Journal of Experimental Botany, Vol. 52, No. 357, pp. 801±809, April 2001
The high oxygen atmosphere toward the end-Cretaceous;
a possible contributing factor to the K/T boundary
extinctions and to the emergence of C4 species
Joseph Gale1,3, Shimon Rachmilevitch1, Joseph Reuveni1 and Micha Volokita2
1
2
Department of Plant Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Blaustein Institute for Desert Research, Ben Gurion University of the Negev, 84990, Israel
Received 22 June 2000; Accepted 20 October 2000
Abstract
Introduction
Angiosperm plants were grown under either the
present day 21 kPa O2 atmosphere or 28 kPa, as
estimated for the end-Cretaceous (100±65 MyBP).
CO2 was held at different levels, within the 24±60 Pa
range, as also estimated for the same period. In C3
Xanthium strumarium and Atriplex prostrata, leaf
area and net photosynthesis per unit leaf area,
were reduced by the high O2, while the whole-plant
respirationuphotosynthesis ratio increased. The high
O2 effects were strongest under 24 Pa, but still
significant under 60 Pa CO2. Growth was reduced
by high O2 in these C3 species, but not in Flaveria sp.,
whether C3, C4, or intermediary grown under light
intensities -350 mmol m 2 s 1 PPF. Photosynthesis
of C3 Flaveria sp. was reduced by high O2, but only
at light intensities )350 mmol m 2 s 1 PPF. It is concluded that the high O2 atmosphere at the endCretaceous would have reduced growth of at least
some of the vegetation, thus adversely affecting
dependent fauna. The weakened biota would have
been predisposed to the consequences of volcanism
and the KuT boundary bolide impact. Conversely,
photosynthesis and growth of C4 Zea mays and
Atriplex halimus were little affected by high, 28 kPa,
O2. This suggests an environmental driver for the
evolution of C4 physiology.
High ambient oxygen and the KuT boundary extinctions
Key words: KuT boundary, extinctions, paleo-atmosphere,
oxygen, C4 emergence.
3
65 million years ago (MyBP) mass disappearances of
some 70% of all biotic species, delineated the CretaceousTertiary (KuT) boundary (Hallam and Wignall, 1997).
Alvarez et al. proposed that the causal agent was a
climatic cataclysm, resulting from the impact of a bolide
(Alvarez et al., 1980). This has now been widely accepted
(Macleod and Keller, 1996). However, one conundrum
remains: the decline of certain ¯ora and fauna species
which commenced some ten million years before the KuT
event (Sloan et al., 1986; Johnson and Hickey, 1990;
Sweet et al., 1990; Johnson, 1993; Macleod et al., 1997).
These and other ®ndings have suggested more gradually
developing biosphere degrading factors, such as extreme
volcanism (Hallam, 1987; Venkatesen et al., 1993;
Courtillot, 1999).
Models of the paleo-atmosphere indicate that there have
been periods of comparatively high oxygen such as the
40 kPa event in the Carboniferous (Berner and Can®eld,
1989; Berner et al., 2000). High levels of CO2, such as
were prevalent in most of Phanerozoic eon, tend to
counteract the photorespiration-inducing, photosynthesisreducing effect of high O2, although decreased activation
of other stromal enzymes has also been reported
(Leegood and Walker, 1982). Oxygen reduced net photosynthesis is signi®cant even at the present atmospheric
level of 21 kPa O2 (von Caemmerer and Farquhar, 1981).
At the end-Cretaceous, oxygen is estimated to have
reached ;28 kPa, while CO2 was comparatively low,
with estimates ranging from 23 Pa (Lasaga et al., 1985) to
the more recent 30±90 Pa (Berner, 1997). It is suggested
To whom correspondence should be addressed. Fax: q972 2 579 0252. E-mail: [email protected]
ß Society for Experimental Biology 2001
802
Gale et al.
that the high oxygen atmosphere at the KuT boundary
may also have been involved in the pre-KuT boundary
decline of both ¯ora and dependent fauna.
Beerling (Beerling, 1994) calculated rates of leaf
photosynthesis for the O2uCO2 atmospheric composition
and temperatures of the late Cretaceous to the present
time, using the Farquhar et al. and von Caemmerer and
Farquhar leaf biochemistry models (Farquhar et al.,
1980; von Caemmerer and Farquhar, 1981). His calculations showed a strong effect of high O2. Interpolating in
Beerling's Fig. 6, for the late Cretaceous, the calculated
rate of photosynthesis is seen to decline from 100 to
60 MyBP by 40±60%. However, these models do not take
into account possible changes in leaf area (see below) and
gas conductance (Rachmilevitch et al., 1999) in response
to elevated O2. The effect on plants of the very high
(35 kPa) O2 event calculated for the Carboniferous period
(345±280 MyBP) has been studied (Beerling et al., 1998).
After 6 weeks growth under 35 Pa CO2 and either high or
present-day O2, photosynthesis per unit leaf area was
reduced 29% by the high O2.
High ambient oxygen and the evolution of C4 plants
13
Cu12C isotope evidence has been reported for the
presence of C4 plants from strata dated to 15 MyBP
(Cerling et al., 1994, 1998; Kingston et al., 1994). Isotope
evidence for C4 plants from the CenomanianuTuranian
(CuT) boundary period (100 MyBP) has been reported
(Kuypers et al., 1999). However, these ®ndings may be
confounded by CAM species, which could produce
similar 13Cu12C values.
Most discussions of environmental conditions which
encouraged the evolution of C4 from C3 physiology
emphasize the effects of low atmospheric CO2, high
temperatures and water stress to which C4 species are
relatively well adapted (Ehleringer et al., 1997; Ehleringer
and Monson, 1993; Cerling et al., 1998; Collatz et al.,
1998). The present study relates to the potential
advantage of C4 plants under relatively high ambient O2.
The following hypothesise were tested: (a) that the
primary production of C3 angiosperms would have been
adversely affected by the high oxygen composition
of the late-Cretaceous, and (b) that the same high
ambient oxygen would not affect C4 plants and may
therefore have provided an environmental driver for their
evolutionary emergence.
Materials and methods
Plant species
Two C3 species, Xanthium strumarium L., and Atriplex prostrata
D.C. and two C4 species, Zea mays L. and Atriplex halimus L.
were studied. Initial growth studies and some photosynthesis
measurements were also carried out on Flaveria pringlei
Table 1. O2 and CO2 composition of the growth chambersimulated atmospheres used in this study
O2
(kPa)
CO2
(Pa)
Period simulated
21
21
21
28c
28c
28c
24a
35b
60
24
35b
60
Pre-industrial
Present ambient
Present ambient O2 with high CO2 estimate
End-Cretaceous O2 with low CO2 estimate
End-Cretaceous O2 with mid-range CO2 estimate
End-Cretaceous O2 with high CO2 estimate
a
This is lower than the immediate `pre-industrial' (;27 Pa) CO2. It was
used for comparison with low-end estimates of the end-Cretaceous
atmosphere.
b
The present average global CO2 is above 36 Pa. 35 Pa was used,
which is closer to the level found within photosynthesising plant stands
or aerated, plant-®lled growth chambers.
c
28 kPa is taken as the oxygen partial pressure of the end-Cretaceous.
This is a high-end estimate.
Gandoger (C3), F. trinervia C.Mohr (C4) and the C3±C4
intermediates F. ¯oridana J.R. Johnston and F. sonorensis
A.M. Powell. All species studied were forage angiosperms.
Angiosperms are thought to have emerged and diversi®ed in the
early Cretaceous (Crane et al., 1995). Fast growing species were
chosen in order that plant parts used for physiological
measurements would have developed under the atmospheric
composition being studied and in order that signi®cant growth
response could be measured in relatively short periods (weeks).
All observations were limited to vegetative growth.
Growth conditions
The growth chamber system for control of ambient O2 and CO2
partial pressures was as described previously (Rachmilevitch
et al., 1999). Plant growth and physiological parameters were
studied under six atmospheric compositions shown in Table 1.
CO2 was controlled to "3 Pa and O2 to "0.5 kPa. Light
intensity was 380"20 mmol m 2 s 1 PPF at the plant tops
for 12 h d 1, obtained from a mixture of ¯uorescent and
incandescent lamps. Air temperatures were 24 and 19"0.5 8C
in the day and night periods, respectively. Air humidity was
above 70% RH at all times, but below the dew point.
Gas exchange measurements
Carbon dioxide exchange of whole young plants (roots and
shoots) each about 20 cm tall, was measured, over periods of
days, in mini-chambers (Reuveni and Gale, 1985). Environmental conditions, including light, temperature, humidity, and
atmospheric composition were the same as in the growth
chambers. The ratio of net daily respirationugross-photosynthesis
(RuP) was calculated assuming that respiration in the light
was 30% of the average dark period values (a reasonable but
arbitrary value, see Kromer, 1995). Gas exchange parameters of
single, fully developed, attached leaves were determined with an
ADC (UK) Model LCA-2 system. For these measurements,
different mixtures of oxygen, nitrogen and carbon dioxide were
obtained with two cascaded Wosthoff Co (Germany) gas-mixing
pumps (Model 2G-18±2F). Gas exchange was measured and
exchange parameters were calculated by standard procedures
(von Caemmerer and Farquhar, 1981). CO2 assimilationuinternal
leaf CO2 partial pressure (AuCi) curves were derived from gas
exchange measurements made under 900 mmol m 2 s 1 photosynthetic photon ¯ux (PPF). Data curves for the AuCi and
KuT-atmosphere, extinction, C4
AuPPF data were smoothed with right-angle hyperbolae
regressions. When measuring the rapid effect of high O2 on leaf
photosynthesis, O2 pressure was switched between 21 and
28 kPa at different levels of PPF, and photosynthesis was
measured after 14±16 min.
Growth rates
Relative growth rate (RGR) was calculated as (ln F±ln I)ut,
where F is the ®nal dry wt in grams, I the initial dry wt,
estimated from the initial fresh weights and the fresh wtudry wt
ratio of parallel samples, and t, the elapsed time in days.
Results
Response of C3 plants to the high (28 kPa) O2
composition of the end-Cretaceous period,
when grown under low-end CO2 estimates
(24 or 35 Pa) for the same period
In the C3 species X. strumarium and A. prostrata,
grown under a medium level estimate of CO2 (35 Pa)
803
photosynthesis per unit leaf area was much reduced by
high O2, albeit to different degrees in the two species. As
shown in Fig. 1, photosynthesis as expressed in the AuCi
curve of X. strumarium was much suppressed in plants
grown and measured under 28 versus 21 kPa O2. This is
evident both in the initial slope (an indication of Rubisco
activity) and in the maximum rate of assimilationÐ
considered an indication of a limitation in ribulose
bisphosphate regeneration. However, in A. prostrata only
the maximum rate of photosynthesis was reduced under
high O2.
An example of the effect of ambient oxygen (28 or
21 kPa) on whole plant CO2 exchange of A. prostrata and
X. strumarium, grown under a low level estimate of CO2
(24 Pa), over a 4 d period, is shown in Fig. 2.
As seen in Fig. 2, high, 28, versus low, 21 kPa O2,
reduced CO2 uptake of both these C3 species by 25±40%
when grown under 24 Pa CO2. However, night-time CO2
ef¯ux was not proportionally reduced, as could be
expected (Amthor, 1989). This resulted in an increased
Fig. 1. Effect of the assumed ambient O2 (28 kPa) of the end-Cretaceous atmosphere on the assimilationuinternal leaf-CO2 (AuCi) curves of C3 species
grown under a medium level estimate of CO2 (35 Pa) for the same period. Note: plants were grown and measurements made at the same O2 partial
pressure.
Fig. 2. Whole plant carbon dioxide exchange of C3 species as affected by the assumed 28 kPa O2 of the end-Cretaceous atmosphere, when grown
under a low-end estimate (24 Pa) for CO2, for that period. Note: plants were grown and measurements made at the same O2 and CO2 levels.
804
Gale et al.
Table 2. Leaf area and whole plant respirationuphotosynthesis
ratio of C3 Xanthium strumarium and Atriplex prostrata, as
affected by the 28 kPa O2 atmosphere of the end-Cretaceous, when
grown under low and mid-level estimates of CO2 (24 or 35 kPa)
Species
Ambient air
partial pressures
Leaf area (cm2) RuP
Average
Oxygen Carbonper leaf
(kPa)
dioxide (Pa) n ˆ 10
X. strumarium 21
28
21
28
24
24
35
35
A. prostrata
24
24
35
35
21
28
21
28
(per unit
dry weight)
nˆ4
19.6"1.2 a
13.7"0.35 a
26.6"1.85 b
19.8"0.96 a
12.00"0.69
10.85"1.12
16.2"0.72
11.95"1.14
b
b
a
b
0.38"0.04
0.48"0.05
0.33"0.04
0.39"0.05
b
a
b
b
0.40"0.03
0.56"0.06
0.31"0.03
0.37"0.03
b
c
a
b
Note: numbers are averages"standard deviations. RuP, integrated
daily whole plant respirationugross-photosynthesis (see `Materials and
methods'). Species and columns were analysed separately by ANOVA:
same letter, difference n.s. (P)0.05); same letters, one with ± difference
signi®cant (P-0.05); different letters, difference highly signi®cant
(P-0.01).
respirationugross-photosynthesis (RuP) (Table 2). A similar result was obtained for plants grown under 35 Pa
CO2. Plant carbon gain is determined by leaf area, by
photosynthesis per unit leaf area and by the overall RuP
ratio. As shown in Table 2, leaf area was also reduced
by 28 versus 21 kPa O2 under both 24 and 35 Pa CO2,
in both species. Leaf number per plant (data not shown)
was not affected.
As shown above (Figs 1, 2; Table 2) at low and
medium level estimates of the ambient end-Cretaceous
CO2 there was a strong effect of elevated O2 on photosynthesis, leaf area and RuP ratio of the C3 species
X. strumarium and A. prostrata. This was expressed in
overall growth (RGR). Data for RGR are given below,
in Fig. 5, together and for comparison with those of
the C4 species. Contrary to these C3 species, the four
Flaveria species studied (including C3, C4 and intermediary C3±C4 species (see `Materials and methods')
showed no RGR effect of ambient oxygen, when grown
under 24 Pa CO2 and either 28 or 21 kPa O2 (data not
shown). This was unexpected, especially for the C3
species, as high O2 greatly increased the leaf gas
conductance of all the Flaveria species (Rachmilevitch
et al., 1999). This anomaly was resolved when photosynthesis of Flaveria was measured as a function of light
intensity. An example of such an AuPPF function is given
for the C3 Flaveria pringlei (Fig. 3).
As seen in Fig. 3, 28 versus 21 kPa O2 signi®cantly
reduced the photosynthesis of leaves of F. pringlei, only
at PPF)350 mmol m 2 s 1. The 12 h daytime light
intensity in the chambers in which the plants were grown,
Fig. 3. Rapid effect of 28 versus 21 kPa O2 on the photosynthesisulight
intensity function (AuPPF ) in attached leaves of Flaveria pringlei plants
(C3) grown under 21 kPa O2 and medium level (35 Pa) CO2. Note: at
each level of PPF the partial pressure of O2 was switched between 21 and
28 kPa (see Materials and methods).
was only 380 mmol m 2 s 1 PPF, at the plant tops, and
considerably less within the canopy.
Response of C3 plants to the high ambient
O2 (28 kPa) of the end-Cretaceous, when grown
under a high-end estimate for CO2 (60 Pa) for the
same period
As noted above, recent estimates of the atmospheric CO2
partial pressure toward the end-Cretaceous, suggest a
level of about double the pre-industrial 27 Pa, although
con®dence limits are very wide (Berner, 1994, 1997). As
relatively high CO2 counteracts at least one negative
effect of high O2 on C3 plants, namely competition with
CO2 for the Rubisco enzyme (von Caemmerer and
Farquhar, 1981), the response of the C3 species X.
strumarium and A. prostrata to high (28 kPa) O2 when
grown under 60 Pa CO2 was studied (Fig. 4).
As seen in Fig. 4, in A. prostrata plants grown under
60 Pa CO2, high, 28 kPa O2, had a smaller effect on
photosynthesis than in plants grown under 24 Pa CO2
(Fig. 1). However, for X. strumarium the effect was
essentially the same.
As noted above, there was a signi®cant depression of
RGR in the two C3 species, when grown under 28 versus
21 kPa O2 (Fig. 5). This depression was larger in plants
grown under the lower levels of CO2 (24 or 35 Pa), but
still present in plants grown at 60 Pa CO2. (Also shown in
Fig. 5, for comparison, are similar RGR data for two C4
speciesÐsee below.)
KuT-atmosphere, extinction, C4
805
Fig. 4. Effect of the assumed ambient O2 (28 kPa) of the end-Cretaceous atmosphere on the assimilationuinternal leaf-CO2 (AuCi) curves of C3 species,
grown under a high-end estimate of CO2 (60 Pa) for the same period.
growth under 28 versus 21 kPa O2 (data not shown).
Consequently and as could be expected from these results,
there was no effect of 28 versus 21 kPa O2 on the growth
(RGR) of the two C4 species, when ambient CO2 was held
at 24, 35, or 60 Pa (Fig. 5). This is in contrast to the
deleterious effect of high (28 kPa) ambient O2 on the
growth of the C3 species.
Leaf diffusion conductance of C3 and C4
species, grown under the atmospheric
composition of the end-Cretaceous
Fig. 5. Effect of the assumed 28 kPa ambient O2 of the end-Cretaceous
atmosphere on the growth (RGR) of two C3 species, Xanthium
strumarium and Atriplex prostrata and two C4 species, Zea mays and
Atriplex halimus, grown under a range of ambient CO2 levels, as
estimated for the same period. Note: plants were grown in growth
chambers with a maximum light intensity of 380 mmol m 2 s 1 PPF, at
the plant tops. Vertical bars s.e.
Response of C4 species to the high (28 kPa) O2
atmosphere of the end-Cretaceous
Leaf area and photosynthesis (the latter as expressed by
AuCi and AuPPF functions) of the C4 species Atriplex
halimus and Zea mays showed no response to elevated O2
(28 versus 21 kPa) when plants were grown under either
low (24) or high end (60 Pa) CO2 (data not shown). Daily
carbon exchange and RuP values for C4 plants (as in
Fig. 2 and Table 2) also failed to show any response to
A study of the response of leaf water vapour diffusion
conductance (gs) to the oxygen level prevailing during the
KuT boundary period has been reported previously
(Rachmilevitch et al., 1999). In these experiments ambient
CO2 had been held at the lower estimates of 24 or 35 Pa.
Growth under 28 versus 21 kPa O2 resulted in much
higher values of gs. This was found for the C3 species
Atriplex prostrata, Xanthium strumarium and Flaveria
pringlei, the C3±C4 intermediate F. sonorensis, and the
C4 species F. trinervia. Only the C3-C4 intermediate
F. ¯oridana showed just a small increase in gs, in response
to the elevated O2. In view of the above noted recent
higher estimate for the end-Cretaceous atmospheric CO2,
the study was extended to include gs response to 28 kPa
O2 under 60 Pa CO2. To this were added gs measurements
of the two C4 species Atriplex halimus and Zea mays.
Results are presented in Fig. 6. For comparison some of
the previous data for gs of X. strumarium and A. prostrata
grown under the lower estimates for atmospheric CO2
(Rachmilevitch et al., 1999) have been included.
In the two C3 species grown under the lower ambient
levels of CO2 (24 and 35 Pa) high 28 versus low, 21 kPa
O2 much increased gs (Fig. 6). This effect was particularly
pronounced in A. prostrata. However, when grown under
60 Pa CO2, gs of plants grown under 21 kPa O2 unexpectedly increased in both species, as compared to that of
806
Gale et al.
such as the Z. mays studied here, have almost certainly
had their photosynthesisugrowth characteristics modi®ed.
However, Rubisco, the main enzyme involved in the
response of C3 plants to ambient CO2 and O2 has been
highly conserved. Such changes as have been found,
such as subunit composition or O2uCO2 speci®city, are for
species originating in periods separated by hundreds of
millions of years (Jordan and Ogren, 1981; Badger and
Andrews, 1987). Theoretical models of the response
of leaf photosynthesis to ambient O2 and CO2 are based
on known biochemical properties of Rubisco (Farquhar
et al., 1980; Beerling, 1994). Responses found here were
generally as could be expected from these models.
However, hitherto unreported effects have also been
found of high O2 on leaf area, leaf diffusion±conductivity,
and the respirationuphotosynthesis (RuP) ratio.
Response of C3 plants to the simulated
end-Cretaceous atmosphere
Fig. 6. Effect of the assumed 28 kPa ambient O2 of the end-Cretaceous
atmosphere on leaf diffusion conductance (gs) of two C3 and two C4
plant species, grown under three different CO2 levels, as variously
estimated for the same period. Note: for comparison, some of the
previous data for C3 plants grown under the lower level estimates of CO2
are included (Rachmilevitch et al., 1999). Vertical bars s.e.
plants grown under 35 Pa CO2. Under 60 Pa CO2 there
was no further signi®cant effect (P)0.05) of oxygen level
(28 versus 21 kPa) on gs in either of these C3 species. In
the two C4 species A. halimus and Zea mays, stomatal
conductance, gs, generally showed no signi®cant response
to high O2. An exception was for A. halimus grown under
24 Pa CO2 in which gs was some 50% higher when plants
were grown under 28 versus 21 kPa O2 (P-0.01).
Discussion
Crucial factors of this study are the actual partial
pressures of carbon dioxide and oxygen prevalent at the
end-Cretaceous. All estimates have wide con®dence limits
(cf. Berner and Can®eld, 1989; Berner et al., 2000). Just
one high-end estimate for O2, namely 28 kPa, has been
used, and a range of carbon dioxide values between 24
and 60 Pa (Lasaga et al., 1985; Berner, 1997).
Care is needed when projecting the characteristics of
ancient plants from the response of their extant representatives. There is an inherent assumption that physiological responses to environmental parameters have not
changed signi®cantly with time, in this case the last 150
million years. Moreover, modern, highly bred plants,
An early paper on soybean (Glycine max) reports the
effect of medium levels of O2 (;30 kPa) at low CO2
(-35 Pa) on plant photosynthesis (Forrester et al., 1966).
Data from their Fig. 7 indicate a 45% drop in the rate of
photosynthesis per unit leaf area, when going from
(present day) 36 Pa CO2 and 21 kPa O2 to the low end
CO2 estimate for the end-Cretaceous of 24 Pa and the
high end O2 estimate of 28 kPa. This is similar to what
was obtained for the C3 species studied here and is in
general accord with the report of Beerling et al. (Beerling
et al., 1998) working with a simulated Carboniferous
atmosphere of 35 kPa O2 and 35 Pa CO2. As in this
study, the expected reduction in photosynthesis (see
Introduction) was found and it was reported that there
was no adaptation of plants to the high O2 atmosphere.
As shown in Fig. 1, in C3 plants grown under a
medium level estimate for CO2 (35 Pa) photosynthesis
was reduced by ambient 28 versus 21 kPa O2. However,
respiration was not reduced proportionally (Fig. 2), as
could be expected (Amthor, 1989). This may have been
a result of a relative increase in the rate of respiration
in response to high O2 (Gale, 1974). Consequently the
ratio of daily respiration to gross photosynthesis (RuP)
increased very signi®cantly (Table 2). Leaf area was
reduced by about 25% in the high O2-grown plants,
irrespective of whether the plants were grown under
low or medium CO2 (24 or 35 Pa) (Table 2). There was,
therefore, a combination of at least three factors; all of
which tend to reduce primary productivity: reduced leaf
area, reduced photosynthesis per unit leaf area and higher
RuP ratio.
Under low and medium level estimates of CO2 for the
end-Cretaceous (24 and 35 Pa), high O2 was detrimental
to factors affecting primary productivity of the C3 species
KuT-atmosphere, extinction, C4
X. strumarium and A. prostrata. The same effects were
also present, but to a lesser degree, when they were grown
under the higher (60 Pa) CO2 estimate. As before, the two
species differed in degree of response. This can be seen in
the leaf AuCi curves of plants grown under 60 Pa CO2 and
either 21 or 28 Pa O2 (Fig. 4). Whereas in A. prostrata the
high O2 effect was smaller than when the plants had been
grown under low ambient CO2 (Fig. 1), in X. strumarium
the O2 response was just as large.
The above effects of high O2 translated into a large
reduction of RGR in the C3 plants grown under 28 versus
21 kPa O2 at the lower (24 and 35 Pa) ambient CO2
levels (Fig. 5). In plants grown under 60 Pa CO2 RGR
reduction was much smaller, but still signi®cant. It should
be borne in mind that vegetative growth is essentially
exponential. Consequently, over extended periods, even a
small drop in RGR becomes a large percentage reduction
in primary productivity. As could be expected from the
variability in physiological responses, there was considerable species variability in growth response (as shown in
Fig. 5 and discussed below, for C4 species). Of special
signi®cance in respect to C3 species variability was the
lack of growth response of the C3 and C3±C4 intermediate
Flaveria species to high (28 kPa) O2 (data not shown).
This was found when they were grown under relatively
low (24 Pa) ambient CO2 and a maximum daytime light
intensity of ;380 mmol m 2 s 1 PPF, at the plant tops.
This lack of growth response was found despite the large
increase in gs of all the Flaveria species studied (C3, C4
or C3±C4 intermediates) when grown under high O2
(Rachmilevitch et al., 1999). However, as shown in Fig. 3,
at high PPF ()350 mmol m 2 s 1) leaves of the C3
Flaveria species showed a clear, rapid (within minutes)
20% reduction in photosynthesis in response to high O2
(28 versus 21 kPa). This suggests that there may have
been reduced growth of Flaveria species in geographic
regions of high solar insolation. However, it should
be noted that plants growing under high solar radiation and not, as here, in a growth cabinet, may adapt,
ontogenetically.
In the present study, plant response to the putative
end-Cretaceous atmosphere was compared to that of
today. However, in most of the Cretaceous period, CO2
levels were much higher than today and dropped to a low
at the KuT boundary (Berner, 1994). Consequently, the
percentage reduction in primary productivity, in the tens
of millions of years preceding the KuT boundary, may
have been even more signi®cant than found here.
Response of C4 plants to the simulated
end-Cretaceous atmosphere
Photosynthesis and growth of the C4 species studied here
(Z. mays, A. halimus and F. trinervia) did not respond to
high 28, versus low, 21 kPa O2. This was the case even
807
when they were grown under 24 Pa CO2, the low-end
estimate for the end-Cretaceous period. Apart from
A. halimus grown under low (24 Pa) CO2, there appeared
to be no gs response to high O2 in Z. mays and A. halimus
(Fig. 6). This differs from what was found for the C4
species F. trinervia in which gs much increased in high O2,
low CO2-grown plants (Rachmilevitch et al., 1999). High
gs sensitizes plants to water stress. The reduction in leaf
area in response to elevated O2 in C3 plants (Table 2) may
have been a result of high gs-induced water stress. However, this was not measured, or expected, as air humidity
was very high in the growth chambers (see Materials and
methods). If increased gs in response to high O2 were
indeed more common in C3 than in C4 plants, this would
be a further factor favouring the evolution of C4 physiology. However, the variance found here among the few
species studied, requires a further survey of C3 and C4
species gs response to O2, before conclusions can be
drawn.
Lack of evidence of C4 plants in a given stratum does
not necessarily mean that they had not yet emerged, only
that they were not widespread. Until recently the earliest
indication of the presence of C4 plants was reported for
strata dated to ;15 MyBP (Kingston et al., 1994) with
earlier indications for 100 MyBP (Kuypers et al., 1999).
Today, C4 species contribute only ;18% to world plant
productivity mainly, but with exceptions (see Sage and
Monson, 1998) in regions of high solar insolation
(Ehleringer et al., 1997; Collatz et al., 1998). It is possible
that the relatively low ambient O2 of today (21 kPa) is
one reason for the lack of more widespread distribution
of extant C4 species.
Conclusions
The paleo-atmosphere and the KuT boundary extinctions
The C3 species studied here are considered to be representative of important angiosperm forage plants of the late
Cretaceous period. However only a few species were
tested and the degree of their reactions varied. Even so the
results obtained strongly suggest that for some millions
of years prior to the bolide impact of 65 MyBP, primary
production of many plant species would have been
severely diminished. Reduced plant production would
have adversely affected the sustenance of dependent
herbivores (insect and animal) with the effect ampli®ed
in carnivores and along the ecological food chain. This,
together with other factors of biosphere degradation
at the end-Cretaceous period (see Introduction) may
explain the decline in many biota species which occurred
prior to the 65 MyBP KuT boundary catastrophe. As
ventured by Hallam and Wignall, the bolide impact may
have been `only the ®nal coup de grace' (Hallam and
Wignall, 1997). It is intriguing to note that according to
808
Gale et al.
the models of Berner and Can®eld and Berner et al.
(Berner and Can®eld, 1989; Berner et al., 2000), the endPermian, the greatest of all the mass extinctions
(Courtillot, 1999) was preceded (albeit by some tens of
millions of years) by the highest ever O2 event (;40 kPa),
with concomitant low, ;25 Pa, CO2 (Berner, 1994).
The paleo-atmosphere and the emergence of C4 plants
Contrary to the response of the C3 species, the primary
productivity of the C4 species was unaffected by the
(assumed) 28 kPa O2 of the end-Cretaceous, even at the
lowest estimate for CO2 (24 Pa) for that period, which
was tested. This suggests that high O2 may have constituted a strong driver for the evolution of C4 from C3 plants,
at least as far back as about 100 MyBP, the earliest date
for which evidence has been found suggesting their presence (Kuypers et al., 1999). This is in addition to other
C4 evolution-inducing environmental factors, such as low
CO2, high temperatures and water stress (Ehleringer and
Monson, 1993; Cerling et al., 1994, 1998).
Acknowledgements
This project was carried out under a grant from the Bi-National,
US Israel Research Fund (BSF). Partial support was received
from the Aaron Beare Foundation, South Africa. Special thanks
are due to Professor Robert Pearcy for his co-operation and for
extending the use of facilities at UC Davis, CA, to JG, for the
Flaveria studies.
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