Bot. Bull. Acad. Sin. (2003) 44: 107-112

Wang & Yeh — d¹³C of marine macroalgae from Taiwan

d¹³C values of marine macroalgae from Taiwan

Wei-Lung Wang¹ and Hsueh-Wen Yeh^2,*

¹Department of Biology, National Changhua University of Education, Changhua 500, Taiwan

²Institute of Earth Sciences, Academia Sinica, Taipei 115, Taiwan

(Received April 18, 2002; Accepted January 10, 2003)

Abstract. The natural abundance of stable carbon isotope values (d¹³C) of organic matter of marine macroalgae collected from Taiwan and its offshore islands (Penghu Islands) were analyzed in this study. These values ranged from -10.5 to -29.5‰. The highest d¹³C value came from green algae (-10.5‰), Ulva pertusa while most algae exhibited values in the -14 to -19‰ range. On average, the d¹³C values of red algae (-17.7‰) are slightly more negative than those of green (-16.5‰) and brown (-13.6‰) algae. The carbon isotope record from organic matter of marine algae and its implications for marine algae physiology are also discussed.

Keywords: Marine macroalgae; d¹³C; Taiwan.

Introduction

Carbon isotope fractionation is associated with photosynthesis, and non-photosynthetic physical-chemical processes can also discriminate, e.g. lipid synthesis from carbohydrates (Farquhar et al., 1989). The carbon isotope composition of plants is employed to indicate photosynthetic pathways in terrestrial plants (Bender, 1968; Troughton, 1979). The d¹³C values for terrestrial C₃, C₄, and CAM plants range from -22 to -38‰, -11 to -19‰, and -13 to -34‰, respectively. The lower d¹³C values in the range for CAM plants refers to facultative CAM plants functioning as C₃ plants. Carbon isotope combinations measured in marine algae range between -8.8‰ and -34.7‰, potentially leading to the mistaken impression that both C₃ and C₄ photosynthetic pathways are present (Raven et al., 1982; Stephenson et al., 1984; Dunton and Schell, 1987).

Many studies on the pathway of CO₂ assimilation during photosynthesis in plants have demonstrated that differences arise in metabolism of the carbon isotopes ¹³C and ¹²C (Park and Epstein, 1960, 1961; Bender, 1968, 1971; Tregunna et al., 1970; Smith and Epstein, 1971; Troughton, 1979; Farquhar et al., 1989; Sackett, 1991; Johnston and Raven, 1992; Maberly et al., 1992; Raven, 1993). With terrestrial C₃ and C₄ plants, the degree of discrimination against ¹³CO₂ is distinctive and falls in two clearly separable ranges (Bender et al., 1973; Smith et al., 1973; Yeh and Wang, 2001). The ranges for both C₃ as well as C₄ plants can largely be explained by variations in d¹³C values of source CO₂ and/or in environmental conditions, in addition to probable species effect (Yeh and Wang, 2001).

However, with plants which assimilate CO₂ via the CAM pathway, the extent of discrimination stretches over the entire range covering both in C₃ and C₄ plants (Bender et al., 1973; Osmond et al., 1973). Other work has shown that much of the isotope discrimination during photosynthetic CO₂ uptake is by ribulose-bisphosphate carboxylase-oxygenase (RUBISCO) in terrestrial C₃ plants (Park and Epstein, 1961; Whelan et al., 1973) while the carboxylation catalyzed by phosphoenolpyruvate carboxylase (PEPCase) shows only a slight isotopic discrimination in terrestrial C₄ plants (Whelan et al., 1973). Since these are the two known carboxylases involved in photosynthetic CO₂ assimilation, CO₂ assimilation can be assessed via PEPCase or RUBISCO or both in a given tissue by studying the d¹³C value of the tissue. Reiskind and Bowes (1991) report that phosphoenolpyruvate carboxykinase (PEPCK) fixes CO₂ in the "C₄-like" marine green algae Udotea flabellum, and are involved in the high rates of dark CO₂ fixation in diatoms and brown algae. Thus, an index of CO₂ assimilation in an organism can be theoretically obtained by knowing the d¹³C value.

For subtidal algae two carbon sources are available for photosynthesis: CO₂ and HCO₃^-. The marine macroalgae with d¹³C values lower than -30‰ are mainly subtidal red algae, with some shaded intertidal red algae and a few green algae, and those examined in the laboratory rely on diffusive CO₂ entry (Raven, 1997; Raven et al., 2002). Some red algae with very low d¹³C values and diffusive CO₂ entry live in warmer, high intertidal habitats, with significant CO₂ gain from the atmosphere. Marine benthic organisms with very positive d¹³C values are mainly green macroalgae and seagrass, with some red and brown macroalgae, with a high d¹³C that can be accommodated by CO₂ use without discrimination in favor of d¹³C (Raven et al., 2002). All of these organisms are able to use HCO₃^-.

*Corresponding author. Tel: 886-2-2783-9910 ext. 619; Fax: 886-2-2783-9871; E-mail: hwyeh@earth.sinica.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Most species of marine benthic photolithotrophs have d¹³C values ranging from -10 to -30‰. The d¹³C value cannot distinguish between HCO₃^- and CO₂ use although other evidence suggests that most of these organisms can use HCO₃^-. The photosynthesis of organisms with d¹³C values more positive than that of CO₂ in seawater (-10‰) must involve HCO₃ use. It is mainly unknown what proportion of the different carbon sources are taken up by the different species of macroalgae, which obviously complicates the interpretation of the data. A more extensive investigation of the d¹³C values in marine algae from Taiwan was undertaken to understand more fully the carbon isotope record and its implications for marine algae physiology.

Materials and Methods

Marine macroalgae were collected from Taiwan and its offshore islands (Penghu Islands) (Figure 1). Collection sites for the algae examined are cited where data on d¹³C are given.

The samples were initially dried in an oven at 50°C. The dried samples were pulverized in an agate mortar with a pestle. For carbonate-containing samples, each of the

powdered samples was treated with hydrochloric acid (1.2 M) for 48 h, or until effervescence stopped, to remove carbonates. The completely decalcified sample was washed with distilled water and then dried at 50°C. Around 4 mg of the powder sample was placed in a Pt crucible admixed with purified CuO. The CuO had been treated with pure oxygen gas of slightly greater than one atmosphere pressure at about 1100°C for one week. The crucible with the mixture was combusted at 1100°C under vacuum in a quartz reaction vessel (Yeh et al., 1995). The CO₂ was separated from other combustion products by condensation and evaporation under the control of liquid nitrogen and dry ice. The carbon-isotope compositions of the CO₂ samples were determined in an isotope ratio mass-spectrometer (McKinney et al., 1950). The isotope radio mass spectrometer employed in this study was a VG SIRA-10. The results are reported in d¹³C_PDB values (Craig, 1957). The overall reproducibility is better than 0.1 permil at the 90% level of confidence (Yeh et al., 1995). The statistic is derived from results of repeated analysis of a spectrographic graphite powder standard for more than two decades and random reanalysis of a sample. Normally, one aliquote of the graphite standard was analyzed after every six samples were.

Results and Discussion

There is a large range of d¹³C values for the marine macroalgae studied here (Figure 2) and elsewhere (Figure 3) (Craig, 1953; Parker, 1964; Smith and Epstein, 1970; Smith and Epstein, 1971; Black and Bender, 1976; DeNiro and Epstein, 1981; Fry et al., 1982; Kerby and Raven, 1985; Raven et al., 1987; Dauby, 1989; Fenton and Ritz, 1989; Surif and Raven, 1990; Wiencke and Fischer, 1990; Raven, 1991; Raven and Johnston, 1991; Ye et al., 1991; Fischer and Wiencke, 1992; Raven and Osmond, 1992; Maberly et al., 1992; Raven et al., 1994; Raven et al., 1995; Raven et al., 2002). The d¹³C values of the marine algae obtained in this study range from -11.7 to -29.5‰ (Table 1). For the Chlorophyta, the values range from -10.5 to -21.2‰ (Figure 2) with a mean of -16.5‰. For the Phaeophyceae, the values range from -11.7 to -15.8‰ (Figure 2) with a mean of -13.6‰. For the Rhodophyta, the values range from -12.4 to -29.5‰ (Figure 2) with a mean of -17.7‰. The highest d¹³C value is from the green algae Ulva pertusa (-10.5‰) whereas most algae exhibit the d¹³C values in the -13.4 to -17.2‰ range (Figure 2). Fry et al. (1982) reported high d¹³C values for a number of marine algae, which corresponds to results presented herein. On average, the d¹³C values of red algae are slightly more negative than those of green and brown algae. Black and Bender (1976), Fry et al. (1982), Maberly et al. (1992) and Raven et al. (1995) also reported some marine red algae with relatively low d¹³C values. The cultivated alga, Halymenia microcarpa, in this study is somewhat more reduced in ¹³C than samples of this alga collected subtidally (Table 1). The work of Wiencke and Fischer (1990) involved algae cultured in the laboratory, with possibly a less constrained source inorganic d¹³C values than is found in the sea. The d¹³C val

Figure 1. Map showing collecting localities (·) in Taiwan and its offshore islands (Penghu Islands).

Wang & Yeh — d¹³C of marine macroalgae from Taiwan

ues of the inorganic carbon in the source is crucial in interpreting discrimination. In the sea, values are believed to fairly constant, but they may be variable locally; variation is particular likely in culture. The implication is that the inorganic carbon in the culture tank may be more negative ¹³C than the sea. No relationship is apparent between the d¹³C of the algae and their phylogeny.

The d¹³C values for the marine algae in Figure 2 indicate that these algae discriminate against ¹³C in their assimilation of carbon. These values are in good agreement with the results from tropical and subtropical algal values (Figure 3)(Craig, 1953; Park and Epstein, 1961; Parker, 1964; Black and Bender, 1976; Fry et al., 1982). Since terrestrial C₃ plants have d¹³C values between -22 and -38‰ and some of the marine algae in Table 1 show a similar range, we could conclude that these marine algae d¹³C are comparable to terrestrial C₃ plants (Kerby and Raven, 1985).

The d¹³C values of the marine macroalgae from previous works (including work on cultured algae) are shown in Figure 3. Apparently, two groups can be delineated for the Chlorophyta, and the values range from -8.8 to -21.3 ‰ and from -25.7 to -32‰. Only one group can be distinguished for the Phaeophyceae and Rhodophyta, and the values range from -8.8 to -38.3‰ and from to -9.6 to -34.7 ‰, respectively. The d¹³C value range of marine algae do

Figure 3. d¹³C values of marine green, brown, and red macroalgae from previous works. Data from Craig (1953), Parker (1964), Smith and Epstein (1970), Smith and Epstein (1971), Black and Bender (1976), DeNiro and Epstein (1981), Fry et al. (1982), Kerby and Raven (1985), Raven et al. (1987), Dauby (1989), Fenton and Ritz (1989), Surif and Raven (1990), Wiencke and Fischer (1990), Raven (1991), Raven and Johnston (1991), Ye et al. (1991), Fischer and Wiencke (1992), Raven and Osmond (1992), Maberly et al. (1992), Raven et al. (1994), Raven et al. (1995), Raven et al. (2002).

not fit into the photosynthetic pathways type in terrestrial plants. It would be helpful to point out that no seaweed is known to perform CAM (except for a few fucoid brown algae where the contribution to organic C is only a few percentage points) and only one seaweed (Udotea flabellum) is known to have C₄-like photosynthesis although with PEPCK as its (C₃+C₁) carboxylase, this enzyme has a much greater ¹³C/¹²C discrimination than does PEPC (Raven et al., 1995). However, some marine algae in Table 1 do not fit into the range commonly observed with terrestrial plants. In particular Ulva pertusa, a green algae, has a d¹³C of about -10.5‰ (Table 1). Other marine algae, Colpomenia sinuosa, Padina sanctae-crucis, Enteromorpha intestinalis and Acetabularia sp., have similar d¹³C values (-8.8, -9.5, -8.8 and -9.4‰)(Figure 3), which, when compared to terrestrial plants, are at the upper extreme of d¹³C values for C₄ plants and below the lower extreme for C₃ plants (Bender, 1968). Thus, a definite conclusion on the major pathway of CO₂ assimilation in these

Figure 2. d¹³C values of marine green, brown and red macroalgae from Taiwan.

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

organisms cannot be reached, except to state that they should be a subject of future research with emphasis on C₄-type metabolism utilizing the major detectable carboxylase PEPCK, which uses phosphenolpyruvate and ADP and produces oxaloacete and ATP.

Most d¹³C values for marine algae clearly correspond to either limitation by CO₂ diffusion or by a low-discrimination HCO₃^- active transport process. In a few cases, however, no such process seems to be limiting, and neither CO₂ diffusion nor HCO₃^- active transport are limiting.

Wang & Yeh — d¹³C of marine macroalgae from Taiwan

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Acknowledgments. This study was supported by National Science Council Grant NSC 86-2116-M-001-012. We thank Miss H. T. Yang and C. H. Lu for technical assistance. Comments of Drs. Chi-Ming Yang and J. A. Raven and an anonymous reviewer contributed to the betterment of this manuscript, and we are grateful.

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