Bot. Bull. Acad. Sin. (1997) 38: 45_48

Kao — M. transmorrisonensis in a mountain grassland

Contribution of Miscanthus transmorrisonensis to soil organic carbon in a mountain grassland: Estimated from stable carbon isotope ratio

Wen-Yuan Kao¹

Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China

(Received March 18, 1996; Accepted Novmeber 27, 1996)

Abstract. To estimate the contribution of Miscanthus transmorrisonensis to soil organic matter in a high mountain grassland at Tartarchia Anpu (altitude of 2,600 m) in Taiwan, I analyzed the stable carbon isotope ratios (d¹³C) of dominant plant species and soil organic matter. The d¹³C analysis reveals that among the five dominant plant species M. transmorrisonensis (d¹³C of -12.37‰) is the only species that uses C₄ photosynthetic pathway. In contrast, the d¹³C values of other plants species ranged from -29.21 to -23.3 ‰ (average of -26.25‰), typical values for terrestrial plants with C₃ photosynthetic pathway. The average d ¹³C value of soil samples collected from ten locations over the grassland was -18.56 ± 0.36 ‰ again indicating that the vegetation contains a mixture of C₃ and C₄ type plants. Using two-end member linear mixing models, I then estimated that about 55% of soil organic carbon in this grassland came from M. transmorrisonensis.

Keywords: C₃ and C₄ photosynthetic pathways; Grassland; Miscanthus transmorrisonensis; Soil organic carbon; Stable carbon isotopes.

Introduction

The concentration of greenhouse gasses, mainly carbon dioxide, methane, and nitrous oxide in the atmosphere has been increasing since the Industrial Revolution. As a consequence, the earth warms up and climates change (Houghton et al., 1990). It has been suggested that C₄ plants evolved as an adaptation to low CO₂ concentration in Late Tertiary (Ehleringer et al., 1991). The CO₂ concentrating mechanism of C₄ pathway confers a higher productivity, better water use efficiency, and greater temperature tolerance as compared with C₃ plants, however, the advantages may be diminished at elevated CO₂. Thus, concern about the potential threat posed to plants containing the C₄ photosynthetic pathway by increasing atmospheric carbon dioxide has been raised (Henderson et al., 1994). To evaluate the potential effect of changes in climate on the distribution of C₄ plants, it is hence essential to study the current distribution of C₄ plants in the various ecosystems.

Stable carbon isotopes have been used in agricultural and ecological research for decades (Tieszen and Boutton, 1989). The two stable carbon isotopes are ¹²C and ¹³C, which comprise 98.89 and 1.11%, respectively, of all carbon in nature (Ehleringer and Rundel, 1989). Currently, the carbon isotope ratio (d¹³C) of atmospheric CO₂ is about -7.5‰ (Keeling et al., 1984). Plants contain less ¹³C than the atmosphere because the physical and chemical pro

cesses involved in CO₂ uptake discriminate against the isotope (Berry, 1989). During photosynthetic uptake of CO₂, the carboxylation enzyme ribulose bisphosphate carboxylase discriminates against ¹³C more than phosphoenol pyruvate carboxylase (O'Leary, 1981; Roeske and O'Leary, 1984). As a consequence, C₃ plants have less ¹³C than C₄ plants. For example, d¹³C values of C₃ plants vary from -23 to -34 ‰, whereas C₄ plants have values of approximately -10 to -15 ‰ (O'Leary, 1988). Therefore, ¹³C analysis has become an accepted method for determining the pathway of CO₂ fixation of terrestrial plants.

In natural ecosystems, soil organic carbon is derived predominantly from the residues of native vegetation, hence the stable carbon isotope ratio of the vegetation has a direct impact on the d¹³C of the soil organic matter (d¹³C_som). It has been shown that the natural isotopic abundance of soil organic matter corresponds closely to that of the vegetation cover from which it originated (Nadelhoffer and Fry, 1988). Thus, the characteristic d¹³C of the vegetation serves as a marker to indicate the origin of soil organic matter (Balesdent et al., 1987; Rao et al., 1994; Kessel et al., 1994). For example, mass-balance calculations based on the d¹³C values of a geological sample and assumed end-member d¹³C values for C₃ and C₄ plants have been used to estimate the relative proportion of C₃- and C₄- derived carbon in a sample of organic carbon (Bird et al., 1994).

The objective of this study was to analyze the carbon isotopes ratio of soil organic carbon to estimate the relative contribution of C₃ and C₄ plants to soil organic carbon in a mountain grassland.

¹Fax: 886-2-782-7954.

Botanical Bulletin of Academia Sinica, Vol. 38, 1997

Study Site

Tartarchia Anpu grassland (altitude of 2,600 m) is located in Yushan National Park (23°29' N, 120°48' E), Nantou county, in central Taiwan. It is classified as a mountain temperate grassland. The mean monthly air temperature ranges from 6°C in December to 14°C in July (Climatological data annual report, Central Weather Bureau, ROC). The mean annual precipitation is approximately 4,000 mm. A visual examination of the vegetation indicated that Miscanthus transmorrisonensis, Baeothryon subcapitatum (Thwaites) T. Koyama, Pinus taiwanensis Hayata, Yushania nittakayamensis, and Lycopodium cernuum Linn. are the five most dominant plant species of the vegetation composition.

Materials and Methods

Leaf materials were collected from five individuals of each of the five dominant species. Individuals of each species were sampled at least 1 m away from each other. Leaves were dried at 70°C for at least 48 h and then ground to a fine powder using a mortar and pestle. Ten soil sampling points were chosen over the grassland, the distance between each sampling points was at least 1 m. To minimize the effect of progressive modification of isotopic composition of soil organic matter by microbial degradation (Nadelhoffer and Fry, 1988), at each sample point the top 5 cm of soil of a 0.02 m plot was collected. Soil samples were air-dried, after removal of coarse litter and roots, and sieved through a 0.5 mm mesh (Der Shuenn, Taiwan). The pH value of the soil was determined using a pH meter (Suntex TS-2, Taiwan) by mixing soil with distilled water in a proportion of 1 to 10. Total carbon contents were determined with an elemental analyzer (NA1500, Fisons, Italy).

Inorganic carbon was removed from soil samples prior to isotopic analysis by pre treatment with 1 M HCl at room temperature overnight, rinsed with distilled water, then oven dried for 12 h.

Two to 3 mg of dried plant material or 10 to 15 mg of soil sample were sealed with 1 g of copper oxide wire (Merck) and 1 piece of silver foil (2 mm × 10 mm) under vacuum in a 6 mm quartz tube and heated to 850°C for 4 h (Ehleringer and Osmond, 1989). The tubes were then allowed to cool slowly for 10 h. After combustion, the sealed tube contained CO₂, H₂O, and N₂. The tube was then cracked under vacuum, and the gasses were separated by passing them through an ethanol-dry ice trap to remove H₂O and a liquid nitrogen trap to collect CO₂ (Ehleringer and Osmond, 1989). The carbon isotope ratio of the purified CO₂ was determined on an isotope ratio mass spectrometer (SIRA10, VG Instruments, Oxford, UK). The carbon isotope ratio of the organic matter was expressed as

d¹³C (‰) = [(R_sample/R_PDB)- 1] × 1000, where R = ¹³C/¹²C.

Thus, d¹³C is the difference in carbon isotope ratios between a sample (R_sample) and the PDB standard (R_PDB), in

thousandths (‰) of the isotope ratio in the standard, and PDB refers to the belemnite carbonate standard of the Peedee Formation, South Carolina, USA, which is accepted as the international standard. Measurements of organic standards were reproducible to ±0.05‰.

The following equation was then used to calculate the proportion of the soil carbon from C₄ plants:

d¹³C_som = d¹³C_C4 × f + d¹³C_C3 × (1-f) (eqn. 1)

where

d¹³C_som = carbon isotope ratio of the soil organic matter,

d¹³C_C3 = carbon isotope ratio of C₃ plants,

d¹³C_C4 = carbon isotope ratio of C₄ plants,

f = proportion of carbon from C₄ plants, and

1-f = proportion of carbon from C₃ plants.

Results and Discussion

d¹³C Values of Vegetation and Soil Organic Matter

Results of the d¹³C values of the five dominant species in the Tartarchia Anpu grassland are presented in Table 1. According to these values, the five dominant species can be divided into two groups. The d¹³C value of M. transmorrisonensis is within the range of typical C₄ plants (d¹³C of -14 to -10 ‰) indicating that they use the C₄ photosynthetic pathway. In contrast, L. cernuum, P. taiwanensis, Y. nittakayamensis, and B. subcapitatum have d¹³C values ranging from -29.21 to -23.33 ‰, which are typical of plant species with the C₃ photosynthetic pathway, indicating that these species are C₃ plants.

The average pH value and total carbon content of ten soil samples were 4.5 and 8.0 ± 0.7 (mean ± S.E. ), respectively. The d¹³C values of these samples range from -19.02 to -18.11 ‰ (Table 2). The d¹³C values of the soil samples were between that of C₃ plants and C₄ plants, implying that soil organic carbon of this grassland came from a mixture of C₃ and C₄ vegetation.

Estimate of Soil Carbon Derived from M. transmorrisonensis

The primary influence of d¹³C of soil organic matter is the relative contribution of C₃ versus C₄ plants to the total net primary productivity of the community under study. The distinct difference in d¹³C value of M. transmorrisonensis from other dominant plant species

Table 1. The d¹³C values (mean ± S. E., n=5) of the dominant species in the Tartarchia Anpu grassland.

Species d¹³C_PDB (‰)

Miscanthus transmorrisonensis -12.37 ± 0.14

Baeothryon subcapitatum (Thwaites)

T. Koyama -23.33 ± 0.26

Pinus taiwanensis Hayata -26.03 ± 0.22

Yushania nittakayamensis -26.41 ± 0.31

Lycopodium cernuum Linn. -29.21 ± 0.13

Kao — M. transmorrisonensis in a mountain grassland

Table 2. d¹³C values (‰), total carbon content and pH of the soil organic matter and the calculated contribution of C₄ plants (according to eqn.1 and the values of Table 1) in the Tartarchia Anpu grassland.

Location pH C (%) d¹³C_PDB (‰) % of C₄

1 4.5 7.7 -18.23 57.8

2 4.3 12.2 -18.11 58.6

3 4.2 7.1 -18.07 58.9

4 4.4 9.3 -18.58 55.3

5 4.4 8.2 -19.02 52.1

6 4.5 9.4 -18.98 52.4

7 4.5 5.5 -18.78 53.8

8 4.6 6.2 -18.39 56.6

9 4.6 5.6 -18.94 52.7

10 4.5 9.1 -18.45 56.2

mean ± S.E. 4.5± 0.0 8.0 ± 0.7 -18.56 ± 0.36 55.4 ± 0.8

(Table 1) serves as a marker to trace the contribution of this species to soil organic carbon. Thus using the mass-balance equation (eqn. 1) and assuming two end-members of -12.37 (the average d¹³C value of M. transmorrisonensis) and -26.26 ‰ (the average d¹³C value of dominant C₃ plants), it was estimated that M. transmorrisonensis contributes about 55% of the total soil organic carbon in this mountain grassland (Table 2).

In a steady-state system, d¹³C of the soil organic matter should be nearly identical to that of the plant community from which the organic matter was derived, unless the isotopic composition of plant tissue is altered during decomposition (Boutton, 1996). Direct measurements of five dominant plant species in a woodland indicate that d¹³C of plant tissue remains relatively constant during the early stages of decomposition (Boutton, 1996). Wedin et al. (1995) found small (0.6 to 1.5 ‰) shifts in the d¹³C of litter from four grass species. In addition, some indirect evidence also suggests that fractionation during decomposition is small (from < 1 to 2‰ ) especially in terrestrial ecosystems (Nadelhoffer and Fry, 1988; Boutton, 1996). The average d¹³C value of M. transmorrisonensis is about 14‰ higher than that of C₃ plants. Therefore, even if there is discrimination during decomposition, the isotopic discrimination would be very small compared to the large d¹³C difference in C₃ and C₄ plants. Though fractionation during decomposition was not measured in this study, the estimate of the relative contribution of M. transmorrisonensis versus other C₃ plants to soil organic carbon based on the analysis of carbon isotopic composition is still valid.

Chou et al. (1991) had previously studied the vegetation succession in the same grassland, finding that the coverage percentage of M. transmorrisonensis after a surface clearing varied in three measurements, from 3% to 22%. In addition, seasonal variation in contribution to primary production by C₃ and C₄ plants has been found in a mixed prairie (Ode et al., 1980). These results indicate that an estimate of the relative contribution of C₃ versus C₄ plants to primary production based on a one-time measurement of the vegetation coverage could result in an erroneous conclusion. In contrast, analysis of the d¹³C of the soil organic matter pool reflects the long-term integrated d¹³C of

the plant communities that contributed to the soil organic matter at a given site.

In conclusion, analysis of the stable carbon isotope ratio of soil organic carbon provides quantitative evidence of the relative contribution of C₃ versus C₄ plants to a community's net primary productivity.

Acknowledgments. The author is grateful to the following: The Yu-shan National Park Bureau for allowing the author access to the study site; Dr. Chung-Ho Wang for providing the mass spectrometer; Ms. Yu-Shiun Chiou and Mr. Kuo-Wei Chang for their assistance; and Dr. Chang-Huang Chou for the inspiration for this study. Drs. George R. Waller and H. H. Cheng, who provided constructive suggestions in improving the manuscript. This research was supported by the Academia Sinica, Taiwan, ROC.

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