Physiological differences between free-floating and periphytic filamentous algae, and specific submerged macrophytes induce proliferation of filamentous algae: A novel implication for lake restoration

Chemosphere 239 (2020) 124702

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Physiological differences between free-floating and periphytic filamentous algae, and specific submerged macrophytes induce proliferation of filamentous algae: A novel implication for lake restoration Weizhen Zhang a, b, Hong Shen a, *, Jia Zhang a, b, Jia Yu a, c, Ping Xie a, Jun Chen a, ** a

Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China b University of Chinese Academy of Sciences, Beijing, 100049, China c College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Filamentous algae in periphytic lifestyle possess better physiological advantages.
Macrophytes play an important role in the proliferation of filamentous algae.
Filamentous algae show speciesspecific physiological responses to macrophytes.
It is of practical importance to select proper macrophytes during lake restoration.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 April 2019 Received in revised form 27 August 2019 Accepted 27 August 2019 Available online 28 August 2019

Restoration of submerged macrophytes is widely applied to counteract eutrophication in shallow lakes. However, proliferation and accumulation of filamentous algae (possessing free-floating and periphytic life forms) hamper growth of submerged macrophytes. Here, we explored factors triggering the excessive proliferation of filamentous algae during lake restoration using field investigations and laboratory experiments. Results showed that, compared with free-floating Oscillatoria sp. (FO), periphytic Oscillatoria sp. (PO) showed faster growth rate, greater photosynthetic capacities and higher phosphorus (P) affinity. Therefore, PO was physiologically competitively superior to FO under low P concentration and improved light conditions. And proliferation of filamentous algae was mainly manifested in periphytic life form. Besides, field results showed that density of filamentous algae in water column might be related to substrate types. Some macrophyte (Ceratophyllum oryzetorum and Potamogeton crispus) might provide proper substrates for proliferation of filamentous algae. Further physiological experiments found that Oscillatoria showed specific eco-physiological responses to different macrophyte species. Hydrilla verticillata and C. oryzetorum promoted growth and photosynthetic activity of Oscillatoria, while Potamogeton malaianus inhibited growth and P uptake of PO. Myriophyllum spicatum exhibited no impact on growth of Oscillatoria. Our results revealed the intrinsic (physiological differences between free-floating and periphytic life forms of filamentous algae) and extrinsic (different macrophytes) factors affect the

Handling Editor: Tsair-Fuh Keywords: Filamentous algal bloom Oscillatoria sp. Periphytic life form Free-floating life form Macrophytes Ecological restoration

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Shen), [email protected] (J. Chen). https://doi.org/10.1016/j.chemosphere.2019.124702 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

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proliferation of filamentous algae, which are important for guidance on planting of submerged macrophytes during lake restoration. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Re-establishing dominance of submerged macrophytes in shallow eutrophic lakes is widely used to control eutrophication and improve water quality (Hilt et al., 2006; Xu et al., 2014; Tang et al., 2018; Zhang et al., 2018). However, filamentous algae have frequently bloomed in lakes where macrophyte restorations have been applied, such as Cladophora glomerata in the Laurentian Great Lakes (Higgins et al., 2008), Mougeotia in Lake Zwemlust (Gulati and van Donk, 2002) and Melosira varians and Oscillatoria sp. in West Lake (Fu et al., 2017). Excess proliferation of filamentous algae has greatly impeded lake restoration, as filamentous algae hampers the growth of macrophytes by shading (Irfanullah and Moss, 2004; Tiling and Proffitt, 2017). In addition, decomposition of filamentous algae results in anoxic conditions, disappearance of benthic animals (Green and Fong, 2016), intense bacterial activity, and even production of noxious odors and toxic substances (Chun et al., 2013). Many filamentous algae have two life forms e free-floating and periphytic forms. The free-floating filaments are distributed evenly in water column, while the periphytic algae attach on surfaces including sediment and macrophytes. During bloom of filamentous algae, the periphytic filamentous algae thrive, decay, and detach from the surface, and then float to the water surface due to the buoyancy effect of gas bubbles produced by photosynthesis (Berry and Lembi, 2000). Switch between free-floating and periphytic life forms occurs under certain conditions including suitable illumination mode (Fattom and Shilo, 1984). Studies on filamentous algal blooms have been conducted mainly in pelagic habitats, but rarely in benthic or periphytic habitats (Quiblier et al., 2013). Thus, knowledges about factors influencing growth of free-floating and periphytic forms of filamentous algae are important for understanding mechanisms underlying outbreak of filamentous algal blooms, which can guide our future decisions in the practices of lake restoration utilizing submerged macrophytes. To successfully re-establish a macrophyte-dominated state in eutrophic lakes, it is necessary to understand complex interactions between submerged macrophytes and filamentous algae. Some macrophytes not only provide substrates for filamentous algae, but also alter the photosynthetic activity (Zhu et al., 2010) and nutrient utilization of filamentous algae (Wium-Andersen et al., 1982; Mjelde and Faafeng, 1997). In previous studies, different effects of macrophytes on growth of filamentous algae have been observed, including inhibition, enhancement or no effect (Table S1). So far, numerous studies have reported the effects of macrophytes on freefloating algae, but effects on periphytic algae are largely unknown (Allen et al., 2016). It still remains unclear how the two life forms of filamentous algae respond eco-physiologically to the presence of macrophytes. Considering the facts that filamentous algae have different life forms and that filamentous algae proliferate following the planting of some submerged macrophytes, we suggest the following hypotheses: (1) different physiological characteristics of free-floating and periphytic filamentous algae affect formations of filamentous algal mats, (2) substrates affect the attachment, photosynthesis and growth of filamentous algae, and (3) submerged macrophytes have species-specific eco-physiological influences on filamentous algae. Oscillatoria sp. is a typical species of filamentous algae with dual

forms d FO and PO, respectively (Fattom and Shilo, 1984). To test hypothesis (1), we compared the growth, photosynthesis traits and P uptake kinetics of FO and PO in laboratory. To test hypotheses (2) and (3), we conducted both field study and laboratory experiments. The field study was carried out in West Lake in Hangzhou, China, during its vegetation restoration. We investigated the distribution of filamentous algae in the water column and on the surfaces of different substrates (sediments and submerged macrophytes), and then we analyzed the impacts of different substrates and physicochemical factors on filamentous algae. In the laboratory experiments, we further measured the physiological responses of FO and PO to different abiotic and macrophytes substrates. 2. Materials and methods 2.1. Field study 2.1.1. Study site The study areas were situated at Maojiabu and Xilihu (30130  30 140 N, 120 70 -120 80 E), two sub-lakes in the western littoral zone of West Lake in Hangzhou, China. West Lake is an ornamental shallow lake, with a mean water depth of 2.3 m. It was added to the UNESCO World Heritage List in 2011. Water diversion from the Qiantang River to West Lake began since 1986 for improving water quality. To combat eutrophication, submerged macrophytes cultivation were also implemented in West Lake. Submerged macrophytes (including Potamogeton crispus, Vallisneria natans, Myriophyllum spicatum, Ceratophyllum oryzetorum and Najas major) were successfully restored in 2012, with annual mean biomass of 637 ± 239 g m2 (fresh weight m2) and coverage of 27 ± 8% (Zeng et al., 2017). The hydraulic retention time of West Lake was approximately one month and total phosphorus (TP) concentrations decreased to below 0.05 mg L1 (Wang et al., 2017). In recent years, severe filamentous algal blooms occurred in the western area where vegetation resotred (Fu et al., 2017). Two enclosed vegetated areas in Maojiabu (M1, 30 m  15 m; and M2, 36 m  24 m) and an unenclosed non-vegetated area in Xilihu (X) (Fig. S1) were selected as study sites in this study. 2.1.2. Sample collection and measurement Monitoring of macrophyte, sampling of water and phytoplankton and periphytic algae (including epipelon and epiphyton living on sediments and macrophytes, respectively) were conducted in areas M1, M2, and X in April 2014, October 2014, January 2015, April 2015 and August 2015. The sampling and measurement of macrophyte biomass were performed according to previously described methods (Wang et al., 2017). Three sampling units were randomly distributed in each area for sampling of overlying water, phytoplankton and epipelon. Overlying water and phytoplankton samples were collected from the 0e0.5 m layer using a 5 L Schindler sampler. For epiphyton sampling, 3 replicates (each replicate containing at least 5 plants) of each species of macrophyte in each area (without parts living in sediments) were collected into a plastic bag. Periphytic algae on the surface of macrophytes and sediments were collected according to Steinman et al. (2006). Phytoplankton and periphytic algae were preserved by acetic Lugol's solution. Algal identification and counts were performed

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under 400  magnification using an Olympus BX41 microscope (Olympus, Tokyo, Japan) according to Hu and Wei (2006). The density of phytoplankton and periphytic algae was calculated in cells L1 and cells cm2, respectively. Water temperature, dissolved oxygen (DO), redox potential (ORP), total dissolved solids (TDS) and pH were monitored in situ using a multi-parameter water quality detector (YSI Incorporated, Yellow Springs, OH, USA). Water quality parameters including TP, soluble reactive phosphorus (SRP) and total nitrogen (TN) were measured immediately after sampling according to Huang et al. (2000). 2.2. Laboratory experiment 2.2.1. Algae and culture condition Oscillatoria sp. FACHB-278 was obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences. The algae were cultured in BG11 medium (Rippka et al., 1979) at 25 ± 1  C under cool white fluorescent lamps at an illumination of 50 mmol photons m2 s1 with a 12:12-h light: dark cycle. Uniform culture of algal filaments used for inoculation was obtained by stirring and dispersion during the exponential growth phase in 5 L flasks. All glassware was soaked in 0.1 M HCl for 24 h and then rinsed with ultrapure water before the experiment. The medium was prepared using analytical grade reagents and ultrapure water. Flasks and BG11 medium were sterilized at 121  C for 30 min. 2.2.2. Substrates and pretreatments Four abiotic substrates and four macrophyte species were selected to test the physiological differences of Oscillatoria sp. in responses to abiotic and different macrophyte substrates. The abiotic substrates included pebble (long-axis of 3e6 cm, short axis of 2e4 cm and thickness of 0.5e1 cm), rough rock (length of 4e6 cm, width of 3e4 cm and height of 3e4 cm), PVC (poly-vinyl chloride, 2 cm  2 cm  1 mm) and PET (poly-ethylene terephthalate, 2 cm  2 cm  2 mm). The macrophyte species included Hydrilla verticillata, Potamogeton malaianus, Ceratophyllum oryzetorum and Myriophyllum spicatum. These four species of macrophyte are widely used for ecological restoration in eutrophic lakes (Ye et al., 2009; Rodrigo et al., 2013; Zeng et al., 2017). The abiotic substrates were carefully scrubbed and then sterilized at 121  C for 30 min (pebble and rough rock) or rinsed repeatedly with 75% alcohol (PVC and PET) before use. Pretreatment of macrophytes were conducted according to

Fig. 1. Relative cell density of filamentous algae in planktonic (phytoplankton) and periphytic (epipelon and epiphyton) forms in vegetated areas during bloom and nonbloom periods and in non-vegetated area in the West Lake.

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previously described methods (Nakai et al., 1999; Korner and Nicklisch, 2002; Wu et al., 2007; Wolters et al., 2019). Selected aquatic macrophytes were cultivated in tap water in a glass tank under natural light for 1 month. One day before the experiment, plants (top shoots with a length of 10e20 cm) were carefully rinsed under tap water to remove attached organisms and adhesive materials, followed by 10 min sonication in an ultrasonic bath. Pilot experiments showed that this did not affect the viability of plant.

2.2.3. Experimental design Co-culture of macrophytes and filamentous algae was used to simulate their real symbiotic state in natural waters. Macrophytes were rinsed repeatedly with sterilized water and planted in 500 mL beakers containing 300 mL of autoclaved BG11 medium at biomass densities of 2.5e3 g fw L1 according to those occurring in lakes (Zeng et al., 2017). FO in the exponential growth phase were inoculated with the four abiotic substrates (rocky: pebble and rough rock; plastic: PVC and PET) and four species of macrophyte (H. verticillata, P. malaianus, C. oryzetorum and M. spicatum). A pure culture of Oscillatoria sp. without any abiotic or macrophyte substrate was set as a control. To prevent nutrient limitation, low initial cell densities of uniform FO (2.18 ± 0.52 mg L1 Chl a) were set in each treatment, and the Chl a of PO was 0 mg L1. In our pilot experiments, FO and PO reached the stationary phase after 13 and 17 d of incubation, respectively. Thus, the co-culture experiment lasted 17 d.

2.2.4. Determination of algal growth Chlorophyll a (Chl a) concentration was used to monitor the growth of FO and PO. FO and PO were collected at day 0, 4, 7, 10, 13 and 17 for determination of algal growth. Algal suspension of FO was collected and filtered through a 0.45-mm pore size membrane filter (HA, Millipore). PO was carefully brushed and collected from surfaces of both substrates (abiotic substrates or macrophytes) and walls of the beakers for measurement of Chl a concentration. Chl a concentration was measured according to the method described by Stein (1973). Regular inspection with microscope showed that there were no other algae in cultures at the beginning of the experiment and that the biomass of other algae in the cultures never exceeded 8% of total algal biomass during the experiment. 2.2.5. Chlorophyll fluorescence parameters A small fraction of the light absorbed by algae is re-emitted as fluorescence, and in vivo chlorophyll fluorescence is an effective probe for algal photosynthesis (Krause and Weis, 1991). After incubation for 4 and 17 d, chlorophyll fluorescence parameters of FO and PO were measured to describe algal photosynthetic capacity. As FO was uniformly distributed in the medium, 5 mL of shaken medium was sampled. Three small pieces of biofilm with PO were randomly sampled. Chlorophyll fluorescence parameters were measured according to method described before (Shi et al., 2015).

2.2.6. P uptake kinetics Four abiotic substrates (pebble, rough rock, PVC, and PET) and M. spicatum showed no obvious effects on growth of Oscillatoria. H. verticillata and C. oryzetorum promoted growth of Oscillatoria, while P. malaianus inhibited growth of Oscillatoria. Therefore, after cultivation for 17 d, rough rock, M. spicatum, H. verticillata, and P. malaianus were selected to further explore effects of different substrates on P uptake kinetics of FO and PO. P uptake kinetics experiments were performed according to method described before (Shen and Song, 2007).

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Fig. 2. Linear regressions between macrophytes (A. biomass of C. oryzetorum and P. crispus, B. percentage of V. natans biomass) and logarithmic density of filamentous algae in water column in the West Lake.

2.3. Statistical analysis Origin 8.5.1 (Microcal Software, Northampton, MA, USA) was used for graphical plotting and curve fitting and SPSS 18.0 (Windows software, Inc., Chicago, IL, USA) was used for statistical analyses. Independent-sample T tests were used to compare differences in algal growth, photosynthetic parameters and P uptake parameters between FO and PO in control. Analysis of variance (ANOVA) followed by post-hoc Duncan's tests were used to analyze significant differences of data among different groups. All the data were tested for normality before performing independent-sample T tests and ANOVA. If necessary, data were log-transformed to approximate normality. Non-parametric analyses were carried out if data could not meet the normality even after transformation. Pearson's correlations analysis and linear regression were used to test for correlations of filamentous algal density in the water column with physico-chemical factors and with biomass of submerged macrophytes, respectively. Significant differences were set at the level of p ¼ 0.05.

influenced by other factors with similar physico-chemical levels. The data of macrophyte biomass were cited from Wang et al. (2017) (Fig. S4). Submerged macrophytes including M. spicatum, H. verticillata, V. natans, C. oryzetorum, N. major and P. crispus grew at M1 and M2, while no macrophyte grew at X. Meanwhile, nearly no filamentous algae were found at site X (non-vegetated area) in phytoplankton, epipelon, and epiphyton. Although relative densities of filamentous algae (Fig. 1) in phytoplankton, epipelon, and epiphyton were all high during the bloom, their relative density during non-bloom period were higher in epiphyton than in phytoplankton (p < 0.01) and epipelon (p < 0.001). This suggested that macrophytes might provide substrates for the attachment and growth of filamentous algae. Furthermore, the linear regression analysis (Fig. 2) indicated that logarithm of the filamentous algal density in water column was positively correlated with biomass of C. oryzetorum and P. crispus (p < 0.05), and negatively correlated with percentage of V. natans biomass (p < 0.01). However, no significant correlations were found between filamentous algae and other macrophyte species (N. major, H. verticillata and M. spicatum) (p > 0.05). This implied that specific plants may promote the proliferation of filamentous algae. Filamentous algal blooms could occur under a wide range of nutrient concentrations (Kjeldsen et al., 1996; Berry and Lembi, 2000; Gulati and van Donk, 2002; Ruley and Rusch, 2002; Irfanullah and Moss, 2004; Vis et al., 2008; Cattaneo et al., 2013; Quiblier et al., 2013; Frossard et al., 2014; Hudon et al., 2014). This suggest that other factors may play a more important role than nutrients in regulation the proliferation of filamentous algae. For example, Cladophora glomerata blooms occurred before and after implementation of P loading reductions along the shores of the North America's Great Lakes. Researches indicated that the former happened due to the high concentration of soluble P induced by anthropogenic eutrophication, and the latter happened because Zebra mussel invasion changed the substrate availability, water transparency and P cycle of plankton-benthic habitats at ecosystem level (Higgins et al., 2008). In our study, many environmental changes which affect the growth of filamentous algae have occurred in the West Lake, including decreased concentration of

3. Results & discussion 3.1. Factors influencing bloom of filamentous algae in eutrophication restoration waters During the field study, severe filamentous algal blooms occurred in vegetation sites at M1 and M2 in Apr 2014 and Apr 2015, respectively. Dominant species of the bloom were Oscillatoria sp. (72.55% of biomass) and M. varians (Fig. S2, 10.93%) in M1, and Spirogyra sp. (50.82%), Oscillatoria sp. (19.2%) and Cladophora sp. (5.07%) in M2. The cell density reached 7.6  108 cells L1 at M1 in Apr 2014 and 1.3  107 cells L1 at M2 in Apr 2015, respectively. During the bloom, waterbody was dark green and turbid with a layer of flocculent algal mat floating on the surface of the water (Fig. S3). However, during non-bloom period, algal density in water column was only 104-106 cells L1 (Table S2), and water was clear with visible bottom almost at all the study sites. The physico-chemical features of sampling sites were listed in Table S3. The physico-chemical features were similar between bloom and non-bloom sites in vegetation areas. Results of pearson's pairwise correlation analysis (Table S4) showed that physicochemical factors including water temperature, pH, DO, ORP, TN, TP and SRP showed no significant correlations with density of filamentous algae in water column (p > 0.05). These results indicated that the proliferation of filamentous algae might be

Fig. 3. Growth curves (A) of free-floating Oscillatoria (FO) and periphytic Oscillatoria (PO) in controls from day 0e17, and P uptake parameters (B and C) and chlorophyll fluorescence parameters (D, E and F) on day 17 (mean ± SD, n ¼ 3). Statistical significances are indicated as: *p  0.05, **p  0.01.

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Table 1 Kinetic constants for P uptake of free-floating Oscillatoria (FO) and periphytic Oscillatoria (PO) with no substrate (control), abiotic (rough rock) and biotic (M. Spicatum, H. verticillata, and P. malaianus) substrates (25  C) on day 17 (mean ± SD, n ¼ 3). Substrates

Vm (mmol P mg Chl a1 h1) FO

Control Rough rock H. verticillata M. spicatum P. malaianus

Km (mM P) PO

a

49.742 ± 6.613 46.355 ± 9.903a 59.613 ± 2.774a 190.387 ± 21.839b 154.226 ± 13.581c

FO d

8.935 ± 0.194 8.194 ± 0.742d 14.581 ± 0.581e 5.323 ± 0.161f 2.774 ± 0.129g

PO a

12.194 ± 2.677 12.871 ± 2.129a 11.548 ± 2.161a 24.258 ± 4.742b 23.968 ± 2.871b

6.387 ± 0.903c 7.29 ± 2.065cd 10.323 ± 1.774d 15.645 ± 1.29e 7.774 ± 1.419cd

Differences in lowercase letters indicate significant differences in Vm and Km values among treatments obtained from a two-way ANOVA and post hoc multiple comparisons with the Duncan’s test at p < 0.05.

nutrients (mainly P) (Schindler et al., 2016), improved light conditions (Asaeda and Van Bon, 1997) and restored macrophytes (Hilt et al., 2006). In terms of external factors, restoration of aquatic vegetation can provide new attaching substrates for filamentous algae. It's well known that Oscillatoria can form biofilms on attached substrates (Hassan and Lim, 2012). Thus, plants in the West Lake could provide substrates for Oscillatoria to attach on, and Oscillatoria was thought to create a periphytic layer on these plants. The correlation between macrophytes and filamentous algae was tentatively explored in our field study above, and the physiological mechanism behind these results was further explored in our following experiments. Internal factors speaking, filamentous algae possess free-floating and periphytic life forms, and the latter accounted for majority of the biomass. We also determined whether and how they respond to changes in nutrient and light in laboratory.

3.2. Different physiological characteristics between FO and PO To date, few studies have compared physiological differences between free-floating and periphytic filamentous algae. The layers of periphytic Oscillatoria were formed on the surfaces of 4 macrophytes and 4 abiotic substrates after inoculation of Oscillatoria filaments. Oscillatoria twined around the macrophyte leaves and formed a layer of biofilm with reticulated structures. PO showed significantly higher (p < 0.001) growth potential than FO, and PO were responsible for most biomass of Oscillatoria (Fig. 3A). Chlorophyll fluorescence parameters on day 17 showed that PO had significantly higher ETRmax and a values than FO (p < 0.01, p < 0.05, respectively) (Fig. 3E and F). FO had a higher (p < 0.05) maximum P uptake rate (Vm) than PO (Fig. 3B), while PO had a stronger affinity for P (lower Km) than FO (Fig. 3C). In summary, compared with FO, PO showed faster growth rate, greater photosynthetic capacities

Fig. 4. Growth (Log (Chl aþ1), mg L1) of free-floating Oscillatoria (FO) (A), periphytic Oscillatoria (PO) (B) and total Oscillatoria (C) with no substrate (control), abiotic materials (plastic: PET, PVC; rocky: pebble, rough rock) and submerged macrophytes (H. verticillata, C. oryzetorum, M. spicatum and P. malaianus) as substrates (mean ± SD, n ¼ 3). Statistical significance is indicated as: *p  0.05 vs controls, **p  0.01 vs controls, ***p  0.001 vs controls.

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and higher P affinity. PSII electron transport (ETRmax values) can match well with CO2 fixation in the dark reaction of photosynthesis (Genty et al., 1989). Furthermore, different efficiencies in capturing light energy (a values) and CO2 fixation directly determine the variations in algal biomass (Atta et al., 2013). Thus, higher photosynthetic activity of PO might explain its faster growth. Also, higher P affinity of PO provides a competitive advantage, especially under P-limited conditions. It is known that periphytic algae are competitively superior in utilizing nutrients from sediments, whereas planktonic algae have a competitive advantage with respect to light (Hansson, 1988; Vadeboncoeur et al., 2002). Thus, pelagic algae might be at a disadvantage at low nutrient concentrations, while periphytic algae might no longer be subject to light limitation if water clarity is improved (Hansson, 1992). Moreover, bottom sediments provide a rich supply of nutrients for the proliferation of algal biofilms (Gainswin et al., 2006; Rydin et al., 2017). Therefore, our results indicate that physiological superiority of PO under low P concentrations and improved light conditions could facilitate the rapid proliferation of PO and thus the formation of algal mats, ultimately triggering the bloom of Oscillatoria. 3.3. Different effects of abiotic and macrophyte substrates on physiological characteristics of FO and PO Our laboratory experiment further explored whether and why filamentous algae respond differently to different substrates (abiotic substrates and macrophytes). Overall, results showed that macrophyte species played a much more important role than abiotic substrates in the proliferation of filamentous algae, and that influences of macrophytes on FO and PO were species-specific. On day 17 (logarithmic growth phase), compared with control, all the abiotic substrates exerted no significant effects on growth, photosynthetic activity and P uptake property of FO and PO (p > 0.05), except a significant promotion of rough rock on the growth of FO (p < 0.01) and a decrease of smooth pebble on Fv/Fm value of FO (p < 0.05) (Figs. 4, 5, S5 and Table 1). Results of previous studies

show that periphytic filamentous algae grow faster on rough substrates than on smooth ones (Schneck et al., 2011; Hassan and Lim, 2012). Similar effects on growth of total filamentous algae were also observed between rough rock and smooth pebble. These may be due to larger surface area of rough rock and it is easier for filamentous algae to attach on surface of rough substrates. However, no obvious effects of rough rock on PO were observed. These might be explained by switch of PO to FO. Compared with the controls, physiological features of FO and PO in the four macrophyte substrates showed significant differences. On day 17, compared with control, growth of both FO (p < 0.05) and PO (p < 0.001) and photosynthetic activity (Fv/Fm value of PO, p < 0.001) were significantly increased by H. verticillata and C. oryzetorum (Figs. 4 and 5). In our field investigation, biomass of C. oryzetorum were positively correlated with density of filamentous algae in water column, while no significant correlation between filamentous algae and H. verticillata was found. This might be due to the fact that the low occurrence frequency of H. verticillata (only once throughout the investigation). Earlier studies indicated that the leaves of some submerged macrophytes with fine branches can provide complex habitats for epiphytic algae and promote their growth (Toporowska, 2008; Wolters et al., 2019). This may explain the results in our study. In laboratory experiment, M. spicatum did not affect (p > 0.05) growth of Oscillatoria sp. compared with control (Fig. 4). Although M. spicatum notably increased Fv/Fm value of PO (p < 0.05) and maximum P uptake velocity (Vm) of FO (p < 0.05), Vm of PO (p < 0.05) and P affinity (higher Km) of both FO and PO (p < 0.05) were significantly decreased (Fig. 5 and Table 1). This was consistent with our field observation that M. spicatum had no significant correlation with density of filamentous algae in water column. M. spicatum had allelopathic effect on the growth of some filamentous cyanobacteria, such as Aphanizomenon flos-aquae, Planktothrix agardhii and Limnothrix redekei (Korner and Nicklisch, 2002). Conversely, some studies indicate that increasing surface roughness of substrates increases abundance of Oscillatoria sp. and other filamentous algae attached on the substrates (Schneck et al., 2011;

Fig. 5. Fv/Fm values for free-floating life form of Oscillatoria (FO) and periphytic life form of Oscillatoria (PO) with abiotic materials (PET, PVC, pebble and rough rock) and submerged macrophytes (H. verticillata, C. oryzetorum, M. spicatum and P. malaianus) as substrates after incubation for 4 (A) and 17 (B) d (mean ± SD, n ¼ 3). Values of control (no substrates) were set to 100% for comparison. Statistical significances are indicated as: *p  0.05 vs control, ***p  0.001 vs control.

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Hassan and Lim, 2012). Thus, the complex habitats provided by the fine branching structure of M. spicatum could promote growth of Oscillatoria sp. Therefore, our results might be due to a trade-off between allelopathic inhibition and growth promotion of Oscillatoria sp. by M. spicatum. P. malaianus inhibited growth and phosphorus uptake of Oscillatoria sp. due to higher sensitivity of PO to P. malaianus than that of FO. According to laboratory results, P. malaianus exerted no effect (p > 0.05) and inhibition (p < 0.05) on growth of FO and PO, respectively (Fig. 4). Moreover, P. malaianus significantly increased (p < 0.05) and decreased (p < 0.05) Vm of FO and PO, respectively (Table 1). This was contradicted by a previous study which showed that epiphytic algae were less sensitive towards allelochemicals released by plants than planktonic algae due to co-evolution (Hilt and Gross, 2008). However, in another study, allelopathic effects of macrophyte extracts on epiphytic algae were recognized and a mass of almost epiphyte-free macrophytes were found (WiumAndersen, 1971). Therefore, in our study, higher sensitivity of PO to P. malaianus than that of FO might be ascribed to a closer distance of PO than FO to harmful substances released by the macrophyte. 3.4. Suggestions for selecting appropriate macrophyte species for restoration It's known that benthic filamentous algae often form mats at the bottom of water bodies and appear on the surface of the lakes in the afternoon, especially when the atmospheric pressure drops (Speziale et al., 1991; Hudon et al., 2014). However, our study found another source for such mats. Both field investigations and laboratory experiments showed that certain macrophyte species could promote the proliferation of Oscillatoria by providing substrates. Moreover, our laboratory results implied that P reduction and transparency improvement in restored lakes could promote the rapid formation of Oscillatoria biofilms both on the sediment and on the macrophytes, which could trigger their accumulation on the surface. These also suggest the importance of appropriately selecting macrophyte species to prevent filamentous algal bloom in restoration of eutrophic lakes. Considering the synchrony between macrophyte germination and filamentous algal occurrence in early spring, it is not recommended, in this season, to harvest some macrophytes (Quilliam et al., 2015; Tang et al., 2017) (eg. H. verticillata and C. oryzetorum) even which can promote filamentous algae. Therefore, in the early spring, we should avoid planting of H. verticillata and C. oryzetorum that promote growth of filamentous algae, while P. malaianus which can inhibit filamentous algae can be recommended as a pioneer species. Furthermore, some macrophytes like M. spicatum, H. verticillata and C. oryzetorum have strong capability of nutrients removal (Dai et al., 2012; Wan et al., 2016; Lu et al., 2018) and allelopathic inhibition to phytoplankton (Mohamed, 2017). Thus, these species can be transplanted during the vigorous growth period of macrophytes and phytoplankton when development of filamentous algae is not suitable. In this regard, further studies are needed to evaluate the influences of other macrophyte species on the growth of filamentous algae. 4. Conclusions Our study indicated that PO was physiologically superior to FO in photosynthetic activity and P uptake, which might stimulate proliferation of PO and quick formation of Oscillatoria mats under improved light condition and reduced P level during eutrophication restoration. Moreover, macrophytes might be suitable substrates for colonization of filamentous algae. Some macrophyte substrate could promote obvious growth of Oscillatoria but others cause inhibition. These species-dependent influences of macrophytes on

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filamentous algae are of practical importance in the restoration and management of lake ecosystems. Acknowledgments The authors are grateful to two anonymous reviewers for their valuable comments and suggestions. We thank Professors Zhenbin Wu and Biyun Liu for their help on sample collections, Drs. Wulai Xia, Li Wang, Feng Chen, Fenli Min and Lei Zeng for their help on the field study and laboratory experiments. We thank Dr. Hong Fu for generously providing micrograph of filamentous algae and scene photos. We thank Dr Jeffrey S. Owen for his professional editing. We appreciate Dr Liang Chen for his help in paper revision. This work was jointly supported by the National Key Research and Development Program of China [grant number 2017YFA0605201], the National Major Science and Technology Program for Water Pollution Control and Treatment [grant number 2017ZX0740100301], the Featured Institute Service Projects from Institute of Hydrobiology, Chinese Academy of Sciences [grant number Y85Z061601] and the State Key Laboratory of Freshwater Ecology and Biotechnology [grant number 2019FBZ03]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124702. Disclosure statement None. References Allen, J.L., Ten-Hage, L., Leflaive, J., 2016. Allelopathic interactions involving benthic phototrophic microorganisms. Environ. Microbiol. Rep. 8, 752e762. Asaeda, T., Van Bon, T., 1997. Modelling the effects of macrophytes on algal blooming in eutrophic shallow lakes. Ecol. Model. 104, 261e287. Atta, M., Idris, A., Bukhari, A., Wahidin, S., 2013. Intensity of blue LED light: a potential stimulus for biomass and lipid content in fresh water microalgae Chlorella vulgaris. Bioresour. Technol. 148, 373e378. Berry, H.A., Lembi, C.A., 2000. Effects of temperature and irradiance on the seasonal variation of a Spirogyra (chlorophyta) population in a midwestern lake (USA). J. Phycol. 36, 841e851. Cattaneo, A., Hudon, C., Vis, C., Gagnon, P., 2013. Hydrological control of filamentous green algae in a large fluvial lake (Lake Saint-Pierre, St. Lawrence River, Canada). J. Great Lakes Res. 39, 409e419. Chun, C.L., Ochsner, U., Byappanahalli, M.N., Whitman, R.L., Tepp, W.H., Lin, G., Johnson, E.A., Peller, J., Sadowsky, M.J., 2013. Association of toxin-producing Clostridium botulinum with the macroalga Cladophora in the Great lakes. Environ. Sci. Technol. 47, 2587e2594. Dai, Y., Jia, C., Liang, W., Hu, S., Wu, Z., 2012. Effects of the submerged macrophyte Ceratophyllum demersum L. on restoration of a eutrophic waterbody and its optimal coverage. Ecol. Eng. 40, 113e116. Fattom, A., Shilo, M., 1984. Hydrophobicity as an adhesion mechanism of benthic cyanobacteria. Appl. Environ. Microbiol. 47, 135e143. Frossard, V., Versanne-Janodet, S., Aleya, L., 2014. Factors supporting harmful macroalgal blooms in flowing waters: a 2-year study in the Lower Ain River, France. Harmful Algae 33, 19e28. Fu, H., Xu, J., Xiao, E., He, F., Xua, P., Zhou, Q., Wu, Z., 2017. Application of dual stable isotopes in investigating the utilization of two wild dominant filamentous algae as food sources for Daphnia magna. J. Freshw. Ecol. 32, 339e351. Gainswin, B.E., House, W.A., Leadbeater, B.S.C., Armitage, P.D., Patten, J., 2006. The effects of sediment size fraction and associated algal biofilms on the kinetics of phosphorus release. Sci. Total Environ. 360, 142e157. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87e92. Green, L., Fong, P., 2016. The good, the bad and the Ulva: the density dependent role of macroalgal subsidies in influencing diversity and trophic structure of an estuarine community. Oikos 125, 988e1000. Gulati, R.D., van Donk, E., 2002. Lakes in The Netherlands, their origin, eutrophication and restoration state-of-the-art review. Hydrobiologia 478, 73e106. Hansson, L.A., 1988. Effects of competitive interactions on the biomass development of planktonic and periphytic algae in Lakes. Limnol. Oceanogr. 33, 121e128. Hansson, L.A., 1992. Factors regulating periphytic algal biomass. Limnol. Oceanogr.

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