Tree Physiology 28, 1525–1533
© 2008 Heron Publishing—Victoria, Canada
Analyses of δ13C and δ18O in tree rings of Callitris columellaris
provide evidence of a change in stomatal control of photosynthesis in
response to regional changes in climate
LOUISE E. CULLEN,1 MARK A. ADAMS,1,2 MARTI J. ANDERSON3 and PAULINE
F. GRIERSON1,4
1
Ecosystems Research Group, School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia
2
Present address: Lawson-Paterson Centre, Faculty of Agriculture, Food and Natural Resources, The University of Sydney, NSW 2006, Australia
3
Department of Statistics, University of Auckland, Private Bag 92019, Auckland, New Zealand
4
Corresponding author ([email protected])
Received March 10, 2008; accepted June 4, 2008; published online August 1, 2008
Summary We examined relationships between stable isotopes of carbon (δ13C) and oxygen (δ18O) in tree rings of
Callitris columellaris F. Muell in the semi-arid Pilbara region
of north-western Australia. To test the hypothesis that stomatal
control of photosynthesis decreases during drier periods, we
developed δ13C and δ18O chronologies spanning 1919–1999,
and used a permutation regression approach to relate a 21-year
running correlation between δ13C and δ18O to rainfall and temperature at Marble Bar and our study site. The relationship between δ13C and δ18O switched from being always negative before 1955 to being consistently positive after 1976, suggesting
an increase in stomatal control of photosynthesis in recent decades. Changes in the δ13C–δ18O relationship reflected changes
in rainfall, which has increased in the region by 30% since
1976. The correlation between δ13C and δ18O was positively related to the 21-year running mean of normalized rainfall anomalies at both the study site (P = 0.045, Adj. r 2 = 0.47) and Marble Bar (P = 0.046, Adj. r 2 = 0.48). In addition, the δ13C–δ18O
correlation was negatively related (P = 0.047, Adj. r 2 = 0.61) to
temperatures at Marble Bar. Our interpretation of the role of
changes in climate affecting the relationship between tree-ring
δ13C and δ18O is supported by evidence from the isotope composition of foliage samples: foliar δ13C and δ18O were negatively correlated with log stomatal conductance (δ13C,
r = –0.41; δ18O, r = –0.42), whereas the correlation between foliar δ13C and δ18O was positive (r = 0.63, P = 0.027) after the
summer wet period. Our data indicate that stomatal control of
photosynthesis in Callitris adjusts to region-wide changes in
climate and that, in a warmer and drier world, trees might adapt
by increasing non-stomatal control of photosynthesis.
Keywords: dendrochronology, non-stomatal limitations, Pilbara, rainfall, semi-arid, temperature.
Introduction
Stomata regulate both water loss (via transpiration) and carbon
gain (via photosynthesis) (e.g., Farquhar and Sharkey 1982),
but there remains some debate about the dominance of stomatal limitations to photosynthesis (Flexas and Medrano
2002). The degree of stomatal limitation is highly dependent
on environmental conditions, but appears to be most important
during drought (Flexas and Medrano 2002, Macfarlane et al.
2004). For example, drought reduced stomatal limitations to
photosynthesis in studies of physiological responses to seasonal or interannual changes in water availability (Teskey et
al. 1986, Wilson et al. 2000) and in manipulative experiments
(e.g., Giorio et al. 1999). However, long-lived species such as
trees experience multi-decadal shifts in environmental conditions that alter stomatal properties. For example, an elevated
CO2 concentration reduces stomatal density, with the effect
being more noticeable at decadal or longer time-scales than in
short-term field experiments (Hetherington and Woodward
2003).
Alterations in the physical properties of stomata (size, density) with environmental changes are fairly well documented;
however, questions remain about how stomatal limitations to
photosynthesis respond to long-term variation in climate. In
particular, we lack information for inland continental semiarid or arid environments (up to 500 or 250 mm rainfall per annum), whereas there have been numerous studies on stomatal
limitations in species from temperate (e.g., Kaiser and Kappen
2000, Gunderson et al. 2002, Tissue et al. 2005) and Mediterranean (e.g., Gulías et al. 2002, Medrano et al. 2002, Grassi
and Magnani 2005) regions. Arid and semi-arid environments
are characterized by highly variable rainfall and both the
amount of rainfall and its variability have significant effects on
biological processes (Noy-Meir 1973). Improved understanding of how climate variability influences stomatal control of
carbon exchange between trees and the atmosphere is also of
wider significance given the climate change predictions of reduced rainfall and pronounced cycles of drought and heavy
rain for many areas (IPCC 2001).
Measurements of the abundance of stable isotopes of carbon
1526
CULLEN, ADAMS, ANDERSON AND GRIERSON
(δ13C) and oxygen (δ18O) in tree rings are increasingly used to
reconstruct climates of the recent past and their influence on
physiological processes, especially carbon fixation (Leavitt
and Long 1989, Robertson et al. 1997, Barbour et al. 2002, Adams and Kolb 2004). Interpretations of isotope ratios in wood
owe much to our knowledge of the effects of leaf-level physiological processes on isotope ratios in plants (Farquhar et al.
1982, Yakir 1992, Barbour and Farquhar 2000, Barbour et al.
2000). Nevertheless, the well-described dependence of δ13C
on the ratio of intercellular to atmospheric CO2 concentrations
(Ci /Ca) (Farquhar et al. 1982) provides limited information
about the strength of stomatal control of Ci and photosynthesis
(A), because a change in Ci (inferred from δ13C) could be the
result of a change in either stomatal conductance (gs ) or A.
Greater understanding of stomatal processes can be gleaned
from simultaneous analyses of δ13C and δ18O, because the latter is unaffected by photosynthetic activity (Yakir 1992). Instead, δ18O composition of plant matter reflects not only that
of source water and isotopic exchange between organic compounds and plant water, but also the effect of humidity on
evaporative enrichment of leaf water (Yakir 1992, Roden and
Ehleringer 2000, Barbour 2007). More significantly for studies focused on stomatal limitations, δ18O is often negatively
correlated with gs, because changes in gs and transpiration with
humidity alter leaf temperatures and evaporative enrichment
(Barbour et al. 2000).
The negative relationship between δ18O and gs has significant potential to help disentangle the causes of variation
in δ13C (Farquhar et al. 1998, Adams and Grierson 2001,
Barbour 2007). If gs is the dominant limitation, then δ13C and
δ18O will be positively correlated. As a simple example, an increase in gs will decrease evaporative enrichment in 18O by
cooling the leaf and increase discrimination against 13C (more
negative δ13C), because the CO2 supply is greater (Farquhar et
al. 1998). However, if photosynthetic capacity drives variation
in δ13C, then variation in gs can be detected from δ18O and
the two isotopes will be either negatively or not correlated
(Farquhar et al. 1998). The potential for δ18O to help separate
biochemical from stomatal conductance effects on δ13C appears greatest in water-limited environments. Source water
δ18O can explain up to 99% of the variation in δ18O of wood for
species in temperate environments (Roden and Ehleringer
1999). However, Ferrio and Voltas (2005) concluded that, for
δ18O of wood in the drought-adapted Mediterranean conifer
Pinus halepensis Mill., changes in δ18O of rainfall in the order
of 1.5‰ were masked by a strong transpiration effect on leaf
water δ18O. Furthermore, numerous studies have indicated that
simultaneous measurements of δ13C and δ18O in leaves or
tree-rings can be used to investigate relative stomatal limitations to photosynthesis by inferring stomatal behavior (Sternberg et al. 1989, Saurer et al. 1997, Scheidegger et al. 2000,
Barbour et al. 2002, Barbour 2007).
We used a dendrochronological (tree ring) approach, combined with recently developed analytical techniques (Biondi
2000, Biondi and Waikul 2004), to interpret correlations between δ13C and δ18O in tree rings of Callitris columellaris
F. Muell (Cupressaceae) growing in the semi-arid Pilbara re-
gion of north-western Australia (Figure 1). Callitris columellaris (formerly C. glaucophylla: revised by Farjon (2005))
is one of the few species in mainland Australia suitable for
an investigation of multi-decadal adjustment in physiological
processes in semi-arid environments. The species is relatively
long-lived (250 years), has clear annual rings that can be
cross-dated (LaMarche et al. 1979) and used for isotope analysis (Cullen and Grierson 2006, 2007). The Pilbara has hot
summers, low annual rainfall (310 mm) and high variability in
rainfall both within and among years (Bureau of Meteorology
1995). We aimed to determine whether control of stomatal
conductance on photosynthesis in C. columellaris varied
through time in response to changes in climate over several decades. We hypothesized that relative stomatal control of photosynthesis, as indicated by a positive correlation between
tree-ring δ13C and δ18O, is greater during wetter periods. Conversely, during dry periods, stomatal conductance will be suppressed and photosynthetic capacity more limiting, the result
being that the two isotopes are either negatively correlated, or
show no relationship. Hence, we expected correlations between δ13C and δ18O to be positively related to rainfall and relative humidity and negatively related to temperature. We provide support for our interpretations of changes in the correlation between tree-ring δ13C and δ18O based on measurements
of these isotopes in foliage samples collected just after the
summer wet period and during the following dry period as a
modern-day analogue for historical changes in water availability.
Figure 1. Location of study site in the central Pilbara region, northwestern Australia.
TREE PHYSIOLOGY VOLUME 28, 2008
CLIMATE CHANGE AND STOMATAL CONTROL OF PHOTOSYNTHESIS
Materials and methods
Study site and field sampling
In the Pilbara region of north-western Australia, C. columellaris is primarily confined to south-facing gullies and gorges,
where it is relatively well protected from the frequent fires of
the floodplains and upper slopes. We selected a representative
gully (22°51′05″ S, 118°37′40″ E), about 150 km north-west
of the township of Newman containing a stand of C. columellaris (Figure 1). Mean yearly rainfall at Newman is 313 mm,
over 60% of which falls between December and March. Rainfall is often associated with cyclones or rain-bearing low-pressure belts (Bureau of Meteorology 1995). Daytime temperatures at Newman in summer (late November to March) range
from 26 to 40 °C, whereas winter temperatures are between 12
and 26 °C. Similar seasonal changes in climate are observed at
several other meteorological stations in the inland Pilbara.
In April and May 2003, 11 cores of 5-mm diameter were extracted from eight C. columellaris trees in the gully. Foliage
samples were collected from 12 trees in May 2004 and nine
trees in July 2004. Rainfall at the study site in the 3 months before May averages 136 mm and is more than twice that of the
3-month period leading up to the end of July (65 mm). To examine the relationship between foliar isotope composition and
stomatal conductance, we measured foliage gas exchange in
May 2004 with a CIRAS-1 Infrared Gas Analyzer equipped
with a Parkinson automatic PLC(broad) leaf cuvette (PPSystems, Amesbury, MA).
Stable isotope analysis
Cores were air dried and hand-sanded with sand paper up to
1200 grit. Visual cross-matching and skeleton plots were used
to assign a calendar date to each ring (Stokes and Smiley
1968). Wood from each year of each core was separated with a
razor blade, oven-dried (60 °C) and then combined on a drymass basis. In C. columellaris, removal of non-cellulose components in tree rings appears necessary to produce δ13C and
δ18O chronologies that have stable relationships with climate
(Cullen and Grierson 2006). Consequently, resins, lignin and
hemicellulose were removed from the wood material with
diglyme-HCl followed by bleaching with acidified chlorite
(Cullen and Macfarlane 2005).
Finely ground subsamples of bulk foliage and extracted cellulose (2–2.5 mg) were weighed into tin capsules for δ13C
analysis and corresponding subsamples of 0.22–0.27 mg were
weighed into silver capsules for δ18O analysis. Isotope analyses were undertaken at the Western Australian Biogeochemistry Centre, University of Western Australia in Perth. Carbon
isotope ratios were measured with an ANCA S/L-20/20 mass
spectrometer system (PDZ Europa, UK). The precision of the
ANCA system, calculated as the mean standard deviation of a
minimum of five replicates of an internal laboratory reference
(radish collegate; δ13C = –28.61 ‰) included in each run, was
0.13‰. The δ18O values of foliage and xylem water were measured with a High Temperature Conversion/Elemental
Analyser (TC/EA) coupled to a Finnigan DELTA+XL mass
spectrometer (Thermo Electron Corporation, Bremen, Ger-
1527
many). Internal lab standards for δ18O analysis of foliage were
laboratory-grade sucrose (35.35‰, precision = 0.66‰) and
benzoic acid (20.05‰, precision = 0.41‰). For each batch of
analyses, 10% of the samples were analyzed twice to check
homogeneity of sample preparation: mean standard deviation
of sample replicates was 0.18‰ for δ13C and 0.49‰ for
δ18O. Values of δ13C are reported as the 13C/12C ratio relative to
the Vienna PeeDee Belemnite standard, and δ18O values are
reported as the 18O/16O ratio relative to the Vienna SMOW
standard.
The raw δ13C chronology exhibited a decline in δ13C beginning around 1955, which we attributed to the lowering of δ13C
of air through anthropogenic-related increases in CO2 concentration (the “Suess effect,” see Keeling et al. 2005 and Cullen
and Grierson 2007). We removed this trend in the carbon isotope chronology by using the annual records of past atmospheric δ13C obtained from ice cores (Francey et al. 1999).
Moving correlation between δ13C and δ18O
Biondi (2000) proposed that subtle temporal changes in climate–tree growth relationships could be examined based on
multiple, overlapping periods (moving intervals). For example, using a moving principal component regression model
(response function), Biondi (2000) identified increased sensitivity of growth in Douglas-fir to summer water stress since
the 1970s. We used similar running or moving correlations between δ13C and δ18O to investigate temporal variation in stomatal control of photosynthesis. The correlation between δ13C
and δ18O was calculated for overlapping 21-year periods, with
an advance each time of one year, i.e., 1919–1939, 1920–1940
and so on. The correlation coefficients were then plotted as a
function of the middle year of each period.
Climate data
Rainfall and temperature data were obtained from Marble Bar,
the meteorological station closest to our field site (~220 km
away; Figure 1) with long-term records extending back to at
least 1919. We also obtained climate data for our site, going
back to 1889, using the Data Drill service provided by the
Queensland Department of Natural Resources and Mines.
Data Drill accesses grids of climate data interpolated to a 0.05°
(~5 km) spatial resolution from point observations using
splining and kriging techniques (Jeffrey et al. 2001). Relative
humidity (RH) records at Marble Bar began only in 1939; too
short for use in this study. Instead we used the maximum and
minimum temperature data provided by Data Drill to calculate
monthly RH according to Equation 1, with the minimum
temperature as an estimate of dewpoint temperature:
RH =
es
100
ea
(1)
where es and e a are saturated and actual vapor pressure, respectively. Vapor pressure (e(T )) was calculated as:
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CULLEN, ADAMS, ANDERSON AND GRIERSON
17.27 T
e( T ) = 0.6108 exp T + 273.3
(2)
where T is the maximum temperature for es and the dewpoint
temperature for ea (FAO 1998). Relative humidity, calculated
with Equations 1 and 2, was significantly and positively correlated with RH at Marble Bar over the period common to both
data series, 1939–2004 (r = 0.78).
We normalized mean January–February relative humidity
and maximum (daytime) temperatures and total January–February rainfalls relative to the 1919–1999 mean and standard
deviation for each series. We used data for January and February because tree-ring isotopes in C. columellaris were most
strongly correlated with climate in these summer months (Cullen and Grierson 2007). Annual summer rainfall and temperature anomalies at Marble Bar and the study site were significantly and positively correlated (r = 0.76 and 0.83, P < 0.001).
For each climate series, we calculated 21-year running means
for the same periods as the moving correlation between δ13C
and δ18O.
Relating changes in the δ13C–δ18O correlation to climate
Overlapping time periods in the running correlations between
δ13C and δ18O and the running means of climate variables invalidate the assumption of independence required by parametric regression analysis. We therefore used a permutation approach (Manly 1991, Anderson and Legendre 1999) to investigate relationships between climate and the δ13C–δ18O correlation. The permutation method provides a test of a statistic
such as t (produced from a regression analysis) by calculating
the probability of getting a value equal to or higher than the observed t by recalculating t after random re-orderings (permutations) of exchangeable units under a relevant null hypothesis
(Anderson 2001a).
Based on a null hypothesis that correlations between δ13C
and δ18O are not related to climate, all possible pairings of the
isotope and climate data are equally likely (Manly 1991, Anderson 2001a). The significance, or P-value, of the test is
calculated as:
P=
No. of t * ≥ t
Total no. of permutations
(3)
where t* is the value of t calculated from the regression analysis using the permuted data, t is the value of the observed statistic and the total number of permutations includes the original ordering of the observed data.
Construction of a permutation test requires a decision as to
which units are to be reordered, but exchanging units under the
null hypothesis assumes that the observations are independent
(Anderson 2001b). Hence, for the analysis of the relationship
between the isotope correlation and climate, the permutable
units are the two isotope series, not the running correlation between them. However, the raw isotope data are ordered as
a time-series and are, therefore, inherently not interchangeable
(Manly 1991). To remove the autocorrelation in the isotope
data, we fitted an autoregressive (AR) model to each series:
for both series, the selected model based on the Akaike Information Criterion was an AR(1) process. The resulting residuals obtained after fitting the AR(1) model were then used as
the permutable units.
We calculated the regression coefficients, r 2 and the t-statistic from a simple linear regression between the running correlation between the residual isotope series and each of the
21-year running means of normalized climate anomalies. The
residual δ13C and δ18O isotope series were each separately
reordered, the running correlations recalculated, and the regression analysis repeated, with a total of 4999 permutations
of the residuals. The P-value of each test was calculated with
Equation 3.
Results
Tree-ring isotope correlations
Although individual correlations between δ13C and δ18O were
significant (P < 0.05) for just two periods (Figure 2a, black
line), there was, nonetheless, a striking change in the running
correlation between tree-ring δ13C and δ18O over the last
80 years. The running correlation changed from being always
negative from 1929 to 1955, moving through a transition phase
during 1956–1975 and then became consistently positive after
1976 (Figure 2a). Specifically, between 1929 and 1955, the
correlation between δ13C and δ18O averaged –0.32, whereas
between 1949 and 1952 the correlation reached its most negative and was significant, ranging from –0.45 to –0.47. After
1976, the relationship between the isotopes averaged +0.34.
The shift in the correlation between the isotopes is not a result
of trivial occasional discrepancies in isotope values or trends
(Figure 2b). In particular, if the 13C and 18O data for 1942 were
replaced with the mean for 1922 to 1962, the correlation between the isotopes is still always negative before 1955 (Figure 2a, gray line). Up until 1955, above-average δ13C values
were matched by below-average δ18O (and vice versa). The
positive correlation after 1976, however, reflects general reductions in δ13C and δ18O, as well as coherency from
year-to-year in terms of the direction of change (Figure 2b).
Tree-ring δ13C ranged from –18.80 to –24.95‰, whereas tree
ring δ18O ranged from 29.49 to 38.97‰ (see Cullen and
Grierson 2007 for full isotope chronologies).
Relationship between the δ13C–δ18O correlation and climate
The relationship between δ13C and δ18O was significantly and
positively related to rainfall. The permutation test of the linear
regression between rainfall at the study site or Marble Bar and
the δ13C–δ18O correlation provides evidence to reject our null
hypothesis of no relationship between the δ13C–δ18O correlation and rainfall (Figure 3). Instead, the shift from a negative to
a positive correlation between δ13C and δ18O reflects an increase in rainfall in the region. Between 1919 and 1955, when
the correlation between δ13C and δ18O was negative, summer
(January–February) rainfall at the study site was 20% (27 mm)
below average (Figure 4a), whereas at Marble Bar it was
around 12% (20 mm) below average (Figure 4b). Further-
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Figure 2. (a) Running correlation between
tree-ring δ13C and δ18O: black line is the
correlation calculated based on measured
isotope data, and the gray line is the correlation when the data for 1942 were replaced with the mean for 1922 to 1962.
Correlations were calculated for overlapping 21-year periods, with an advance
each time of 1 year. The correlation coefficients are plotted as a function of the middle year of each period. The vertical
dashed lines indicate the points where the
correlation between δ13C and δ18O began
to increase (1955) and where the correlation between the isotopes became consistently positive (1976). The horizontal
dashed lines indicate the 0.05 significance
level. (b) The δ13C and δ18O (‰) chronologies, expressed as anomalies from their
1919–1999 mean and standard deviation,
developed from tree rings of
C. columellaris in the central Pilbara.
more, between 1949 and 1952, when the relationship between
δ13C and δ18O was significantly negative, summer rainfall was
over 80 mm below average at both the study site and Marble
Bar. During much of the transition period in the δ13C–δ18O relationship, summer rainfall was average, and began to increase
only in the 1970s (Figures 2 and 4). Since 1976, rainfall has
been 30% (40 mm) above the long-term mean at the study site
and at Marble Bar, corresponding to the period of consistently
positive correlations between δ13C and δ18O. Replacing the
isotope data for 1942 with the mean of 1922–1962 had only a
small effect on the regression results (study site: y = 0.87x +
0.12, P = 0.058, Adj. r 2 = 0.43; Marble Bar: y = 0.88x + 0.09, P
= 0.015, Adj. r 2 = 0.61).
There was no evidence of a significant influence of relative
humidity at the study site on the δ13C–δ18O relationship (observed t = 1.93, P = 0.337, Adj. r 2 = 0.04). This reflects the
clear difference between temporal patterns in relative humidity compared with the δ13C–δ18O relationship (Figures 2 and
4c). Unlike rainfall, periods of above- or below-average relative humidity over the last 80 years do not coincide with
changes in the relationship between tree-ring δ13C and δ18O.
The δ13C–δ18O relationship was significantly and negatively related to temperature at Marble Bar (Figure 5a). If the
13
C and 18O data for 1942 are substituted with the corresponding means for 1922–1962, the relationship between Marble
Bar temperature and the isotope correlation is still negative,
but non-significant (Observed t = –7.80, P = 0.074, Adj. r 2 =
0.50). A multiple regression with temperature and rainfall at
Marble Bar as the predictors did not explain much additional
variation in the δ13C–δ18O correlation (Adj. r 2 = 0.69). Summer temperatures at Marble Bar have varied over the last
80 years in an opposite manner to rainfall and the two variables
display a significant negative linear relationship (r = –0.78,
P < 0.05). Before 1955, mean summer maximum temperatures
were 0.5 °C above the long-term mean of 40.4 °C, whereas
since 1976, temperatures at Marble Bar have been 0.46 °C below the long-term mean (Figure 6). Unlike Marble Bar, temperature anomalies during January–February at the study site
exhibit little long-term variation (Figure 6). Correspondingly,
a permutation test provided no evidence to reject our null hypothesis that the δ13C–δ18O correlation and temperature are
not related at the study site (Figure 5b).
Figure 3. Relationships between the
21-year running correlation between the
δ13C and δ18O residual series and the
21-year running mean of total summer
(January–February) rainfall anomalies at
(a) the study site and (b) Marble Bar. The
δ13C and δ18O residual series were produced from the raw isotope series by fitting an autoregressive (AR) model of
order 1.
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CULLEN, ADAMS, ANDERSON AND GRIERSON
Figure 4. January–February rainfall anomalies at the (a) study site, based on data
provided by the SILO Data Drill service
and (b) Marble Bar. (c) January–February
relative humidity (RH) anomalies at the
study site, calculated with Equations 2
and 3. Anomalies (gray lines) were calculated as the difference between the individual value and the mean of the entire
series divided by the standard deviation of
the series. Black lines are smoothing
splines with a frequency response cut-off
of 40 years to indicate longer-term
(multi-decadal) trends. The vertical dashed
lines indicate the points where the correlation between δ13C and δ18O began to increase (1955) and where the correlation
between the isotopes became consistently
positive (1976) (see Figure 2).
Discussion
Foliage isotope correlations and gas exchange
Consistent with theoretical expectations, δ C and δ O composition of bulk foliage collected in May 2004 were negatively
correlated with log stomatal conductance (loge gs ) (δ13C,
r = –0.41; δ18O, r = –0.42): trees with higher gs (which varied
6-fold between trees) tended to have foliage more depleted in
18
O and 13C. Also, and consistent with our interpretation that
the change from drier and hotter summers to wetter and cooler
summers in the last two decades has caused the change from a
negative to a positive correlation between tree-ring δ13C and
δ18O, foliage δ13C and δ18O in May 2004 (just after the summer wet period) were positively and significantly correlated
(r = 0.63, P = 0.027; Figure 7a), whereas in July (midwinter
dry period) there was a negative albeit nonsignificant relationship between foliage δ13C and δ18O (r = –0.41, P = 0.264; Figure 7b).
13
18
Long-term changes in relative stomatal limitations to
photosynthesis
Over the past 80 years, stomatal control of photosynthesis in
Callitris columellaris has seemingly varied, with a clear shift
in the middle of the 20th century. Between 1919 and 1955, tree
rings that were relatively enriched in 18O were more depleted
in 13C and vice versa. We interpret the consistently negative
correlation between the isotopes before 1955 as indicating that
stomatal conductance, gs, was not the only source of variation
in tree-ring δ13C. Instead, variation in δ13C and, therefore, CO2
concentration within leaves (Ci ) was also partly a result of
varying demand for CO2 by photosynthesis (Farquhar et al.
1998). In the second half of the 20th century, and after 1976 in
particular, tree-rings have been consistently either enriched or
Figure 5. Relationships between the
21-year running correlation between the
δ13C and δ18O residual series and the
21-year running mean of the January–February daytime temperature anomalies at
(a) Marble Bar and (b) the study site. The
δ13C and δ18O residual series were produced from the raw isotope series by fitting an autoregressive (AR) model of
order 1.
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Figure 6. January–February temperature
anomalies at (a) study site and (b) Marble
Bar. Anomalies (gray lines) were calculated as the difference between the individual value and the mean of the entire
series divided by the standard deviation of
the series. Black lines are smoothing
splines with a frequency response cut-off
of 40 years to indicate longer-term
(multi-decadal) trends. The vertical
dashed lines indicate the points where the
correlation between δ13C and δ18O began
to increase (1955) and where the correlation between the isotopes became consistently positive (1976) (see Figure 2).
depleted in both 18O and 13C. The shift to a consistently positive correlation between tree-ring δ13C and δ18O suggests increased stomatal control of Ci and, therefore, of photosynthesis.
Our data suggest variation in photosynthetic capacity still
exerts a controlling influence on Ci because the correlations
between the isotopes were not strong. If photosynthetic capacity were unimportant, then more of the variation in δ13C would
be attributed to gs and tree-ring δ13C and δ18O would be highly
correlated. We did not detrend our data to remove non-climatic
influences on isotope composition, such as the increase in δ13C
with tree age (Fessenden and Ehleringer 2002). Consequently,
increased noise in the dataset may have dampened the correlation between the isotopes. In addition, recent studies have
placed a renewed emphasis on the role of mesophyll conductance (affecting the movement of CO2 from the internal cavities within leaves to the sites of carboxylation within chloroplasts) in limiting photosynthesis (Flexas et al. 2008, Siebt et
al. 2008). In particular, Siebt et al. (2008), in a reanalysis of
published 13C data, found that variation in mesophyll conductance can be large enough to account for temporal, species or
site differences in 13C. In our case, variation in internal conductance of CO2 may contribute to year-to-year differences in
tree-ring 13C, also potentially dampening the correlation be-
tween 13C and 18O. Nevertheless, we argue that, according to
our simple model and relative to the period before 1955,
stomatal limitations to photosynthesis in C. columellaris have
increased in the last 40 years. In support of this assertion, we
found that foliage (and trees) with greater gs was more depleted in 18O and 13C, as predicted by theory (Farquhar et al.
1982, 1998, Yakir 1992). A positive relationship between δ13C
and δ18O has been linked to a negative relationship between
leaf δ13C and δ18O and gs in wheat (Barbour et al. 2000), cotton
(Barbour and Farquhar 2000) and European beech (Keitel et
al. 2006).
Changes in stomatal limitations to photosynthesis with
climate
At the scale of decades, the isotopic evidence for a change in
stomatal control of photosynthesis in C. columellaris matches
clear shifts in climate: the δ13C–δ18O correlation was positively related to rainfall, but negatively related to temperature.
Both (1) the negative correlation between the isotopes and (2)
enrichment of tree-ring δ18O before 1955, imply that lower
rainfall and higher temperatures dampened stomatal conductance to limit water loss⎯a typical response of plants to water
stress (Chaves et al. 2003). Consistent with this interpretation,
we found foliar δ13C and δ18O tended to be negatively related,
Figure 7. Relationship between δ13C and
δ18O of foliage in (a) May and (b) July
2004.
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1532
CULLEN, ADAMS, ANDERSON AND GRIERSON
albeit not significantly, during the winter dry period (August).
This response to water stress suggests that, before 1955, gs was
unlikely to be the main cause of the year-to-year variations in
photosynthesis and δ13C ratios. Similarly, in black spruce
seedlings, repeated drought reduced both the sensitivity of
stomatal conductance to drought and stomatal control of photosynthesis (Stewart et al. 1994). Our results are consistent
with those of Flexas and Medrano (2002), who concluded
that metabolic processes are progressively inhibited during
drought to the point that metabolic impairment becomes the
major limitation to photosynthesis, notwithstanding stomatal
closure.
The switch to a positive relationship between tree-ring δ13C
and δ18O after 1955 coincides with an increase in summer rainfall, and a corresponding decrease in temperature in the region
beginning around 1960 (Hennessy et al. 1999). Positive relationships between δ13C and δ18O are expected when water is
less limiting because there a reduced need to limit water loss
and stomata can operate over a wide range of water availability
(Scheidegger et al. 2000). Previous studies have related positive δ13C–δ18O relationships to gradients in soil water content
(Saurer et al. 1997), changes in humidity of canopy air associated with decreased land-use intensity (Scheidegger et al.
2000), vapor pressure deficits (Barbour et al. 2002) and phase
changes in atmospheric circulation that, in turn, alter the importance of summer rains for plant growth (Welker et al.
2005). Although in our study a directional change in the 18O
value of Pilbara rainfall could explain the decline in tree-ring
18
O since 1960, this appears unlikely. Rainfall over the northwest of Australia is largely oceanic in origin, being generally
associated with cyclones or rain-bearing low-pressure belts
(Bureau of Meteorology 1995). Hence, the local Meteoric Water Line, including 18O signatures, is unlikely to have changed.
Rather, in the Pilbara region, increases in rainfall and humidity
after 1960 appear to have increased stomatal conductance and
its control of photosynthesis.
Retrospective analyses such as this study are clearly unable
to quantify stomatal limitations to photosynthesis directly
(sensu Farquhar and Sharkey 1982). Nonetheless, the change
in the relationship between carbon and oxygen isotopes in tree
rings demonstrates significant adjustment of physiological
characteristics over decades in response to region-wide
changes in climate. Based on this study, we suggest that nonstomatal limitations to photosynthesis are likely to become increasingly important in areas where rainfall is expected to decrease.
Acknowledgments
This research was funded by the Australian Research Council in collaboration with Pilbara Iron Pty. Ltd. (LP0214150). Thank you to the
Ecosystems Research Group for field and lab assistance and Pilbara
Iron for assistance with field logistics. We also thank Mark Westoby
for statistical advice.
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