INTRODUCTION
Intertidal seaweeds commonly experience desiccation and nutrient limitation stress associated with the tidal cycle (Brawley and Johnson, 1991; Davison and Pearson, 1996). The emersion stress reduces macroalgal growth, photosynthesis, and disrupts cell membranes, and the levels of stress increase with the elevation of shore heights (Schonbeck and Norton, 1980, Dring and Brown, 1982; Mabery and Madsen, 1990). Macroalgal propagules are bottleneck because they are unable to survive in the harsh environmental conditions, and their physiological responses to emergence are clearly related to the subsequent distribution and abundance (Brawley and Johnson, 1991; Vadas et al., 1992; Davison et al., 1993). Generally, dense germlings are able to survive under desiccation condition, where well-spaced individuals would be killed as tested on the shore (Ang and De Wreede, 1992; Andrew and Viejo, 1998) and in simulated tidal regimes in the laboratory (Hruby and Norton, 1979). However, the survival and growth of crowded germlings were negatively correlated, irrespective of monoculture or mixed culture under favorable environmental conditions (Reed et al., 1991; Creed et al., 1996, 1997; Choi and Norton, 2005a; 2005b).
One can reasonably speculate that juvenile and adult macroalgae are more tolerant for the stress than germlings of the species, and the physiological responses of germlings differ between species. Therefore, we could expect that the outcome of competition in mixtures of two species will be changed by the degree of the environmental stressors, such as nutrient depletion and emersion stress. There is evidence that the outcome of competition in mixtures of two species in the adult stage has been altered by temperature, irradiance (Enright, 1979) and nutrient availability (Fujita, 1985; Peckol and Rivers, 1995; Ceccherelli and Cinelli, 1997) but no studies have done in the germling stage.
On many rocky shores in northern Europe, fucoid algae have clear vertical distribution: Pelvetia canaliculata from the top shore followed by Fucus spiralis, F. vesiculosus and/or Ascophyllum nodosum with F. serratus at the lowest levels (Lewis, 1964). F. vesiculosus and A. nodosum have overlapping reproductive period in spring and their mixed stands are occasionally found in the mid intertidal zone (Knight and Parke, 1950). The two species are air-exposed periodically and emersion is an important physical stress. Thus, F. vesiculosus and A. nodosum are suitable to examine the effect so interactions between varying degrees of desiccation stress and the density of the two competitors. The outcome of competition in mixtures will also be examined with F. vesiculosus and A. nodosum under two different degrees of desiccation and nutrient sat a wide range of density and frequency. We tested the following hypotheses: 1) Crowding of germlings protects them from desiccation, irrespective of species involved. 2) The outcome of competition can be changed in mixtures of A. nodosum and F. vesiculosus by emerged stress and nutrient levels.
MATERIALS AND METHODS
1.Study Site
Twenty female and five male plants of F. vesiculosus and A. nodosum were collected separately from Port St. Mary ledges (N 54° 0', W 4° 44') in May 1999. Receptacles were cut from the male and female plants, washed several times using filtered seawater, dried for 5 h, and submerged in filtered seawater to induce gamete release. After 1 h, the receptacles were removed and clean zygote suspensions were prepared as described by Creed et al.(1996).
2.Methods
1)Effects of periodic emersion on the competition of germlings
A total forty-six Petri dishes, each containing 8 slides (2.5 × 2.0 cm) and 20 mℓ of filtered seawater, were prepared and allocated to 23 treatments (Table 1). To achieve the settlement density required (Table 1), four different zygote concentrations (100, 1,000, 5,000 and 15,000 zygotes mℓ) for each species were prepared and 5, 5, 10 and 20 mℓ of zygote suspensions were in oculated to achieve the log-scale settlement densities (Table 1).
After 24 hours, twelve from 16 slides for each treatment were chosen and four slides were discarded. Of these twelve slides four were used for determining settlement density and eight for the culture experiment. Settlement density was determined after setting up the culture experiment and is shown in Table 1.
Twenty-three slides representing each treatment were put on two steps in each culture tank of four tanks (25 l), continuously submerged and 6 h emerged every 12 h tidal cycle in the artificial tidal tanks as described in detail by Kim et al. (2011), and referred to as “submerged” and “emerged” respectively. An emergent period of 6 h was chosen because it is similar to the average submerged/ exposed period of the two species on the shore (Schonbeck and Norton, 1978).
Germlings were cultured in culture medium (Kain and Jones, 1964) for a period of 3 weeks at 10 ± 1°C, 16:8 h LD and 120 μmol photons/m2/s. The culture medium was not changed during the culture period. The final mortality and lengths of the germlings were measured. The lengths of total 25 germlings were measured on each of four replicate slides. In mixtures, the germlings of Fucus vesiculosus (hereafter Fucus) were clearly distinguished from those of Ascophyllum nodosum (hereafter Asocophyllum) by the presence of apical hairs. Percentage mortality of germlings was monitored after measuring lengths. One hundred germlings on each slide were counted to determine the mortality of germlings.
To test whether the relative growth of the Fucus and Ascophyllum in mixtures is altered by the submergence period, Log10 output ratios (Fv/An = Fucus vesiculosus / Ascophyllum nodosum) for mean length was calculated.
When intra- and inter-specific competition is equivalent between the two competing species, log output ratio is 0. If relative growth of the two species is not altered by the submergence period, their final log output ratios should overlap between the two submerged conditions.
2)Effects of nutrient on the competition of germlings
The effects of nutrients on the performance of germlings were examined with Fucus and Ascophyllum. This experiment was set up with the same settlement density of germlings as described above.
Eight 5 l plastic tanks were used, half of which were filled with 2 l of culture medium (Kain and Jones, 1964) and the others with 2 l of filtered (0.25 μm) seawater. Twenty-three slides bearing germlings of different densities and proportions of the two species (Table 1) were put into each tank. Germlings were cultured for a period of 3 weeks at 10 ± 1 °C, 16:8 h LD, and 120 μmol photons/m2/s. The media were changed weekly and after three weeks the mortality and lengths of the germlings were measured.
To test whether the relative growth rates of Fucus and Ascophyllum in mixtures is altered by nutrient conditions, Log10 output ratios (Fv/An) for mean length were calculated and the results interpreted as described above.
3)Data analyses
Data were analyzed using one-way and two-way ANOVA. Homogeneity of variances was tested by Cochran’s test. Where necessary, data were transformed before analysis to meet the assumptions of parametric tests (Sokal and Rohlf, 1981). The significance of the differences between means was tested with the Tukey HSD test. The regressions for the growth and mortality of germlings were compared with ANCOVA.
RESULTS
1.Effects of periodic emersion on the competition of germlings
The mortality of germlings increased with density and emergence periods both in the monoculture and mixtures (Figure 1). Once compared the mortality over the range of density levels of monocultures, the mortality of Ascophyllum was significantly higher than that of Fucus both in submerged and emerged treatments (Figure 2, Table 2). For both species, the duration of emergence influenced the mortality of germlings. The results of ANCOVA revealed that the mortality was significantly higher in emerged rather than submerged treatments in both species (Fucus : F1,29 = 50.89, p<0.001; and Ascophyllum : F1,29 = 71.41, p<0.001). In mixtures, the identification of dead germlings to species was impossible and only total mortality was measured and it was com pared between two emergence treatments. Mortality was significantly higher in emerged compared to submerged treatments (Figure 2, Table 2).
The mean length of the two species varied at a wide range of density and frequency of Fucus and Ascophyllum, and in two submerged treatments. The growth of both species declined with increasing density and duration of emergence (Figure 3). Fucus always grew faster than Ascophyllum in both monoculture and mixture, and in submerged and emerged treatments (Figure 3). There was a significant difference in mean length between the two species (Table 3). When comparing the effects of density on two species, the growth of Fucus rapidly declined with in creasing density compared to Ascophyllum.
The effects of duration of emergence on the growth of germlings were compared for each species. Germlings grew faster in submerged than emerged condition both in monoculture and mixtures over a wide range of density and frequency. In monoculture, the growth of germlings was significantly greater in submerged compared to emerged treatments for Fucus was (ANCOVA, F1,29 = 152.35, p<0.001), but not for Ascophyllum (ANCOVA, F1,29 = 0.43, p>0.05). In mixtures, log output ratio was different between submerged and emerged treatments (Figure 4). The values of log output ratio were more than 0.1, indicating that Fucus prevailed over Ascophyllum in the competitive battles in both treatments. The output ratios of submerged and emerged treatments were separately plotted, showing that the relative growth of the two species was altered by the duration of submergence (Table 4). It was greater in submerged than emerged treatments, indicating that the growth of Fucus is more enhanced by submergence than that of Ascophyllum as in monocultures.
2.Effects of nutrient on the competition of germlings
The mortality of Fucus and Ascophyllum increased with density and nutrient concentration both in the monocultures and the mixtures. In monoculture, the mortality of Ascophyllum was significantly higher than that of F. vesiculosus in high nutrients but not in low nutrients (Table 5). In monoculture of each species, the mortality of germlings was significantly influenced by nutrient concentration for Ascophyllum (ANCOVA, F1,29 = 7.23, p<0.05) but not for Fucus (F1,29 = 0.17, p = 0.68). In mixtures, total mortality of germlings was significantly higher at high than low nutrients (Table 5).
Nutrients influenced the growth of Fucus and Ascophyllum cultured at different densities and relative frequencies of the two species. Both in high and low nutrients, Fucus always grew significantly faster than Ascophyllum both in monoculture and mixtures (Table 6), and the effect of density on the growth was greater in Fucus than Ascophyllum.
In monocultures of each species, the growth of germlings was better in high nutrients for Fucus (ANCOVA, F1,29 = 196.32, p<0.001), whereas in low nutrients for Ascophyllum (ANCOVA, F1,29 = 52.82, p<0.001). In mixtures, log output ratio was different between low and high nutrients (Figure 5). The values of log output ratio were more than 0.1, indicating that Fucus ‘won’ in the competitive battles with Ascophyllum at both nutrient levels. The output ratios of low and high nutrients were separately plotted, showing that the relative growth of the two species was altered at two nutrient levels (Table 7). Log output ratio was greater in high than in low nutrients, indicating that the growth of Fucus is more enhanced than that of Ascophyllum in high nutrients.
DISCUSSION
On the mid shore, the vertical distribution of Ascophyllum is wider than that of Fucus vesiculosus (Lewis, 1964; Schonbeck and Norton, 1978) and the juveniles of the former are occasionally found on the their uppermost areas (Lewis, 1964). In the present study, Ascophyllum is likely to benefit when competing with Fucus both under periodical emergence and under low nutrient levels. The results well agree with the responses of adult plants. Ascophyllum recovers rapidly than Fucus when they are resubmerged after desiccation (Brinkhuis et al., 1976; Strömgren, 1977). Ascophyllum grows well in summer when nutrient levels are minimal (Mathieson et al., 1976; Vadas et al., 1976; Hardwick-Witman and Mathieson, 1986) and on sheltered shores, where nutrient circulation is less than exposed shores (Lewis, 1964; Keser et al., 1981; Keser and Larson, 1984). In contrast, the growth of Fucus is higher in high nutrients than in low nutrients and produces two types of hairs, apical and hyaline, which serve to enhance nutrient uptake at low nutrient concentrations (Schonbeck and Norton, 1978; Galvin, 1988).
Mortality and growth of germlings in monoculture and mixtures of A. nodosum and F. vesiculosus were density-dependent, indicating that intra- and interspecific competition occurred between germlings, irrespective of cohorts or competitors. The presence of intraspecific and interspecific competition between germlings of the two species were identified with are placement experimental design (density is constant with 500 zygote/cm2 but relative proportion of the two species is changed) by Choi and Norton(2005b). They also reported the facilitation between the two species in the germling stage that F. vesiculosus enhanced the survival of A. nodosum germlings under stressful field condition. In the present study, germling competition was more severe at higher density than at lower density but positive density effects were not found even in the emerged treatment. Recently, intraspecific and interspecific competition between germlings of Pelvetia canaliculata and Fucus spiralis were also found in artificial tidal tanks, and Pelvetia grew better at 6 h exposure treatment than at continuous submerged treatment (Kim et al., 2011). So far, growth responses of germlings were species specific under various environmental conditions and more detailed physiological research for germlings should be performed.
In the present study, the outcome of interspecific competition between germlings of F. vesiculosus and A. nodosum was slightly altered by duration of emergence and nutrient concentration, but not to such an extent as to change the outcome as found in F. spiralis and P. canaliculata (Kim et al., 2011). There are many stressful environmental factors such as high-temperature and irradiance on the rocky shore, and eutrophication and climate change (i.e., ocean acidification and sea water temperature increase) by human activity, these environmental factors might change seaweed community structure. Thus, to understand tiny germling worlds, we need to study on the growth and survivorship of germlings in order to know the combined effects of various environmental factors. Present results revealed that the responses of germlings to environmental stresses are different between fucoid species but we have to study in detail to understand their distribution on the shore.
