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High tolerance to fluctuating salinity allows Sargassum thunbergii germlings to survive and grow in artificial habitat of full immersion in intertidal zone
Journal of Experimental Marine Biology and Ecology 412 (2012) 66–71
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High tolerance to fluctuating salinity allows Sargassum thunbergii germlings to survive and grow in artificial habitat of full immersion in intertidal zone Shao Hua Chu, Quan Sheng Zhang ⁎, Yong Zheng Tang, Shu Bao Zhang, Zhi Cheng Lu, Yong Qiang Yu Ocean School, Yantai University, Yantai 264005, PR China
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 18 October 2011 Accepted 22 October 2011 Available online 17 November 2011 Keywords: Germling Salinity Sargassum thunbergii Tolerance
a b s t r a c t In this study, a four-way factorial experimental design (three temperatures, four salinities, two frequencies and three durations of osmotic stress) was used to test the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to osmotic stress. Results showed that the growth and survival of germlings were significantly affected by salinity and duration of osmotic stress, rather than by osmotic frequency. In addition to the main effects, all two-way, three-way and four-way interactions between the four stresses on the growth were significant. Germlings showed quick growth with relative growth rate (RGR, % day−1) over 16% when cultured at 32 psu combined with 30 °C. Although growths of germlings subjected to hypo-osmotic and hyper-osmotic conditions were significantly inhibited, RGRs over 12% were obtained except for those at 35 °C with the RGRs below 10%. In comparison to growth, survival was more tolerant to osmotic stress. Germlings showed high survivals over 60% when exposed to most osmotic stresses, by the end of experiment. Only under extreme conditions (12 psu for 4 h or 8 h at 35 °C) were the survivals reduced to below 10%. Although the effects of osmotic stress on the growth and survival were significant, germlings exhibited quick growth and high survival under most conditions. And those extreme conditions which had great destructive effect on germlings barely exist in field. Therefore, the construction of artificial tanks is feasible for the restoration of S. thunbergii beds in the intertidal zone. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Seaweed beds in the intertidal zone are communities consisting of macroalgae, and they play important roles in the coastal ecosystem, such as providing food and habitat for coastal marine life (Largo and Ohno, 1993; Terawaki et al., 2001). In recent years, however, human activities have caused a rapid decline in seaweed beds (De Jonge and De Jong, 2002; Rodríguez-Salinas et al., 2010; Terawaki et al., 2003; Zhao et al., 2008). In order to achieve sustainable development of the intertidal zone, effective measures for the restoration of seaweed beds are urgently required. Previous algal colonizations induced by the dispersal of microscopic life-stages and transplantation of juveniles or adults have been demonstrated on artificial seaweed beds or reefs in the benthic or subtidal zone of many regions (Carter et al., 1985; Falace et al., 2006; Hernández-Carmona et al., 2000; Terawaki et al., 2003). However, restoration in the intertidal zone is more challengeable due to daily exposure at low tide results in daily fluctuation in a range of important environmental factors (Bell, 1993; Davison and Pearson, 1996; Helmuth, 2002; Helmuth and Hofmann, 2001; Wright et al., 2004). The success of restoration in the intertidal zone
⁎ Corresponding author. Tel.: + 86 535 6706011; fax: + 86 535 6706299. E-mail address: [email protected] (Q.S. Zhang). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.10.025
is more dependent on the ability of survival and growth of the transplants in the stressful environment. The common brown alga Sargassum thunbergii which forms extensive beds in the intertidal zones is an appropriate species for the restoration of intertidal seaweed beds due to its wide ecological amplitude, high economic and ecological value (Kim et al., 2007; Padilha et al., 2005; Park et al., 2005; Zhang et al., 2009; Zhao et al., 2007). However, the natural populations of S. thunbergii along the northern coast of China have been depleted (Zhao et al., 2007, 2008). Sargassum germlings exhibited wide tolerance to environmental stress. Sargassum muticum germlings survived exposure to salinities as low as 5 psu, even during the first week after fertilization (Steen, 2004). Sargassum horneri germlings grew over a wide range of temperatures, from 10 °C to 25 °C (Choi et al., 2008). Our previous study showed that germlings of S. thunbergii exhibited strong tolerance to high temperature and low salinity; however, the effect of desiccation on the survival and growth of germlings was especially prominent (Chu et al., 2011). Therefore, it is most important to eliminate desiccation for the restoration of intertidal seaweed beds. The construction of artificial tanks is an effective way to avoid desiccation. Nevertheless, this measure may cause other problems, such as reduced salinity in the tank resulted from the heavy rain and increased salinity resulted from high temperature. Hence it is essential to investigate the effect of salinity fluctuation on the survival and growth of S. thunbergii germlings. In this investigation, germlings shortly released from fertile thalli of S. thunbergii were collected and cultured in the laboratory. The
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stress resistance of germlings to adverse environmental conditions including high temperature, reduced and increased salinity, high frequency and long duration of osmotic stress, and their interactive effects on growth and survival were tested. Relevant results will be applied to the restoration of intertidal seaweed beds. 2. Materials and methods 2.1. Collection of germlings Fertile female and male specimens of S. thunbergii were collected on July, 2010, in an intertidal zone of Xiaoheishan Island (37°58′N, 120°39′ E), located at the northern side of Shandong Peninsula, China. Selected thalli were healthy and yellowish-brown in appearance with intact and inflated receptacles which had no obvious shedding. Thalli of 2 kg were cleaned several times with seawater to remove surface epiphytes. They were then packed in plastic bags and stored in a plastic foam box full of crushed ice until transported to the laboratory where they were subsequently transferred to a plastic tank filled with 75 L filtered seawater which was continuously aerated. The tank containing thalli was kept at 22 °C, 10 μmol photons m− 2 s − 1 and a 12L: 12D (light: dark cycle) photoperiod. At 24 h post-fertilization, released germlings sank to the bottom of tank. After removal of thalli from the tank, the remaining seawater containing germlings was filtered with an 80-mesh nylon sieve, followed by filtration with a 200-mesh nylon sieve. A total of 3.0×105 germlings were obtained. To remove fouling, dross and epiphytes, collected germlings were repeatedly washed in tap seawater in the 200-mesh nylon sieve. Germlings were subsequently transferred to a 3 L glass tank filled with sterile filtered seawater. Following stirring even, the germling suspension was immediately poured into each Petri dish (60 mm×15 mm). A total of 220 Petri dishes with germlings of about 700 in each were obtained and used for our experiment. 2.2. Stress treatments After 24 h, germlings attached to the Petri dishes and were cultured in three light- and temperature-controlled incubators (Zhujiang, China) with 72 dishes in each, on the basis of the following factorial experimental design. Samples were cultured under three temperatures conditions: 15 °C, 20 °C, and 25 °C at night, and 25 °C, 30 °C and 35 °C during the day to mimic the temperature difference between nigh and day; four levels of salinity: 12 psu, 21 psu, 32 psu and 50 psu; two frequencies of osmotic stress: once a day and every other day; and three durations of osmotic stress: 2 h, 4 h and 8 h in every 24 h, for a total of 72 different combinations of temperature, salinity, frequency and duration. Three independent replicates were used for each combination. All treatments were cultured for 12 days at an irradiance of 60 μmol photons m − 2 s − 1 with a 10L: 14D light: dark cycle. Various concentrations of seawater were prepared by diluting sterile seawater with distilled water or adding NaCl and measured with a salinity hydrometer (GM Manu-facturing Co.). Sterile filtered seawater with four levels of salinity was used as the culture medium and changed according to the experimental design (twice a day or every other day). Therefore, contamination problems were avoided by frequent change of sterile culture medium. No distinct detachment of germlings followed the solution change due to the developed rhizoids. Irradiance within the wavelength range 400–700 nm was measured using a Li-Cor LI-250 light meter equipped with a LI-190SA quantum sensor. The ranges of all the factors chosen were in accordance with the fluctuations of conditions in the natural habitat of S. thunbergii. In summer, the temperature ranged from 23.5 to 25.6 °C and the extreme maximum temperature ranged from 32.0 to 34.0 °C in the northern o ffshore areas of Shandong Peninsula (Li and Zhao, 2002). The salinity of nature seawater measured was 32. However, about half of the annual precipitation fell during the period between June and August (i.e. the
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sexual reproductive period of S. thunbergii), and the salinity generally fell (Song et al., 2009). 2.3. Measurements of length and survival rate Growths of germlings were measured in terms of length, excluding rhizoids at the end of the experiment. The lengths of 4 germlings were measured using a microscope with ocular micrometer respectively in 5 areas arranged in a cross pattern (the upper, lower, left and right periphery and the center), for a total of 20 germlings in each dish. The initial mean length of germlings in four dishes was also measured before the stress treatment for the estimation of each relative growth rate (RGR, % day− 1). RGR was calculated as 100 (ln (L1)−ln (L0))/t, where L0 is the initial mean length of germlings, and L1 is the length at the end of various treatments, respectively, and t is the length of treatment period in days (Hunt, 1978). Prior to the stress treatment, the mean survival rate of germlings in four dishes was measured as the initial survival rate. Survival rates were calculated under a stereoscopy microscope every other day. In each dish germlings were counted respectively in 5 areas, as mentioned above, for a total of about 200 germlings for the calculation of each survival rate. Germlings were classified as dead when they became collapsed or structurally fragmented. 2.4. Statistical analysis All data were analyzed using SPSS for Windows 13.0. Prior to all statistical analyses, the homogeneity of variances was verified with Levene's test. A univariate analysis of variance was performed to test the significance of the main effects and interactions on the growth of germlings, with temperature, salinity, frequency and duration of osmotic stress as fixed factors, and RGR as dependent variable. Irradiance was used as the covariate to remove any confounding influence of irradiance, because the irradiances of various Petri dishes were slightly different. Main effects and interactions of temperature, salinity, frequency and duration of osmotic stress on the survival of germlings were analyzed by a three-way repeated measures ANOVA, with temperature, salinity, frequency and duration of osmotic stress as between-subjects factors, survival rates over the day as a within-subject factor, and irradiance as a covariate. Partial eta squared (η2) was used as a measure of effect size (Vila-Gispert et al., 2002). Similarly to r2, partial eta squared is the proportion of variation explained for a certain effect (effect SS/effect SS
Table 1 ANOVA for effects of temperature, salinity, frequency and duration of osmotic stress on the relative growth rate (RGR, % day− 1) of germlings. T: temperature, S: salinity, F: frequency, D: duration. η2 was used as a measure of effect size. Variable
df
Mean square
F
p
η2
Irradiance Temperature (T) Salinity (S) Frequency (F) Duration (D) T×S T×F S×F T×D S×D F×D T×S×D T×S×F T×F×D S×F×D T×S×F×D Error
1 2 3 1 2 6 2 3 4 6 2 12 6 4 6 12 4247
0.004 0.830 0.187 b 0.001 0.012 0.047 0.080 0.006 0.006 0.008 0.020 0.012 0.004 0.002 0.010 0.004 b 0.001
9.969 2241.393 505.774 0.758 32.75 125.697 215.082 16.352 16.392 21.593 52.82 33.728 10.075 6.467 26.014 10.992
0.002 b 0.001 b 0.001 0.384 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
0.002 0.514 0.263 b 0.001 0.015 0.151 0.092 0.011 0.015 0.03 0.024 0.087 0.014 0.006 0.035 0.03
Dependent variable (RGR) was untransformed and the assumption of homogeneity met Levene's test (F = 1.076, p = 0.381).
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+ error SS). Partial eta squared has the advantage over eta squared (effect SS/total SS) that it does not depend on the ANOVA design used because it does not use total SS as the denominator (Tabachnick and Fidell, 2000). Tukey's tests were used for the post-hoc comparisons. The differences were considered to be statistically significant under a probability value of 5% (p b 0.05). 3. Results 3.1. Effects of osmotic stress on growth of germlings Prior to the stress treatment, the initial mean length of germlings measured was 120 μm. At the end of the experiment, final lengths
A
12 psu 21 psu 20
32 psu 50 psu
Duration: 2h Frequency: once a day
ranged from 122.2 to 1050 μm due to the significant effects of temperature and osmotic stress (Table 1). Although the growth of germlings was significantly affected by salinity (Table 1), nearly all the RGRs were over 7% when cultured at 21 psu and 50 psu. Only at 12 psu combined with 35 °C for 4 h or 8 h, the RGRs were below 6% (Fig. 1C–F). The duration of osmotic stress exhibited significant effects on the growth. However, it showed minor influence with low η2 (Table 1). When cultured at moderate temperature, germlings showed quick growth even exposed to long duration of osmotic stress. For example, when cultured at 12 psu, 21 psu or 50 psu for 8 h during daily exposure to 25 °C or 30 °C, the RGRs were over 10% (Fig. 1E). There was no significant main effect of osmotic frequency on the growth of germlings (Table 1). Its effect on the growth was reflected in the interactions (Table 1). As compared to the frequency
B
16
RGR (% day-1)
RGR (% day-1)
16 12 8 4 0
Duration: 2h Frequency: tw ice a day
20
12 8 4
25
30
o
0
35
25
Tempreture ( C)
C
Duration: 4h Frequency: once a day
20
D
RGR (% day-1)
RGR (% day-1)
8 4
12 8 4
25
30
0
35 o
25
Tempreture ( C)
E
30
o
35
Tempreture ( C)
Duration: 8h Frequency: once a day
20
F
Duration: 8h Frequency: tw ice a day
20 16
RGR (% day-1)
16
RGR (% day-1)
35
16
12
12 8 4 0
o
Duration: 4h Frequency: tw ice a day
20
16
0
30
Tempreture ( C)
12 8 4
25
30
o
Tempreture ( C)
35
0
25
30
o
35
Tempreture ( C)
Fig. 1. Relative growth rate (RGR; % day− 1) of germlings exposed to various temperatures (25 °C, 30 °C and 35 °C for 10 h during the day), salinities (12 psu, 21 psu, 32 psu and 50 psu), frequencies (once a day and twice a day) and durations of osmotic stress (2 h, 4 h and 8 h) at an irradiance of 60 μmol photons m− 2 s− 1. A, B, C, D, E and F denote various frequency and duration of osmotic stress. The top left key denotes the salinity. Values are means ± SE (n = 3).
S.H. Chu et al. / Journal of Experimental Marine Biology and Ecology 412 (2012) 66–71
and duration of osmotic stress, the salinity showed greater effects on the growth of germlings on the basis of η2 (Table 1). The three-way interaction between salinity, frequency and duration of osmotic stress had significant effect with low η2 (Table 1). RGRs over 10% were obtained when exposed to the combination of three stresses with the exception of those at 35 °C (Fig. 1A–F).
3.2. Effects of osmotic stress on survival of germlings The salinity showed significant effect on the survival of germlings; however, when cultured at 21 psu and 50 psu, the survival rates were over 70% (Fig. 2). The results of Tukey's tests showed that although the effect of 12 psu on the survival of germlings was significant in comparison with 32 psu (post-hoc tests: pb 0.001), there was no significant difference between the effects of 50 psu and 32 psu (post-hoc tests: p=0.999). These results suggested that the damage of hypo-osmotic stress was more severe than hyper-osmotic stress. The duration of osmotic stress exhibited significant effects on the survival with low η2 (Table 2). When cultured at moderate temperature (25 °C or 30 °C), germlings showed high survival (over 90%) even exposed to long duration of osmotic stress (for 8 h) (Fig. 2E). Based on η2, the salinity exhibited greater effects on the survival (Table 2). The survival of germlings was not significantly affected by the three-way interaction between salinity, frequency and duration of osmotic stress (Table 2).
A
12 psu 21 psu
Duration: 2h Frequency: once a day
32 psu 50 psu
80 60 40 20 0
3.3. Order of effect size The growths of germlings were significantly affected by all the stresses and their interactions except for the main effect of osmotic frequency (Table 1). However, for the survival, the main effect of osmotic frequency and its interactions with other stresses showed no significant effects (Table 2). On the other hand, 12 psu, 21 psu and 50 psu significantly inhibited the growth of germlings in comparison with 32 psu (post-hoc tests: p b 0.001); however, the survival rates of germlings cultured at 21 psu and 50 psu were not significantly decreased when compared with those at 32 psu (post-hoc tests: p= 0.823 and p= 0.999, respectively). These results indicated that the survival of germlings was more tolerant to osmotic stress than the growth. Compared with η 2 values estimated, the effects which contributed to the growth of S. thunbergii germlings, in order of effect size,were:temperature>salinity>temperature×salinity>temperature× frequency>temperature × salinity × duration > salinity × frequency × duration > salinity × duration > temperature × salinity × frequency × duration > frequency × duration > duration > temperature × duration > temperature×salinity×frequency>salinity×frequency>temperature× frequency×duration (Table 1). The effects which contributed to the survival of germlings, in order of effect size, were: temperature × salinity > salinity> temperature × salinity × duration > temperature> temperature × duration > salinity × duration > duration (Table 2).
B
25
30
o
Duration: 2h Frequency: twice a day
100
Survival (%)
Survival (%)
100
80 60 40 20 0
35
25
Tempreture ( C)
C
60 40 20 25
30
60 40 20 30
o
Tempreture ( C)
35
35
80 60 40 20 0
25
30
o
Tempreture ( C)
F
35
Duration: 8h Frequency: twice a day
100
Survival (%)
80
25
o
Duration: 4h Frequency: twice a day
100
35
Duration: 8h Frequency: once a day
100
Survival (%)
o
Tempreture ( C)
E
0
D Survival (%)
Survival (%)
80
0
30
Tempreture ( C)
Duration: 4h Frequency: once a day
100
69
80 60 40 20 0
25
30
o
35
Tempreture ( C)
Fig. 2. Survival rates of germlings exposed to various temperatures (25 °C, 30 °C and 35 °C for 10 h during the day), salinities (12 psu, 21 psu, 32 psu and 50 psu), frequencies (once a day and twice a day) and durations of osmotic stress (2 h, 4 h and 8 h) at an irradiance of 60 μmol photons m− 2 s− 1. A, B, C, D, E and F denote various frequency and duration of osmotic stress. The top left key denotes the salinity. The initial mean survival rate was 94.08%. Values are means ± SE (n = 3).
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3.4. Appropriate and extreme conditions for the growth and survival of germlings The germlings of S. thunbergii showed quick growth with RGRs over 16% when cultured at 32 psu combined with 30 °C (Fig. 1). Although the growths of germlings subjected to moderate osmotic stresses were significantly inhibited, RGRs over 12% were obtained (Fig. 1). Only at 35 °C were the RGRs below 10% (Fig. 1). Germlings showed high survivals over 80% under appropriate conditions (25 °C and 30 °C), and over 60% at moderate salinities, by the end of experiment (Fig. 2). Only under extreme conditions (12 psu for 4 h or 8 h at 35 °C) were the survivals reduced to below 10% (Fig. 2C–F).
4. Discussion As in many organisms on land and in the sea, early life stages likely represent the most vulnerable phases in the population development of seaweeds (Santelices, 1990; Vadas et al., 1992). Macroalgal propagules and germlings are delicate structures often strongly responding toward changes in their abiotic environment (Santelices, 1990). However, our results showed that under full immersion conditions the germlings of S. thunbergii exhibited a high tolerance to osmotic stress. The responses of different stages of an organism's life-cycle to different environments can have important implications for their persistence in those habitats (Wright et al., 2004). Therefore, the ability of S. thunbergii germlings to
Table 2 Repeated measures ANOVA for effects of temperature, salinity, frequency and duration of osmotic stress on the survival rate of germlings. T: temperature, S: salinity, F: frequency, D: duration. η2 was used as a measure of effect size. Variable
df
Mean square
F
p
η2
Within-subjects Time Time × irradiance Time × T Time × S Time × F Time × D Time × T × S Time × T × F Time × S × F Time × F × D Time × T × D Time × S × D Time × T × S × D Time × T × S × F Time × T × F × D Time × S × F × D Time × T × S × F × D Error (time) Irradiance T S F D T×S T×F S×F F×D T×D S×D T×S×D T×S×F T×F×D S×F×D T×S×F×D Error
2.210 2.210 4.420 6.630 2.210 4.420 13.259 4.420 6.630 4.420 8.840 13.259 26.519 13.259 8.840 13.259 26.519 316.015 1 2 3 1 2 6 2 3 2 4 6 12 6 4 6 12 143
20.751 97.257 1324.691 1399.456 38.372 299.800 1809.265 69.650 34.584 23.462 560.305 441.418 323.876 51.512 50.173 73.637 46.573 45.537 166.353 4674.764 4030.048 20.109 1024.431 4765.424 117.576 58.711 50.933 1427.461 868.498 829.838 66.665 32.809 91.046 58.356 56.970
0.456 2.136 29.091 30.733 0.843 6.584 39.732 1.530 0.759 0.515 12.304 9.694 7.112 1.131 1.102 1.617 1.023
0.654 0.114 b 0.001 b 0.001 0.441 b 0.001 b 0.001 0.188 0.615 0.743 b 0.001 b 0.001 b 0.001 0.331 0.361 0.077 0.437
0.003 0.015 0.289 0.392 0.006 0.084 0.625 0.021 0.016 0.007 0.256 0.289 0.374 0.045 0.030 0.064 0.079
2.920 82.057 70.740 0.353 17.982 83.649 2.064 1.031 0.894 25.057 15.245 14.566 1.170 0.576 1.598 1.024
0.090 b 0.001 b 0.001 0.553 b 0.001 b 0.001 0.131 0.381 0.411 b 0.001 b 0.001 b 0.001 0.326 0.681 0.152 0.430
0.020 0.534 0.597 0.002 0.201 0.778 0.028 0.021 0.012 0.412 0.390 0.550 0.047 0.016 0.063 0.079
Dependent variable (survival) was untransformed and the assumption of homogeneity met Levene's test (F = 2.747, p = 0.271). Once Mauchly's tests of sphericity were invariably significant, the Huynh–Feldt adjustment was used for hypothesis testing.
resist these physical stresses reflected the feasibility of restoration of intertidal seaweed beds by seeding germlings in artificial tanks. Intertidal seaweeds are subject to salinity stress when exposed to low tide or trapped in tide pools, where fresh water from rain may lower salinity, or where evaporation may raise salinity (Macler, 1988). The tolerance of seaweeds to salinity stress may vary from one life stage to another, and increase with algal age (Norton, 1977; Steen, 2004). Our results showed that even as the early life history stage, germlings of S. thunbergii also had a broad tolerance to salinity from 12 psu to 50 psu (Figs. 1 and 2). The RGR of S. muticum germlings decreased with decreasing salinity below 25 psu during the week of exposure at various salinities; however, germlings that had been exposed to 20 psu achieved the same RGR as the germlings in the control dishes within one week during the recovery period at 30 psu (Steen, 2004). This is similar to S. thunbergii germlings observed in the present study. Our results showed that both growth and survival of germlings had high tolerance to the frequency of osmotic stress, suggesting that high frequent rains in the rainy season would not significantly affect the growth, especially not the survival of germlings. The duration of osmotic stress exhibited a significant but minor effect on the growth and survival of germlings, as mentioned above. Most survival rates and RGRs reached over 80% and 7% respectively when exposed to osmotic stress for 8 h (Figs. 1E–F and 2E–F). It suggested that the tidal level, i.e. the duration of osmotic stress would not have dominant influence on the growth and survival of S. thunbergii germlings in the intertidal zone. Intertidal macroalgae usually experience multiple stresses simultaneously (Li and Brawley, 2004). Environmental stress in the upper distributional limit of the rocky intertidal zone is driven by a synergistic combination of factors (Petes et al., 2007). Our results showed that multiple stress factors interacted significantly on survival and growth of S. thunbergii germlings (Tables 1 and 2). In comparison with the main effects, the combinations of temperature, salinity, frequency and duration of osmotic stress caused a more substantial decline (Figs. 1 and 2). Nevertheless, as mentioned above, the survival of germlings was more tolerant than the growth, suggesting that even if the growth of S. thunbergii germlings was severely inhibited due to the long duration of hypo-osmotic or hyper-osmotic conditions at high temperature, germlings can still maintain high survival. In the present study, the extreme level of combined stresses (35 °C and 12 psu for over 4 h) had great destructive effect on germlings (Figs. 1C–F and 2C–F). However, the low salinity is usually resulted from strong rainfall which also causes the decrease of temperature. Therefore, high temperature and long duration of low salinity can generally not be concurrent with each other. In contrast with the extreme conditions, germlings exhibited high survival and quick growth in other multiple stress treatments (Figs. 1 and 2). Combining stress factors results in interactions ranging from stress intensification (additive) through increased tolerance (antagonistic) to stress (Gamon and Pearcy, 1990; Larcher et al., 1990). For example, tolerance of heat stress was improved by simultaneous exposure of embryos to hypersaline seawater in Fucus vesiculosus and Fucus spiralis (Li and Brawley, 2004). It is also reported that there is a positive correlation between the ability of seaweeds to tolerate different stresses (Davison and Pearson, 1996). Consequently, it is not surprising that S. thunbergii germlings showed a high tolerance to combined stress. Our results indicated that the optimal condition for germlings was a salinity of 32 psu and a maximum ambient temperature of 30 °C. Hence better restoration will be achieved when seeding S. thunbergii germlings under this condition. Germlings at about 24 h postfertilization will be collected in the laboratory and then seeded in tanks. Such germlings which were used in the present study have immature rhizoids so that they can attach to the tanks. Germlings were greatly impaired only by high temperature together with long duration of low salinity which can generally not be concurrent with each other as mentioned above. Under the other conditions which probably exist in the intertidal zone, S. thunbergii germlings showed quick growth, at least high survival rates. Therefore, construction of
S.H. Chu et al. / Journal of Experimental Marine Biology and Ecology 412 (2012) 66–71
artificial tanks in natural habitat is both effective and feasible. This work is in progress to further restore the seaweed beds in the intertidal zone. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (no. 31070376) and the Developmental Program of Science and Technology of Shandong Province, China (no. 2009GG10005008). [ST] References Bell, E.C., 1993. Photosynthetic response to temperature and desiccation of the intertidal alga Mastocarpus papillatus. Mar. Biol. 117, 337–346. Carter, J.W., Jesse, W.N., Foster, M.S., Carpenter, A.L., 1985. Management of artificial reefs designed to support natural communities. Bull. Mar. Sci. 37, 114–128. Choi, H.G., Lee, K.H., Yoo, H.I., Kang, P.J., Kim, Y.S., Nam, K.W., 2008. Physiological differences in the growth of Sargassum horneri between the germling and adult stages. J. Appl. Phycol. 20, 729–735. Chu, S.H., Zhang, Q.S., Liu, S.K., Tang, Y.Z., Zhang, S.B., Lu, Z.C., Yu, Y.Q., 2011. Tolerance of Sargassum thunbergii germlings to thermal, osmotic and desiccation stress. Aquat. Bot. doi:10.1016/j.aquabot.2011.09.002. Davison, I.R., Pearson, G.A., 1996. Stress tolerance in intertidal seaweeds. J. Phycol. 32, 197–211. De Jonge, V.N., De Jong, D.J., 2002. Ecological restoration in coastal areas in the Netherlands: concepts, dilemmas and some examples. Hydrobiologia 478, 7–28. Falace, A., Zanelli, E., Bressan, G., 2006. Algal transplantation as a potential tool for artificial reef management and environmental mitigation. Bull. Mar. Sci. 78, 161–166. Gamon, J.A., Pearcy, R.W., 1990. Photoinhibition in Vitis californica: the role of temperature during high-light treatment. Plant Physiol. 92, 487–494. Helmuth, B., 2002. How do we measure the environment? Linking intertidal thermal physiology and ecology through biophysics. Integr. Comp. Biol. 42, 837–845. Helmuth, B.S.T., Hofmann, G.E., 2001. Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal zone. Biol. Bull. 201, 374–384. Hernández-Carmona, G., García, O., Robledo, D., Foster, M., 2000. Restoration techniques for Macrocystis pyrifera (Phaeophyceae) populations at the southern limit of their distribution in México. Bot. Mar. 43, 273–284. Hunt, R., 1978. Plant Growth Analysis. Edward Arnold, London. Kim, Y.H., Kim, E.H., Lee, C., Kim, M.H., Rho, J.R., 2007. Two new monogalactosyl diacylglycerols from brown alga Sargassum thunbergii. Lipids 42, 395–399. Larcher, W., Wagner, J., Thammathaworn, A., 1990. Effects of superimposed temperature stress on in vivo chlorophyll fluorescence of Vigna unguiculata under saline stress. J. Plant Physiol. 136, 92–102. Largo, D.B., Ohno, M., 1993. Constructing an artificial seaweed bed. In: Ohno, M., Critchley, A.T. (Eds.), Seaweed Cultivation and Marine Ranching. JICA, Tokyo, pp. 113–130.
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Li, R., Brawley, S.H., 2004. Improved survival under heat stress in intertidal embryos (Fucus spp.) simultaneously exposed to hypersalinity and the effect of parental thermal history. Mar. Biol. 144, 205–213. Li, R.S., Zhao, S.L., 2002. Marine Resources and Environment in Shandong. Ocean Press, Beijing. Macler, B.A., 1988. Salinity effects on photosynthesis, carbon allocation, and nitrogen assimilation in the red alga, Gelidium coulteri. Plant Physiol. 88, 690–694. Norton, T.A., 1977. Ecological experiments with Sargassum muticum. J. Mar. Biol. Assoc. U. K. 57, 33–43. Padilha, F.P., Franca, F.P., Costa, A.C.A., 2005. The use of waste biomass of Sargassum sp. for the biosorption of copper from simulated semiconductor effluents. Bioresour. Technol. 96, 1511–1577. Park, P.J., Heo, S.J., Park, E.J., Kim, S.K., Byun, H.G., Jeon, B.T., Jeon, Y.J., 2005. Reactive oxygen scavenging effect of enzymatic extracts from Sargassum thunbergii. J. Agric. Food Chem. 53, 6666–6672. Petes, L.E., Menge, B.A., Murphy, G.D., 2007. Environmental stress decreases survival, growth, and reproduction in New Zealand mussels. J. Exp. Mar. Biol. Ecol. 351, 83–91. Rodríguez-Salinas, P., Riosmena-Rodríguez, R., Hinojosa-Arango, G., Muñiz-Salazar, R., 2010. Restoration experiment of Zostera marina L. in a subtropical coastal lagoon. Ecol. Eng. 36, 12–18. Santelices, B., 1990. Patterns of reproduction, dispersal and recruitment in seaweeds. Oceanogr. Mar. Biol. Annu. Rev. 28, 177–276. Song, W.B., Warren, A., Hu, X.Z., 2009. Free-living ciliates in the Bohai and Yellow seas, China. Science Press, Beijing. Steen, H., 2004. Effects of reduced salinity on reproduction and germling development in Sargassum muticum (Phaeophyceae, Fucales). Eur. J. Phycol. 39, 293–299. Tabachnick, B.G., Fidell, L.S., 2000. Computer-Assisted Research Design and Analysis. Allyn and Bacon, Boston. Terawaki, T., Hasegawa, H., Arai, S., Ohno, M., 2001. Management-free techniques for restoration of Eisenia and Ecklonia beds along the central Pacific coast of Japan. J. Appl. Phycol. 13, 13–17. Terawaki, T., Yoshikawa, K., Yoshida, G., Uchimura, M., Iseki, K., 2003. Ecology and restoration techniques for Sargassum beds in the Seto Inland Sea. Jpn. Mar. Pollut. Bull. 47, 198–201. Vadas, R.L., Johnson, S., Norton, T.A., 1992. Recruitment and mortality of early postsettlement stages of benthic algae. Br. Phycol. J. 27, 331–351. Vila-Gispert, A., Moreno-Amich, R., García-Berthou, E., 2002. Gradients of life-history variation: an intercontinental comparison of fishes. Rev. Fish. Biol. Fish. 12, 417–427. Wright, J.T., Williams, S.L., Dethier, M.N., 2004. No zone is always greener: variation in the performance of Fucus gardneri embryos, juveniles and adults across tidal zone and season. Mar. Biol. 145, 1061–1073. Zhang, Q.S., Li, W., Liu, S., Pan, J.H., 2009. Size-dependence of reproductive allocation of Sargassum thunbergii (Sargassaceae, Phaeophyta) in Bohai Bay, China. Aquat. Bot. 91, 194–198. Zhao, F.J., Wang, X.L., Liu, J.D., Duan, D.L., 2007. Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers. J. Appl. Phycol. 19, 409–416. Zhao, Z.G., Zhao, F.J., Yao, J.T., 2008. Early development of germlings of Sargassum thunbergii (Fucales, Phaeophyta) under laboratory conditions. J. Appl. Phycol. 20, 475–481.
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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
High tolerance to fluctuating salinity allows Sargassum thunbergii germlings to survive and grow in artificial habitat of full immersion in intertidal zone Shao Hua Chu, Quan Sheng Zhang ⁎, Yong Zheng Tang, Shu Bao Zhang, Zhi Cheng Lu, Yong Qiang Yu Ocean School, Yantai University, Yantai 264005, PR China
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 18 October 2011 Accepted 22 October 2011 Available online 17 November 2011 Keywords: Germling Salinity Sargassum thunbergii Tolerance
a b s t r a c t In this study, a four-way factorial experimental design (three temperatures, four salinities, two frequencies and three durations of osmotic stress) was used to test the tolerance of Sargassum thunbergii germlings shortly released from fertile thalli to osmotic stress. Results showed that the growth and survival of germlings were significantly affected by salinity and duration of osmotic stress, rather than by osmotic frequency. In addition to the main effects, all two-way, three-way and four-way interactions between the four stresses on the growth were significant. Germlings showed quick growth with relative growth rate (RGR, % day−1) over 16% when cultured at 32 psu combined with 30 °C. Although growths of germlings subjected to hypo-osmotic and hyper-osmotic conditions were significantly inhibited, RGRs over 12% were obtained except for those at 35 °C with the RGRs below 10%. In comparison to growth, survival was more tolerant to osmotic stress. Germlings showed high survivals over 60% when exposed to most osmotic stresses, by the end of experiment. Only under extreme conditions (12 psu for 4 h or 8 h at 35 °C) were the survivals reduced to below 10%. Although the effects of osmotic stress on the growth and survival were significant, germlings exhibited quick growth and high survival under most conditions. And those extreme conditions which had great destructive effect on germlings barely exist in field. Therefore, the construction of artificial tanks is feasible for the restoration of S. thunbergii beds in the intertidal zone. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Seaweed beds in the intertidal zone are communities consisting of macroalgae, and they play important roles in the coastal ecosystem, such as providing food and habitat for coastal marine life (Largo and Ohno, 1993; Terawaki et al., 2001). In recent years, however, human activities have caused a rapid decline in seaweed beds (De Jonge and De Jong, 2002; Rodríguez-Salinas et al., 2010; Terawaki et al., 2003; Zhao et al., 2008). In order to achieve sustainable development of the intertidal zone, effective measures for the restoration of seaweed beds are urgently required. Previous algal colonizations induced by the dispersal of microscopic life-stages and transplantation of juveniles or adults have been demonstrated on artificial seaweed beds or reefs in the benthic or subtidal zone of many regions (Carter et al., 1985; Falace et al., 2006; Hernández-Carmona et al., 2000; Terawaki et al., 2003). However, restoration in the intertidal zone is more challengeable due to daily exposure at low tide results in daily fluctuation in a range of important environmental factors (Bell, 1993; Davison and Pearson, 1996; Helmuth, 2002; Helmuth and Hofmann, 2001; Wright et al., 2004). The success of restoration in the intertidal zone
⁎ Corresponding author. Tel.: + 86 535 6706011; fax: + 86 535 6706299. E-mail address: [email protected] (Q.S. Zhang). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.10.025
is more dependent on the ability of survival and growth of the transplants in the stressful environment. The common brown alga Sargassum thunbergii which forms extensive beds in the intertidal zones is an appropriate species for the restoration of intertidal seaweed beds due to its wide ecological amplitude, high economic and ecological value (Kim et al., 2007; Padilha et al., 2005; Park et al., 2005; Zhang et al., 2009; Zhao et al., 2007). However, the natural populations of S. thunbergii along the northern coast of China have been depleted (Zhao et al., 2007, 2008). Sargassum germlings exhibited wide tolerance to environmental stress. Sargassum muticum germlings survived exposure to salinities as low as 5 psu, even during the first week after fertilization (Steen, 2004). Sargassum horneri germlings grew over a wide range of temperatures, from 10 °C to 25 °C (Choi et al., 2008). Our previous study showed that germlings of S. thunbergii exhibited strong tolerance to high temperature and low salinity; however, the effect of desiccation on the survival and growth of germlings was especially prominent (Chu et al., 2011). Therefore, it is most important to eliminate desiccation for the restoration of intertidal seaweed beds. The construction of artificial tanks is an effective way to avoid desiccation. Nevertheless, this measure may cause other problems, such as reduced salinity in the tank resulted from the heavy rain and increased salinity resulted from high temperature. Hence it is essential to investigate the effect of salinity fluctuation on the survival and growth of S. thunbergii germlings. In this investigation, germlings shortly released from fertile thalli of S. thunbergii were collected and cultured in the laboratory. The
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stress resistance of germlings to adverse environmental conditions including high temperature, reduced and increased salinity, high frequency and long duration of osmotic stress, and their interactive effects on growth and survival were tested. Relevant results will be applied to the restoration of intertidal seaweed beds. 2. Materials and methods 2.1. Collection of germlings Fertile female and male specimens of S. thunbergii were collected on July, 2010, in an intertidal zone of Xiaoheishan Island (37°58′N, 120°39′ E), located at the northern side of Shandong Peninsula, China. Selected thalli were healthy and yellowish-brown in appearance with intact and inflated receptacles which had no obvious shedding. Thalli of 2 kg were cleaned several times with seawater to remove surface epiphytes. They were then packed in plastic bags and stored in a plastic foam box full of crushed ice until transported to the laboratory where they were subsequently transferred to a plastic tank filled with 75 L filtered seawater which was continuously aerated. The tank containing thalli was kept at 22 °C, 10 μmol photons m− 2 s − 1 and a 12L: 12D (light: dark cycle) photoperiod. At 24 h post-fertilization, released germlings sank to the bottom of tank. After removal of thalli from the tank, the remaining seawater containing germlings was filtered with an 80-mesh nylon sieve, followed by filtration with a 200-mesh nylon sieve. A total of 3.0×105 germlings were obtained. To remove fouling, dross and epiphytes, collected germlings were repeatedly washed in tap seawater in the 200-mesh nylon sieve. Germlings were subsequently transferred to a 3 L glass tank filled with sterile filtered seawater. Following stirring even, the germling suspension was immediately poured into each Petri dish (60 mm×15 mm). A total of 220 Petri dishes with germlings of about 700 in each were obtained and used for our experiment. 2.2. Stress treatments After 24 h, germlings attached to the Petri dishes and were cultured in three light- and temperature-controlled incubators (Zhujiang, China) with 72 dishes in each, on the basis of the following factorial experimental design. Samples were cultured under three temperatures conditions: 15 °C, 20 °C, and 25 °C at night, and 25 °C, 30 °C and 35 °C during the day to mimic the temperature difference between nigh and day; four levels of salinity: 12 psu, 21 psu, 32 psu and 50 psu; two frequencies of osmotic stress: once a day and every other day; and three durations of osmotic stress: 2 h, 4 h and 8 h in every 24 h, for a total of 72 different combinations of temperature, salinity, frequency and duration. Three independent replicates were used for each combination. All treatments were cultured for 12 days at an irradiance of 60 μmol photons m − 2 s − 1 with a 10L: 14D light: dark cycle. Various concentrations of seawater were prepared by diluting sterile seawater with distilled water or adding NaCl and measured with a salinity hydrometer (GM Manu-facturing Co.). Sterile filtered seawater with four levels of salinity was used as the culture medium and changed according to the experimental design (twice a day or every other day). Therefore, contamination problems were avoided by frequent change of sterile culture medium. No distinct detachment of germlings followed the solution change due to the developed rhizoids. Irradiance within the wavelength range 400–700 nm was measured using a Li-Cor LI-250 light meter equipped with a LI-190SA quantum sensor. The ranges of all the factors chosen were in accordance with the fluctuations of conditions in the natural habitat of S. thunbergii. In summer, the temperature ranged from 23.5 to 25.6 °C and the extreme maximum temperature ranged from 32.0 to 34.0 °C in the northern o ffshore areas of Shandong Peninsula (Li and Zhao, 2002). The salinity of nature seawater measured was 32. However, about half of the annual precipitation fell during the period between June and August (i.e. the
67
sexual reproductive period of S. thunbergii), and the salinity generally fell (Song et al., 2009). 2.3. Measurements of length and survival rate Growths of germlings were measured in terms of length, excluding rhizoids at the end of the experiment. The lengths of 4 germlings were measured using a microscope with ocular micrometer respectively in 5 areas arranged in a cross pattern (the upper, lower, left and right periphery and the center), for a total of 20 germlings in each dish. The initial mean length of germlings in four dishes was also measured before the stress treatment for the estimation of each relative growth rate (RGR, % day− 1). RGR was calculated as 100 (ln (L1)−ln (L0))/t, where L0 is the initial mean length of germlings, and L1 is the length at the end of various treatments, respectively, and t is the length of treatment period in days (Hunt, 1978). Prior to the stress treatment, the mean survival rate of germlings in four dishes was measured as the initial survival rate. Survival rates were calculated under a stereoscopy microscope every other day. In each dish germlings were counted respectively in 5 areas, as mentioned above, for a total of about 200 germlings for the calculation of each survival rate. Germlings were classified as dead when they became collapsed or structurally fragmented. 2.4. Statistical analysis All data were analyzed using SPSS for Windows 13.0. Prior to all statistical analyses, the homogeneity of variances was verified with Levene's test. A univariate analysis of variance was performed to test the significance of the main effects and interactions on the growth of germlings, with temperature, salinity, frequency and duration of osmotic stress as fixed factors, and RGR as dependent variable. Irradiance was used as the covariate to remove any confounding influence of irradiance, because the irradiances of various Petri dishes were slightly different. Main effects and interactions of temperature, salinity, frequency and duration of osmotic stress on the survival of germlings were analyzed by a three-way repeated measures ANOVA, with temperature, salinity, frequency and duration of osmotic stress as between-subjects factors, survival rates over the day as a within-subject factor, and irradiance as a covariate. Partial eta squared (η2) was used as a measure of effect size (Vila-Gispert et al., 2002). Similarly to r2, partial eta squared is the proportion of variation explained for a certain effect (effect SS/effect SS
Table 1 ANOVA for effects of temperature, salinity, frequency and duration of osmotic stress on the relative growth rate (RGR, % day− 1) of germlings. T: temperature, S: salinity, F: frequency, D: duration. η2 was used as a measure of effect size. Variable
df
Mean square
F
p
η2
Irradiance Temperature (T) Salinity (S) Frequency (F) Duration (D) T×S T×F S×F T×D S×D F×D T×S×D T×S×F T×F×D S×F×D T×S×F×D Error
1 2 3 1 2 6 2 3 4 6 2 12 6 4 6 12 4247
0.004 0.830 0.187 b 0.001 0.012 0.047 0.080 0.006 0.006 0.008 0.020 0.012 0.004 0.002 0.010 0.004 b 0.001
9.969 2241.393 505.774 0.758 32.75 125.697 215.082 16.352 16.392 21.593 52.82 33.728 10.075 6.467 26.014 10.992
0.002 b 0.001 b 0.001 0.384 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
0.002 0.514 0.263 b 0.001 0.015 0.151 0.092 0.011 0.015 0.03 0.024 0.087 0.014 0.006 0.035 0.03
Dependent variable (RGR) was untransformed and the assumption of homogeneity met Levene's test (F = 1.076, p = 0.381).
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+ error SS). Partial eta squared has the advantage over eta squared (effect SS/total SS) that it does not depend on the ANOVA design used because it does not use total SS as the denominator (Tabachnick and Fidell, 2000). Tukey's tests were used for the post-hoc comparisons. The differences were considered to be statistically significant under a probability value of 5% (p b 0.05). 3. Results 3.1. Effects of osmotic stress on growth of germlings Prior to the stress treatment, the initial mean length of germlings measured was 120 μm. At the end of the experiment, final lengths
A
12 psu 21 psu 20
32 psu 50 psu
Duration: 2h Frequency: once a day
ranged from 122.2 to 1050 μm due to the significant effects of temperature and osmotic stress (Table 1). Although the growth of germlings was significantly affected by salinity (Table 1), nearly all the RGRs were over 7% when cultured at 21 psu and 50 psu. Only at 12 psu combined with 35 °C for 4 h or 8 h, the RGRs were below 6% (Fig. 1C–F). The duration of osmotic stress exhibited significant effects on the growth. However, it showed minor influence with low η2 (Table 1). When cultured at moderate temperature, germlings showed quick growth even exposed to long duration of osmotic stress. For example, when cultured at 12 psu, 21 psu or 50 psu for 8 h during daily exposure to 25 °C or 30 °C, the RGRs were over 10% (Fig. 1E). There was no significant main effect of osmotic frequency on the growth of germlings (Table 1). Its effect on the growth was reflected in the interactions (Table 1). As compared to the frequency
B
16
RGR (% day-1)
RGR (% day-1)
16 12 8 4 0
Duration: 2h Frequency: tw ice a day
20
12 8 4
25
30
o
0
35
25
Tempreture ( C)
C
Duration: 4h Frequency: once a day
20
D
RGR (% day-1)
RGR (% day-1)
8 4
12 8 4
25
30
0
35 o
25
Tempreture ( C)
E
30
o
35
Tempreture ( C)
Duration: 8h Frequency: once a day
20
F
Duration: 8h Frequency: tw ice a day
20 16
RGR (% day-1)
16
RGR (% day-1)
35
16
12
12 8 4 0
o
Duration: 4h Frequency: tw ice a day
20
16
0
30
Tempreture ( C)
12 8 4
25
30
o
Tempreture ( C)
35
0
25
30
o
35
Tempreture ( C)
Fig. 1. Relative growth rate (RGR; % day− 1) of germlings exposed to various temperatures (25 °C, 30 °C and 35 °C for 10 h during the day), salinities (12 psu, 21 psu, 32 psu and 50 psu), frequencies (once a day and twice a day) and durations of osmotic stress (2 h, 4 h and 8 h) at an irradiance of 60 μmol photons m− 2 s− 1. A, B, C, D, E and F denote various frequency and duration of osmotic stress. The top left key denotes the salinity. Values are means ± SE (n = 3).
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and duration of osmotic stress, the salinity showed greater effects on the growth of germlings on the basis of η2 (Table 1). The three-way interaction between salinity, frequency and duration of osmotic stress had significant effect with low η2 (Table 1). RGRs over 10% were obtained when exposed to the combination of three stresses with the exception of those at 35 °C (Fig. 1A–F).
3.2. Effects of osmotic stress on survival of germlings The salinity showed significant effect on the survival of germlings; however, when cultured at 21 psu and 50 psu, the survival rates were over 70% (Fig. 2). The results of Tukey's tests showed that although the effect of 12 psu on the survival of germlings was significant in comparison with 32 psu (post-hoc tests: pb 0.001), there was no significant difference between the effects of 50 psu and 32 psu (post-hoc tests: p=0.999). These results suggested that the damage of hypo-osmotic stress was more severe than hyper-osmotic stress. The duration of osmotic stress exhibited significant effects on the survival with low η2 (Table 2). When cultured at moderate temperature (25 °C or 30 °C), germlings showed high survival (over 90%) even exposed to long duration of osmotic stress (for 8 h) (Fig. 2E). Based on η2, the salinity exhibited greater effects on the survival (Table 2). The survival of germlings was not significantly affected by the three-way interaction between salinity, frequency and duration of osmotic stress (Table 2).
A
12 psu 21 psu
Duration: 2h Frequency: once a day
32 psu 50 psu
80 60 40 20 0
3.3. Order of effect size The growths of germlings were significantly affected by all the stresses and their interactions except for the main effect of osmotic frequency (Table 1). However, for the survival, the main effect of osmotic frequency and its interactions with other stresses showed no significant effects (Table 2). On the other hand, 12 psu, 21 psu and 50 psu significantly inhibited the growth of germlings in comparison with 32 psu (post-hoc tests: p b 0.001); however, the survival rates of germlings cultured at 21 psu and 50 psu were not significantly decreased when compared with those at 32 psu (post-hoc tests: p= 0.823 and p= 0.999, respectively). These results indicated that the survival of germlings was more tolerant to osmotic stress than the growth. Compared with η 2 values estimated, the effects which contributed to the growth of S. thunbergii germlings, in order of effect size,were:temperature>salinity>temperature×salinity>temperature× frequency>temperature × salinity × duration > salinity × frequency × duration > salinity × duration > temperature × salinity × frequency × duration > frequency × duration > duration > temperature × duration > temperature×salinity×frequency>salinity×frequency>temperature× frequency×duration (Table 1). The effects which contributed to the survival of germlings, in order of effect size, were: temperature × salinity > salinity> temperature × salinity × duration > temperature> temperature × duration > salinity × duration > duration (Table 2).
B
25
30
o
Duration: 2h Frequency: twice a day
100
Survival (%)
Survival (%)
100
80 60 40 20 0
35
25
Tempreture ( C)
C
60 40 20 25
30
60 40 20 30
o
Tempreture ( C)
35
35
80 60 40 20 0
25
30
o
Tempreture ( C)
F
35
Duration: 8h Frequency: twice a day
100
Survival (%)
80
25
o
Duration: 4h Frequency: twice a day
100
35
Duration: 8h Frequency: once a day
100
Survival (%)
o
Tempreture ( C)
E
0
D Survival (%)
Survival (%)
80
0
30
Tempreture ( C)
Duration: 4h Frequency: once a day
100
69
80 60 40 20 0
25
30
o
35
Tempreture ( C)
Fig. 2. Survival rates of germlings exposed to various temperatures (25 °C, 30 °C and 35 °C for 10 h during the day), salinities (12 psu, 21 psu, 32 psu and 50 psu), frequencies (once a day and twice a day) and durations of osmotic stress (2 h, 4 h and 8 h) at an irradiance of 60 μmol photons m− 2 s− 1. A, B, C, D, E and F denote various frequency and duration of osmotic stress. The top left key denotes the salinity. The initial mean survival rate was 94.08%. Values are means ± SE (n = 3).
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3.4. Appropriate and extreme conditions for the growth and survival of germlings The germlings of S. thunbergii showed quick growth with RGRs over 16% when cultured at 32 psu combined with 30 °C (Fig. 1). Although the growths of germlings subjected to moderate osmotic stresses were significantly inhibited, RGRs over 12% were obtained (Fig. 1). Only at 35 °C were the RGRs below 10% (Fig. 1). Germlings showed high survivals over 80% under appropriate conditions (25 °C and 30 °C), and over 60% at moderate salinities, by the end of experiment (Fig. 2). Only under extreme conditions (12 psu for 4 h or 8 h at 35 °C) were the survivals reduced to below 10% (Fig. 2C–F).
4. Discussion As in many organisms on land and in the sea, early life stages likely represent the most vulnerable phases in the population development of seaweeds (Santelices, 1990; Vadas et al., 1992). Macroalgal propagules and germlings are delicate structures often strongly responding toward changes in their abiotic environment (Santelices, 1990). However, our results showed that under full immersion conditions the germlings of S. thunbergii exhibited a high tolerance to osmotic stress. The responses of different stages of an organism's life-cycle to different environments can have important implications for their persistence in those habitats (Wright et al., 2004). Therefore, the ability of S. thunbergii germlings to
Table 2 Repeated measures ANOVA for effects of temperature, salinity, frequency and duration of osmotic stress on the survival rate of germlings. T: temperature, S: salinity, F: frequency, D: duration. η2 was used as a measure of effect size. Variable
df
Mean square
F
p
η2
Within-subjects Time Time × irradiance Time × T Time × S Time × F Time × D Time × T × S Time × T × F Time × S × F Time × F × D Time × T × D Time × S × D Time × T × S × D Time × T × S × F Time × T × F × D Time × S × F × D Time × T × S × F × D Error (time) Irradiance T S F D T×S T×F S×F F×D T×D S×D T×S×D T×S×F T×F×D S×F×D T×S×F×D Error
2.210 2.210 4.420 6.630 2.210 4.420 13.259 4.420 6.630 4.420 8.840 13.259 26.519 13.259 8.840 13.259 26.519 316.015 1 2 3 1 2 6 2 3 2 4 6 12 6 4 6 12 143
20.751 97.257 1324.691 1399.456 38.372 299.800 1809.265 69.650 34.584 23.462 560.305 441.418 323.876 51.512 50.173 73.637 46.573 45.537 166.353 4674.764 4030.048 20.109 1024.431 4765.424 117.576 58.711 50.933 1427.461 868.498 829.838 66.665 32.809 91.046 58.356 56.970
0.456 2.136 29.091 30.733 0.843 6.584 39.732 1.530 0.759 0.515 12.304 9.694 7.112 1.131 1.102 1.617 1.023
0.654 0.114 b 0.001 b 0.001 0.441 b 0.001 b 0.001 0.188 0.615 0.743 b 0.001 b 0.001 b 0.001 0.331 0.361 0.077 0.437
0.003 0.015 0.289 0.392 0.006 0.084 0.625 0.021 0.016 0.007 0.256 0.289 0.374 0.045 0.030 0.064 0.079
2.920 82.057 70.740 0.353 17.982 83.649 2.064 1.031 0.894 25.057 15.245 14.566 1.170 0.576 1.598 1.024
0.090 b 0.001 b 0.001 0.553 b 0.001 b 0.001 0.131 0.381 0.411 b 0.001 b 0.001 b 0.001 0.326 0.681 0.152 0.430
0.020 0.534 0.597 0.002 0.201 0.778 0.028 0.021 0.012 0.412 0.390 0.550 0.047 0.016 0.063 0.079
Dependent variable (survival) was untransformed and the assumption of homogeneity met Levene's test (F = 2.747, p = 0.271). Once Mauchly's tests of sphericity were invariably significant, the Huynh–Feldt adjustment was used for hypothesis testing.
resist these physical stresses reflected the feasibility of restoration of intertidal seaweed beds by seeding germlings in artificial tanks. Intertidal seaweeds are subject to salinity stress when exposed to low tide or trapped in tide pools, where fresh water from rain may lower salinity, or where evaporation may raise salinity (Macler, 1988). The tolerance of seaweeds to salinity stress may vary from one life stage to another, and increase with algal age (Norton, 1977; Steen, 2004). Our results showed that even as the early life history stage, germlings of S. thunbergii also had a broad tolerance to salinity from 12 psu to 50 psu (Figs. 1 and 2). The RGR of S. muticum germlings decreased with decreasing salinity below 25 psu during the week of exposure at various salinities; however, germlings that had been exposed to 20 psu achieved the same RGR as the germlings in the control dishes within one week during the recovery period at 30 psu (Steen, 2004). This is similar to S. thunbergii germlings observed in the present study. Our results showed that both growth and survival of germlings had high tolerance to the frequency of osmotic stress, suggesting that high frequent rains in the rainy season would not significantly affect the growth, especially not the survival of germlings. The duration of osmotic stress exhibited a significant but minor effect on the growth and survival of germlings, as mentioned above. Most survival rates and RGRs reached over 80% and 7% respectively when exposed to osmotic stress for 8 h (Figs. 1E–F and 2E–F). It suggested that the tidal level, i.e. the duration of osmotic stress would not have dominant influence on the growth and survival of S. thunbergii germlings in the intertidal zone. Intertidal macroalgae usually experience multiple stresses simultaneously (Li and Brawley, 2004). Environmental stress in the upper distributional limit of the rocky intertidal zone is driven by a synergistic combination of factors (Petes et al., 2007). Our results showed that multiple stress factors interacted significantly on survival and growth of S. thunbergii germlings (Tables 1 and 2). In comparison with the main effects, the combinations of temperature, salinity, frequency and duration of osmotic stress caused a more substantial decline (Figs. 1 and 2). Nevertheless, as mentioned above, the survival of germlings was more tolerant than the growth, suggesting that even if the growth of S. thunbergii germlings was severely inhibited due to the long duration of hypo-osmotic or hyper-osmotic conditions at high temperature, germlings can still maintain high survival. In the present study, the extreme level of combined stresses (35 °C and 12 psu for over 4 h) had great destructive effect on germlings (Figs. 1C–F and 2C–F). However, the low salinity is usually resulted from strong rainfall which also causes the decrease of temperature. Therefore, high temperature and long duration of low salinity can generally not be concurrent with each other. In contrast with the extreme conditions, germlings exhibited high survival and quick growth in other multiple stress treatments (Figs. 1 and 2). Combining stress factors results in interactions ranging from stress intensification (additive) through increased tolerance (antagonistic) to stress (Gamon and Pearcy, 1990; Larcher et al., 1990). For example, tolerance of heat stress was improved by simultaneous exposure of embryos to hypersaline seawater in Fucus vesiculosus and Fucus spiralis (Li and Brawley, 2004). It is also reported that there is a positive correlation between the ability of seaweeds to tolerate different stresses (Davison and Pearson, 1996). Consequently, it is not surprising that S. thunbergii germlings showed a high tolerance to combined stress. Our results indicated that the optimal condition for germlings was a salinity of 32 psu and a maximum ambient temperature of 30 °C. Hence better restoration will be achieved when seeding S. thunbergii germlings under this condition. Germlings at about 24 h postfertilization will be collected in the laboratory and then seeded in tanks. Such germlings which were used in the present study have immature rhizoids so that they can attach to the tanks. Germlings were greatly impaired only by high temperature together with long duration of low salinity which can generally not be concurrent with each other as mentioned above. Under the other conditions which probably exist in the intertidal zone, S. thunbergii germlings showed quick growth, at least high survival rates. Therefore, construction of
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