Responses of the submerged macrophyte Vallisneria natans to elevated CO 2 and temperature

Inorganic carbon and temperature are 2 important factors that regulate the growth of submerged macrophytes. However, experimental evidence regarding the eco-physiological changes that occur in submerged macrophytes in response to elevated CO2 and temperature is still limited. To investigate how the submerged macrophyte Vallisneria natans (Hydrocharitaceae), a common species in the waters of the middle and lower reaches of the Yangtze River, responds to these factors, we conducted a mesocosm experiment using simulated CO2 elevation (by bubbling CO2 into experimental water) and ambient temperature warming systems. During the 60 d experiment, CO2 elevation significantly increased the inorganic carbon concentration in the water column. The warming systems elevated average water temperature by approximately 3°C. The elevation of CO2 levels significantly enhanced the photosynthetic performance, growth and clonal propagation of V. natans. When combined with an increase in CO2, elevated temperatures also promoted photosynthesis and growth. The individual ramet biomass of V. natans decreased with increasing temperature, but only significantly under ambient CO2 levels. CO2 elevation increased both stolon elongation and bud number. At elevated CO2 concentration, more biomass was allocated to the stolons, roots and buds, while less biomass was allocated to the leaves. These results indicate that the eco-physiological responses of V. natans should increase its stress tolerance in aquatic plant communities under future spatial and temporal variation in CO2 levels, however, further research is required.


INTRODUCTION
Since the first records in 1958, the concentration of CO 2 in the atmosphere has increased at an annual rate of 0.45%, and the present atmo spheric CO 2 concentration is > 360 ppm (Parry et al. 2007).If no emission reduction measures are taken, the atmo spheric CO 2 concentration is estimated to reach 450 ppm by 2040 and 700 ppm by 2100 (Houghton et al. 1996(Houghton et al. , 2001)).According to the IPCC (2001,2007), the greenhouse effect caused by such high CO 2 concentrations will increase the global average temperature by 1.4 to 5.8°C.Sustained increases in inorganic carbon lev-els and temperature impact the photosynthesis and carbon balance of plants, thereby influencing their growth, development and functioning (Ahuja et al. 2010, Wang et al. 2012).However, most previous studies have focused on the impacts of elevated atmospheric CO 2 and ambient temperature in terrestrial habitats, while relatively few studies have considered such processes in aquatic habitats (Olesen & Madsen 2000).Although the effects of each factor alone on submerged macrophytes have been studied extensively (i.e.Rooney & Kalff 2000, Madsen & Maberly 2003, Lacoul & Freedman 2006, Pagano & Titus 2007), studies on the influence of the interaction between these 2 factors on submerged macrophytes are rare.
Submerged macrophytes are important to aquatic ecosystems and play an important role in regulating the structure and function of these systems (Cook 1990, Bornette & Puijalon 2011).Macrophytes are one of the primary producers in aquatic ecosystems and are an important part of the aquatic food chain (van Donk et al. 1990).Furthermore, the complex habitats created by submerged macrophytes offer food resources and shelter to organisms in and around the water body (Sheldon 1987).The effect of temperature on submerged macrophytes is well known (Madsen & Brix 1997, Rooney & Kalff 2000).Most macrophytes require a temperature higher than 10°C to maintain growth (Lacoul & Freedman 2006).Barko & Smart (1981) studied 3 common submerged macrophytes and demonstrated that they increased production of aboveground biomass as temperature increased, thus increasing their total biomass.Potamogeton crispus begins turion production at temperatures above 20°C (Rogers & Breen 1980).You et al. (2013b) found that as temperatures increase, the invasive ability (i.e.production of large stem bases) of Eichhornia crassipes increases, with its distribution spreading from southern China into central and northern China.
All submerged macrophytes can directly use the CO 2 dissolved in the water column/sediment porewater, and some can also use bicarbonate as an alternative carbon source (Spence & Maberly 1985, Sand-Jensen & Gordon 1986, Madsen & Sand-Jensen 1991, Yan et al. 2006, Xie et al. 2013).Submerged macrophytes whose carbon source is restricted to free CO 2 in the water column have a higher affinity for CO 2 than bicarbonate users (Maberly & Madsen 1998, Pagano & Titus 2007).Additionally, increased CO 2 availability enhances the growth of submerged species such as Vallisneria americana, V. spiralis and Myriophyllum verticillatum (Titus & Andorfer 1996, Madsen & Maberly 2003, Nishihara & Ackerman 2007).However, other studies have also shown that utilization of bicarbonate increases when plants are grown under conditions of low CO 2 availability (Sand-Jensen & Gordon 1986, Jones 2005).These results indicate that the inorganic carbon content of water is an important resource for submerged macrophytes, and that macrophytes vary in their ability to exploit carbon sources under inorganic carbon enrichment (Lacoul & Freedman 2006, Bornette & Puijalon 2011, Wang et al. 2012).
The increase of inorganic carbon content in water caused by CO 2 elevation can significantly increase the photosynthetic rate of submerged macrophytes (Madsen et al. 1996, Zimmerman et al. 1997, Maberly & Madsen 1998, Pagano & Titus 2007).Similarly, temperature can also directly influence the metabolism of submerged macrophytes.For example, elevated temperatures increase photorespiration because of the reduced solubility of CO 2 compared with O 2 , and the reduced specificity of rubisco for CO 2 at higher temperatures (Sage & Monson 1999, Wang et al. 2012).However, in contrast to terrestrial plants, the responses of submerged macrophytes to in creases in CO 2 and temperature are poorly understood (Wang et al. 2012).
Vallisneria natans (Lour.)Hara (Hydrocharitaceae) is a perennial submerged macrophyte that is widely distributed in the waters of the middle and lower Yangtze River basin in China (Xie et al. 2005, Ke & Li 2006, Cao et al. 2011).V. natans can produce stolons, spread horizontally above the sediment surface, and form ramets at the nodes.Each ramet has a basal rosette of leaves which may extend to the surface but does not form a canopy.It is widely used in ecological restoration because of its extensive adaptability, high reproductive capacity and high adsorption capacity for pollutants and nutrients (Ke & Li 2006, Cao et al. 2011).In addition to CO 2 , V. natans can also use bicarbonate as a carbon resource (Dou et al. 2013).In this study, V. natans was used to examine the ecophysiological responses of submerged macrophytes to CO 2 and ambient temperature elevation.Two hypotheses were tested: (1) V. natans can adapt to elevated CO 2 levels and temperature via alteration of photosynthetic performance and growth plasticity, and (2) elevated CO 2 levels and temperature promote plant growth and vegetative production.

Experimental design
In March 2013, similarly sized buds of Vallisneria natans were collected from Gonghu Bay (31°31' N, 120°16' E) in Taihu Lake, the third largest freshwater lake in China.Upon collection, the buds were placed in aluminum pans filled with sand substratum for sprouting in a laboratory at Nanjing Forestry University in Jurong City, Jiangsu Province, China.After sprouting, 24 plantlets of similar sizes (length: 4.8 ± 0.24 cm; fresh weight: 1.0 ± 0.031 g) were selected as experimental material.To ensure the full development of the creeping stems, one individual plantlet was placed into each mesocosm block (L × W × H = 50 × 30 × 30 cm) containing 8 cm of substratum (sand and clay mixture); the mesocosms were then left in situ to acclimate for 1 wk before the experiment began.
The study was conducted at an experimental site of Nanjing Forestry University.The experiment lasted for 60 d, from 21 May until 20 July 2013, which was consistent with the growing season of V. natans in eastern China.A 2-way factorial experimental design was employed which included 2 levels of CO 2 (ambient and elevated CO 2 ) and 2 levels of temperature (ambient and elevated temperature) in 6 blocks, with 1 replicate per block.Elevated CO 2 concentration (1000 ppm) was obtained by bubbling ambient air (CO 2 concentration: 370 ppm) mixed with CO 2 stored in gas cylinders (99.99% purity) (Yan et al. 2006) into a semi-enclosed system; the control treatment was obtained by bubbling am bient air into the system (see Fig. 1 for detailed ex perimental setup).In both treatments, the air velocity was 4 l min −1 , and the gases (both ambient air and air mixed with CO 2 ) were pumped into the water system.The elevated CO 2 levels significantly increased the free CO 2 and bi carbonate concentrations in the water column, and reduced the pH and CO 3 2− concentrations (p < 0.001, Table 1).The dissolved inorganic carbon concentrations (CO 2 , HCO 3 − and CO 3 2− ) in our experiment were consistent with previous in situ studies in many lakes in China (590 to 2000 µmol l −1 ) (Xiao et al. 2007b, Zhai et al. 2007, Zhang et al. 2009).The ambient temperature elevation was simulated using far infrared quartz heating tubes (15 cm from the mesocosm, 2000 W, 12 h d −1 ).The water temperature was increased by approximately 3°C compared with the control treatment (in situ water temperature) during the experimental period.The temperature elevation did not have a significant effect on water pH (p > 0.05, Table 1).The treatments were replicated 6 times, for a total of 24 mesocosms.The water depth in each mesocosm was 20 cm (Fig. 1; to minimize the influence of phytoplankton, the water was filtered using a plankton net) (Cao et al. 2012).The physical and chemical characteristics of the water during the experimental period are given in Table 1.Water quality characteristics were analyzed using Hach ® portable water quality meters.The soluble inorganic carbon level was analyzed using a carbon dioxideselective electrode (Orion™,9502BNWP,Thermo Scientific).All water quality characteristics (i.e.temperature, pH, dissolved oxygen, turbidity, CO 2 , HCO 3 − and CO 3 2− concentrations) were re corded at noon every third day.

Plant harvest
One week before harvest, the photosynthetic activity of V. natans under different treatments was measured using a Diving-PAM fluorometer (Walz), and the data were collected using WinControl software (Walz).Healthy leaves on the second-youngest of the ramets in each daughter plant were chosen and dark-acclimated for 20 min using leaf clips (Diving-LC, Walz) (You et al. 2013a).After the acclimation period, the rapid light curves (RLCs) were measured.The photosynthetic photon flux density (PPFD) was increased in 9 steps (0,11,30,56,96,147,275,419 and 715 µmol photons m −2 s −1 ) over a 90 s period.The relative electron transport rates (rETRs, calculated as rETR = ΦPSII × PPFD, where ΦPSII is the quantum efficiency of PSII) were used to determine the following photosynthetic parameters: α (photosynthetic efficiency under nonsaturating irradiance), E k (irradiance at the onset of saturation) and rETR max (maximum rETR to E k ).These parameters were estimated using the following model proposed by Platt et al. (1980): , where P m is the maximum photosynthetic rate, α is the initial slope of rETR−PAR response curve, and β is the slope of the rETR−PAR response curve after saturation.E k was calculated by dividing P m (maximum electron rate) by α (Mvungi et al. 2012).

Statistical analysis
Before analysis, all data were log-transformed to meet the conditions of homogeneity of variance and normal distribution of residuals.No significant differences were detected among blocks (p > 0.05), therefore the effects of CO 2 and elevated temperature on photo synthesis, growth and reproduction of V. natans (e.g. total plant biomass, ramet number, individual ramet biomass, creeping stem length and bud number) were analyzed by 2-way ANOVA.A 2-way multivariate ANOVA (MANOVA) was employed to investigate the effects of CO 2 and elevated temperature on the biomass allocation para meters.When a significant treatment effect was detected, post hoc pairwise comparisons of means were conducted to examine the differences between treatments using a studentized Tukey's HSD test for multiple comparisons at the 0.05 significance level.All data were analyzed using SPSS v.18.0.

RESULTS
During the experimental period, the values of all photosynthetic parameters significantly increased with elevated CO 2 levels.The RLCs of the plants under elevated CO 2 were significantly higher than those of the plants in ambient air (Fig. 2).However, the effects of elevated temperature were only significant when interacting with the effects of elevated CO 2 (Fig. 2).The equation fitting revealed that elevated CO 2 levels significantly increased P m , α and E k of the RLCs (Table 2).When interacting with the elevated CO 2 levels, temperature elevation also increased the photosynthetic parameters (α and E k , but not P m ) (Table 2).Elevation of CO 2 increased total biomass and ramet number.Elevated temperature increased total biomass accumulation and ramet number but re duced the individual ramet biomass (only significant at ambient CO 2 ) (Fig. 3a−c, Table 3).Creeping stem length and bud number were significantly higher under elevated CO 2 , but these traits were not affected by temperature or by the interaction between temperature and elevated CO 2 (Fig. 3d,e, Table 3).
Generally, the biomass allocation pattern of Vallisneria natans was only affected by elevated CO 2 (Table 4a).Under increased levels of CO 2 , leaf allocation was significantly reduced (Fig. 4a, Table 4b), and more biomass was allocated to the production of creeping stems, roots and buds (Fig. 4b−d, Table 4b).However, neither elevated temperature nor its interaction with elevated CO 2 had a significant effect on biomass allocation (Fig. 4, Table 4).

DISCUSSION
Compared with terrestrial plants, submerged macrophytes can use a wider range of carbon sources.Many submerged macrophytes can use both free CO 2 and bicarbonate in the water column as carbon sources for photosynthesis (Madsen et al. 1996, Maberly & Madsen 1998, Jones 2005, Cavalli et al. 2012).However, submerged macrophytes always prefer CO 2 due to the additional metabolic cost of bicarbonate utilization (Jones 2005).The use of bicarbonate is believed to be an active process that requires a protein in the plasmalemma membrane to 'pump' in bicarbonate ions (Prins & Elzenga 1989).In the present study, the elevation of ambient CO 2 significantly increased the total biomass and clonal growth (ramet number) of Vallisneria natans, which is consistent with previous studies suggesting that CO 2 enrichment stimulated Vallisneria species (bicarbonate user) (Titus et al. 1990, Titus & Andorfer 1996).Moreover, changes in aquatic pH values can also alter the inorganic carbon balance, indirectly affecting the growth and reproduction of submerged macrophytes (Schippers et al. 2004, Baattrup-Pedersen et al. 2013).
For submerged macrophytes that also use bicarbonate, internal (or recycled) CO 2 concentration is the dominant component that causes a reduction of CO 2 loss, thus maintaining high CO 2 concentration (Madsen & Maberly 2003).Therefore, the ability to use bicarbonate may increase the internal concentration of CO 2 , and hence suppress or reduce the oxygenation reactions of rubisco, which may promote the photosynthetic activity of bicarbonate users such as V. natans (Maberly & Madsen 1998, Cavalli et al. 2012, Xie et al. 2013).In addition, abiotic and biotic factors (e.g.water velocity and shoot morphology) also affect CO 2 diffusion through a boundary layer around the leaves of submerged macrophytes (Nielsen et al. 2006) Additionally, the moderate increase in water temperature in combination with elevated CO 2 may also have increased the activity of photosynthetic enzymes and carbonic anhydrase (Olesen & Madsen 2000).However, the maximum temperature dependence of growth has been observed only at saturating inorganic carbon availability (Olesen & Madsen 2000); as the temperature increases, the proportion of carbon lost via photorespiration increases, and the positive effect of temperature on enzymatic processes and inorganic carbon diffusion is counterbalanced or even reversed (Long 1991).Furthermore, dark respiration rates also increase with temperature; to maintain growth, photosynthesis should increase in response to warmer temperatures at elevated CO 2 conditions (Morison & Lawlor 1999).
Growth and clonal reproduction of V. natans were significantly promoted under elevated CO 2 conditions, as indicated by the higher biomass, higher ramet production and longer creeping stems.These effects likely resulted from the increased supply of inorganic carbon in the water, which likely increased the available photosynthetic substrate and thus enhanced plant biomass accumulation (Maberly & Madsen 1998).Our results agree with those of many previous studies (e.g.Maberly & Madsen 1998, Nielsen et al. 2006, Pagano & Titus 2007).Titus & Andorfer (1996) found that the aboveground biomass of submerged macrophytes increased by 5.5 to 7 times when the CO 2 level was elevated in an acidic aquatic environment (pH 5).Indeed, a CO 2 increase of 2 to 10 times the ambient level was previously shown to promote biomass accumulation of submerged macrophytes in multiple previous studies at various pH levels (Titus 1992, Olesen & Madsen 2000, Yan et al. 2006, Malheiro et al. 2013).Clonal reproduction is the major reproduction and dispersal method of Vallisneria species (Xiao et al. 2006(Xiao et al. , 2007a(Xiao et al. , 2011)).In the present study, longer creeping stems and a greater ramet number were observed under elevated CO 2 conditions, which is consistent with previous studies; this clonal growth enables Vallis neria species to occupy more habitats (Yan et al. 2006).
An increase in temperature also increased growth rate and ramet number in V. natans.Temperature elevation can increase the activity of photosynthetic   enzymes and carbonic anhydrase, which likely caused the increases of biomass and ramet number in our experiment.These results are in agreement with a previous study in which higher temperatures increased growth under higher inorganic carbon availability (Olesen & Madsen 2000).Additional inorganic carbon resources can be used only if other resources (e.g.water soluble nutrients) are concurrently available (Xie et al. 2013).In addition, the temperature increase in the present experiment was relatively minor (approximately 3°C), which may have been insufficient to affect the composition and balance of inorganic carbon in the water column and thus may have further influenced the utilization of inorganic carbon in V. natans.A recent study produced similar results, indicating that soil warming had little effect on the gas exchange of plants at the alpine treeline (Streit et al. 2014).
The individual ramet biomass of V. natans decreased with elevated temperature, but only significantly at ambient CO 2 .CO 2 elevation may promote the assimilation process, as reflected by the observation of increased ramet sizes, which reflects a positive response to an increase in inorganic carbon levels in the water (Titus & Andorfer 1996, Yan et al. 2006, Xie et al. 2013).However, tem perature ele-    Biomass allocation in V. natans was significantly affected by CO 2 concentration, which demonstrated the plant's plasticity to some extent.Under the elevated CO 2 condition, more biomass was allocated to the roots, ramets and buds, while less biomass was allocated to the leaves.When inorganic carbon is no longer a limiting factor, nutrient intake will likely be more important than photosynthesis as a result of carbohydrate production, and roots may therefore be emphasized over leaves (Titus & An dorfer 1996, Geng et al. 2004).Additionally, greater resource allocation to creeping stems may allow V. natans to capture more resources, and reduce competition for soil nutrients with neighboring plants (Xiao et al. 2006, 2007a, Yan et al. 2006), and more buds will likely lead to higher productivity in waters with high CO 2 concentrations.
Different macrophytes have different underwater carbon utilization abilities (Madsen et al. 1993, Xie et al. 2013), which influence the distribution and structure of aquatic plants (Lacoul & Freedman 2006, Bornette & Puijalon 2011).However, studies on the influence of interactions between CO 2 and temperature on submerged macrophyte communities remain limited, and further research is needed.

122Fig. 1 .
Fig. 1.Experimental design showing one of the experimental blocks.Vallisneria natans plantlets were placed in mesocosms that were subjected to different treatments.Two CO 2 levels (ambient and elevated) and 2 temperature levels (ambient and elevated; elevation achieved with far infrared quartz heating tubes) were employed (n = 6)

124Fig. 3 .
Fig. 3. Differences in (a) total plant biomass (per mesocosm), (b) ramet number, (c) individual ramet biomass, (d) creeping stem length and (e) bud number of Vallisneria natans under different CO 2 and temperature conditions (n = 6).Bars with different letters indicate significant differences among the treatments (studentized Tukey's HSD) vation reduced the individual ramet size of V. natans.Many studies have evaluated the relationship between temperature and individual size, employing theoretical frameworks such as Bergmann's rule, James' rule and the temperature− size dependent rule.Daufresne et al. (2009) studied ectothermic aquatic organisms (bacteria, phyto-and zooplankton and fish) from the individual scale to the community scale and found that global warming increased the proportion of small and young individuals, thereby decreasing the size−age relationship.Individual miniaturization may therefore be a third general rule for the re sponse of aquatic ecosystems to global warming (Daufresne et al. 2009).

Table 2 .
Effects of elevated CO 2 and elevated temperature on light response curve parameters in Vallisneria natans.Significant differences (p < 0.05) among treatments are indicated by different letters; P m = maximum photosynthetic rate, α = initial slope of rETR−PAR response curve, E k was calculated by dividing P m by α; R 2 is regression coefficient of rETR−PAR response curves 123 Fig. 2. Relative electron transport rate (rETR) curves for Vallisneria natans under ambient and elevated CO 2 and temperature.Each curve represents the mean of 6 replicate samples