Natural abundance of 13C in tropical grasses from the INPA, Instituto
Nacional de Pesquisas da Amazônia, herbarium
ERNESTO MEDINA1, LUIZ ANTONIO MARTINELLI2, EDELCILIO
BARBOSA3 and REYNALDO LUIZ VICTORIA2
(recebido em 30/10/97; aceito em 30/09/98)
ABSTRACT - (Natural abundance of 13C in tropical grasses from the INPA, Instituto
Nacional de Pesquisas da Amazônia, herbarium). The stable carbon isotopic
composition of 165 grass species was determined with the objective of verifying their
photosynthetic pathway (C3 and C4). The samples, taken from the INPA herbarium,
were mainly collected in the North of Brazil. Approximately 60% of the species proved
to be of the C4 type, with  13C values ranging from -13.6 to -9.5‰, while the
remainder 40% belonged to the C3 type, with values ranging from -34.7 to -23.4‰.
This relatively high proportion of C3 species is probably due to the high relative
humidity of the sites where the species were collected.
RESUMO - (Abundância natural de isótopos de carbono em gramíneas tropicais do
herbário do INPA, Instituto Nacional de Pesquisa da Amazônia). Foi determinada a
composição isotópica natural do carbono em 165 espécies de gramíneas encontradas no
herbário do INPA com o intuito de se determinar o tipo de ciclo fotossintético (C3 ou
C4). Aproximadamente 60% das espécies foram classificadas como sendo do tipo C4,
com valores de  13C variando entre -13,6 a - 9,5‰, enquanto as restantes 40% foram
classificadas como sendo espécies do tipo C3, com valores de  13C variando de -34,7 a
-23,4‰. A proporção relativamente elevada de espécies do tipo C3, provavelmente, é
conseqüência da umidade relativa alta encontrada nos locais em que as espécies foram
coletadas.
Key words - Grasses, Amazon,  13C, photosynthetic cycle, C3, C4
Introduction
The grass family (Poaceae) is highly diversified in terms of photosynthetic types. In this
family C3 and C4 species co-occur, with a clear tendency for the C3 species to
dominate in relatively more humid and/or cooler areas. These ecological separations of
C3 and C4 grass species have been shown across latitudinal gradients (Ellis et al. 1980,
Hattersley 1983, 1992), altitudinal gradients in the tropics (Meinzer 1978, Tieszen et al.
1979, Rundel 1980), and gradients of water availability and seasonality (Medina &
Motta 1990).
The general C4 pathway of photosynthesis is characterized by the primary CO2 fixation
mediated by phospho-enol-pyruvate-carboxylase that occurs in the cell of the
mesophyll. The result of this fixation is the synthesis of malic and/or aspartic C4 acids.
These acids are subsequently translocated to the bundle-sheath cells, where they are
decarboxylated and the resulting CO2 is fixed through the normal carbon reduction
pathway mediated by Rubpcarboxylaseoxigenase. The C4-grasses can be further
differentiated in more or less discrete groups according to the enzymes mediating the
decarboxylation of organic acids in the bundle-sheath (Farquhar et al. 1989). Those
groups are: a) malic enzyme NAD-dependent (NAD-ME), b) phospho-enolcarboxykinase (PCK), and c) malic enzyme NADP-dependent (NADP-ME). In the
NAD-ME group the main acid synthesized after the primary fixation of CO2 is
aspartate, while in the NADP-ME group the main acid is malate. In the PCK type both
aspartate and malate are produced in the primary reaction (for a detailed description see
Hattersley 1992).
The measurement of the natural abundance of carbon-13 ( 13C) is a straightforward
method to assess the basic photosynthetic pathway in terrestrial higher plants (Farquhar
et al. 1989). The isotope technique is becoming increasingly used, especially in
environmental studies. However, there is still a lack of large scale survey in grasslands
of many parts of the world.
This paper reports on the  13C values of grass species growing naturally in lowlands in
northern Brazil. The frequency distribution of  13C values, both in C3 and C4 grasses,
and their distribution among taxonomic well differentiated groups will, hopefully,
stimulate further research on the ecophysiological properties of tropical grass species.
Material and methods
The species have been separated according to their taxonomic position as recognized by
Watson & Dallwitz (1992). The assignment of C4 grasses to biochemical types follows
Hattersley (1987) and is only a first approximation.
Isotopic technique - The carbon isotopic composition expresses the relative abundance
of atoms of 13C (less abundant) in relation to atoms 12C (more abundant). The carbon
isotopic composition may be expressed in terms of  13C (‰) notation, defined as: 13C
= [(Rsample/Rstd)-1] . 100, where: R is the ratio 13C:12C of the sample and standard (std),
respectively. The standard reference material is a limestone (PDB - Pee Dee formation).
The following equation was developed by Farquhar et al. (1982) to estimate the
abundance of stable carbon isotopes in C3 plants:  13C3 =  13CO2-atm - a - (b-a) .
(ci/ca), where:  13C3 is the stable isotopic composition of a C3 plant;  13CO2-atm is the
stable isotopic composition of the atmospheric CO2 (-7 to - 8‰ for free atmospheric
CO2); ca is the concentration of atmospheric CO2 and ci is the concentration inside the
leaf intercellular space. The constant a is the fractionation that occurs due to diffusion of
CO2 from the atmosphere to the stomata. This value is relatively constant and equals to
4.4‰ (Farquhar et al. 1989). Finally, b is the net fractionation caused by carboxylation
(approximately constant and equals to 30‰).
According to Farquhar et al. (1989), the  13C of C4 plants ( 13C4) may be estimated by
the following simplified equation:  13C4 =  13CO2-atm a - (b4 + b . Ø - a) . (ci/ca),
where: b4 is the effective discrimination by PEP carboxylase, approximately -5.7‰,
and Ø is the "leakness" of CO2 and HCO3 fromthe bundle sheath to mesophyll cells. The
value of this leakness is most of the time equals to 0.34, making the term (b4 + b . Ø - a)
equals to zero, which simplifies the above equation to:  13C4 =  13CO2-atm- a.
Collection of herbarium material - All dried leaves of grass species were taken from the
herbarium of the National Institute of Amazon Research (INPA), comprising a total of
168 samples. From this total, 69 samples were collected in the Amazon region (mainly
from the central part of the Basin), six in the Rio de Janeiro state, one near the city of
Brasília and one in the Chapada dos Guimarães (MT). The remainder samples (91) did
not have specified sampling sites.
Measurement of carbon isotopic composition - The dried leaves were ground and
sieved. This material was combusted at 900°C in the presence of CuO in sealed and
evacuated Pyrex tubes for 12 hours. Carbon stable isotope analyses were performed on
the CO2 produced by this combustion in a mass spectrometer Finnigan-Delta E, fitted
with double inlet and double collector systems. As already mentioned, results are
expressed in  13C ‰ relative to the PDB standard. Samples were analyzed at least in
duplicate with a maximum difference of 0.3‰ between replicates.
Results and Discussion
During the photosynthetic process, plants promote isotopic fractionation of the CO2
atmospheric source. They tend to favor the lighter isotope (12C) in relation to the heavier
(13C). Therefore, plants will have less 13C in relation to the atmospheric CO2. In terms
of  13C notation, plants will have more negative values in relation to the atmospheric
CO2. The extent of this isotopic discrimination is different between C3 and C4 plants.
The C3 group of plants has more negative values in relation to C4 group (table 1). This
important difference enables the use of carbon isotopic composition to distinguish C3
and C4 plants (Smith & Epstein 1971).
The isotopic composition of the atmospheric CO2 ( 13CO2-atm) and its concentration are
environmental factors that influence the abundance of stable carbon isotopes of C3
plants ( 13C3). On the other hand, the CO2 concentration inside the leaf intercellular
space (ci) is a physiological factor that controls the  13C3 values.
The values of  13C3 of tree leaves vary from -25 to -35‰. This large variability is due
primarily to variations in the balance of stomatal conductance to photosynthesis that, in
turn, affect the ci/ca ratio and, in a lesser extent, due to variations in the  13C values of
the atmospheric CO2. Generally, C3 plants in the tropics had  13C values smaller than
C3 plants in temperate areas (table 1). Typically, the  13C values of plants in the tropics
vary from -29 to -34‰ (e.g. Medina & Minchin 1980, Medina et al. 1991, Ducatti et al.
1991, Kapos et al. 1993, Martinelli et al. 1994, Fischer & Tieszen 1995, Buchmann et
al. 1997a, Martinelli et al. 1998), while in temperate areas typical  13C values vary
from -25 to -29‰ (e.g. Schleser 1992, Flanagan et al. 1996, Hanba et al. 1997,
Buchmann et al. 1997b). This difference observed in  13C values of leaves found in
tropical and temperate areas is mainly due to relatively wetter conditions found in the
tropics, which lead to higher ratios of stomatal conductance to photosynthesis in the
tropics, increasing the ci/ca ratio. This, in turn, leads to lower  13C values in these areas
(Farquhar et al. 1989).
The different set of grasses that were taken for carbon isotope analyses display the
expected distribution of natural abundance of 13C (table 2). The subfamilies Pooideae
and Bambusoideae are homogeneously of the C3 photosynthetic type, while the
Chloridoideae are homogeneously C4. The Arundinoideae contain C3 (Gynerium) and
C4 (Aristida) genera. Within the large group of Panicoideae the tribes Arundinellae,
Andropogonae and Maydeae contain only C4 species. The tribe Paniceae on the
contrary, contain exclusively C4 (the best represented genera being Axonopus,
Brachiaria, Digitaria, Echinochloa, and Paspalum) and C3 genera (such as
Ichnanthus). The genus Panicum has both C3 and C4 species (Hattersley 1987).
The  13C values of C3 grasses averaged -28.9 ± 2.5‰ (n = 65) and have a wide range
of 11.3‰ (figure 1). The average  13C values of C3 grasses collected in the Amazon
region was equal to -28.9 ± 2.3‰ (n = 25) and have a range of 9.0‰. The average  13C
of C3 grasses samples from unknown sites was equal to -28.7 ± 2.4‰ (n = 33), having a
range of 10.3‰. These averages are not statistically different from each other (t-test for
unequal variance). As already pointed out, this range may be a consequence of the
ecological conditions during leaf development.
Figure 1. Histogram showing the distribution of 13C (0/00) among grass species. F is the
frequency.
In comparison with  13C values of leaves from tropical trees, the average  13C of C3
grasses is less negative (table 1). On the other hand, the average  13C values of C3
grasses found in this study was more negative in relation to the average  13C values
found for nine C3 grasses samples collected at border areas of the Lobo Dam (São
Paulo state), for which the average value was -26.1 ± 1.3‰ (Mozeto et al. 1996). These
differences are difficult to explain, mainly due to the fact that plants listed in table 2
have not their habitat fully characterized. Therefore, the environmental conditions, to
which these plants were exposed during their growth, are not known. However,
independently of local environmental conditions, it is likely that tree leaves collected
from a forest have less light available during their life, in relation to leaves of C3
grasses. This, in turn, leads to a higher ratio of stomatal conductance to photosynthesis
(high ci/ca ratio) in leaves from the forests, leading to more negative  13C values
(Farquhar et al. 1989). In addition, the  13C of the atmospheric CO2, which is the
source for photosynthesis, is more negative inside forests (-11 to -16‰), in relation to
the free atmosphere (-8 to -9‰) (Medina et al. 1986, Sternberg et al. 1989, Grace et al.
1995, Lloyd et al. 1996, Kruijt et al. 1996). This also contributes to decrease the value
of C3 leaves from forest trees, although at a lesser extent than the light factor
(Broadmeadow & Griffiths 1993, Sternberg 1997).
The C4 grass species sampled averaged -11.7 ± 0.9‰ (n = 103), and their range of
values was only 4.1. The average  13C values of C4 grasses collected in the Amazon
region was equal to -11.8 ± 0.9‰ (n = 44) and have a range of 3.7‰. The average  13C
of C4 grasses samples from unknown sites was equal to -11.7 ± 0.8‰ (n = 58), having a
range of 4.1‰. These averages are not statistically different from each other (t-test for
unequal variance) and are in the range expected for C4 plants (table 1).
The most remarkable of this list of tropical grasses is the relatively high proportion of
C3 species (~40%) either for samples collected in the Amazon region and from
elsewhere. For the plants from the Amazon region, this is probably the result of the
relatively humid environments where grasses were sampled. Amazonian areas in
northern Brazil receive annual rainfalls above 2000 mm with minimum of 50 to 200
mm in July and maximum between 200 and 350 mm in January. Supporting this
hypothesis, from 21 species collected at wetlands in the Lobo Dam (São Paulo state),
30% were C3 plants (Mozeto et al. 1996).
Most of the Bambusoideae genera are associated with forest environments (Chusquea,
Olyra, and Lithachne) while others occupy seasonally flooded sites (Leersia, Luziola,
and Oryza). A similar situation is observed within the Panicoideae, with C3 species
associated with wet and flooded sites (Hymenachne and C3 species of Panicum).
However, flooded sites in Amazonian areas are frequently dominated by C4 species of
the Panicoideae subfamily. Notable examples are Echinochloa polystachia, Panicum
elephantipes, P. fasciculatum, and several species of Paspalum (Junk 1970, Medina et
al. 1976, Escobar & Gonzáles 1979, Haase 1989, Medina & Motta 1990, Piedade et al.
1992).
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1. Instituto Venezolano de Investigaciones Científicas (IVIC), Centro de Ecología y
Ciencias Ambientales, Aptdo 21827, Caracas, 1020-A, Venezuela.
2. Centro de Energia Nuclear na Agricultura (CENA), Av. Centenário 303, 13416-000
Piracicaba, SP, Brazil.
3. Instituto Nacional de Pesquisas da Amazônia (INPA), Alameda Cosme Ferreira 1756,
69000-083 Manaus, AM, Brazil.
Correspondence to: L.A. Martinelli.