31, 2011
Peak midday A covaried with Rn across species, and the
­relationship between A and Rn was correlated with leaf N per
unit area and soluble sugar accumulation. Both light-saturated
A and Rn increased with leaf N per unit area, as has been
observed in a broad range of species (Reich et al. 1998,
Burton et al. 2002, Wright et al. 2006). A often shows a strong
positive relationship with leaf N, reflecting the large proportion
of leaf N that is in the chloroplast (Field and Mooney 1986,
Evans 1989, Reich et al. 1998), while Rn increases with leaf N
because leaf N often reflects overall enzyme concentrations in
leaves (Reich et al. 1998, Wright et al. 2006). A and Rn also
covaried with daytime soluble sugar accumulation, but not
starch accumulation. A produces soluble sugars, which are the
primary substrate for Rn. Accordingly, soluble sugar accumulation with increasing A would increase the substrate pool and
subsequently Rn. Consistent with this, daytime soluble sugar
accumulation increased with A, and Rn increased with daytime
soluble sugar accumulation. Across species, soluble sugar
depletion overnight increased with increasing Rn, as has been
observed in other studies (Bouma et al. 1995), suggesting that
in these six Eucalyptus species, Rn may play a key role in regulating the size of the soluble sugar pool.
Xeric species often exhibit higher maximum A and R
­compared with mesic species, associated with greater LMA in
Photosynthesis and respiration covary in Eucalyptus 1003
Table 2. ​Area-based leaf N, soluble sugars, starch, total non-structural carbohydrates (TNC) and leaf mass per area (LMA) at dawn (07:00), sunset
(19:00) and the following pre-dawn sampling period (04:00) for six Eucalyptus species on 11 and 12 January 2008.
Variable
E. agrophloia
Leaf N (g m−2)
​ ​07:00
2.34 ± 0.07
​ ​19:00
2.32 ± 0.07
​ ​04:00
2.29 ± 0.15
Soluble sugars (g m−2)
​ ​07:00
4.51 ± 0.26
​ ​19:00
6.21 ± 0.51
​ ​04:00
4.65 ± 0.39
Starch (g m−2)
​ ​07:00
3.35 ± 0.28
​ ​19:00
5.43 ± 0.75
​ ​04:00
4.59 ± 0.29
TNC (g m−2)
​ ​07:00
7.86 ± 0.29
​ ​19:00
11.64 ± 0.89
​ ​04:00
9.24 ± 0.55
LMA (g m−2)
​ ​07:00
134.6 ± 6.5
​ ​19:00
139.3 ± 6.1
​ ​04:00
127.8 ± 6.2
E. camaldulensis
E. dunnii
E. grandis
E. sideroxylon
E. tereticornis
3.35 ± 0.19
3.11 ± 0.08
3.42 ± 0.14
2.02 ± 0.13
1.72 ± 0.09
2.07 ± 0.09
2.14 ± 0.18
2.60 ± 0.08
2.41 ± 0.09
2.89 ± 0.07
2.68 ± 0.15
3.04 ± 0.10
2.59 ± 0.18
2.29 ± 0.11
2.57 ± 0.09
3.40 ± 0.40
5.01 ± 0.46
3.52 ± 0.66
5.86 ± 0.77
8.99 ± 0.70
5.93 ± 0.34
5.24 ± 0.91
5.38 ± 0.81
3.90 ± 0.54
2.81 ± 0.36
4.42 ± 0.36
2.95 ± 0.58
4.38 ± 0.74
6.61 ± 0.54
4.07 ± 0.62
3.06 ± 0.41
4.27 ± 0.39
4.18 ± 0.24
7.72 ± 0.91
7.65 ± 1.23
7.34 ± 0.89
5.05 ± 0.46
4.86 ± 0.25
4.78 ± 0.85
2.58 ± 0.50
3.59 ± 0.45
3.35 ± 0.58
4.05 ± 0.55
5.12 ± 0.47
3.78 ± 0.84
6.47 ± 0.15
9.29 ± 0.69
7.70 ± 0.77
13.38 ± 1.41
16.64 ± 1.25
13.27 ± 1.03
10.29 ± 0.92
10.25 ± 0.79
8.68 ± 0.79
5.39 ± 0.37
8.01 ± 0.46
6.31 ± 0.47
8.43 ± 0.44
11.73 ± 0.74
7.85 ± 0.90
104.2 ± 5.2
95.3 ± 2.3
107.7 ± 4.1
125.5 ± 9.0
157.4 ± 5.3
133.3 ± 6.7
169.8 ± 3.2
161.1 ± 10.3
183.6 ± 1.7
140.5 ± 5.7
129.5 ± 2.0
139.6 ± 2.6
198.3 ± 3.3
187.1 ± 4.0
204.8 ± 7.3
Data are presented as means ± 1 SE. n = 3–4.
xeric species (Abrams et al. 1994, Niinemets 2001, Warren
et al. 2006, Mitchell et al. 2008). In this study, LMA generally
was higher in xeric species and the phreatophyte. Increased
LMA was associated with increased leaf N per unit leaf area,
and both A and Rn increased with leaf N per unit area.
Accordingly, our results suggest that differences among
­species in A and Rn reflected species differences in leaf N,
associated with differences in LMA. However, within species,
the relationship between A and Rn did not clearly vary with leaf N.
This may be because Rn primarily reflected variation in N, but A
also covaried with daytime gs. These patterns suggest that
broad relationships in A and Rn in these Eucalyptus species are
driven by leaf N, but within species, A is also regulated by diurnal patterns in gs, even under the high soil moisture, low vapour
pressure deficit conditions in this study.
Relationships between daytime carbon dynamics and gs
Peak midday A varied with gs, and increases in peak midday A
were associated with increased soluble sugar accumulation.
The close relationship between A and gs was consistent with a
wide range of studies that suggest that A and daytime gs are
closely linked (Medlyn et al. 2001, Lewis et al. 2002a,
Kreuzwieser and Gessler 2010), reflecting the role of gs in regulating the supply of CO2 to the site of carboxylation. Increasing
soluble sugar accumulation with increasing A is consistent with
a short-term (minutes to hours) source:sink limitation (Herold
1980, Lewis et al. 2002b). Short-term carbohydrate accumulation in leaves may also occur as an osmoregulatory mechanism
(Arndt et al. 2008). Eucalyptus species from mesic habitats
have been shown to differ from Eucalyptus species from xeric
habitats in their patterns of carbohydrate accumulation for
osmoregulation (Merchant et al. 2006). Regardless of the
mechanisms regulating soluble sugar accumulation, these
results suggest that gs, coupled with leaf N per unit area, primarily regulates diurnal patterns in A in these six species under
the high soil moisture conditions of our study; gs accounted for
93% of the variation in A within species. Our previous work on
these species indicates that vapour pressure deficit (D) is a
key factor regulating nocturnal patterns in sap flow, transpiration and gs at this site (Phillips et al. 2010). In turn, variation in
A resulting from changes in gs may drive differences in carbohydrate accumulation among these species.
Nocturnal gs, leaf chemistry and photosynthesis
Understanding physiological differences between mesic and
xeric species requires an understanding of leaf-level carbon
dynamics and water fluxes over a full 24-h (diel) period.
Nocturnal sap flux occurs in a wide range of plant species
(Darwin 1898, Levitt 1967, Gindel 1970), and may account
for up to 20% of total diel water flux in woodlands (Dawson
et al. 2007). In this study, nocturnal gs ranged from 5 to 20%
of peak midday gs. The adaptive significance of nocturnal gs,
­particularly under dry conditions, is unclear (Phillips et al.
2010, Zeppel et al. 2011). Some studies have linked nocturnal gs with increased N uptake by plants (McDonald et al.
2002, Snyder et al. 2003, Scholz et al. 2007, Cramer et al.
2009). Although leaf N would not be expected to respond to
short-term variation in gs, it may reflect variation in nocturnal
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1004 Lewis et al.
Figure 4. ​Relationships between net photosynthetic rates (A) at 10:00, dark respiration rates at night (Rn) at 22:00 and leaf [N] on an area basis
(a, b), changes in soluble sugar (SS) concentrations on an area basis (c, d) and changes in starch concentrations on an area basis (e, f) across six
Eucalyptus species. Species are differentiated by discrete symbols as in Figure 1. The slope (m) and coefficient of determination (r 2) are shown for
each significant regression.
gs if differences in nocturnal gs were related to longer-term
differences in nutrient acquisition (Caird et al. 2007, Dawson
et al. 2007, Scholz et al. 2007). However, although leaf [N]
varied across species, nocturnal gs exhibited limited interspecific variation, both during our study period and during a
longer-term study using the same trees (Phillips et al. 2010).
These results suggest that nocturnal gs in these Eucalyptus
species is substantial, but may not have a significant effect on
N uptake, consistent with studies on other species (Ludwig
et al. 2006, Caird et al. 2007, Howard and Donovan 2007,
Marks and Lechowicz 2007, Scholz et al. 2007, Howard and
Donovan 2010).
Tree Physiology Volume 31, 2011
Some studies have indicated that nocturnal gs may increase
total daily carbon uptake by rapidly increasing A at very low
light levels in the period after dawn (Oren et al. 1999, Dawson
et al. 2007). However, our results suggest that variation in
early-morning A was not related to pre-dawn gs. The highest gs
values were observed in E. grandis, which had relatively low
early-morning A; gs was relatively uniform across the other five
species. Although increasing pre-dawn gs has been related to
faster stomatal opening (Mansfield and Heath 1961) and
increased early-morning A (Oren et al. 1999, Dawson et al.
2007), there is limited evidence that pre-dawn gs affects A or
gs during the following day (Caird et al. 2007).
Photosynthesis and respiration covary in Eucalyptus 1005
Conclusions
Leaf carbon balance reflects the relationship between photosynthesis (A) and dark respiration at night (Rn). In this study, A
accounted for 44% of the variation in Rn across species, indicating that A and Rn share a general relationship in these six
Eucalyptus species. The use of a common-garden experimental
approach allowed us to differentiate between genetic and environmental control of physiological differences among species.
Given the similarity across species in the physiological
response to environmental conditions, our results suggest that
the A and Rn relationship was primarily regulated by environmental factors. Within these eucalypt species, A and Rn
increased with higher leaf N per unit area and higher daytime
leaf soluble sugar accumulation. A also covaried with gs, which
accounted for 93% of the variation in A within species. These
results suggest that A and Rn are linked through leaf N and
carbohydrates. Further, the xeric species and the phreatophyte
have higher A and Rn compared with the mesic species due to
greater LMA, associated with greater leaf N, and carbohydrates. In summary, the relationship between A and Rn across
species suggests that differences between mesic and xeric
species observed in other studies may be primarily driven by
environmental factors, such as soil moisture, rather than reflect
inter-specific genetic variation.
Acknowledgments
We thank Renee Attard, Renee Smith and Dr Kaushal Tewari for
their assistance with the field and laboratory campaigns, and
Drs Mike Ryan and Melanie Zeppel for their comments on earlier drafts of this manuscript. This is contribution number 253
from the Louis Calder Center and Biological Station, Fordham
University.
Funding
Discovery Project Number DP0879531 (B.L., N.P. and D.T.) of
the Australian Research Council; University of Western Sydney
International Research Initiatives Scheme (Grant Number
71827 to N.P. and 71846 to J.L.); Boston University; Fordham
University; the Roberts Fund at Bowdoin College (C.H.).
References
Abrams, M.D. 1994. Genotypic and phenotypic variation as stress
adaptations in temperate tree species: a review of several case
studies. Tree Physiol. 14:833–842.
Abrams, M.D., M.E. Kubiske and S.A. Mostoller. 1994. Relating wet and
dry year ecophysiology to leaf structure in contrasting temperate
tree species. Ecology 75:123–133.
Amthor, J.S. 2000. The McCree–de Wit–Penning de Vries–Thornley
respiration paradigms: 30 years later. Ann. Bot. 86:1–20.
Anekonda, T.S., R.S. Criddle, M. Bacca and L.D. Hansen. 1999.
Contrasting adaptation of two Eucalyptus subgenera is related to
differences in respiratory metabolism. Funct. Ecol. 13:675–682.
Arndt, S.K., S.J. Livesley, A. Merchant, T.M. Bleby and P.F. Grierson.
2008. Quercitol and osmotic adaptation of field-grown Eucalyptus
under seasonal drought stress. Plant Cell Environ. 31:915–924.
Austin, M.P., T.M. Smith, K.P. Van Niel and A.B. Wellington. 2009.
Physiological responses and statistical models of the environmental
niche: a comparative study of two co-occurring Eucalyptus species.
J. Ecol. 97:496–507.
Ayub, G., R.A. Smith, D.T. Tissue and O. Atkin. 2011. Impacts of drought
on leaf respiration in darkness and light in Eucalyptus saligna
exposed to industrial-age atmospheric CO2 and growth ­temperature.
New Phytol. 190:1003–1018.
Azcon-Bieto, J. and C.B. Osmond. 1983. Relationship between photosynthesis and respiration. The effect of carbohydrate status on the
rate of CO2 production by respiration in darkened and illuminated
wheat leaves. Plant Physiol. 71:574–581.
Bouma, T.J., R. De Visser, P.H. Van Leeuwen, M.J. De Kock and H.
Lambers. 1995. The respiratory energy requirements involved in
nocturnal carbohydrate export from starch-storing mature source
leaves and their contribution to leaf dark respiration. J. Exp. Bot.
46:1185–1194.
Burton, A.J., K.S. Pregitzer, R.W. Ruess, R.L. Hendrick and M.F. Allen.
2002. Root respiration in North American forests: effects of nitrogen concentration and temperature across biomes. Oecologia
131:559–568.
Cai, T., L.B. Flanagan and K.H. Syed. 2010. Warmer and drier conditions stimulate respiration more than photosynthesis in a boreal
peatland ecosystem: Analysis of automatic chambers and eddy
covariance measurements. Plant Cell Environ. 33:394–407.
Caird, M.A., J.H. Richards and L.A. Donovan. 2007. Nighttime stomatal
conductance and transpiration in C3 and C4 plants. Plant Physiol.
143:4–10.
Carter, J.O. and D. White. 2009. Plasticity in the Huber value contributes to homeostasis in leaf water relations of a mallee Eucalypt with
variation to groundwater depth. Tree Physiol. 29:1407–1418.
Cramer, M.D., H.-J. Hawkins and G.A. Verboom. 2009. The importance
of nutritional regulation of plant water flux. Oecologia 161:15–24.
Darwin, F. 1898. Observations on stomata. Philos. Trans. R. Soc. Lond.
B 190:531–621.
Dawson, T., S. Burgess, K. Tu, R. Oliveira, L. Stantiago, J. Fisher, K.
Simonin and A. Ambrose. 2007. Nighttime transpiration in woody
plants from contrasting ecosytems. Tree Physiol. 27:561–576.
Dijkstra, P. and H. Lambers. 1989. Analysis of specific leaf area and
photosynthesis of two inbred lines of Plantago major differing in relative growth rate. New Phytol. 113:283–290.
Evans, J.R. 1989. Photosynthesis and nitrogen relationships in leaves
of C3 plants. Oecologia 78:9–19.
Farquhar, G.D. and T.D. Sharkey. 1982. Stomatal conductance and
photosynthesis. Annu. Rev. Plant Physiol. 33:317–45.
Field, C., and H.A. Mooney. 1986. The photosynthesis nitrogen relationship in wild plants. In On the Economy of Form and Function. Ed. T.J.
Givnish. Cambridge University Press, Cambridge, UK, pp 25–55.
Gindel, I. 1970. The nocturnal behaviour of xerophytes grown under
arid conditions. New Phytol. 69:399–404.
Herold, A. 1980. Regulation of photosynthesis by sink activity: the
missing link. New Phytol. 86:131–144.
Howard, A.R. and L.A. Donovan. 2007. Helianthus nighttime conductance and transpiration respond to soil water but not nutrient availability. Plant Physiol. 143:145–155.
Howard, A.R., and L.A. Donovan. 2010. Soil nitrogen limitation does
not impact nighttime water loss in Populus. Tree Physiol.
30:23–31.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1006 Lewis et al.
Kreuzwieser, J., and A. Gessler. 2010. Global climate change and tree
nutrition: influence of water availability. Tree Physiol. 30:1221–1234.
Levitt, J. 1967. The mechanism of stomatal action. Planta (Berl.)
74:101–118.
Lewis, J.D., M. Lucash, D.M. Olszyk and D.T. Tingey. 2002a. Stomatal
responses of Douglas-fir seedlings to elevated carbon dioxide and
temperature during the third and fourth years of exposure. Plant Cell
Env. 25:1411–1421.
Lewis, J.D., X.Z. Wang, K.L. Griffin and D.T. Tissue. 2002b. Effects of age
and ontogeny on photosynthetic responses of a determinate annual
plant to elevated CO2 concentrations. Plant Cell Env. 25:359–368.
Ludwig, F., R.A. Jewitt and L.A. Donovan. 2006. Nutrient and water
addition effects on day- and night-time conductance and transpiration in a C3 desert annual. Oecologia 148:219–225.
Mansfield, T.A., and O.V.S. Heath. 1961. Photoperiodic effects on stomatal behaviour in Xanthium pennsylvanicum. Nature 191:974–975.
Marks, C.O. and M.J. Lechowicz. 2007. The ecological and functional
correlates of nocturnal transpiration. Tree Physiol. 27:577–584.
McDonald, E.P., J.E. Erickson and E.L. Kruger. 2002. Can decreased
transpiration limit plant nitrogen acquisition in elevated CO2? Funct.
Plant Biol. 29:1115–1120.
Medlyn, B.E., C.V.M. Barton, M.S.J. Broadmeadow et al. 2001. Stomatal
conductance of forest species after long-term exposure to elevated
CO2 concentration: a synthesis. New Phytol. 149:247–264.
Merchant, A., M. Tausz, S.K. Arndt and M.A. Adams. 2006. Cyclitols
and carbohydrates in leaves and roots of 13 Eucalyptus species suggest contrasting physiological responses to water deficit. Plant Cell
Environ. 29:2017–2029.
Merchant, A., A. Callister, S. Arndt, M. Tausz and M. Adams. 2007.
Contrasting physiological responses of six Eucalyptus species to
water deficit. Ann. Bot. 100:1507–1515.
Mitchell, P., E. Veneklaas, H. Lambers and S. Burgess. 2008. Using
multiple trait associations to define hydraulic functional types in plant
communities of south-western Australia. Oecologia 158:385–397.
Ngugi, M.R., D. Doley, M.A. Hunt, P. Ryan and P. Dart. 2004.
Physiological responses to water stress in Eucalyptus cloeziana and
E. argophloia seedlings. Trees—Struct. Funct. 18:381–389.
Niinemets, Ü. 2001. Global-scale climatic controls of leaf dry mass per
area, density and thickness in trees and shrubs. Ecology
82:453–469.
Oren, R., N. Phillips, B. Ewers, D. Pataki and J. Megonigal. 1999. Sapflux-scaled transpiration responses to light, vapour pressure deficit,
and leaf area reduction in a flooded Taxodium distichum forest. Tree
Physiol. 19:337–347.
Phillips, N.G., J.D. Lewis, B.A. Logan and D.T. Tissue. 2010. Inter- and
intra-specific variation in nocturnal water transport in Eucalyptus.
Tree Physiol. 30:586–596.
R Development Core Team. 2009. R: a language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna, Austria.
Reich, P.B., M.B. Walters, D.S. Ellsworth, J.M. Vose, J.C. Volin, C.
Gresham and W.D. Bowman. 1998. Relationships of leaf dark respiration to leaf nitrogen, specific leaf area and leaf life-span: a
test across biomes and functional groups. Oecologia
114:471–482.
Scholz, F.G., S.J. Bucci, G. Goldstein, F.C. Meinzer, A.C. Franco and F.
Miralles-Wilhelm. 2007. Removal of nutrient limitations by long-term
Tree Physiology Volume 31, 2011
fertilization decreases nocturnal water loss in savanna trees. Tree
Physiol. 27:551–559.
Sefton, C.A. 2003. Genetic and environmental variation in leaf
­morphology of juvenile eucalypt leaves. Ph.D. thesis, University of
Western Sydney, Richmond, NSW, Australia.
Shapiro, J.B., K.L. Griffin, J.D. Lewis and D.T. Tissue. 2004. Response of
Xanthium strumarium leaf respiration in the light to elevated CO2
concentration, nitrogen availability and temperature. New Phytol.
162:377–386.
Snyder, K.A., J.H. Richards and L.A. Donovan. 2003. Night-time
­conductance in C3 and C4 species: do plants lose water at night? J.
Exp. Bot. 54:861–865.
Tissue, D.T. and S.J. Wright. 1995. Effect of seasonal water availability
on phenology and the annual shoot carbohydrate cycle of tropical
forest shrubs. Funct. Ecol. 9:518–527.
Tissue, D.T., J.D. Lewis, S.D. Wullschleger, J.S. Amthor, K.L. Griffin and
O.R. Anderson. 2002. Leaf respiration at different canopy positions
in sweetgum (Liquidambar styraciflua) grown in ambient and elevated concentrations of carbon dioxide in the field. Tree Physiol.
22:1157–1166.
Tjoelker, M.G., J. Oleksyn, P.B. Reich and R. Zytkowiak. 2008. Coupling
of respiration, nitrogen, and sugars underlies convergent temperature acclimation in Pinus banksiana across wide-ranging sites and
populations. Glob. Change Biol. 14:782–797.
Turnbull, M.H., D.T. Tissue, R. Murthy, X. Wang and K.L. Griffin.
2004. Nocturnal warming increases photosynthesis at elevated
CO2 partial pressure in Populus deltoides. New Phytol.
161:819–826.
Warren, C.R., E. Dreyer, M. Tausz and M.A. Adams. 2006. Ecotype
adaptation and acclimation of leaf traits to rainfall in 29 species of
16-year-old Eucalyptus at two common gardens. Funct. Ecol.
20:929–940.
Warren, C.R., I. Aranda and F.J. Cano. 2011. Responses to water stress
of gas exchange and metabolites in Eucalyptus and Acacia spp. Plant
Cell Environ. doi:10.1111/j.1365-3040.2011.02357.x.
Webb, W.L., W.K. Lauenroth, S.R. Szarek and R.S. Kinerson. 1983.
Primary production and abiotic controls in forests, grasslands, and
desert ecosystems in the United States. Ecology 64:134–151.
Weltzin, J.F., M.E. Loik, S. Schwinning, et al. 2003. Assessing the
response of terrestrial ecosystems to potential changes in precipitation. Bioscience 53:941–952.
Whitehead, D., K.L. Griffin, M.H. Turnbull, D.T. Tissue, V.C. Engel, K.J.
Brown, W.S.F. Schuster and A.S. Walcroft. 2004. Response of
total night-time respiration to differences in total daily photosynthesis in a canopy of Quercus rubra L. Glob. Change Biol.
10:925–938.
Wright, I.J., P.B. Reich, O. Atkin, C.H. Lusk, M.G. Tjoelker and M.
Westoby. 2006. Irradiance, temperature and rainfall influence leaf
dark respiration in woody plants: evidence from comparisons across
20 sites. New Phytol. 169:309–319.
Zeppel, M.J.B., D. Tissue, D.T. Taylor, C. Macinnis-Ng and D. Eamus.
2010. Rates of nocturnal transpiration in two evergreen temperate
woodland species with differing water-use strategies. Tree Physiol.
30:988–1000.
Zeppel, M.J.B., J.D. Lewis, B.E. Medlyn, et al. 2011. Interactive effects
of elevated CO2 and drought on nocturnal water fluxes in Eucalyptus
saligna. Tree Physiol. 31:932–944.