Journal of Experimental Botany, Vol. 56, No. 422, pp. 3033–3039, December 2005
doi:10.1093/jxb/eri300 Advance Access publication 1 November, 2005
RESEARCH PAPER
Oxygen isotope enrichment (D18O) as a measure of
time-averaged transpiration rate
M. S. Sheshshayee1,*, H. Bindumadhava1, R. Ramesh2, T. G. Prasad1, M. R. Lakshminarayana3 and
M. Udayakumar1
1
Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India
2
Planetary and Geosciences Division, Physical Research Laboratory, Ahmedabad, 380 009, India
Department of Physics, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India
3
Received 1 July 2005; Accepted 26 August 2005
Abstract
Experimental evidence is presented to show that the
18
O enrichment in the leaf biomass and the mean (timeaveraged) transpiration rate are positively correlated
in groundnut and rice genotypes. The relationship
between oxygen isotope enrichment and stomatal conductance (gs) was determined by altering gs through
ABA and subsequently using contrasting genotypes of
cowpea and groundnut. The Peclet model for the 18O
enrichment of leaf water relative to the source water is
able to predict the mean observed values well, while it
cannot reproduce the full range of measured isotopic
values. Further, it fails to explain the observed positive
correlation between transpiration rate and 18O enrichment in leaf biomass. Transpiration rate is influenced
by the prevailing environmental conditions besides the
intrinsic genetic variability. As all the genotypes of both
species experienced similar environmental conditions,
the differences in transpiration rate could mostly be
dependent on intrinsic gs. Therefore, it appears that
the D18O of leaf biomass can be used as an effective surrogate for mean transpiration rate. Further, at
a given vapour pressure difference, D18O can serve as
a measure of stomatal conductance as well.
Key words: ABA, groundnut, mean transpiration rate,
enrichment, rice, stomatal conductance.
18
O
Introduction
Plant biomass production is determined by the total water
used and the water use efficiency (WUE, ratio of net CO2
assimilation rate to the transpiration rate), especially under
water-limited conditions (Passioura, 1976). Since water
availability is the most important constraint, particularly in
the semi-arid tropics, increasing WUE is regarded as one of
the potential approaches to improve crop yields. Recent
efforts in selecting wheat genotypes with improved WUE
resulted in higher productivity under water-limited conditions (Condon et al., 2002, Rebetske et al., 2002; Richards
et al., 2002). In these genotypes, the higher WUE was
achieved through a reduction in stomatal conductance (gs).
The gs regulates CO2 entry for photosynthesis besides controlling transpiration rate. Hence when gs is reduced, although transpiration decreases, it also hinders CO2 entry.
Most often an increase in WUE is associated with reduced
gs which can be counterproductive in terms of biomass
accumulation (Udayakumar et al., 1998a; Sheshshayee
et al., 2003). Therefore, from the agricultural point of view,
it is essential to increase WUE without compromising
transpiration. Such genotypes would possess superior mesophyll efficiency to assimilate CO2 and hence need to be
identified so as to have a distinct advantage in improving
agricultural productivity.
To this end it becomes imperative simultaneously to
determine the genetic variability in WUE and transpiration
in plants. Carbon isotope discrimination (D13C) has been
well established as a time-averaged surrogate for WUE
(Farquhar and Richards, 1984; Farquhar et al., 1989), The
application of the carbon isotope discrimination technique
to assess the genetic variability in WUE has been examined
and validated in container and field experiments in several
crop species such as cowpea (Ashok et al., 1999; Aniya and
Herzog, 2004), wheat (Condon and Hall, 1997; Richards
et al., 2002; Condon et al., 2004), groundnut (Wright
* To whom correspondence should be addressed. Fax: +91 80 23636713. E-mail: [email protected]
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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3034 Sheshshayee et al.
et al., 1988; Rao, et al., 1995; Bindumadhava et al., 2003),
and rice (Sheshshayee et al., 2003; Impa et al., 2005).
Similarly, the enrichment of the heavy isotope of oxygen in
leaf water (or that of the biomass) relative to the source water
is being adopted to assess variations in transpiration rate and
stomatal conductance (gs). Although the theory explaining
the phenomenon of oxygen isotopic enrichment during the
evaporation of water from the ocean surface has been known
for almost three decades (Craig and Gordon, 1965), the
application of this theory to predict differences in gs and
transpiration rate has been fairly recent (Flanagan et al.,
1991b, 1994; Farquhar and Lloyd, 1993; Bindumadhava
et al., 1999; Bindumadhava, 2000). However, discrepancy
between the Craig–Gordon prediction and the measured
d18O of the leaf water has been reported (White et al., 1994;
Buhay et al., 1996). Further, the relationship between
stomatal conductance and leaf water 18O enrichment has
remained equivocal (see Discussion), although increased
transpiration has clearly been shown to enrich leaf water 18O
(Gonfiantini et al., 1965; DeNiro and Epstein, 1979).
The major objective of the investigation was to show that
the 18O enrichment in the biomass can be used as a surrogate for the mean transpiration rate by a re-examination of
the relationship between D18O and stomatal conductance.
This relationship was tested both under field and growth
chamber conditions.
Materials and methods
Plants were grown under well-watered conditions in containers
(60345330 cm) having 20 kg of red sandy loam and farmyard
manure mixed in a 3:1 (v/v) proportion. The containers were arranged
randomly in open field conditions, adequate plant nutrients were
supplied once a month, and prophylactic measures were taken as and
when required to raise healthy plants. All experiments were conducted at the Department of Crop Physiology, GKVK Campus,
University of Agricultural Sciences, Bangalore, India (128 589 N and
778 359 E).
Alterations in stomatal conductance using
abscisic acid (ABA)
Petioles of sunflower (KBSH-1) leaves from 35-d-old plants were
excised under water and immediately placed in a test tube containing
15 ml of ABA solution (cis-trans ABA; Sigma, USA). Sets of four
leaves were maintained at different ABA concentrations (ranging
between 104 M and 107 M and a control without ABA) and
allowed to transpire in a controlled growth chamber under a vapour
pressure deficit (VPD) of 15 mbar and a PPFD of 1200 lmol m2 s1
(400–700 nm). The leaves were initially allowed to transpire up to
10 ml of the solution to flush out the leaf water. The time taken for
leaves to transpire an additional 10 ml of the solution was recorded and transpiration rate was calculated after measuring the leaf
area. The leaf water was extracted immediately for determining
its 18O. The 18O enrichment in leaf water (D18O) relative to that of
the distilled water (d18O of distilled water was 13.3&).
Genotypic variations in stomatal conductance among
cowpea genotypes
Based on the results of a previous experiment (Sheshshayee, 1998),
a few cowpea genotypes with variable gs and transpiration rates were
identified and raised in containers under well-watered condition in an
open field. Stomatal conductance (gs) and transpiration rate of the
third fully expanded leaf from the apex of 45-d-old plants were
measured using a portable gas exchange system (LCA-4, ADC,
Hoddesdon, EN11, ODB, UK). The temperature and RH of the leaf
chamber were maintained close to those of ambient air. The mean
natural light intensity was 1600 lmol m2 s1. All observations were
recorded between 09.00 h and 11.30 h (Indian standard time).
Extraction of leaf water
Immediately after determining gas exchange, holes were punched,
avoiding the major veins, and placed in tapering plastic tubes and
immediately closed using rubber stoppers. The tubes were purged
with pure, dry nitrogen gas and frozen to the liquid nitrogen temperature of 196 8C for 20 min. The tubes were transferred to a hot
water bath (80 8C) and, after 10 min of thawing, the tubes were
centrifuged at 5000 rpm for 5 min. The leaf water that collected at the
tapering end of the tube was drawn out using a syringe and an aliquot
of 200 ll was immediately introduced into a 10 ml vacutainer tube
(Becton Dickinson vacutainer systems, USA). The tubes were airtight and all the leaf water was collected instantly; care was taken to
keep the fractionation that might occur during the extraction process
to be minimal. Unlike the ‘classical’ method of extraction of leaf
water by pumping, the present method does not cause significant
isotopic fractionation due to incomplete extraction.
Determination of D18O of leaf water
The 18O composition of the leaf water was determined by CO2equilibration technique (Scrimgeour, 1995). CO2 gas of known
oxygen isotopic composition was introduced to the head-space
volume of the vacutainer tube containing leaf water and equilibrated
overnight at 30 8C. The CO2 was then introduced into the mass
spectrometer (Tracermass, PDZ-Europa, UK) for the determination
of d18O on a continuous flow mode. The d18O of the source water
(used for irrigating the plants) was also similarly determined. The
analytical uncertainty was typically 60.15&.
The 18O enrichment in the leaf water over the source water
(D18Olw) was computed as follows.
D18 Olw = D18 Olw d18 Oiw
where d18O is the isotopic composition compared with VSMOW
(Vienna-Standard Mean Ocean Water) and the subscripts lw and iw
refer to leaf water and irrigation water, respectively (for the experiment with ABA solution the d18O of the distilled water was used).
Gravimetric determination of variability in mean transpiration
rate (MTR) in rice and groundnut genotypes
The mean transpiration rate (MTR) was determined gravimetrically
(Udayakumar et al., 1998b) in selected genotypes of rice and groundnut in separate experiments. Both the experiments were carried out
between January and April 2002. The experimental season was characterized with a mean temperature of 30.6 8C (maximum), 17.2 8C
(minimum) and a VPD of 15 mbar. The natural photon flux density
was 1600 lmol m2 s1. Briefly, the gravimetric method involved
weighing the containers daily using a mobile weighing device for
a period of 30 d. The container weight was brought back to field
capacity daily by adding water. The amount of water added over the
experimental period was summed to arrive at the total evapotranspiration (ET ). A mobile rain out shelter was moved over the
experimental area during nights and rain episodes to maintain
a specific water regime in the containers. The soil surface of all the
containers was covered with plastic pieces to minimize surface
evaporation. Simultaneously, ‘bare’ containers (without plants) were
also weighed to quantify the evaporation component (Es) of ET.
18
O enrichment and transpiration rate
Cumulative water transpired (CWT ) over the experimental period
was calculated as the difference between ET and Es. The MTR was
computed from the ratio of CWT to the leaf area duration (LAD=
(LA1+LA2)/2330 d, where, LA1 and LA2 are leaf areas at the beginning and end of the experiment, respectively).
Determination of d18O in leaf biomass
The leaves of rice and groundnut genotypes that matured during
the experimental period were separately harvested and oven-dried at
70 8C for 72 h). Finely ground dry leaf powder (0.8–1.2 mg) was taken
to determine d18Olb by on-line pyrolysis using TC/EA interfaced
with an IRMS (Delta Plus, ThermoFinnigan, Bremen, Germany)
through a continuous flow device (Conflo-III, ThermoFinnigan,
Bremen, Germany) at the National Facility for Stable isotope studies, Department of Crop Physiology, University of Agricultural
Sciences, Bangalore, India. The analytical uncertainty of isotope
measurements was less than 0.2&.
The 18O enrichment in leaf biomass (D18Olb) was computed as
follows:
D18 Olb ð&Þ = d18 Olb d18 Oiw
Statistical analysis
Analysis of variance (ANOVA) for all the experiments was computed for a completely randomized design using MSTAT-C software.
The geometric mean regression (Model-II) was used to plot all
relationships.
Results
Stomatal conductance (gs) and
18
O enrichment
The plant hormone abscisic acid (ABA) induces stomatal closure and, accordingly, excised leaves of sunflower (KBSH-1)
fed with the highest concentration of ABA (104 M)
recorded the lowest transpiration rate, which increased as
the ABA concentration decreased. D18Olw also showed a
very similar pattern. A strong positive correlation between
transpiration rate (TR) and D18Olw was evident (Fig. 1).
To examine this relationship further, the stomatal conductance of leaves of a few contrasting cowpea genotypes
was determined with a gas exchange system. A significant
genotypic variability in gs and D18Olw was noticed in this
set of cowpea genotypes (Table 1). The D18Olw showed
a significant positive relationship with gs (R2=0.90; P <
0.05; n=5) reconfirming the stomatal control of 18O
enrichment.
Genetic variability in D18O, stomatal conductance and
mean transpiration rate
Gas exchange parameters are snap-shot measurements and
do not integrate the diurnal as well as the day to-day variations in transpiration rate and gs. Similarly, the 18O
composition of the leaf water also varies significantly in
time. The transfer of the 18O signature from leaf water into
cellulose/biomass has been well elucidated (Sternberg
et al., 1986). The 18O enrichment of the leaf biomass
relative to source (D18Olb) can serve as a measure of mean
3035
14
12
10
8
6
4
2
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Transpiration Rate
Fig. 1. Relationship between D18Olw (&) and transpiration rate (mmol
m2 s1). Transpiration rate was altered by inducing stomatal closure
using ABA (filled diamonds, control, filled squares, 107 M, open
triangles, 106 M, closed triangles, 105 M, open diamonds, 104 M) in
excised sunflower leaves. The leaf water oxygen isotopic enrichment
(D18Olw) was computed relative to distilled water (d18Odw= 13.3&)
used for preparing ABA solutions. The set-up was kept in a growth
chamber with 15 mbar VPD and 1200 lmol m2 s1 light intensity in
PAR range. Each value is a mean of three replicates (y=2.23x+0.461;
R2=0.74, P <0.001; n=15).
(time-integrated) transpiration rate (MTR). The latter was
gravimetrically determined for rice and groundnut in
separate experiments over an extended period of 30 d.
Significant genotypic variability in MTR was noticed in
both the crop species (Table 2). Since the biomass formation is continuous, the D18Olb should be a time integrated
measure of D18Olw. Therefore, the relationship between the
MTR and D18Olb among the genotypes was examined and
a significant positive relationship was found (Fig. 2). As the
leaf biomass and not the cellulose was analysed, it could be
argued that the differences in organic composition among
genotypes could possibly account for the observed variability in D18Olb especially in the long-term experiment.
However, this can be safely ruled out because (i) the bulk of
the dry matter of the leaf (>70%) is cellulose and (ii) the
remaining components are not isotopically very different,
especially in the same species of plants. It was apparent
from Fig. 2 that the slopes of the regression between D18O
and MTR were different for groundnut and rice. The
difference could arise due to significant variations in the
leaf structural composition and architecture of these two
species.
Discussion
The first mechanistic explanation for the evaporative
enrichment of 18O in large water bodies was provided by
Craig and Gordon (1965). This theory was extended by
several models to predict the 18O enrichment in leaf water
3036 Sheshshayee et al.
Table 1. Genotypic variability in stomatal conductance, transpiration rate and D18Olw among cowpea genotypes
Transpiration rate and stomatal conductance were recorded using a portable photosynthesis system (ADC, LCA4, UK). The leaf water 18O enrichment
(D18Olw) over the irrigation water was determined by the CO2 equilibration method using an IRMS. Each value is a mean of three replicates. The d18O
value of irrigation water was 3.7&.
Genotype
Stomatal conductance
(mmol m2 s1)a
Transpiration rate
(mmol m2 s1)a
D18Olw (&)a
APC-40 GC-20
APC-123-V-683
V-585
APC-4125
APC-121-P-132
CDb (P=0.05)
290611
260617
22766
230610
28065
11.02
10.362
8.261.5
7.862
7.661
9.261
0.19
33.660.60
31.260.52
30.360.22
30.560.26
33.160.10
0.98
a
b
Mean value 6standard deviation.
CD is the critical difference required to conclude that any two values of a parameter are significantly different from each other.
Table 2. Genotypic variability in mean transpiration rate and D18Olb in rice and groundnut
Mean transpiration rate was determined by gravimetry and the leaf biomass 18O enrichment over the irrigation water (D18Olb) was then assessed using an
IRMS. Each value is a mean of five replicates.
Rice
Groundnut
D Olb (&)
Genotype
MTR (mol m2 d1)
D18Olb (&)
140.11
180.56
138.56
123.28
131.44
175.61
159.22
136.83
137.78
152.89
116.39
34.06
33.26
33.77
29.64
31.44
33.44
33.60
32.12
31.20
31.90
26.20
TNAU262
LI-1
JAL-18
ATG-17
JL-24
TIR-17
VRI-4
ALR-2
ICGS-11
CO-3
CO-1
Sen Nghe An
144.78
10.67
31.88
0.95
177.61
196.83
266.67
245.72
211.72
214.67
156.56
208.83
249.89
296.22
267.67
231.06
218.61
56.11
31.27
31.2
37.66
31.70
29.98
32.69
28.87
30.28
34.94
29.89
34.21
32.98
32.14
3.94
Genotype
MTR (mol m
IET15297
IET16348
IET16364
ALM6
JAYA
IET15924
Kirwana
IET16347
IET15963
MRB-2
MRB-1
Mean
CDa (P=0.05)
a
2
1
d )
18
CD is the critical difference required to conclude that any two values of a parameter are significantly different from each other.
(Flanagan et al., 1991b, 1994; Flanagan, 1993; Farquhar
and Lloyd, 1993; Roden and Ehleringer, 1999).
18
18
D Oe = e + ek + ðD Ov ek Þea =ei
where, D18Oe is the 18O enrichment in the leaf water at
the site of evaporation relative to source water, e* and ek are
the equilibrium and kinetic (oxygen isotopic) fractionation factors, respectively (expressed in per million units),
D18Ov, the 18O enrichment of the atmospheric water vapour
relative to the source water, and ea, ei, the partial pressures
of water outside and inside the leaf, respectively.
However, it was observed that the actual oxygen enrichment in bulk leaf water (D18Olw) was less than that
predicted by the Craig–Gordon theory (Allison et al.,
1985; Leaney et al., 1985; Flanagan et al., 1991a, b;
Wang and Yakir, 1995; Barbour et al., 2002). To explain
this discrepancy, several modifications have been attempted. Leaney et al. (1985) considered ‘two pools’ of water in
a leaf. The first pool is an enriched fraction due to
evaporation and the second, the unfractionated xylem water
pool. The ‘Peclet model’ of Farquhar and Lloyd (1993) and
Barbour et al. (2000) account for the progressive variation
of leaf water enrichment along the leaf mesophyll tissue due
to convective and diffusive mixing of the enriched water
and the unfractionated xylem water. The ‘string-of-lakes’
model (Gat and Bowser, 1991; Yakir, 1992; Helliker and
Ehleringer, 2000, 2002) provides for the spatial variation in
d18O across the entire leaf surface. The string-of-lakes
model argues that the enriched leaf water would gradually
flow into the xylem thus spatially altering the source water
isotopic composition. It is to be noted that, in general, (i)
all these models deal with steady-state fluxes of water from
the stem to the leaf, from the leaf to the boundary layer,
from the boundary layer to the atmosphere, and (ii) they
do not address the effect of stomatal response to the
atmospheric water vapour deficit, unlike Lindroth and
Halldin (1986) who suggest an empirical relation. Recently,
18
O enrichment and transpiration rate
a non-steady-state model was proposed by Farquhar and
Cernusak (2005); according to these authors, the model ‘is
less important during the day and hence for determining the
18
O enrichment in organic matter’.
In some earlier experiments it was observed that the 18O
enrichment in the leaf water was directly proportional to the
transpiration rate (Walker et al., 1989; Yakir et al., 1990;
Yakir, 1998). Gan et al. (2002) showed that the leaf water
as well as the leaf organic matter D18O was significantly
higher in the leaves exposed to lower RH, which indicated
that the 18O enrichment linearly increased with transpiration rate. A lower stomatal conductance (gs), at a given
VPD, is also known to reduce the transpiration rate and
hence 18O enrichment (Fig. 1 of Wang and Yakir, 1995).
Our results are consistent with these, but they contrast with
the trend reported for the relationship between leaf water
enrichment and transpiration among wheat genotypes
(Barbour et al., 2000).
A
39
37
35
33
3037
A simplified Peclet model developed by Fraquhar and his
coworkers (Barbour and Farquhar, 2000) was obtained
from Dr Margaret Barbour and the predicted D18O was
determined. Several input parameters such as fractionation
caused during diffusion through stomata (32&) and
through the boundary layer (21&) were considered (Cappa
et al., 2003) in the model. The source water of the GKVK
tube well used for irrigation was 3.7&. The oxygen
isotopic enrichment over the source water was estimated
using the model. Figure 3 clearly demonstrates that the
mean groundnut and rice values of the predicted D18Olb
(31.7& and 31.7&) and the measured D18Olb (32.1& and
31.9&), respectively, match very well. However, the model
is unable to account for the full range of values: the
predicted values lie in a narrow range of 30–33&, whereas
the observed values vary from 26–38&. A possible reason
could be that several parameters used as inputs in the Peclet
model, such as leaf and air temperatures, relative humidity,
boundary layer and stomatal conductances, were measured
at one single instance during the day. These values are
known to fluctuate considerably diurnally and hence would
influence the 18O enrichment in the leaf biomass in a
cumulative fashion. Thus the measured D18O would vary
more than that predicted by the model.
Even though the mean values are predicted well by the
model, the observed positive correlation between transpiration rate and 18O enrichment in the leaf biomass (Fig. 2)
cannot be explained by it. Buhay et al. (1996) showed that
31
29
27
150
39
170
190
210
230
250
270
290
MTR
B
37
35
37
33
35
31
33
29
31
27
25
29
27
25
110
130
150
170
190
MTR
Fig. 2. Relationship between MTR (mol m2 d1) and D18Olb (&)
among selected genotypes of (A) groundnut (y=0.072x+16.33; R2=0.69;
P <0.001; n=12) and (B) rice (y=0.114x+15.36; R2=0.46; P <0.05; n=11).
MTR was determined gravimetrically and the d18Olb using an IRMS
interfaced with TC/EA. The analytical uncertainty of oxygen isotope
measurement was less than 0.2&. The leaf biomass oxygen isotopic
enrichment relative to that of irrigation water (d18Oiw= 3.7&) was
computed. Each value is a mean of five replicates.
25
27
29
31
33
35
37
39
Fig. 3. Relationship between predicted and measured D18Olb (&) in
groundnut (open symbols) and rice genotypes (closed symbols). A
simplified Peclet model to obtain the predicted D18O was provided by Dr
Margaret Barbour. The diffusion fractionation through stomata was
considered as 32& and that through the boundary layer as 21& (Cappa
et al., 2003). The equilibrium fractionation between C=O and water for
carbonyl exchange (27&) and for the whole leaf biomass (8&) was as
per Barbour and Farquhar (2000). Other values used: d18Ov= 4.0&,
d18Osw= 3.7&, RH=55%, Tair =28 8C, boundary layer conductance
(gb)=1.0 mmol m2 s1, effective length for Peclet effect=0.018 m,
proportion of exchangeable oxygen in cellulose=0.56, and proportion
of xylem water in meristem=0.8. Measured leaf temperatures were used
for the calculation.
3038 Sheshshayee et al.
ek and gb depend on the nature of the boundary layer,
influenced by wind speed and leaf temperature, while the
shape/area of the leaf is also important. In this study’s
experiments all plants experienced the same wind speed
and air temperature. The shapes of the leaves are also comparable within species. The dependence of the leaf water
isotopic composition on the leaf temperature is of a small
magnitude (Majoube, 1971). Therefore, it appears necessary to modify existing models to incorporate the stomatal
response to changing VPD naturally.
Conclusion
In this work, it has been shown experimentally that there is
a positive correlation between the transpiration rate (caused
by stomatal conductance) and the oxygen isotope enrichment in leaf biomass in groundnut and rice genotypes that
contrast with the observations of Barbour and Farquhar
(2000). Transpiration rate can increase either because of
increased stomatal conductance or when the vapour pressure difference between the leaf and air is increased. When
the transpiration rate was altered by inducing differential
stomatal closure through ABA, the D18Olw closely followed the changes in transpiration rate (Fig. 1). In addition,
the genotypes varying in stomatal conductance showed
similar variations in mean transpiration rate. The D18Olb, an
integrated measure of the leaf water D18O values, showed
a significant positive relationship with mean transpiration
rate (Fig. 2). These results suggest that the D18Olb is a good
time integrated measure of stomatal conductance. Existing
models (such as the Peclet model) need to be modified to
explain the observed positive correlation between transpiration rate and 18O enrichment in leaf biomass.
Acknowledgements
The authors thank two anonymous referees for constructive suggestions. Dr Anura V Kurpad, Head, Nutrition Division, Department
of Physiology, St John’s Medical College, Bangalore is thanked for
his help during the initial stages of this work and Mr Shashidhar, G
and Ms Impa S Muttappa for the help in conducting the groundnut
and rice experiments. This work was funded by the Department of
Science and Technology (DST), in the form of a Young Scientist
project to MSS (HR/OY/B-03/96) and CSIR in the form of Senior
Research Fellowship to HB [9/271(51)/96-EMR-1]. Financial assistance by DST and DBT (SP/IO/LF-01/98; BT/IS/06/004/98) is acknowledged. The assistance of Mr Nagabhushana and Mr A Ramesha
in sample preparation and isotope analysis is acknowledged. Special
thanks to Mr Ram Mhatre, of Thermo-electron for the maintenance
of the IRMS.
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