Bot. Bull. Acad. Sin. (2005) 46: 197-203

LIU et al. — Effect of ulvoid algae on Thalassia hemprichii

The effect of ulvoid macroalgae on the inorganic carbon utilization by an intertidal seagrass Thalassia hemprichii

Shao-Lun LIU¹, Wei-Lung WANG^1,*, Danilo T. DY², and Cheng-Chang FU³

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

²Marine Biology Section, Department of Biology, University of San Carlos, Cebu City 6000, Philippines

³Oldinburgh Company, Ltd., Taipei 100, Taiwan

(Received October 12, 2004; Accepted January 27, 2005)

Abstract. Blooms of ulvoid macroalgae (mainly Enteromorpha and Ulva) have covered 80% of the intertidal seagrass bed at Wanlitung, southern Taiwan, effectively shading the seagrasss species Thalassia hemprichii resulting in a decrease in photosynthetic performance and low inorganic carbon (C_i) uptake. We looked for evidence of C_i limitation and investigated the C_i utilization characteristics of ulvoid-free and ulvoid-covered T. hemprichii. The rapid light curve (RLC) function of the Diving-PAM (Diving-PAM, Walz, Germany) was used to measure in situ photosynthetic performance (based on the effective quantum yield of PSII [Y] values) of intact seagrasses that were placed in small incubating chambers. Significantly, a lower RETR_max (maximum relative electron transport rate) and E_k (light intensity at the onset of saturation) were noted in the ulvoid-covered compared to the ulvoid-free T. hemprichii, suggesting that the former has acclimatized to the low light environment becoming a "shade type" plant. The ulvoid-covered T. hemprichii showed some evidence of C_i limitation since a significant increase in RETR_max (up to 46%; P < 0.05) was noted after an increase in the concentration of NaHCO₃ from 2.2 (normal seawater) to 6.2 mM. In terms of C_i utilization characteristics, T. hemprichii could directly absorb HCO₃^- as the major C_i source but partially depended on the extracellular carbonic anhydrase (CA) to convert HCO₃^- to CO₂ prior to uptake in the ulvoid-free, high light-adapted populations. A wastewater stream with a high nutrient load coming from the urbanized area may have caused the frequent blooms of ulvoid macroalgae.

Keywords: Bicarbonate; Photosynthesis; Taiwan; Ulvoid bloom.

Abbreviations: AF, absorption factor; AZ, acetazolamide; CA, carbonic anhydrase; C_i, inorganic carbon; DIN, dissolved inorganic nitrogen; DIP, dissolved inorganic phosphate; E_k, the light intensity at the onset of photosynthetic saturation; RETR, relative electron transport rate; HEPES, N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; F, minimal chlorophyll-a fluorescence in the light-adapted state; Fm', maximum chlorophyll-a fluorescence in the light-adapted state; PAM, pulse amplitude modulated; PPFD, photosynthetic photon flux density; RETR_max, maximum relative electron transport rate; PSII, photosystem II; Y, the effective quantum yield of PSII; a_RETR, the initial slope of the light-limited relative electron transport rate.

Introduction

Seagrasses are submerged marine angiosperms that comprise about 60 species worldwide (Den Hartog, 1970; Green and Short, 2003), seven of which have been recorded from Taiwan (Yang et al., 2002). To date, very few studies on seagrasses in Taiwan have appeared (Mok et al., 1993; Shieh and Yang, 1997; Lin and Shao, 1998; Yang et al., 2002). Lin and Shao (1998) reported that Thalassia hemprichii (Ehrenberg) Ascherson from southern Taiwan was threatened by anthropogenic activities due to excessive nutrient input. The nutrient over-enrichment of coastal waters can lead to the proliferation of bloom-forming "ephemeral" macroalgae which end up shading seagrass populations and eventually displacing seagrass as the dominant benthic autotrophs (McGlathery, 2001).

Manipulative experiments on a naturally occurring bloom of Ulvaria obscura (Kützing) Gayral in Armitage Bay, Blakely Island, Washington State, USA resulted in reduced seagrass shoot density (Nelson and Lee, 2001). Competition with other macrophytes for one or more resources, such as light, O₂, or inorganic carbon (C_i) could be behind the seagrass growth reduction (Coffaro and Bocci, 1997) suggesting some form of resource limitation.

At Wanlitung (N 21º99'60", E 120º70'50'') in southern Taiwan frequent blooms of ulvoid macroalgae (mainly Enteromorpha spp. and Ulva spp.) were observed in the intertidal habitat covering some portion of the flourishing seagrass, Thalassia hemprichii. A continuous wastewater stream from the urbanized area was observed to drain into the seagrass habitat. Dissolved inorganic nitrogen (DIN) was present at 0.51 ppm during low tide and 0.49 ppm during high tide. Dissolved inorganic phosphorus (DIP) was 0.24 ppm during low tide and 0.03 ppm during high tide (Liu and Wang, unpublished data). The DIN:DIP

*Corresponding author. E-mail: wlwang@cc.ncue.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

ratio was 2.1 at low tide and 16.3 at high tide. Clearly, DIP was very high during low tide. This could partly explain the bloom of ulvoid macroalgae that covered about 80% of the seagrass bed.

Schwarz et al. (2000) observed that photosynthesis in Cymodocea serrulata (R. Brown) Ascherson et Magnus and Halophila ovalis (R. Brown) Hooker f. inhabiting the subtidal habitat (10-12 m depth) had photosynthetic rates limited by ambient C_i concentrations depending on the irradiance available. The same species in the intertidal habitat were not limited by C_i concentration at any irradiance up to 1500 µmol photons m^-2 s^-1. The latter result was contrary to a previous conclusion regarding C_i limitation in intertidal plants (Björk et al., 1997), and Schwarz et al. (2000) highlighted the need for performing photosynthetic investigation in situ.

We hypothesized that the shading of seagrass by ulvoid macroalgae could have detrimental effects on T. hemprichii, lowering its photosynthetic performance and thus its C_i requirement. In addition, an excessive amount of ulvoid algae could deplete the surrounding C_i concentration, thereby also affecting the pH of the surrounding medium. Beer (1996) suggested two possible mechanisms for C_i transport in seagrass: (1) direct uptake of HCO₃^- and (2) extracellular dehydration of HCO₃^- to CO₂ as catalyzed by membrane-associated carbonic anhydrase (CA).

In the ulvoid-covered population, we expect T. hemprichii to exhibit some degree of C_i limitation and adopt a mechanism of Ci utilization slightly different to what it would use in an ulvoid-free population. In this study, we tested for evidence of C_i limitation and investigated differrences in C_i utilization characteristics between ulvoid-free and ulvoid-covered T. hemprichii. We also used an underwater PAM fluorometer (Diving-PAM) for in situ measurements of photosynthetic performance of T. hemprichii.

Materials and Methods

Study Site

Our study area was on the intertidal reef flats at Wanlitung (N 21°99'60", E 120°70'50"), southern Taiwan (Figure 1). The seagrass bed covered about 0.002 ha and was found at 0-50 cm depth between low tide and high tide. To get a sense of the ambient environmental conditions around the ulvoid-free and ulvoid-covered seagrass populations during low tide and high tide, irradiance and pH was determined with a spherical quantum sensor and pH meter, respectively. To get a rough idea on the coverage of T. hemprichii and the ulvoid algae, the transect quadrat method after Lin and Shao (1998) was used. A 0.25 m² quadrat (50 × 50 cm) divided into 25 squares (10 × 10 cm) was placed on the substratum at five-meter intervals along a 50-m transect line, and the coverage of T. hemprichii and ulvoid algae was estimated. In each quadrat, only five squares (10 × 10 cm²) were randomly selected for shoot density estimation of T. hemprichii.

Figure 1. Map showing the study site at the southern tip of Taiwan.

Counting of shoot density was done manually. Simultaneously, five squares (10 × 10 cm²) were also randomly selected for estimating the biomass of the ulvoid algae (Enteromorpha-Ulva spp.). The ulvoid algal samples were collected and dried using silica gel. The silica gel was changed every 2 h for 24 h. The use of silica gel was done to facilitate the drying of algal samples while in the field.

Experimental Protocol

The in situ experimental protocol for studying the C_i utilization characteristics of T. hemprichii (not covered and covered with ulvoid macroalgae) followed that of Schwarz et al. (2000). The rapid light curve (RLC) function of the Diving-PAM (Diving-PAM, Walz, Germany) was used to measure the in situ photosynthetic performance of seagrass samples (Beer et al., 1998). Materials were placed in Perspex incubating chambers (20 ml) (Schwarz et al., 2000) and tested for evidence of C_i limitation by adding a known amount of NaHCO₃ in the incubating chamber and measuring the photosynthetic performance of the seagrass sample before and after the addition. All RLC's were performed under normal seawater conditions. An initial RLC was performed under ambient HCO₃^- concentrations (ca. 2.2 mM C_i, mainly in the form of HCO₃^-). Then, 0.4 ml of 100 mM NaHCO₃ solution was injected from a 2.5 ml syringe to increase by about 2 mM the C_i concentration in the chamber (4.2 mM), and the second RLC was measured. Prior to the third RLC measurement, a further 0.4 ml of 100 mM NaHCO₃ was injected to give a final C_i concentration of 6.2 mM in the chamber.

To determine whether the mechanism of C_i transport for T. hemprichii was direct HCO₃^- uptake or external CA-me

LIU et al. — Effect of ulvoid algae on Thalassia hemprichii

diated HCO₃^- utilization, photosynthesis of the seagrass sample inside an incubating chamber was measured (using the RLC function of the diving PAM) before and after the addition of 1 ml 1 M HEPES buffer solution (pH 8.8, final concentration 50 mM). The aim was to change the equilibrium between the different C_i forms within the chamber, resulting in a CO₂ concentration that was 0.11% and an HCO₃^- concentration that was 67% of the total seawater C_i content. In addition, to demonstrate evidence for an external CA-mediated HCO₃^- uptake, photosynthesis of a separate seagrass sample inside an incubating chamber was measured (using the RLC function of the Diving-PAM) before and after the addition of 0.2 ml 0.001 mM acetazolamide (AZ) solution (a membrane-impermeable CA inhibitor, final concentration 100 µM). All experimental protocols were performed thrice.

Chlorophyll Fluorescence Measurements

The photosynthetic performance of seagrass samples was assessed based on the effective quantum yield of PSII (Y) values provided by the Diving-PAM's RLC function. Y was measured in darkness, and eight irradiance intensities were provided by the internal halogen lamp of the Diving-PAM. Photosynthetic photon flux density (PPFD) ranged from non-saturating to saturating intensities (139, 213, 299, 450, 612, 943, 1376, and 2097 mmol photon m^-2 s^-1) at 10s-intervals. The light intensities we used were within the range established by Ralph and Burchett (1995) in their study on the photosynthetic responses of the seagrass Halophila ovalis. In order to avoid other factors (i.e. age, position, and any lesions of the leaf) and the diurnal variations of fluorescence parameters that might affect the fluorescence yield of the samples, all measurements were done at the middle fresh portion of the second ranked leaf between 10:00 and 16:00 hours (Durako and Kunzelman, 2002, Durako et al., 2003). Due to lack of measurements for leaf absorptance, only relative transport rate was estimated. The RETR at each irradiance intensity of RLCs was estimated by the equation: RETR = Y*PPFD, where Y was the effective quantum yield of PSII (F_PSII = (F_m' - F)/F_m') and PPFD was the photosynthetically available irradiance reaching the leaf (µmol photon m^-2 s^-1). From the RETR-irradiance data, the maximal RETR at light saturation (RETR_max) and the initial slope of the light-limited relationship (a_RETR) was calculated using the hyperbolic tangent equation: RETR = RETR_max tanh(a_RETR · I/RETR_max), where RETR was the relative electron transport rate, RETR_max was the maximum relative electron transport rate, I was the irradiance and a_RETR the initial slope of the light-limited relationship (Jassby and Platt, 1976). An additional parameter, E_k, defined as the light intensity at the onset of saturation was also computed as: E_k = RETR_max/a_RETR.

Statistical Analysis

Means of photosynthetic parameters (i.e., RETR_max, E_k and a_RETR) between ulvoid-free and ulvoid-covered T. hemprichii were compared using a t-test for independent samples at the 95% significance level. We used ANOVA

with repeated measures to test the effect of RETR_max before and after adding the various chemicals (NaHCO₃, HEPES buffer or AZ) to the incubating medium. Initially the data were tested for normality and homoscedasticity. Appropriate transformations were carried when data (i.e., data on effects of C_i concentration and AZ treatment were converted to log base 2) were not normal and heteroscedastic (Zar, 1984).

Results

In seagrass covered with ulvoid algae, the ambient light intensity at the bottom was 168 and 24 µmol photon m^-2 s^-1 during low tide and high tide, respectively. In ulvoid-free seagrass, the ambient light intensity was 780 µmol photon m^-2 s^-1 during low tide and 425 µmol photon m^-2 s^-1 during high tide. The pH of the ambient water in ulvoid-covered seagrasss was 8.75 and 8.32 during high tide and low tide, respectively, while in ulvoid-free seagrasss, the ambient pH was 8.42 and 8.19, respectively. The coverage of the ulvoid algae was about 80% of the coverage of T. hemprichi. The total number of shoot counts in T. hemprichii was 52±4 in five 10 × 10 cm² quadrats, and the total biomass of the ulvoid algae was about 15±4 g dry weight in five 10 × 10 cm² quadrats.

Significantly lower RETR_max and E_k values were recorded for the ulvoid-covered compared to the uncovered T. hemprichii populations (Figure 2, Table 1). There was no significant difference between populations for a_RETR (initial slope of the RETR-irradiance curve). However, it should be noted that our starting light intensity (i.e., 193 µmol photon m^-2 s^-1) was relatively high. There were also few data points below the onset of light saturation (E_k), which may have led to a less precise estimate of a_RETR. Hence, these values should be treated as preliminary and subject to further confirmation in future studies.

Our results also indicated a certain degree of C_i limitation for ulvoid-covered T. hemprichii. A significant in

Figure 2. RETR curves of Thalassia hemprichii at different photosynthetic photon flux density (PPFD, µmol photon m^-2 s^-1) for ulvoid-covered populations (solid circle) and ulvoid-free populations (solid square). Data were obtained under ambient seawater conditions. Bars are standard errors (n = 9).

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

crease in RETR_max (up to 46%; P = 0.043; Tukey HSD test) was noted when ulvoid-covered T. hemprichii were supplied with increased NaHCO₃, from a concentration of 2.2 (normal seawater) to 6.2 mM (Figure 3, Table 2). On the other hand, the 4.4 mM and 6.2 mM HCO₃^- concentrations showed no significant difference. The RETR_max of ulvoid-free T. hemprichii remained within the same levels with the addition of NaHCO₃.

Generally, RETR_max in the HEPES-added medium was higher than in the AZ-added medium. These results seemed to suggest that T. hemprichii was able to directly absorb HCO₃^- as the major C_i source but partially depended on the extracellular carbonic anhydrase (CA) to convert HCO₃^- to CO₂ prior to uptake in the high light-adapted populations. However, there was no significant difference in RETR_max between ulvoid-covered and ulvoid-free T. hemprichii after HEPES buffer was added to the medium (Table 3). A lower RETR_max value in the ulvoid-covered and ulvoid-free seagrass was observed, indicating a lower affinity for direct HCO₃^- uptake. On the other hand, the RETR_max of the ulvoid-free T. hemprichii showed a significant (P = 0.049; LSD test) decrease (about 31%) after the enzyme AZ was added to the medium, suggesting the presence of an external CA-mediated HCO₃^- utilization pathway.

Discussion

The RETR-light curves in Figure 1 strongly suggest that the ulvoid-covered T. hemprichii in the intertidal area at Wanlitung, southern Taiwan have acclimatized to the low light environment and become "shade type" plants as opposed to the "sun type" T. hemprichii living in intertidal habitats. Seagrasses adapted to high light conditions have been shown to have a higher maximum photosynthesis and a higher capacity to use HCO₃^- as a source of C_i (Mercado et al., 2003). Similarly, the RETR_max of ulvoid-free T. hemprichii in this study were higher than those of the ulvoid-covered variety, indicating higher photosynthetic capacity.

Figure 3. RETR_max (mean and SE, n = 3) of (a) ulvoid- covered and (b) ulvoid-free Thalassia hemprichii under different inorganic carbon (C_i) concentrations and after the addition of HEPES buffer solution and acetazolamide (AZ). Identical icons are significant at P < 0.05.

LIU et al. — Effect of ulvoid algae on Thalassia hemprichii

could explain why the ulvoid-covered T. hemprichii populations preferentially take up HCO₃^- without the aid of carbonic anhydrase (CA), enabling maintenance of a high photosynthetic rate. The difference between the ulvoid-covered and the ulvoid-free populations seems to indicate the existence of a dual mechanism in T. hemprichii similar to that reported for U. lactuca (Axelsson et al., 1995). However, for now, such a hypothesis must be treated as preliminary and should be verified by further investigations. Furthermore, testing whether such physiological regulation exists in other species of macroalgae and seagrasses would be worthwhile.

The worldwide decline in seagrass habitats has been attributed mainly to a eutrophication-induced degradation of water quality leading to reduced light for submerged vegetation (Koch and Beer, 1996; McGlathery, 2001). Analogous situations exist for the seagrass habitat at Wanlitung. The eutrophication-induced blooms of the ulvoid green algae seemed to cause two main stresses: namely, light limitation and C_i limitation. Comparing other seagrass sites in southern Taiwan, Lin and Shao (1998) estimated the seagrass beds at Nanwan and Dakwan at about 0.3-0.4 ha while only about 0.0002 ha was observed at Wanlitung. Our data are not sufficient to estimate the extent to which the photosynthetic response of the plants to shade conditions quantitatively affects plant health or to which extent C_i limitation reduces the photosynthetic potential of these plants. Further research on a wider spatial scale and with a seasonal component would help uncover these problems.

Acknowledgements. The authors sincerely thank the two anonymous reviewers for their comments and suggestions. We are also very gratefully to Mr. Liang-Chan Lee at Department of Biology, National Changhua University of Education, Changhua, Taiwan for his field assistance. This study was supported by the National Science Council, Republic of China (NSC92-2611-M-018-001).

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Botanical Bulletin of Academia Sinica, Vol. 46, 2005

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LIU et al. — Effect of ulvoid algae on Thalassia hemprichii