Synergistic effects of altered salinity and temperature on estuarine eelgrass (Zostera marina) seedlings and clonal shoots

Journal of Experimental Marine Biology and Ecology 457 (2014) 143–150

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Synergistic effects of altered salinity and temperature on estuarine eelgrass (Zostera marina) seedlings and clonal shoots Tiina Salo a,b,⁎, Morten Foldager Pedersen a a b

ENSPAC, Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark Department of Biosciences, Åbo Akademi University, Artillerigatan 6, 20520 Åbo, Finland

a r t i c l e

i n f o

Article history: Received 29 January 2014 Received in revised form 30 March 2014 Accepted 5 April 2014 Available online 4 May 2014 Keywords: Hypo-salinity Life stage Multiple stressors Seagrass Stress

a b s t r a c t Salinity and temperature are among the most important factors determining eelgrass distribution and performance. Plants in estuarine environments experience large variations in both on a seasonal basis and exceptionally warm summers have caused massive die-backs of eelgrass in many areas. We investigated experimentally how different combinations of salinity and temperature affect the physiological performance of adult eelgrass (Zostera marina) shoots and seedlings. Plants were exposed to different combinations of salinity (salinity 5, 12.5 and 20) and temperature (15, 20 and 25 °C) in a 5-week aquarium experiment. Plants responded in general negatively to decreasing salinity and increasing temperature and the combination of high temperature and low salinity resulted in markedly higher mortality rates and lower leaf production when compared to plants held at more optimal combinations of salinity and temperature. Seedlings had higher absolute mortality, while adult shoots were relatively more sensitive to unfavorable levels of salinity. Leaf tissue sucrose concentrations in both life stages decreased at low salinity, whereas salinity and temperature resulted in contrasting starch concentrations between seedlings and adult shoots. Our results show that altered salinity and temperature may have negative synergistic effects on eelgrass performance. Future climate changes may thus have serious impacts on eelgrass survival and performance. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Seagrasses are foundation species in many soft-bottom coastal ecosystems, where they form extensive populations that provide ecosystem services such as production of organic matter, sequestering of carbon and serve as habitat for a wealth of associated organisms (Duarte, 2000). Water temperature is an important factor for the range distribution of marine plants and algae (e.g. Lüning, 1990). The temperate seagrass Zostera marina L. (eelgrass) is widely distributed on the northern hemisphere (den Hartog, 1970) where it is abundant in coastal soft-bottom systems (Short et al., 2007). In the North Atlantic, eelgrass occurs across a broad range of water temperatures, from NW Greenland (Nugsuak Head; den Hartog, 1970) where water temperatures range between −1.5 and 7 °C (Krause-Jensen et al., 2012) to the Mediterranean in the NE Atlantic and North Carolina in the NW Atlantic where water temperatures often exceed 25 °C in summer (e.g. Jarvis et al., 2012; Sfriso and Ghetti, 1998). The southern distribution limit of eelgrass is set by the maximum water temperature in summer, but exceptionally high summer temperatures (e.g. during heat waves) can also reduce growth, increase mortality and cause massive die-backs in

⁎ Corresponding author. E-mail address: [email protected] (T. Salo).

http://dx.doi.org/10.1016/j.jembe.2014.04.008 0022-0981/© 2014 Elsevier B.V. All rights reserved.

temperate areas with lower average temperatures (e.g. Moore and Jarvis, 2008; Reusch et al., 2005). Salinity may be important for the regional/local distribution of marine organisms including seagrasses. Although eelgrass is most common in estuarine areas (den Hartog, 1970; Short et al., 2007) it is found across a broad range of salinities ranging from full oceanic strength (salinity 35), across intermediate salinity (salinity 10–20) in many estuaries to brackish waters reaching salinities 5–6 e.g. in the inner Baltic Sea (Boström et al., 2014; den Hartog, 1970). Salinity may undergo considerable spatial and temporal variations in estuaries (e.g. Dickson et al., 1982; Lartigue et al., 2003; Nejrup and Pedersen, 2012) and hypo- and hyper-saline conditions may stress marine plants and algae because energy is spent to maintain turgor-pressure and membrane integrity, thereby reducing overall fitness (Hellebust, 1976; Kirst, 1989; Ritchie, 1988). Despite the ecological importance of eelgrass and the obvious importance of both water temperature and salinity as potential stressors, relatively few studies have investigated experimentally how either temperature or salinity affect eelgrass performance (e.g. growth and survival) in single factor experiments (e.g. Bergmann et al., 2010; Höffle et al., 2011; Nejrup and Pedersen, 2008; Winters et al., 2011). Organisms are rarely stressed by only one environmental factor and low salinity that eelgrass may experience either occasionally or more permanently in estuarine areas may leave the plants more susceptible to thermal

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stress in summer. Multi-factorial experiments are therefore needed to study the potential synergetic effect of these natural stressors. Eelgrass is a clonal plant and vegetative reproduction has long been considered the main process by which eelgrass maintains meadows and increases patch size (Olesen, 1999). A growing number of studies have, however, shown that sexual reproduction is indeed important for meadow maintenance, long distance dispersal and, especially, for re-colonization and re-establishment of eelgrass populations after die-off events (e.g. Greve et al., 2005; Jarvis and Moore, 2010; Moore and Jarvis, 2008). Juvenile life stages of plants (i.e. seedlings) are often hypothesized to be more vulnerable to environmental stressors than later, well-established stages (Peterson and Baldwin, 2004; Schupp, 1995) and a number of studies have indeed shown that eelgrass seedlings suffer from high in situ mortality (e.g. Boese et al., 2009; Olesen, 1999; Valdemarsen et al., 2010). High juvenile mortality may be caused by the small size of seedlings and the limited amount of below-ground tissues (i.e. roots and rhizomes), which make them susceptible to physical stress imposed by burial, wave action and erosion as well as physiological stress induced by unfavorable conditions (e.g. limited light, hypo- or hyper-saline conditions, extreme temperatures). Young seedlings must rely on the limited storage capacity of what is left of the seed reserves until they have developed functional leaves, rhizomes and roots. Many environmental stressors cause a reduction in net photosynthesis, leading to a drain of the internal C-reserves. Limited development of belowground structures (rhizomes and roots) and lack of clonal integration may therefore leave seedlings more sensitive to such stress than older stages (clonal shoots) that have well developed rhizome systems. Only few studies have experimentally investigated how seedling survival and performance are affected by either water temperature or salinity (e.g. Abe et al., 2008; Niu et al., 2012) and only one study has so far studied the potential interaction effect of temperature and salinity on seedlings (Hootsmans et al., 1987). No studies have yet, to our knowledge, tested if eelgrass seedlings are more sensitive to unfavorable levels and/or combinations of temperature and salinity than later life stages. Most previous studies on temperature and salinity effects on eelgrass have been conducted as single factor experiments where plants are exposed to a range of (constant) temperatures or salinities while keeping all other factors constant and optimal. We aimed to investigate how different combinations of temperature and salinity affect the physiological performance of seedlings and later life stages (i.e. large shoots, or clonal fragments, with well-established rhizomes) of eelgrass originating from the Isefjord – Roskilde Fjord complex (Denmark). We hypothesized: 1) that unfavorable combinations of high temperature and low salinity would have a negative synergistic effect on eelgrass performance, and 2) that eelgrass seedlings are more sensitive to altered salinity and temperature than later life stages. Based on previous studies on plants from the same area (Nejrup and Pedersen, 2008), we expected that water temperatures above 15 °C and salinity below 20 would have a negative effect on eelgrass performance. 2. Materials and methods 2.1. Experimental setup Plants for the experiment were collected in the southern (inner) part of Isefjorden, Denmark (N 55°42′44, E 011°47′35) where eelgrass is abundant in the depth range of 0.5–3.5 m. In this area salinity ranges between 16 and 26 (depending on wind-induced changes in water level) and the water temperature varies from 0 to 21 °C depending on season. Plants were collected at ca. 1 m depth in May 2012. Ambient salinity and water temperature at the sampling time and site were 20 and 12 °C, respectively. Established shoots with well-developed rhizomes (i.e. ramets, connected to a clone, hereafter called ‘adult shoots’) were carefully excavated from the sediment and broken of the rhizome mat. Only shoots

bearing a piece of healthy looking rhizome, i.e. with 5–7 internodes and well developed roots were sampled. Seedlings, identified as small solitary shoots with sparse root development, a very short rhizome (if any) and an attached seed coat, were carefully excavated. All plants were immediately transported to the laboratory and transplanted into aquaria. Senescent leaves (as identified from their brown or black color) were removed from seedlings and adult shoots prior to transplantation. Adult shoots were further standardized to have 4 healthy rhizome internodes with attached roots by cutting of older, partly senescent, internodes with a scalpel. The aquaria contained 17 L of water and ca. 3 L of pre-sieved sandy sediment from the sampling site. The initial salinity and water temperature in the aquaria were adjusted to salinity 20 and 15 °C, respectively. Each aquarium contained both life stages of eelgrass: 10 seedlings were transplanted to one half of each of the aquaria and 12 adult shoots to the other half with a distance of N5 cm between the two life stages. We conducted a 3-factorial experiment to study the effects of salinity, temperature and their interaction on the performance of seedlings and adult shoots, respectively. We applied 3 levels of salinity: low, medium and high (salinity 5, 12.5 and 20) and 3 levels of temperature: low, medium and high (15, 20 and 25 °C) and all the possible combinations, yielding 9 different treatment combinations, each with 3 replicate aquaria. The initial salinity and temperature was adjusted by 2‰ and 1 °C per day until the warranted treatment levels were obtained. We considered the treatment with high salinity (salinity 20) and low temperature (15 °C) as control conditions, because this combination of salinity and temperature is close to the ambient conditions at the donor site during late spring and early summer. Different levels of salinity were obtained by mixing seawater from the North Sea (salinity 30) with tap water containing naturally high DIC levels. The pH and DIC-levels at the different salinity treatments varied from 7.93 ± 0.03 to 8.14 ± 0.03 (mean ± 1 SD across replicate aquaria) and from 3.87 ± 0.18 mM to 3.04 ± 0.11 mM DIC, respectively. Salinity was measured in each aquarium every second day and adjusted when necessary. The target temperatures (15, 20 and 25 °C) were obtained by placing the aquaria in larger water baths (3 aquaria in each bath), equipped with heaters (Julabo ED, Julabo Labortechnik GmbH, Seelbach, Germany) that kept temperatures constant within ±1 °C. Light above the aquaria was provided by lamps with halogen spots (OSRAM Decostar 51; 12 V, 35 W) in a 16:8 hour light–dark cycle. Light intensity at the water surface was ca. 120 μmol photons m−2 s−1 PAR which is close to the saturating level (IK) of eelgrass (Marsh et al., 1986; Olesen and Sand-Jensen, 1993). The water in the aquaria was aerated to ensure mixing and two thirds of the water in all aquaria was changed weekly to ensure that DIC and nutrients were not depleted. Plants were exposed to the target combinations of salinity and temperature for 5 weeks after which a number of response variables indicative for plant fitness (i.e. mortality rate, leaf elongation rate, no of new leaves produced and no of leaves per shoot) and/or stress (i.e. degree of necrosis and tissue concentrations of starch and sucrose) were determined. Mortality rate was estimated from the number of surviving plants (adult shoots and seedlings, respectively) in each aquarium over the course of the period with stable salinity and temperature. Leaf necrosis was estimated as the %-wise area with brown-black discolouration of all leaves on each shoot. The production of new leaves and leaf elongation rate was measured over the last 2 weeks of the experiment using the leaf-marking technique (Sand-Jensen, 1975). The leaves of all shoots were marked with a water-resistant pen 3.0 cm (adult shoots) or 1.5 cm (seedlings) above the leaf sheath. Leaf elongation was measured as the distance between these markings with the initial distance (3.0 or 1.5 cm) subtracted. The concentration of sucrose and starch were measured on pooled plant material from two randomly chosen adult shoots and all surviving seedlings from each aquarium, respectively. The plants were divided into leaves and rhizome parts (without roots) and freeze-dried for 48 hours. Soluble sugars were extracted from ground plant tissue by

T. Salo, M.F. Pedersen / Journal of Experimental Marine Biology and Ecology 457 (2014) 143–150

boiling in 96% (v/v) ethanol. The ethanol extracts were evaporated and the residues were dissolved in deionized water for sucrose analysis. Starch was extracted from the ethanol-insoluble residue in 1 N NaOH for 24 h. The sucrose and starch concentrations of the extracts were determined spectrophotometrically (wavelengths 486 and 640 nm, respectively) using resorcinol and anthrone assays with sucrose as a standard (Huber and Israel, 1982; Yemm and Willis, 1954). Due to the limited root and rhizome biomass in seedlings, sucrose and starch concentrations were only analyzed in the leaf tissue of this life stage. In adult shoots sucrose and starch were determined for both shoot and rhizome tissues. 2.2. Statistical analyses The experimental design was a partly nested design with two between plot, crossed main factors (temperature with 3 levels and, salinity with 3 levels, both fixed factors). Aquaria (plot, random factor) were nested under salinity and temperature. The within plot factor lifestage had 2 levels (seedling or adult shoot, fixed) in each aquarium. The results were first analyzed using multivariate permutational analysis of variance to test the effect of the treatments on the composite response of eelgrass, and because survival, leaf elongation, leaf production, no. of leaves per shoot and leaf senescence were measured on the same individuals, these variables may have been inter-correlated. The effects of temperature, salinity and their interaction were tested against the whole-plot error while life stage and interactions between life stage and salinity and temperature were tested against the subplot error (Anderson et al., 2008). The analysis was carried out using PERMANOVA + (v1.0.3) in PRIMER 6 (v6.1.13) on normalized data (mean of each data set divided by sd). Resemblance matrixes were based on Euclidean distances and all permutations were run 9999 times using type III (partial) sum of squares. Significance level of α = 0.05 was used in all the analysis and pairwise permutational comparisons were conducted after significant results (Anderson et al., 2008). Univariate (permutational) ANOVA was subsequently used to evaluate the effect of treatment factors on each individual response variable as recommended by Quinn and Keough (2004). MDS plots and PERMDISP-function were used to inspect the dispersion of the data. Due to high mortality among seedlings in the low salinity-high temperature treatment, we did not have a complete data set for sucrose and starch content in seedlings and adult shoots. Sucrose and starch data for both life stages were therefore excluded in the multivariate analysis. Consequently, sucrose and starch data in the leaves of adult shoots and seedlings were analyzed in univariate permutational ANOVAs where the df's were adjusted for unbalanced design. The concentrations of sucrose and starch in the rhizome tissue of adult shoots were analyzed by two-way permutational ANOVA, with salinity and temperature considered as fixed factors. 3. Results

Table 1 Results for the multivariate and univariate analysis of variance. Bold values indicate significant results at p b 0.05. df

MS

Pseudo-F

Multivariate response Between Temperature (T) Salinity (S) S×T Residual Within Life stage (LS) LS × T LS × S LS × T × S Residual

2 2 4 18 1 2 2 4 26

15.83 10.05 3.61 1.61 58.95 0.96 7.35 2.65 6.69

Mortality rate Between Temperature (T) Salinity (S) S×T Residual Within Life stage (LS) LS × T LS × S LS × T × S Residual

2 2 4 18 1 2 2 4 26

1134 396.6 220.2 68.57 3215 45.7 145.1 88.6 366.1

Leaf elongation rate Between Temperature (T) Salinity (S) S×T Residual Within Life stage (LS) LS × T LS × S LS × T × S Residual

2 2 4 18 1 2 2 4 26

No of new leaves per shoot Between Temperature (T) Salinity (S) S×T Residual Within Life stage (LS) LS × T LS × S LS × T × S Residual No of leaves per shoot Between Temperature (T) Salinity (S) S×T Residual Within Life stage (LS) LS × T LS × S LS × T × S Residual Necrosis Between

3.1. Phenotypic responses Within

The multivariate response of eelgrass (Table 1) was significantly affected by the interaction between salinity and temperature (p = 0.029), showing that the effect of temperature depended on salinity. High temperature affected the multivariate response, but only at the lowest salinity level. The multivariate response of seedlings and adult shoots differed significantly (p = 0.002). The two life stages were similarly affected by both salinity and temperature as indicated by the non-significant interactions of life stage × salinity (p N 0.35) and life stage × temperature (p N 0.98). Mortality rate ranged from 5 to 60% depending on treatment (Fig. 1A, B and Table 1). Univariate analysis revealed that mortality was affected by the interaction between temperature and salinity (p = 0.038). The mortality rate of adult shoots (6.9 ± 1.8%) and seedlings (25.6 ± 3.3%) was

145

Temperature (T) Salinity (S) S×T Residual Life stage (LS) LS × T LS × S LS × T × S Residual

P

9.85 6.26 2.25

b0.001 b0.001 0.029

8.81 0.14 1.10 0.40

0.002 0.980 0.351 0.910

16.54 5.78 3.21

b0.001 0.013 0.038

8.78 0.13 0.40 0.24

0.006 0.887 0.672 0.910

0.002 0.003 0.004 0.002 0.440 0.003 0.005 0.007 0.009

0.96 14.1 1.88

0.398 b0.001 0.165

50.0 0.30 5.33 0.85

b0.001 0.750 0.011 0.507

2 2 4 18 1 2 2 4 26

0.317 0.222 0.067 0.032 0.028 0.004 0.079 0.063 0.140

9.83 6.86 2.09

0.002 0.006 0.124

0.20 0.03 0.56 0.45

0.668 0.976 0.584 0.775

2 2 4 18 1 2 2 4 26

1.26 0.45 0.28 0.058 2.65 0.056 0.511 0.135 0.428

21.80 7.71 4.77

b0.001 0.004 0.010

6.19 0.12 1.19 0.32

0.020 0.874 0.320 0.863

2.18 3.90 0.74

0.140 0.041 0.571

2.64 0.77 1.17 0.55

0.117 0.749 0.324 0.691

2 2 4 18 1 2 2 4 26

91.9 164.5 31.4 42.2 215.9 23.3 96.2 45.4 81.9

relatively low and independent of temperature at the medium and high salinities, while high mortality rates (45-55%) were recorded for both life stages at high temperature combined with low salinity. The mortality rate of seedlings was significantly higher than that of adult shoots (p = 0.006), but we found no significant interaction effects between life stage and other factors.

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Fig. 1. Mortality rate (A,B), leaf elongation rate (C,D), number of new leaves per plant (E,F), number of leaves per plant (G,H) and degree of necrosis (I,J) for adult shoots (to the left) and seedlings (to the right) at different combinations of salinity and temperature. Note the different scale on leaf elongation data for adult shoots and seedlings (C,D). Data are mean values (across replicate aquaria, n = 3) ± 1 SE.

Absolute leaf elongation rate averaged 0.2 ± 0.02 mm shoot−1 d−1 and 0.02 ± 0.001 mm shoot− 1 d− 1 for adult shoots and seedlings, respectively, across all treatments and was affected significantly by salinity (p b 0.001), but not by temperature nor by the temperature × salinity interaction (Fig. 1C,D, Table 1). Overall, elongation rates were lower at the lowest salinity than at medium or high salinity. Adult shoots had higher elongation rates than seedlings (p b 0.001). Adult

shoots responded relatively more to low salinity than seedlings (Life stage × salinity; p = 0.011) with decreased leaf elongation rates in adult plants in all low salinity treatments, while seedlings showed decreased rates only in the low salinity-high temperature combination. The production of new leaves ranged from 0.0 to 1.5 leaves shoot−1 measured over the last 2 weeks of the experiment (Fig. 1E,F). Leaf production was significantly affected by temperature (p = 0.002) and

T. Salo, M.F. Pedersen / Journal of Experimental Marine Biology and Ecology 457 (2014) 143–150

salinity (p = 0.006), but not by their interaction (Table 1). High temperature and low salinity had both a negative effect on leaf production. The production of new leaves was not affected by life stage and nor by any interaction between life stage and other factors (all p N 0.58). The average number of standing leaves per shoot by the end of the experiment ranged from ca. 2 in the low salinity-high temperature treatment to ca. 3.5 in the medium salinity-low temperature treatment (Fig. 1 G,H). The interaction between salinity and temperature had a significant effect on the number of leaves per shoot (p = 0.01, Table 1); the number of leaves per shoot was inversely correlated to temperature at low and medium salinity, while no such pattern was evident at the highest salinity. The number of leaves per shoot was significantly lower among seedlings than among mature shoots (p = 0.02). The degree of necrosis on the leaves varied from ca. 20% in most treatments to 36% in the low salinity-high temperature treatment (Fig. 1I,J) and was significantly affected by salinity (p = 0.04, Table 1). Pairwise comparisons revealed that plants from the low salinity treatments were more necrotic than those from the medium salinity treatments (p = 0.02). Leaves of seedlings were equally necrotic than those from adult shoots (p N 0.1). Calculating the relative response for survival, no of leaves, leaf elongation, necrosis and no of new leaves for seedlings and adult shoots separately (Fig. 2) revealed that even though seedlings in general had lower absolute performance and higher mortality (as indicated above), they were not more sensitive to altered salinity or temperature than adult shoots. Further, the impacts of altered salinity and temperature on plant performance and survival were synergistic; the relative response in the most stressful combination (i.e. low salinity-high temperature) was always larger (2.1–8.7 times larger) than the summed responses of high salinity-high temperature and low salinity-low temperature treatments (Fig. 3).

147

Fig. 3. Synergetic effects of low salinity and high temperature on eelgrass. The responses of adult plants and seedlings are pooled (n = 6) and the relative responses are calculated against the control treatment (high salinity-low temperature). Data are mean values ± 1 SE.

shoots also increased with increasing salinity (Pseudo-F2,18 = 6.74, p = 0.0056; Fig. 4B) while temperature had no effect on the sucrose concentration in the rhizomes. The concentration of starch in the leaves ranged from 3.4 ± 2.8 mg g− 1 DW to 17.5 ± 0.8 mg g−1 DW and was affected by temperature (p = 0.04) with higher concentrations in high temperature compared to medium temperature. Starch concentrations remained unaffected by salinity and by the interaction between salinity and temperature (Fig. 4C, Table 2). However, the significant life stage × salinity interaction (p = 0.032) show that salinity affected the starch content in the leaves of seedlings and adult shoots, but did so differently. The starch concentrations for adult shoots tended to increase with increasing salinity while the opposite was true for seedlings. The starch concentrations of rhizome tissues of adult shoots were unaffected by both salinity and temperature (Fig. 4D).

3.2. Soluble carbohydrates 4. Discussion Sucrose concentrations in the leaf tissue ranged from 50 ± 22 mg g−1 DW to 317 ± 139 mg g−1 DW (Fig. 4A) and decreased with decreasing salinity (p = 0.003; Table 2). Neither temperature nor the interaction between salinity and temperature had any effect on the sucrose levels in the leaves. The sucrose concentration in rhizome tissues of adult

Fig. 2. The relative response of seedlings and shoots to alterations in A) temperature (averaged across all levels of salinity, n = 9) and B) salinity (averaged across all levels of temperature, n = 9). The relative responses are calculated against control temperature (15 °C, a) and salinity (salinity of 20, b), respectively. Data are mean values ± 1 SE.

Seagrasses live at the interface between marine and terrestrial habitats and experience therefore short and long term variations in temperature and other environmental variables. Our results show that two very common stressors in estuarine environments, high temperature and low salinity, can have negative synergistic effects on seagrass performance and survival. Heat stress is harmful to plants and may lead to reduced fitness and enhanced mortality (Wahid et al., 2007). Seagrass fitness is lowered at high, but sub-lethal temperatures due to reduced enzyme activity and destabilization of membranes, which affect photosynthetic performance and disturb metabolism whereas temperatures close to or above the upper tolerance limit lead to denaturation of enzymes and enhanced mortality (Staehr and Borum, 2011). Exposure to high temperature during exceptionally warm summers has accordingly led to massive die off events among seagrasses worldwide (e.g. Moore and Jarvis, 2008; Reusch et al., 2005). The temperature response of eelgrass has mainly been examined through correlative field studies, but also by experimental studies in a few cases (reviewed by Lee et al., 2007). These studies show that optimal temperatures for eelgrass growth and photosynthesis average 15.3 ± 1.6 °C and 23.3 ± 1.6 °C, respectively, but variations are large and optimum temperatures seem to correlate with the geographical origin of the plants (Lee et al., 2007). When averaged across all levels of salinity and life stage, increased temperature had a highly significant and negative effect on eelgrass in the present study. Exposure to high temperature for 5 weeks led to enhanced mortality, reduced formation of new leaves and a lower number of standing leaves per shoot whereas leaf elongation rate, necrosis and carbohydrate levels remained unaffected by elevated temperature. These results correspond well to those found in other experimental studies where exposure of eelgrass to temperatures exceeding 24– 25 °C increased mortality and slowed down growth (Abe et al., 2008;

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Fig. 4. Sucrose (A,B) and starch (C,D) concentrations in leaves (A, C) and rhizome tissue (B,D). Data are mean values (across replicate aquaria, n = 3) ± 1 SE.

Bergmann et al., 2010; Höffle et al., 2011; Nejrup and Pedersen, 2008; Niu et al., 2012). Exposure to heat stress should increase respiration more than gross photosynthesis and, thus, lead to lower net photosynthetic rates (Marsh et al., 1986). We expected therefore that elevated temperature would increase the need for stored carbohydrates and, thus, lower the carbohydrate levels within the plants. We found, however, no significant temperature effect on the levels of sucrose and starch. Zimmerman et al. (1989) hypothesized that lack of carbohydrate depletion at high temperatures were due to inhibition of sucrose breakdown. Gu et al. (2012) found a positive correlation between sucrose levels and temperature in eelgrass and Zostera noltii and suggested that it resulted from a decrease in sucrose breakdown rates at high temperatures as organic osmolytes (e.g. sucrose) may function as chemical chaperones and protect plants against heat stress. Also, the activity of sucrose-P synthase, an enzyme involved in sucrose synthesis, has been observed to increase at Table 2 Statistical results of the ANOVA analyses examining the effect of salinity, temperature and life-stage on the sucrose and starch content in eelgrass leaves. Bold values indicate significant results at p b 0.05.

Sucrose Between

Within

Starch Between

Within

df

MS

Salinity (S) Temperature (T) S×T Residual Life stage (LS) LS × S LS × T LS × S × T Residual

2 2 4 18 1 2 2 3 24

34866 2411 7375 4095 31925 694.3 7095 9697 17160

Salinity (S) Temperature (T) S×T Residual Life stage (LS) LS × S LS × T LS × S × T Residual

2 2 4 18 1 2 2 3 22

5.1 17.8 6.22 4.47 544.5 56.3 34.8 4.81 14.4

Pseudo-F

P (perm)

8.51 0.589 1.80

0.003 0.572 0.171

1.91 0.042 0.42 0.56

0.185 0.96 0.66 0.654

1.14 3.98 1.39

0.334 0.036 0.277

38.8 4.00 2.48 0.33

b0.001 0.032 0.108 0.805

high temperatures (Touchette and Burkholder, 2000). Reduced rates of carbohydrate mobilization or increased carbohydrate production rates may thus explain why we did not observe any decrease in sucrose with increasing temperature. As sucrose is one of the main organic osmolytes in seagrasses (Ye and Zhao, 2003), the level of soluble carbohydrates expectedly varied with salinity, being much lower in plants kept at low salinity. Low sucrose concentrations in plants from the low salinity-high temperature-treatment suggest that these plants were able to mobilize sucrose despite the exposure to high water temperature. Lowering the average (across all levels of temperature and life stage) salinity had a negative effect on eelgrass performance (multivariate response: p b 0.001). The multivariate effect was mainly driven by higher mortality, lower leaf production, lower leaf elongation rate, less standing leaves per shoot and more necrotic tissue at low salinity. These responses correspond also well to the results of Nejrup and Pedersen (2008) where mortality and growth of eelgrass was significantly reduced at salinities below 10–15 depending on the response variable. Experimental studies on the temperature response in eelgrass have most often been conducted by exposing plants to a range on constant temperatures while keeping all other variables constant and as optimal as possible. Organisms are, however, rarely stressed by only one factor at the time in nature. Estuarine, shallow water organisms live in an environment where factors, such as water temperature, salinity and light, may vary considerably over short time-scales. Water temperature changes on a seasonal scale in temperate systems, but may also undergo short-term (hours–days) variations in shallow and sheltered waters while salinity may undergo quick and substantial variations as a result of tides, wind-induced changes in water level and rainfall. High, but sub-lethal, water temperatures will typically reduce net photosynthesis (Marsh et al., 1986) and sub-optimal levels of salinity may also stress marine plants because energy is spent to maintain the turgor-pressure through regulation of ions and osmolytes (Touchette, 2007). Low salinity has also been documented to induce changes in chlorophyll content (Thorhaug et al., 2006) and may decrease photosynthetic capacity due to changes in pigmentation or due to e.g. decreased photosystem I and II functioning or inhibition of electron flow (Touchette, 2007). Concomitant stress caused by high temperature and sub-optimal levels of salinity may therefore increase the competition between carbon demanding

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processes within the plant. As high temperature and low salinity may both increase energy requirements of a plant while simultaneously decreasing or inhibiting photosynthesis, the plant responses should be either additive or synergistic. Our study showed that concurrent exposure to unfavorable levels of temperature and salinity synergistically increased the sensitivity of eelgrass to both stressors. Most response variables seemed thus relatively robust to changes in temperature as long as salinity was kept at an ambient level, whereas the same changes in temperature had a strong negative effect if salinity was low (Fig. 3). The same pattern was evident for salinity (Fig. 3); many response variables remained largely unaffected by changes in salinity as long as the temperature was kept at the ambient level, whereas the same changes in salinity had a strong negative effect at high temperature. Similar results have been published by Hootsmans et al. (1987), who found that the survival of eelgrass seedlings was reduced further at low salinity (salinity 1 and 10) when the temperature was high (20 °C as opposed to 10 °C) whereas the survival at 10 and 20 °C was more or less identical at higher salinity. High summer temperature may thus be harmful to eelgrass, but the heat stress imposed upon plants during summer will be amplified if the plants experience low salinity levels at the same time. Other factors can likely interact with low salinity and/or high temperature and amplify the negative effects of these stressors. For example, high water temperature in combination with low irradiance (Jarvis and Moore, 2010; Moore et al., 2012) or low oxygen levels (Raun and Borum, 2013) can increase mortality in seagrass meadows. We don't know if the surviving plants from our most extreme treatment would have been able to recover if they had been returned to more favorable conditions after the 5 weeks exposure period. Long term experiments with exposure of eelgrass to elevated temperatures and a subsequent recovery period have shown that eelgrass have the potential to recover from exposure to heat stress in summer (Bergmann et al., 2010; Winters et al., 2011). One of the major responses of eelgrass at the most unfavorable levels of temperature and salinity was, however, an increased mortality (increasing from 5–15% to 40–50%), and it is clear that there would be no way that the plants could recover from that. The absolute response of most response variables differed between seedlings and shoots (multivariate response, life stage: p = 0.002). Seedlings had, in general, higher mortality rates, slower growth and more necrotic leaves than adult shoots, but salinity stress did also affect the two life stages differently as indicated from the significant interactions between life stage and salinity (Tables 1 and 2). Seedlings suffer in general from rather high mortality in nature and we hypothesized therefore initially that seedlings would be more sensitive to suboptimal levels of temperature and salinity than shoots. Sensitivity to a stressor is best evaluated from the relative response (i.e. response compared to a control value). Relative changes in the applied variables at different levels and/or combinations of temperature and salinity are illustrated in Fig. 2. It seems clear that both seedlings and adult shoots responded more to high than to medium temperature and, that both life stages were almost equally sensitive to increasing temperatures, although leaves of seedlings tended to become slightly more necrotic than leaves of adult shoots. This pattern was somewhat different when looking at the effects of decreasing salinity. Again, both life stages were more sensitive to a large reduction in salinity, but for most response variables, adult shoots responded more negatively than seedlings, which contradicted our expectations. It is not clear why seedlings should be less sensitive to low salinity than adult shoots, but it is well documented that sprouting of eelgrass seeds is more successful at lower salinity (Hootsmans et al., 1987). When comparing the responses of seedlings and shoots at increasingly stressful levels and combinations of temperature and salinity (i.e. low or medium salinity combined with high or medium temperature versus high salinity-low temperature-treatment), both life stages responded substantially, especially at the most extreme (low salinity-high temperature) treatment, but seedlings did not respond

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systematically different and/or more than shoots. The smaller biomass of seedlings has been suggested to be a key element for the high survival rates of seedlings compared to adult shoots. Small biomass requires less storage carbohydrates for physiological purposes and lack of extensive rhizome system diminishes the need to oxygenate the rhizosphere (Biber et al., 2009). Eelgrass seedlings have also been observed to tolerate shading (Biber et al., 2009) and low water oxygen levels in high water temperatures (Raun and Borum, 2013) equally good or better than adult shoots. Thus, even though seedlings appear to be more sensitive to physical disturbance, and have high in situ mortality, they seem to be equally, or even more, tolerant towards physiological stress than adult shoots. However, when considering the higher “baseline” mortality of seedlings compared to adult shoots, changes in environmental factors such as salinity and temperature combined with physical stress might increase seedling mortality more than mortality of older life stages and impact eelgrass meadow dynamics, with a possible emphasis on vegetative reproduction during stress periods. In conclusion, low salinity in combination with high water temperature affected the survival and performance of eelgrass negatively. Future climate changes are predicted to increase occurrence of extreme stress events, such as heat waves and heavy precipitation. These changes may thus have serious impacts on production capacity of seagrass meadows weakening the ecosystem services provided by these coastal ecosystems. However, increase in salinity, as predicted as one alternative outcome of climate change in some scenarios, could possibly alleviate temperature stress to some degree. Acknowledgements TS was funded by the Department for Environmental, Social and Spatial Change, Roskilde University. MFP was funded by the REELGRASS project. Anne Faarborg and Rikke Guttesen are acknowledged for their assistance in the field and in the laboratory. References Abe, M., Kurashima, A., Maegawa, M., 2008. Temperature requirements for seed germination and seedling growth of Zostera marina from central Japan. Fish. Sci. 74, 589–593. Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA + for PRIMER: Guide to software and statistical methods. PRIMER-E, Plymouth, UK. Bergmann, N., Winters, G., Rauch, G., Eizaguirre, C., Gu, J., Nelle, P., Fricke, B., Reusch, T.B.H. , 2010. Population-specificity of heat stress gene induction in northern and southern eelgrass Zostera marina population under simulated global warming. Mol. Ecol. 19, 2870–2883. Biber, P.D., Kenworthy, W.J., Paerl, H.W., 2009. Experimental analysis of the response and recovery of Zostera marina (L) and Halodule wrightii (Ascher) to repeated lightlimitation stress. J. Exp. Mar. Biol. Ecol. 369, 110–117. Boese, B.L., Kaldy, J.E., Clinton, P.J., Eldridge, P.M., Folger, C.L., 2009. Recolonization of intertidal Zostera marina L. (eelgrass) following experimental shoot removal. J. Exp. Mar. Biol. Ecol. 374, 69–77. Boström, C., Baden, S.P., Bockelmann, A.-C., Dromph, K., Fredriksen, S., Gustafsson, C., Krause-Jensen, D., Möller, T., Nielsen, S.L., Olesen, B., Olsen, J., Pihl, L., Rinde, E., 2014. Distribution, structure and function of Nordic eelgrass (Zostera marina) ecosystems: implications for coastal management and conservation. Aquat. Conserv. Mar. Freshw. Ecosyst. http://dx.doi.org/10.1002/aqc.2424. Den Hartog, C., 1970. The seagrasses of the world. North Holland Publ Co, Amsterdam (275 pp.). Dickson, D.M., Wyn Jones, R.G., Davenport, J., 1982. Osmotic adaptation in Ulva lactuca under fluctuating salinity regimes. Planta 155, 409–415. Duarte, C.M., 2000. Marine biodiversity and ecosystem services: an elusive link. J. Exp. Mar. Biol. Ecol. 250, 117–131. Greve, T.M., Krause-Jensen, D., Rasmussen, M.B., Christensen, P.B., 2005. Means of rapid eelgrass (Zostera marina L.) recolonisation in former dieback areas. Aquat. Bot. 82, 143–156. Gu, J., Weber, K., Klemp, E., Winters, G., Franssen, S.U., Wienpahl, I., Huylmans, A.-K., Zecher, K., Reusch, T.B.H., Bornberg-Bauer, E., Weber, A.P.M., 2012. Identifying core features of adaptive metabolic mechanisms for chronic heat stress attenuation contributing to system robustness. Integr. Biol. 4, 480–493. Hellebust, J.A., 1976. Osmoregulation. Annu. Rev. Plant Physiol. 46, 485–505. Höffle, H., Thomsen, M.S., Holmer, M., 2011. High mortality of Zostera marina under high temperature regimes but minor effects of the invasive macroalgae Gracillaria vermiculophylla. Estuar. Coast. Shelf Sci. 92, 35–46. Hootsmans, M.J.M., Vermaat, J.E., Van Vierssen, W., 1987. Seed-bank development, germination and early seedling survival of two seagrass species from the Netherlands: Zostera marina L. and Zostera noltii Hornem. Aquat. Bot. 28, 275–285.

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