Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes

Journal Pre-proof Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes

Jianfeng Chen, Haojie Su, Gaoan Zhou, Yaoyao Dai, Jin Hu, Yihao Zhao, Zugen Liu, Te Cao, Leyi Ni, Meng Zhang, Ping Xie PII:

S0048-9697(20)30244-8

DOI:

https://doi.org/10.1016/j.scitotenv.2020.136734

Reference:

STOTEN 136734

To appear in:

Science of the Total Environment

Received date:

14 November 2019

Revised date:

10 January 2020

Accepted date:

14 January 2020

Please cite this article as: J. Chen, H. Su, G. Zhou, et al., Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes, Science of the Total Environment (2020), https://doi.org/10.1016/j.scitotenv.2020.136734

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© 2020 Published by Elsevier.

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Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes

Jianfeng Chena,b, Haojie Suc, Gaoan Zhoua, Yaoyao Daia, Jin Hua, Yihao Zhaoa, Zugen

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Liub, Te Caoc, Leyi Nic, Meng Zhangb*, Ping Xiec*

a Poyang Lake Eco-economy Research Center, Jiangxi Province Engineering

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Research Center of Ecological Chemical Industry, Jiujiang University, Jiujiang

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332005, China

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b Jiangxi Academy of Environmental Sciences, Nanchang 330039, P.R. China

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c Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of

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Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese

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Academy of Sciences, Wuhan, 430072, China

* Corresponding author: Meng Zhang, Ping Xie Mailing addresses: Jiangxi Academy of Environmental Sciences, 1131# Hongdu North Road, Nanchang 330039, P.R. China Institute of Hydrobiology, The Chinese Academy of Sciences, 7# Donghu South Road, Wuhan 430072, China Tel.: +86 027 68780056; Fax: + 86 027 68780622 E-mail: [email protected], [email protected]

Author contributions: MZ, PX and JC conceived and designed the study. GZ, JH, YD, YZ and JC

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performed the experiment. JC, HS and MZ did the data statistical analysis. JC wrote the manuscript and LN, TC, PX and ZL revised it. All authors edited the manuscript and gave their approval to the final version of the manuscript.

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Note: The authors declare no competing financial interest.

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Effects of benthivorous fish disturbance and snail herbivory on water quality and two submersed macrophytes

ABSTRACT: Benthivorous fish disturbance and snail herbivory are two important factors that

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determine the community structure of submersed macrophytes. We conducted an outdoor mesocosm experiment to examine the separate and combined effects of these

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two factors on water quality and the growth of two mixed-cultivation submersed

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macrophytes, Vallisneria natans and Hydrilla verticillata, with different growth forms.

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The experiment involved two levels of fish (Misgurnus anguillicaudatus) disturbance

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crossed with two levels of snail (Radix swinhoei) intensity. The results revealed that

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fish activity rather than snail activity significantly increased the overlying water concentrations of total suspended solids (TSS), total nitrogen (TN), ammonia nitrogen

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(N-NH4), total phosphorus (TP) and phosphate phosphorus (P-PO4). However, no differences among treatments were observed for chlorophyll a (chl a) concentrations. Fish disturbance or snail herbivory alone did not affect the relative growth rate (RGR) of H. verticillata, but their combined effects significantly decreased the RGR of H. verticillata. Although snail herbivory alone did not affect the RGR of V. natans, fish disturbance alone and the combined effects of these factors drastically reduced its RGR. Both species exhibited increased free amino acid (FAA) contents and decreased ramet numbers, soluble carbohydrate (SC) contents and starch contents in the presence of the fish. Moreover, compared to H. verticillata, V. natans showed

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exceedingly low ramet numbers and starch contents in the presence of the fish. H. verticillata had a higher RGR and summed dominance ratio (SDR2) than V. natans in all treatments; H. verticillata also displayed a larger competitive advantage in the presence of fish disturbance. The present study suggests that (1) fish disturbance rather than snail activity increases water nutrient concentrations, (2) low snail density

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may be harmful to submersed macrophyte growth when the plants are under other abiotic stress conditions and (3) the competitive advantage of H. verticillata over V.

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natans is more preponderant in a turbid environment.

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Key words: benthivorous fish; snail; cumulative effect; disturbance; competitive

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1. Introduction

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Submersed macrophytes are primary producers in lake ecosystems; however,

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macrophytes in lakes are in decline globally (Sand-Jensen et al., 2000; Zhang et al., 2017). Reconstruction of submersed macrophyte communities is crucial for the

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restoration of lake ecosystems (Jeppesen et al., 1997; Scheffer, 1998). However, it is difficult to restore submersed macrophytes, particularly diverse communities, in an entire lake, and more studies on factors that influence macrophyte restoration are needed (Qin et al., 2007, 2014). Benthivorous fish disturbance and invertebrate herbivory are two important factors that influence the population growth and community diversity of submersed macrophytes (Bakker et al., 2016; Wood et al., 2017; Chen et al., 2019). Benthivorous fish disturbance significantly influences water quality. For example, Cyprinus carpio caused a shift from a clear macrophyte-dominated state to a turbid

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phytoplankton-dominated state in 2-4 weeks (Badiou and Goldsborough, 2015). Fish disturbance also increases the levels of water ammonia nitrogen (N-NH4), total phosphorus (TP), and total suspended solids (TSS), among others (Badiou and Goldsborough, 2015; He et al., 2017). In addition, fish disturbance directly affects the colonization of submersed plants (Ribas et al., 2017), even uprooting submersed

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plants (Zambrano and Hinojosa, 1999). The propagules produced by fish disturbance have a strong regeneration ability and become a vital driving force for submersed

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macrophyte diffusion (Lauridsen et al., 1994; Li et al., 2007a; Ribas et al., 2017).

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However, the ecological effects of benthivorous fish on the growth of submersed

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macrophytes and water quality are species-specific. For instance, Astronotus

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crassipinnis increased the number of plant fragments (Ribas et al., 2017), but

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Carassius carassius did not (Gu et al., 2018); C. carpio uprooted submersed macrophytes during spawning (Zambrano and Hinojosa, 1999), whereas Scardinius

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erythrophthalmus showed no negative effects on submersed macrophyte biomass (Dorenbosch and Bakker, 2012). The impacts of fish on macrophytes also depend on the fish biomass density. Fish impacts on macrophytes can be very negative at high fish biomass densities; however, their impact may be neutral or slightly positive at very low fish biomass densities (Wood et al., 2017). Moreover, some results are inconsistent, such as those regarding the concentrations of phosphate phosphorus (P-PO4) (Badiou and Goldsborough, 2015; Gu et al., 2018) and chlorophyll a (chl a) (Kyeongsik, 2001; Parkos et al., 2003; Wahl et al., 2011; Badiou and Goldsborough, 2015; He et al., 2017; Gu et al., 2018; Chen et al., 2019). Therefore, we need to

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improve our understanding of the conditions under which fish would be expected to have corresponding impacts on submersed macrophyte growth and water quality. Misgurnus anguillicaudatus, a Cobitidae fish, is a widely distributed benthivorous fish species in Chinese lakes (Zhang and Li, 2007) that has a strong disturbance ability (Chen et al., 2019), and the effects of this species should be evaluated.

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Snails, which are common invertebrates in lakes, are considered to be generalist grazers with many food sources, such as organic debris, algae and live aquatic

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macrophytes (Brönmark, 1990; Li et al., 2006, 2007, 2008, 2009a, 2009b; Xiong et al.,

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2010; Ribas et al., 2017; Yang et al., 2019). Pulmonate snail species graze on diverse

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macrophytes, and their effects vary according to the plant species and growth stage of

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the snails, as well as the external environmental conditions. For example, the

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herbivory ability of snails for macrophytes usually shows a negative correlation with snail individual's size within species (Li et al., 2006, 2009b); furthermore, selective

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herbivory by snails correlates with the morphological structural traits of macrophytes, dry matter content and other chemical substances (Bolser, 1998; Elger and Lemoine, 2005; Li et al., 2006, 2007; Xiong et al., 2010; Ribas et al., 2017). In general, selective grazing by snails enables more tolerant species to dominate and thus has different effects on submersed macrophyte population abundance and community structure in lake ecosystems (Sheldon, 1987; Li et al., 2007; Marie et al., 2007; Ribas et al., 2017). In addition, the impacts of snails on macrophytes depend on snail density, with a low density facilitating macrophyte growth and a high density negatively impacting macrophyte biomass (Li et al., 2009; Wood et al., 2017).

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Regardless, as the effects of aquatic animal herbivory on submersed macrophyte growth are magnified when other conditions for macrophyte growth are limiting, such as light limitation caused by high water turbidity (Mitchell and Wass, 1996; Hidding et al., 2016), it is necessary to focus on the impacts of snails on macrophytes in combination with other stress conditions.

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Hydrilla verticillata and Vallisneria natans are two common submersed macrophytes in China and around the world, and they have different growth forms: H.

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verticillata is a canopy-former, and V. natans shows rosette growth (Chambers, 1987).

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Submersed macrophytes with different growth forms usually have different adaptive

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strategies for responding to stress, which are often species-specific. Changes in

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morphological and physiological traits, for example, ensure better adaptation to

et al., 2016).

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various light regimes (Cao et al., 2011; Fu et al., 2012; Chen et al., 2016, 2019; Yuan

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In the present study, we evaluated the effects of M. anguillicaudatus (fish) and Radix swinhoei (snail) on water quality and the growth of two mixed-cultivation submersed macrophytes with different growth forms. We examined the overlying water physicochemical and chlorophyll a characteristics as well as the growth and C/N metabolism indices of the two macrophytes. We also examined whether benthivorous fish disturbance and herbivorous snail grazing cause cumulative, synergistic or antagonistic responses.

2. Materials and methods

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2.1. Experimental material This experiment was performed from 4 July to 12 August 2019 (40 days) at the aquatic plant experiment base of the Poyang Lake Eco-economy Research Center (29.67924° N, 116.009238° E) at Jiujiang University, located in the city of Jiujiang in China. In this experiment, V. natans and H. verticillata were mixed and cultured in 16

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outdoor white polyethylene tanks (200 L), each with a 57 cm inner diameter at the bottom, a 66 cm inner diameter at the top, and a 68 cm depth. The clay sediment [0.92

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mg g-1 total phosphorus (dry weight, DW), 1.73 mg g-1 total nitrogen (DW) and 1.2%

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(DW) total organic content] was collected from a lake in the Lushan Botanical Garden

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in the city of Jiujiang. A 6 cm layer of lake sediment and 60 cm of tap water were

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added to each tank. The sediment was fully mixed before the experiment to ensure

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homogeneity of the sediment conditions.

The seedlings/shoots collected for the experiment were similar in size and

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healthy in appearance. The seedlings/shoots were collected from the Xinyi River in the city of Suqian. For H. verticillata, the apical shoot was 19.78 ± 0.14 cm in length and had a fresh weight of 0.61 ± 0.05 g, with no flowers. For V. natans, intact seedlings (containing roots) were 19.56 ± 1.00 cm in length, with a fresh weight of 1.18 ± 0.14 g and 6.20 ± 0.49 leaves. The seedlings/shoots were carefully washed to remove attachments before being planted in the tanks. M. anguillicaudatus, a common benthivorous fish that is widely found in the mid-lower Yangtze lakes, was purchased from a supermarket near Jiujiang University; the individuals were of similar size (7.94 ± 0.26 cm in length and 2.73 ± 0.29 g wet

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weight) and were captured from the water bodies around Poyang Lake. The fish were acclimatized in tap water for 5 days before the experiment. Individuals of R. swinhoei of similar size (0.18 ± 0.02 g wet weight including shell) were collected from the experimental base of the Jiangxi Institute of Water Sciences in the city of Gongqin. 2.2. Experimental design

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At the beginning of the experiment, four shoots of H. verticillata and four seedlings of V. natans were alternately and evenly planted around a bucket wall and

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approximately 10 cm to the bucket wall; one shoot of H. verticillata and one seedling

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of V. natans were planted approximately 10 cm apart from each other in the centre of

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the tank. After the macrophytes were planted, tap water was added to all tanks to a

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depth of 60 cm. Two days after the seedlings/shoots were planted, the fish and snails

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were added to the corresponding tanks. The 16 tanks were established as follows: four fishes were distributed to four tanks with one fish in each tank (F); 64 snails were

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distributed to four tanks with 16 snails in each tank (S); four fishes and 64 snails were distributed to four tanks with one fish and 16 snails in each tank (F+S); and four tanks were included as the control group (C). Newly hatched snails were removed from the tanks during the experimental period. All tanks were placed under a shelter covered with a black sunshade net to provide an appropriate light environment for plant growth. During the experimental period, the water temperature remained within the range of 23.7 °C to 31.8 °C, and the average temperature in all tanks was 27.9 °C. Tap water was added every day to maintain a constant water level of 60 cm.

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2.3. Sampling Approximately 500 ml of overlying water just below the water surface was collected at 8-8:30 a.m. to determine the total nitrogen (TN), N-NH4, TP, P-PO4 and chl a concentrations on the 33rd day of the experiment. The chl a content was measured using a spectrophotometer (Lorenzen, 1967) after residues on a glass fibre

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filter were extracted in 90% acetone for 24 h. The TN and TP contents of the water samples were measured according to standard Chinese methods (Huang et al., 1999).

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For analysis of P-PO4 and N-NH4 concentrations, water samples were filtered through

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a Whatman GF⁄C glass fibre filter (1.2-μm-pore diameter). P-PO4 was determined

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according to the molybdenum blue method (Golterman, 1969). N-NH4 was analysed

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by the Nessler method (Eaton et al., 2005). All samples were centrifuged for 10 min at

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4000 rpm to eliminate the effects of turbidity on absorbance. TSS was measured on the 33rd day of the experiment by passing a known volume of water through pre-dried

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and pre-weighed Whatman GF/C filter paper, which was dried for 6 h at 70 °C and then weighed. The water temperature (T ºC) was measured using a multifunctional YSI meter (Yellow Springs Instruments, Ohio, USA). Photosynthetically active radiation (PAR) was assessed using a LI-COR UWQ-10057 sensor and a LI-1500 light sensor logger (LI-COR, Lincoln, NE, USA). The PAR was measured near the water surface in the water column and at a water depth of 30 cm. The PAR and water temperature were measured at 11:00 to 12:00 a.m. on the 11th, 16th and 23rd days of the experiment. At the end of the experiment, all plants were harvested and washed carefully

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with tap water for further measurement. In each tank, the total ramets were counted for both species. A shoot that rises from the base was considered a ramet for H. verticillata. The plant height was considered as the maximum shoot length for H. verticillata and maximum leaf length for V. natans. H. verticillata is a typical canopy macrophyte, and the branch number and the height of the lowest branch are vital for

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canopy formation, thus influencing the adaptation of this species. We counted the total branch number and measured the height of the lowest branch for individuals of H.

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verticillata. All V. natans were divided into aboveground and belowground parts,

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dried with bibulous paper, weighed, oven dried at 70 °C to a constant weight and

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weighed to determine the dry weight. For H. verticillata, 4-7 individuals were

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randomly selected to determine the dry weight of leaves and stems, and the rest of the

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values were calculated according to their relative proportions. The total biomass of each species in each tank was the sum of the dry weight of all plant organs. The RGR

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of the plants in each tank was calculated by the following formula: RGR = ln(M2/M1)/days, where M2 and M1 were the plant dry weight at the end and beginning of the experiment, respectively, and days was the duration of the experimental period. Ten shoots and seedlings were randomly selected to measure the dry weight at the beginning of the experiment. The dry weight of each species in each tank was calculated according to its quantitative proportion. The two-factor summed dominance ratio (SDR2) was calculated by the following formula: SDR2 = (D + H)/2%, where D and H were the ratio of the individual plant number to the total plant number and the ratio of the dry biomass of each species to the total dry biomass,

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respectively (Numata, 1966). The dried plant samples of the aboveground parts of each species were ground into a fine powder for the determination of soluble carbohydrate (SC), free amino acid (FAA) and starch contents. Approximately 50 mg of the powder was extracted twice with 5 mL of 80% ethanol at 80 °C for 20 min. The extracts were then pooled and centrifuged at 4000 rpm for 20 min. The FAA and SC

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contents in the supernatant were analysed using glucose and alanine as standards, respectively (Yemm and Willis 1954; Yemm et al., 1955). The residue was used to

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examine the content of starch by the method of Dirk et al. (1999).

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2.4. Statistical analyses

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IBM SPSS 19.0 software was used for all statistical analyses. Values are

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expressed as the mean ± standard error (SE). No outliers were found after checking

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the box diagrams. The Shapiro-Wilk test was used to test the model residual normality. The Levene homogeneity test of variance was used to test the equality of variance.

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One-way analysis of variance (ANOVA) was used to test the statistical significance of plant growth, morphological, physiological, summed dominance ratio (SDR2) and water quality indices. Residuals were normally distributed, and the data were ln(X)-, ln(X + 1)- or Sqrt-transformed to achieve homogeneity of variance if necessary. The effects of macrophyte species, treatment, and their interactions on RGR, SDR2, plant height, ramet number and C/N metabolism indices at the end of the experiment were tested by two-way ANOVA. The residuals were normally distributed, and the starch and SDR2 data were log10-transformed to achieve homogeneity of variance. To examine the type of interaction (cumulative effect, synergistic effect and antagonistic

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effect) between snail herbivory or fish disturbance and the plant relative growth rate (RGR), differences between C and S, C and F and C and F+S were calculated between any two repetitions; t-tests were then used to determine differences between (C-S)+(C-F) and C-(F+S) for the corresponding indices. All results were considered significant at the p < 0.05 level, and post hoc pairwise comparisons of means to

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determine significance were performed using the LSD test (at the 0.05 significance

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level).

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3. Results

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3.1. Overlying water quality

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The PAR at the water surface exhibited no differences among treatments.

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However, the PAR below the water surface was significantly lower in the treatments with fish than in the treatments without fish (Fig. 1a-b). The TP, P-PO4, TN, N-NH4

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and TSS concentrations in the F and F+S treatments were significantly higher than those in the C and S treatments, respectively. However, there were no significant differences in TP, P-PO4, TN, N-NH4 and TSS concentrations between the treatments with fish and the treatments with no fish (Fig. 1c-g), nor was there a significant difference in chl a concentrations among all treatments (Fig. 1h). 3.2. RGR and SDR2 Both RGR and SDR2 responded significantly to macrophyte species, treatment and species × treatment interaction effects (Fig. 2, Table 1). For H. verticillata, the RGR showed no significant difference among the C, S

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and F treatments but exhibited a decrease in the F+S treatment compared to the control. However, the RGR in the F+S treatment decreased by only 8.25% compared with the average value of the other three treatments (Fig. 2a). Regarding V. natans, no significant difference in RGR was found between the C and S treatments, although a decrease was observed in the F and F+S treatments

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compared to the control and S treatments. In addition, the average RGR in the F and F+S treatments decreased by 63.39% compared with the average RGR value in the C

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and S treatments (Fig. 2a).

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In all treatments, H. verticillata exhibited a higher RGR than did V. natans. In the

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absence of fish, the average RGR of H. verticillata was 1.57 times higher than that of

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V. natans; in the presence of fish, the average RGR of H. verticillata was 3.99 times

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higher than that of V. natans (Fig. 2a).

H. verticillata exhibited a higher SDR2 than did V. natans in all treatments.

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Compared to the treatments without fish, H. verticillata exhibited an increase in SDR2, and V. natans exhibited a decrease in SDR2 in the treatments with fish. The average SDR2 values of H. verticillata were 6.61 and 2.73 times higher than those of V. natans in the presence and absence of fish, respectively (Fig. 2b). With regard to the combination treatments, fish disturbance and snail herbivory exhibited cumulative effects (t-test, P = 0.41 for H. verticillata and P = 0.49 for V. natans) on the RGR of both species. 3.3. Plant height and ramet number Except for the interaction effects on ramet number, both plant height and ramet

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number responded significantly to macrophyte species, treatment and species × treatment interaction effects (Fig. 3, Table 1). The plant height of H. verticillata in the treatments with fish was greater than that in the treatments without fish (increased by 85.70%), although no difference among all treatments was observed for V. natans. The plant height of H. verticillata

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was also greater than that of V. natans in each treatment. In treatments with fish, the plant height of H. verticillata was 2.40 times greater than that of V. natans, with

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averages of 112.85 and 46.93 cm, respectively (Fig. 3a). In treatments without fish,

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the plant height of H. verticillata was 1.78 times greater than that of V. natans, with

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averages of 60.77 and 34.23 cm, respectively (Fig. 3a).

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Similar statistical analysis results were obtained for ramet number between H.

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verticillata and V. natans. The ramet number was significantly higher in the treatments without fish than in the treatments with fish for both species. In each

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treatment, the ramet number of H. verticillata was higher than that of V. natans. In the treatments with fish, the ramet number of H. verticillata was 7.66 times higher than that of V. natans, with average numbers of 31.63 and 4.13, respectively (Fig. 3b). In treatments without fish, the ramet number of H. verticillata was 2.59 times higher than that of V. natans, with average numbers of 77.00 and 29.75, respectively (Fig. 3b). 3.4. Branch number and height of the lowest branch in H. verticillata The branch number of H. verticillata showed no difference among all treatments, with an average of 192.75 (Fig. 4a). The branch nearest to the bottom of H.

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verticillata was higher in the treatments with fish than in the treatments without fish (Fig. 4b). 3.5. C/N metabolism A significant effect of macrophyte species was observed only for starch. The treatment had significant effects on all C/N metabolism indices. The interaction effect

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of macrophyte species and treatment showed significance only for SC (Fig. 5, Table 1).

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The SC concentration of H. verticillata showed no statistical significance among

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the C, S and F treatments but decreased in the F+S treatment. For V. natans, the SC

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concentration displayed no statistical significance between the C and S treatments but

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decreased in the F and F+S treatments (Fig. 5a).

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Additionally, similar effects on FAA levels in both species were observed for all treatments; both species showed higher FAA contents in the treatments with fish than

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in the treatments without fish, with no difference in FAA concentration between H. verticillata and V. natans in each treatment (Fig. 5b). In contrast to the FAA concentration, the starch concentration was higher in the absence of fish than in the presence of fish for both species. No difference in the starch concentration was found between H. verticillata and V. natans for the treatments without fish, although the starch concentration was higher in H. verticillata than in V. natans in the treatments with fish (Fig. 5c).

4. Discussion

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Water column nutrient concentrations may be increased by sediment resuspension caused by benthivorous fish disturbance and aquatic animal excretion. The extent of the influence is usually species-specific and density-dependent (Li et al., 2007, 2009; Dorenbosch and Bakker, 2012; Badiou and Goldsborough, 2015; Zhang et al., 2016; Gu et al., 2018). In the present study, the increased nutrient

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concentrations were probably caused by fish disturbance or excretion. The presence of the snails did not increase the overlying water nutrient concentrations, which may be

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explained by the low density of snails. Li et al. (2009) reported no difference in TP,

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P-PO4, TN and N-NH4 concentrations between the control and 80 snails m-2

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treatments. Nonetheless, previous studies have reported increased (Zhang et al., 2016;

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He et al., 2017; Gu et al., 2018), unchanged (Parkos et al., 2003; Chen et al., 2019)

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and decreased (Kyeongsik, 2001; Wahl et al., 2011; Badiou and Goldsborough, 2015) water chl a contents due to fish disturbance. In the present study, the lack of a change

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in chl a concentrations among treatments may be explained by a combination of two mechanisms. First, the relatively low water column P-PO4 and N-NH4 concentrations and competition for nutrients by macrophytes restrict the productivity of phytoplankton. Second, by lowering light availability, high turbidity restricts photosynthesis by phytoplankton at large water depths (Kyeongsik et al., 2001; Badiou and Goldsborough, 2015; Chen et al., 2019). Previous studies have reported different adaptation strategies to low-light environments by different growth forms of submersed macrophytes (Cao et al., 2011; Fu et al., 2012; Chen et al., 2016, 2019; Yuan et al., 2016). Consistent with previous

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studies, canopy-forming macrophytes usually elongate their shoots towards the water surface to compensate for low-light conditions (Goldsborough and Kemp, 1988; Fu et al., 2012; Chen et al., 2016, 2019). In the present study, the substantially increased plant height and lowest branch height ensured that the shoots of H. verticillata easily reached the water surface and formed a canopy to maintain a competitive advantage

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under light competition, as indicated by the large number of branches. In addition, the small reduction in total biomass only in the combination treatment indicated better

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adaptation to low-light conditions in H. verticillata than in V. natans.

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In the present study, the plant height of V. natans showed no significant

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differences among the treatments, which might be explained by two factors. First,

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rosette-form macrophytes usually exhibit a weaker ability to increase plant height in a

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turbid environment (Fu et al., 2012; Chen et al., 2019). The ability of V. natans to increase height is inhibited in low-light environments, as reported by Chen et al.

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(2016). Second, macrophytes with a rosette growth form mainly grow through physiological adjustments to adapt to low-light conditions, for example, by lowering the light compensation point of photosynthesis (Titus and Adams, 1979; Su et al., 2004), adjusting carbon and nitrogen metabolism (Yuan et al., 2013; Chen et al., 2019) and other photosynthetic adjustments, which are more common strategies than increasing plant height in severely low-light environments (Chen et al., 2016). Vallisneria natans has no erect stems, and Titus and Adams (1979) reported that V. americana stored 62% of its leaf biomass within 30 cm at the bottom of the water column. In the present study, the compensation depth of V. natans was 32.59 cm

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according to the light compensation point of photosynthesis reported in the study of Su et al. (2004), and 58.4% of the leaves of V. natans were under the photosynthesis compensation depth. This finding indicates that the growth of V. natans was inhibited by low light and that the decreased ramet number in the treatments with fish was probably an adaptive strategy to low-light stress. The decreased ramet number

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prevented more new ramets from being exposed to the low-light stress zone, and the adult plants were able to conserve more nutrients for tolerance to the low-light stress

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(Hutchings and Wijesinghe, 1997).

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Both macrophyte species survived in the treatments with fish in the present

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relatively short-term experiment, although both species were negatively affected by

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fish disturbance, as indicated by the production of fewer ramets. However, comparing

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these two species revealed that H. verticillata produced more ramets than V. natans and that its adult plants were able to maintain a higher growth rate and height than V.

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natans in the presence of fish. The small number of ramets produced by V. natans is a disadvantage for further population reproduction and expansion. These factors may lead to a greater competitive advantage for H. verticillata over V. natans at longer time scales, which was also indicated by the higher two-factor summed dominance ratio of H. verticillata than V. natans in the presence of fish. Radix swinhoei is a generalist grazer, and it feeds on almost all aquatic plants, such as H. verticillata, V. natans, Potamogeton crispus, Potamogeton maackianus, Potamogeton malaianus and Trapa bispinosa (Li et al., 2005; Li et al., 2006, 2007b, 2008, 2009a, 2009b; Xiong et al., 2010). Snails often exhibit selective herbivory when

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multiple food sources coexist, and selective behaviour correlates with plant tissue nutrient contents and external morphological structure (Elger and Lemoine, 2005; Li et al., 2006; Xiong et al., 2010; Yang et al., 2019). Li et al. (2006, 2009b) reported that R. swinhoei had higher grazing rates on V. natans than H. verticillata due to the larger leaf area of the former, which is convenient for snail grazing. In addition, R. swinhoei

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preferred V. natans over H. verticillata in a mixed-culture experiment (Li et al., 2007b). However, the existence of snails alone did not decrease the RGR of either

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macrophyte species in the present study. This result is most likely explained by the

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low snail density (approximately 47 individuals m-2, i.e., 8.46 g m-2 in the present

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study), which yielded weak effects of herbivory on the macrophyte community. Yang

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et al. (2019) reported increased growth of Vallisneria denseserrulata at 240

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individuals m-2 snail density of R. swinhoei. Li et al. (2009) reported a favourable effect of R. swinhoei on Vallisneria spiral growth at a snail density of 80 individuals

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m-2 and no effect at a density of 160 individuals m-2. In addition, Wood et al. (2017) reported that the results of previous studies have been highly variable, ranging from small positive effects to small negative effects on macrophyte biomass at the present biomass density. Nonetheless, the total biomass of H. verticillata decreased in the combination treatment compared to the control treatment, which may be explained by snail herbivory under a low-light environment. Because the tolerance of plants to herbivory usually correlates with the availability of resources, limiting resources influences the compensatory response of plants to herbivores (Wise and Abrahamson, 2005, 2008).

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In the present study, the low-light conditions induced by fish disturbance likely led to higher susceptibility of the plants to herbivory (Chapin et al., 1987; Huisman and Olff, 1998). Therefore, the present findings that the snail density used in this study could have a negative effect in the presence of fish disturbance suggest that additional environmental factors can change a system from one in which herbivory has little

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impact to one in which herbivory has a greater impact. Cumulative, synergistic and antagonistic effects of multiple stressors on

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ecological communities have been reported, and the interaction type can vary between

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specific stressor pairs and varied intraspecific and interspecific (Crain et al., 2008; He

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et al., 2019). For example, shading and clipping showed synergistic effects on shoot

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length and cumulative effects on the biomass of the invasive alien macrophyte Elodea

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nuttallii (He et al., 2019). In the present study, the cumulative negative effects of fish disturbance and herbivorous snail grazing on macrophyte growth indicated that even

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low-density snail herbivory, which alone showed no negative effect on macrophyte growth, is harmful to submersed macrophyte growth. However, the interaction type observed in the present mesocosm experiment may vary in lakes due to the existence of additional stressors. The addition of a third stressor could change interaction effects significantly (Crain et al., 2008). Carbon (C) and nitrogen (N) metabolism is a central factor influencing submersed macrophyte survival and growth in a low-light environment (Cao et al., 2009; Cao et al., 2011; Yuan et al., 2016; Chen et al., 2019). Indeed, low-light stress generally induces increased FAA concentrations in submersed macrophytes, such as V.

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natans, P. crispus, Ceratophyllum demersum, Potamogeton malaianus, and Myriophyllum spicatum (Cao et al., 2009; Zhang et al., 2010; Cao et al., 2011; Yuan et al., 2016), because the nitrogen assimilated by plants usually accumulates as FAA due to the supply of photosynthesis products in a low-light lake environment (Rabe, 1990; Britto and Kronzucker, 2005). In addition, the decomposition of soluble protein to

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FAAs and the accumulation of FAAs due to decreased photosynthetic C production also led to increased FAA contents in a low-light environment (Barker et al., 1966;

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Elamrani et al., 1994; Yuan et al., 2016). The carbohydrate reservoir is a key

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functional trait related to a plant’s ability to tolerate an adverse environment (Cao et

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al., 2011; Yuan et al., 2016). Low light availability often leads to decreased plant

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tissue SC and starch concentrations (Cao et al., 2011; Yuan et al., 2016; Chen et al.,

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2019), which was also observed in the present study. However, the severely low starch contents of V. natans in the presence of fish may be detrimental to the ability of a

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population to tolerate extreme stress factors and undergo normal reproduction in lakes compared to H. verticillata because macrophyte tolerance of stress factors and normal reproduction consume many photosynthetic products (Goldsborough and Kemp, 1988). Compared to morphological traits, the physiological responses of submersed macrophytes are generally relatively sensitive in responding to environmental stress (Chen et al., 2017), and the significantly altered C/N metabolite contents in our study indicated physiological stress in both species in the presence of fish disturbance. However, compared to V. natans and based on C/N metabolism indices, H. verticillata

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was able to substantially ensure its growth, suggesting better adaptation than V. natans in turbid water. In conclusion, benthivorous fish disturbance significantly increases water nutrient concentrations. Cumulative negative effects exist between fish disturbance and herbivorous grazing, which suggests that low-density snail herbivory is harmful

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to submersed macrophyte growth when the plants are under low-light stress conditions. H. verticillata has a competitive advantage over V. natans and becomes

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more preponderant in turbid environments.

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Acknowledgements

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This work was supported by the Education Department of Jiangxi Province

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(GJJ170968). The authors thank the editor and two anonymous reviewers for their

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Figure legends

Figure 1 The characteristics of photosynthetically active radiation (PAR), chlorophyll

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a (chl a) content and hydrochemical indices in the overlying water. PAR-S and PAR-B

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are the PAR at the water surface in the water column and the PAR at a 30 cm water

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depth, respectively. TP, P-PO4, TN, N-NH4 and TSS represent total phosphorus,

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phosphate phosphorus, total nitrogen, ammonia nitrogen and total suspended solids,

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respectively. C, S, F and F+S represent the following treatments: control, snails alone, fish alone and both snails and fish, respectively. The different letters indicate

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significant differences at the 0.05 significance level determined by the LSD test.

Figure 2 The relative growth rate (RGR) and summed dominance ratio (SDR2) of Hydrilla verticillata and Vallisneria natans in each treatment. C, S, F and F+S represent the following treatments: control, snails alone, fish alone and both snails and fish, respectively. The different letters indicate significant differences at the 0.05 significance level determined by the LSD test.

Figure 3 The plant height and ramet number of Hydrilla verticillata and Vallisneria

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natans in each treatment. C, S, F and F+S represent the following treatments: control, snails alone, fish alone and both snails and fish, respectively. The different letters indicate significant differences at the 0.05 significance level determined by the LSD test.

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Figure 4 The branch number and height of the lowest branch of Hydrilla verticillata in each treatment. C, S, F and F+S represent the following treatments: control, snails

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alone, fish alone and both snails and fish, respectively. The different letters indicate

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significant differences at the 0.05 significance level determined by the LSD test.

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Figure 5 The soluble carbohydrate (SC), free amino acid (FAA) and starch contents

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of Hydrilla verticillata and Vallisneria natans in each treatment. C, S, F and F+S represent the following treatments: control, snails alone, fish alone and both snails and

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fish, respectively. The different letters indicate significant differences at the 0.05 significance level determined by the LSD test.

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The authors declare no competing financial interest.

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Table 1 Two-way ANOVA of the relative growth rate, two-factor summed dominance ratio, plant height, ramet number and C/N metabolism indices at the end of the experiment.

** n.s. n.s. **

3 3 3 3

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46.04 0.51 3.79 26.27

df 3 3 3

14.83 25.3 21.75 30.04

** ** ** **

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1 1 1 1

Pr>F ** ** **

3 3 3 3

1.29 3.82 0.72 2.39

n.s. * n.s. n.s.

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RGR (day ) SDR2 (%) Plant height (cm) Ramet number SC (mg g-1) FAA (mg g-1) Starch (mg g-1)

df 1 1 1

Treatment Species×Treatment F Pr>F df F Pr>F 26.96 ** 3 12.35 ** 5.28 ** 3 13.88 ** 24.01 ** 3 9.28 **

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Species F 373.84 447.17 143.51

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*p < 0.05, ** p < 0.01, n.s. = not significant. The factors are macrophyte species (Hydrilla verticillata and Vallisneria natans) and treatment (control, snail treatment,

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fish treatment and snail + fish treatment). RGR, relative growth rate; SDR2,

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two-factor summed dominance ratio; SC, soluble carbohydrate; FAA, free amino acid.

Journal Pre-proof Highlights: Fish disturbance increases water nutrient but not chl a concentrations.

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Cumulative negative effects exist between fish disturbance and snail grazing.

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H. verticillata has a competitive advantage over V. natans in all treatments.

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