Suppressed growth of the submersed macrophyte Vallisneria natans in a non-rooted suspended state

Abstract

The growth, morphological traits, and biomass accumulation and allocation pattern of Vallisneria natans grown from buds were observed in a non-rooted suspended state in the laboratory. Submersed anchored V. natans were about 229% higher in height, 208% higher in leaf number, 719% greater in total root length, 64% higher in lacunal root volume, and 1473% greater in total biomass dry weight compared with suspended V. natans. However, insignificant differences existed in root diameter, specific root length, and biomass allocation patterns. These results indicate that when V. natans is not anchored to a substrate in the normal mode, the growth will be markedly suppressed.

Keywords:

• suspended state
• suppressed growth
• submersed macrophyte
• shallow aquatic habitats
• disturbance

Introduction

Apart from numerous environmental factors which affect the growth of aquatic plants such as nutrient supply (Sculthorpe 1967), water level (Bornette and Amoros 1996; Yang et al. 2004; Xiao et al. 2006; Li et al. 2010, 2011), and waterfowl grazing (Rodríguez-Villafañe et al. 2007), other natural and anthropogenic disturbances such as current velocity, wave action, and boating activities greatly influence aquatic macrophytes. The direct actions of current velocity, waves, and boating activities have been reported to cause the dislodgment of submersed plants (Nilsson 1987; Strand and Weisner 1996; Eriksson et al. 2004).

For the dislodgment of submersed plants, recent attention has focused on impacts on distribution (Nilsson 1987; Bornette and Amoros 1996; Strand and Weisner 1996) and species richness (Eriksson et al. 2004). Submersed macrophytes are unique among rooted aquatic vegetation because they link the sediment with overlying water (Barko et al. 1991). In shallow aquatic habitats, exposure to current velocity, waves, and boating activities can cause submersed anchored macrophytes to break away from the substrate and become suspended in the water column. However, relatively little attention has been paid in understanding the effects of a non-rooted suspended state on growth, morphology, and biomass allocation patterns. Hence, a straightforward experimental approach was employed to study Vallisneria natans in the non-rooted suspended state in terms of growth, biomass accumulation, and allocation traits compared with the normal rooted V. natans. Changes in growth, morphology, and biomass accumulation and allocation patterns were studied to determine how V. natans responds to being dislodged from the substrate.

Materials and methods

Plant materials

Tubers of V. natans were collected from Liangzi Lake at the end of December 2006. Tubers were transported to Hubei University and stored indoor in a plastic basin with fresh tap water. Fresh tap water was added as necessary to maintain about 3 cm until the experiment was initiated. In the middle of March 2007, tubers were placed in a greenhouse where the temperature was controlled at 20 ± 2°C in the day and at 15 ± 2°C at night. Light was provided by metal halide bulbs at a photo flux density of 400 µmol m⁻² s⁻¹. One week later, the tubers produced buds, and the buds were placed in three plastic basins containing 15 cm of water. The experiment was started on 23 March 2007. Twelve young plants of the same size (three to four leaves) were transplanted into 12 small plastic basins (22 cm in diameter and 50 cm in height) containing 10 cm of either fine sand and nutrient medium or nutrient medium only.

Fine sand sample preparation

Silica sand samples were collected and sieved to 0.105–0.150 mm (fine sand). The sand samples were analyzed using the standard methods (Nanjing Institute of Soil Science 1980; Soil Science Society of America 1982). Sand bulk density was measured using the core method. Sand particle density was determined using a liquid displacement method and was used to calculate the total porosity. The values of bulk density, particle density, and total porosity of the sand were 1.3 ± 0.1 g cm⁻³, 2.6 ± 0.2 g cm⁻³, and 49.3 ± 0.6, respectively. Before the experiment, the sand was treated with 2 mol L⁻¹ of HCl at 25°C for 48 h to remove any organic matter, rinsed to neutrality with distilled water, and dried.

Experimental set-up

The control treatment was replicated six times; individual plants were held in a container with nutrient medium and sand substrate. The experimental treatment, also replicated six times, contained only the nutrient medium. The nutrient medium was 86 µM of (NH₄)₂SO₄, 172 µM of KNO₃, 32 µM of NaH₂PO₄, 500 µM of CaCl₂, 208 µM of MgSO₄, 110 µM of Fe-EDTA, 47 µM of H₃BO₄, 9 µM of MnCl₂, 0.8 µM of ZnSO₄, 0.3 µM of CuSO_4, and 0.1 µM of H₂MoO₄ (Xie et al. 2005). The nutrient medium depth was 45 cm. In the control treatment, plants were anchored in the sand. In the experimental treatment, unanchored plants were simply allowed to suspend in the water. During the experiment, nutrient solutions in all replicates were completely replaced every 7 days.

Harvest and laboratory analyses

Final harvest took place on 23 September 2007. At the final harvest, the number of ramets and branches were counted for each clone. Then, seedlings were divided into leaves, stems, and roots, which were gently washed and blotted dry.

Fresh roots were fixed at 4°C for 48 h with a solution of 1.85% (v/v) formaldehyde, 5% (v/v) acetic acid, and 63% (v/v) ethanol. The fixed tissues were sequentially dehydrated with butanol, embedded in paraffin, sliced into 10 µm sections, stretched onto slides coated with Vectabond Reagent, deparaffinized with xylene and ethanol, rinsed, and stained with 0.05% (w/v) of Toluidine blue O as described previously by Hayakawa et al. (1994). Thin transverse sections of root material were viewed and photographed. Lacunal volume, as a percentage of root volume, was estimated from comparing surface areas made through stereology (Steer 1981) using a 5 mm or 10 mm point/line lattice applied to enlarged photographs of magnified sections (Robe and Griffiths 1998).

Finally, the leaves, stems, and roots were dried to constant weight at 80°C for 72 h and weighed to determine dry weight.

Statistical analyses

All parameters were analyzed using one-way analysis of variance between the two treatment groups using the SPSS statistical package (version 17.0, SPSS, Chicago, Illinois, USA). Independent-sample t-test was applied to estimate the difference between the two treatments.

Results and discussion

The experimental treatments significantly affected plant height. The height of V. natans rooted in fine sandy medium was about 229% higher than the suspended counterparts (Table 1). The treatments had similar effects on the number of ramets and the number of leaves. The mean ramet numbers and leaf numbers were 4 and 0, and 10.4 and 5.0 for the submersed anchored V. natans and the suspended V. natans, respectively. Similar trends were observed in the total root length and lacunal volume of the root; the total root length and the lacunal volume of the root of V. natans rooted in sand were 719% and 64% higher than their counterparts suspended in the culture medium alone (Figure 1). However, insignificant differences in root diameter and specific root length were observed between the experimental treatment and the control.

Figure 1. Light micrographs of the root cross-section of V. natans grown with anchored roots (a) and grown in the suspended treatment (b).

Table 1. Mean comparison (mean values ± SD, n = 6) for V. natans traits grown anchored in sand and in a suspended treatment.

Parameters	Anchored	Suspended	p
Height (cm)	48.8 ± 1.4	21.4 ± 5.1	***
Number of ramets	4.0 ± 1.2	0	***
Number of leaf	10.4 ± 1.1	5.0 ± 0.8	***
Total root length (cm)	626.9 ± 227.9	76.6 ± 27.69	**
Diameter of root (mm)	0.2 ± 0.02	0.2 ± 0.01	ns
Lacunal volume of root (%)	17.2 ± 0.9	10.5 ± 2.9	**
Specific root length (cm g^−1DW)	31133.2 ± 27391.7	29984.6 ± 7363.0	ns
Total biomass (DW)	0.3 ± 0.1	0.02 ± 0.01	**
Leaf dry weight (gDW)	0.2 ± 0.1	0.02 ± 0.01	**
Shoot dry weight (gDW)	0.01 ± 0.004	0.0007 ± 0.0005	**
Root dry weight (gDW)	0.03 ± 0.02	0.003 ± 0.001	**
Leaf dry weight %	87.0 ± 3.2	81.2 ± 6.0	ns
Shoot dry weight %	3.6 ± 0.7	3.8 ± 1.8	ns
Root dry weight %	9.4 ± 2.8	14.9 ± 6.8	ns
Root–shoot ratio (DW)	0.2 ± 0.04	0.2 ± 0.1	ns
Note: ns, p > 0.05; **, p < 0.01; and ***, p < 0.001.

The final leaf biomass, root biomass, shoot biomass, and total biomass were significantly affected by the experimental treatment and were 15, 14, 9, and 15 times rooted in anchored V. natans than in suspended V. natans (Table 1). Total biomass accumulation in V. natans was mainly a result of increased leaf biomass, although smaller increases in root and shoot biomass also occurred. However, biomass allocation patterns including root/shoot ratio, leaf, shoot, and root dry weight percentages were not affected by the treatments.

Although some species of submersed aquatic plants that are usually rooted are able to grow when suspended; these results clearly reveal that this mode has a significantly negative influence on V. natans’ growth and morphology. Suspended V. natans exhibited drastic reduction in plant height and the number of ramets and leaves. The suspended state is disadvantageous for the persistence of V. natans due to the importance of root gravitropism (Ge et al. 2000). Roots of submersed aquatic plants perform two essential functions – anchoring plants to sediment and contributing to the accumulation and transport of nutrients, gases, and plant hormones (Sculthorpe 1967; Agami and Waisel 1986). The main anatomical change in the roots of V. natans in the suspended treatment was the variation of the lacunal system that connects leaves, shoot, and roots. In this experiment, the nitrogen and phosphorus concentrations in the water column were at the upper limits for most lakes in China (Wang and Dou 1998); thus, nutrient availability was sufficient to cover the needs of V. natans in the control and in the suspended treatment. The lower lacunal root volume of suspended V. natans indicates that the need for anchorage strength or uprooting resistance is of importance for root development in hydroponic culture; the high porosity of V. natans roots create high buoyancy and thus a high pull-out force on the root (Bagger and Madsen 2004). However, this study found no changes in leaf, shoot, and root weight ratios or the root/shoot ratio in V. natans during the experiment. The weak response of biomass allocation patterns in the suspended treatment was unexpected. Lack of plasticity in the biomass allocation may indicate that V. natans responded to non-rooted suspended state by changing growth, morphological traits and biomass accumulation strategies, rather than by changing biomass allocation patterns.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant No. 30870260 and 30870428), by the Excellent Youth Foundation of Hubei Scientific Committee (Degradation mechanism and healthy evaluation of Lake Ecosystem), and by the KC Wong Education Foundation and the Knowledge Innovation Program of the Chinese Academy of Science.