Natural abundance of 13C in tropical grasses from the INPA, Instituto Nacional de Pesquisas da Amazônia, herbarium

Abundância natural de isótopos de carbono em gramíneas tropicais do herbário do INPA, Instituto Nacional de Pesquisa da Amazônia

Abstracts

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 <FONT FACE="Symbol">d</font> 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.

Grasses; Amazon; <FONT FACE=Symbol>d</font> 13C; photosynthetic; cycle; C3; C4

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 <FONT FACE="Symbol">d</font> 13C variando entre -13,6 a - 9,5‰, enquanto as restantes 40% foram classificadas como sendo espécies do tipo C3, com valores de <FONT FACE="Symbol">d</font> 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.

Natural abundance of ¹³C in tropical grasses from the INPA, Instituto Nacional de Pesquisas da Amazônia, herbarium

ERNESTO MEDINA¹, LUIZ ANTONIO MARTINELLI², EDELCILIO BARBOSA³and 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 d 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 d ¹³C variando entre -13,6 a - 9,5‰, enquanto as restantes 40% foram classificadas como sendo espécies do tipo C3, com valores de d 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, d 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-enol-carboxykinase (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 (d 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 d ¹³C values of grass species growing naturally in lowlands in northern Brazil. The frequency distribution of d 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 d ¹³C (‰) notation, defined as: d¹³C = [(R_sample/R_std)-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: d ¹³C3 = d ¹³CO_2-atm - a - (b-a) . (ci/ca), where: d ¹³C3 is the stable isotopic composition of a C3 plant; d ¹³CO_2-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 d ¹³C of C4 plants (d ¹³C4) may be estimated by the following simplified equation: d ¹³C4 = d ¹³CO_2-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: d ¹³C4 = d ¹³CO_2-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 d 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 d 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 (d ¹³CO_2-atm) and its concentration are environmental factors that influence the abundance of stable carbon isotopes of C3 plants (d ¹³C3). On the other hand, the CO₂ concentration inside the leaf intercellular space (ci) is a physiological factor that controls the d ¹³C3 values.

The values of d 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 d ¹³C values of the atmospheric CO₂. Generally, C3 plants in the tropics had d ¹³C values smaller than C3 plants in temperate areas (table 1). Typically, the d 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 d 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 d 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 d 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 d ¹³C values of C3 grasses averaged -28.9 ± 2.5‰ (n = 65) and have a wide range of 11.3‰ (figure 1). The average d 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 d 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 d¹³C (⁰/₀₀) among grass species. F is the frequency.

In comparison with d ¹³C values of leaves from tropical trees, the average d ¹³C of C3 grasses is less negative (table 1). On the other hand, the average d ¹³C values of C3 grasses found in this study was more negative in relation to the average d 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 d ¹³C values (Farquhar et al. 1989). In addition, the d 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 d 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 d 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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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.

1. Instituto Venezolano de Investigaciones Científicas (IVIC), Centro de Ecología y Ciencias Ambientales, Aptdo 21827, Caracas, 1020-A, Venezuela.

Instituto Venezolano de Investigaciones Científicas (IVIC), Centro de Ecología y Ciencias Ambientales, Aptdo 21827, Caracas, 1020-A, Venezuela.

Publication Dates

• Publication in this collection
  19 Aug 1999
• Date of issue
  Apr 1999

History

• Received
  30 Oct 1997
• Accepted
  30 Sept 1998