Comparison of growth and nutrient uptake capacities of three dominant species of Qinhuangdao green tides

1. Introduction

Green macroalgae blooms, referred to as green tides, are formed primarily by the accumulation of unattached macroalgal biomass including Ulva, Chaetomorpha, Cladophora, and Ulvaria species (Fletcher, 1996; Nelson et al., 2003). During the past several decades, green tides have occurred regularly in coastal areas around the worldwide (Teichberg et al., 2010). International studies on the causes of green tide formation have shown that green tides usually occur in areas with relatively high eutrophication levels, and eutrophication is considered an important cause of green tides (Kamer et al., 2001; Charlier et al., 2007; Yabe et al., 2009; Teichberg et al., 2010). Compared with other algae, green tide algae, the species causing green tides, have stronger competitive advantages regarding nutrient uptake (Luo et al., 2012). In eutrophic waters, they can rapidly absorb large amounts of nutrients and maintain a high growth rate, resulting in the continuous increase in algae biomass and making it the dominant species (Luo et al., 2012; Wang et al., 2012). Green tide algae can also store excess nutrients to maintain a high growth rate under low-nutrient conditions (Pedersen and Borum, 1997).

Since 2015, a novel green tide has been blooming in Qinhuangdao City (Hebei Province, China) on the western coast of the Bohai Sea (Han et al., 2019; Song et al., 2019b). The dominant species of green tides in offshore waters of Qinhuangdao include Ulva australis, Bryopsis plumosa, and Ulva prolifera, with obvious morphological differences (Song et al., 2019a). In previous publications, Ulva pertusa has been reported to be one of dominant species for the green tide in Qinhuangdao (Song et al., 2019a, 2019b; Han et al., 2019, 2022a). Since this species has been identified as a synonym of U. australis recently (Hughey et al., 2021), we replaced U. pertusa with U. australis in this text. Of these, U. australis is a economic macroalgae widely distributed along the coastal areas of the western North Pacific (Tseng, 1983). It has a slightly yellow color, with wrinkles at the edge and irregular pores on the surface. The species Bryopsis plumosa is a green macroalgae distributed worldwide and a well-known nutrient pollution indicator (Lapointe et al., 2010). Bryopsis plumosa is dark green in color and has a main branch and several irregular branches. In addition,U. prolifera is also the causative species in the world’s largest green macroalgae blooms in the Yellow Sea, China (Liu et al., 2010; Wang et al., 2015). It is bright green in color and tubular, with more branches (Zhang et al., 2013; Song et al., 2019b). There is an obvious succession phenomenon during the occurrence of Qinhuangdao green tides (Han et al., 2019; Song et al., 2019a, 2019b). According to the different dominant species, the occurrence of green tides can be divided into three stages (Song et al., 2019a). The first stage lasts from late April to mid-May, during which the biomass levels are low and U. australis is the main species. In the second stage, biomass increases gradually from mid-May to mid-June, and the dominant species isB. plumosa. The third stage occurs from mid-June to late September, when green algal biomass is highest and the duration is longest. The species U. prolifera is the dominant species in this stage (Han et al., 2019; Song et al., 2019a). However, it is still unclear which environmental factors drive the succession of dominant species of green tides in Qinhuangdao and which physiological characteristics of U. prolifera are involved in the competition with the other two species, making it the dominant species with the largest biomass.

We speculated that the different adaptation strategies of U. australis,B. plumosa, and U. prolifera to environmental factors are responsible for dominant species succession. In this work, we investigated the effects of temperature, irradiance, and nutrients on the growth rates of U. australis, B. plumosa, and U. prolifera and compared their nutrient uptake capacities. By this investigation, it would be possible to answer the following two questions: (1) was the succession of dominant species of green tide in Qinhuangdao related to the dominance of growth of the three dominant species over a range of temperature and light? (2) was the growth and nutrient uptake capacity of U. prolifera higher than that of U. australis and B. plumosa?

2. Materials and methods

2.1 Algal material

During green tides outbreak, vast numbers of suspended macroalgae accumulate primarily on the Jinmenghaiwan Bathing Beach of Qinhuangdao, which is the main area affected by green tides in Qinhuangdao. Macroalgae were sampled from the Jinmenghaiwan Bathing Beach (39°53′42.93″N, 119°32′34.49″E) in July 2019. Morphological characteristics and internal transcribed spacer (ITS) sequences and 5S ribosomal DNA (5S rDNA) spacer sequences were chosen for species identification.

These samples were cleaned thrice using sterilized seawater on site, dried with absorbent paper, stored in liquid nitrogen, then transported to the laboratory within 48 h. In order to minimize the effect of natural environmental conditions on algal physiological traits, each algal sample was cultured for 3–5 d using air stones before the experiment. The condition was kept at 16℃ at an irradiance of 100 μmol/(m²·s) in a 12 h light:12 h dark cycle.

2.2 Competitive growth of the three algae species under different temperature and light conditions

To test the effects of temperature and light on the growth of U. australis, B. plumosa, and U. prolifera, we selected the temperatures of 10℃, 15℃, 20℃, and 25℃, with the light intensities of 50 μmol/(m²·s), 100 μmol/(m²·s), 150 μmol/(m²·s), and 200 μmol/(m²·s). Nutrients were supplemented according to f/2 medium, and the experiment was carried out in a glass flask with 200 mL of f/2 medium, which was added every 5 d during the culture period with 12 h light: 12 h dark cycle. The initial biomass of each algae was 0.2 g. The experiment was divided into two groups, single culture and co-culture. In the single culture, U. australis, B. plumosa, and U. prolifera were cultured separately under different temperature and light conditions. According to the succession rule of dominant species, co-culture groups were defined as follows: (1) U. australis and B. plumosa at 10℃; (2) U. australis and B. plumosa at 20℃; (3) B. plumosa and U. prolifera at 20℃; and (4) B. plumosa and U. prolifera at 25℃. The light intensities for each group were 50 μmol/(m²·s), 100 μmol/(m²·s), 150 μmol/(m²·s), and 200 μmol/(m²·s). Each group consisted of three replicates. The algae were weighed after 10 d, and the relative growth rate (RGR) was calculated as follows: RGR = (lnw₂−lnw₁) /t, where w₁ denotes the initial fresh mass and w₂ the fresh mass after t d (Cui et al., 2015). We selected the RGR as the evaluation indicator of the effect.

2.3 Effects of ${{\boldsymbol {NO}}_3^-} $ , ${{\boldsymbol {NH}}_4^+}$ and ${{\boldsymbol {PO}}_4^{3-}}$ on algal growth rates

The experimental period for investigating the effects of nitrogen and phosphorus on the growth of U. prolifera,U. pertulva, and B. plumosa was 10 d. Nitrogen available for macroalgal uptake in coastal waters can be in the form of nitrate and ammonium, we used two different nitrogen sources, namely nitrate (NaNO₃) and ammonium (NH₄Cl). To prevent phosphate restriction during the experiment, 30 μmol/L phosphate (KH₂PO₄) was added to all experiments. In the nitrogen source experiment, the concentrations of nitrogen ( ${{\rm {NO}}_3^-} $ or ${{\rm {NH}}_4^+} $ ) were 5 μmol/L, 10 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L. NaNO₃, NH₄Cl, and KH₂PO₄ were applied to adjust the nutrient concentrations. In the phosphate experiment, the concentrations of phosphate were 5 μmol/L, 10 μmol/L, 20 μmol/L, 30 μmol/L, and 40 μmol/L. To prevent nitrogen restriction, 50 μmol/L ${{\rm {NO}}_3^-} $ and 50 μmol/L ${{\rm {NH}}_4^+} $ were added to each group of experiments. Briefly, we placed 0.2 g of algae into a 1 L glass bottle which contained 500 mL of sterile artificial sea water and added the corresponding nutrients. We selected the RGR as the evaluation indicator of the effect. The experiment was carried out at 20℃ and 100 μmol/(m²·s) with a 12 h light: 12 h dark cycle. Each group consisted of three replicates. Previous studies have shown that the third stage with U. profilera as the dominant species, which has the largest biomass, tnolhe longest duration, and the greatest impact on the environment, begins in mid-June. According to our field survey, the average temperature and light intensity in the sea area in mid-June were about 20℃ and 100 μmol/(m²·s), therefore, in order to simulate the environmental conditions in the field to the greatest extent, above environmental conditions were designed to explore whether U. profilera may became the dominant species with the largest biomass.

2.4 Effects of ${{\boldsymbol {NO}}_3^-}$ , ${{\boldsymbol {NH}}_4^+}$ and ${{\boldsymbol {PO}}_4^{3-}}$ on nutrient uptake

The nitrogen ( ${{\rm {NO}}_3^-} $ and ${{\rm {NH}}_4^+} $ ) uptake experiment was carried out in 1-L sterile glass bottles, whereas for the phosphate uptake experiment, 1-L sterile plastic bottles were used. Prior to use, all bottles were cleaned with 10% hydrochloric acid. In the nitrogen uptake experiment, the concentrations of ${{\rm {NO}}_3^-} $ or ${{\rm {NH}}_4^+} $ were 5 μmol/L, 10 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L. To avoid phosphorus restriction, 30 μmol/L ${{\rm {PO}}_4^{3-}} $ was added. In the phosphorus uptake experiment, the concentrations of ${{\rm {PO}}_4^{3-}} $ were 5 μmol/L, 10 μmol/L, 20 μmol/L, 30 μmol/L, and 40 μmol/L. To prevent nitrogen restriction, 50 μmol/L ${{\rm {NO}}_3^-} $ and 50 μmol/L ${{\rm {NH}}_4^+} $ were added. The initial biomass for this part of experiment is 0.2 g. Nitrogen starvation incubation was performed for about 3 d before the start of the experiment. In all experiments, 2 mL seawater samples were collected after 1 h, 2 h, 3 h, and 4 h, and the nutrient concentration was analyzed using an automatic nutrient analyzer (AA3, BRAN LUEBBE). The experiment was carried out at 20℃ and 100 μmol/(m²·s) with a 12 h light: 12 h dark cycle. The algal material was oven-dried at 60℃ for 72 h to determine the dry mass (D_M). Uptake rates (V) were calculated from changes in substrate concentrations during each sampling interval using the equation:

where C₀ denotes the initial nutrient concentration, C_t is the concentration after t d, V₀ denotes the initial amount of water, V_t is the amount of water after t d, t denotes the sampling interval time, and D_M represents the dry weight of algal biomass.

The Michaelis-Menten equation is widely used to describe the growth and nutrient uptake of macroalgae. The competitive ability of macroalgae to obtain nutrients can be determined using the kinetic parameter R_max (maximum absorption rate). The parameter R_max is obtained from a hyperbolic plot of absorption rates at a series of concentrations and is used to measure the ability of macroalgae to absorb nutrients at different concentrations. The R_max/K_s ratio describes the relationship between uptake rate and substrate concentration, with higher values suggesting competitive advantages and higher affinity.

2.5 Data analysis

Tests of homogeneity of variance were conducted, and separate two-way analysis of variance (ANOVA) was performed. Differences were considered significant at P<0.05. Data were analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The data of the uptake rate experiment were analyzed using multivariate variance. Tukey’s test was performed for all parameters.

3. Results

3.1 Molecular identification of macroalgae

The Neighbor-Joining Tree based on the ITS gene showed that the 15 green macroalgal samples belonged to 3 clades (Fig. 1). All of the K samples grouped together in the U. pertusa/australis clade. All of the Y samples grouped together in the B. plumosa clade. All of the H samples showed the highest affiliation with the U. prolifera/linza clade. However, according to the phylogeny of the 5S spacer region, the U. prolifera/linza clade in the phylogenetic tree based on 5S sequence was divided clearly into two groups (U. prolifera and U. linza, Fig. 2). All of the H samples grouped in the U. prolifera clade. The sequences of 15 samples have been submitted to NCBI, and the sequence numbers are shown in Table 1.

Figure  1.  Phylogenetic tree determined by analysis of internal transcribed spacer sequences of macroalgae. Numbers at the nodes were bootstrapping support values larger than 50% after 1000 replicates in Neighbor-Joining analyses.

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Figure  2.  Phylogenetic tree determined by analysis of 5S sequences of macroalgae. Numbers at the nodes were bootstrapping support values larger than 50% after 1000 replicates in Neighbor-Joining analyses.

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Table  1.  Accession numbers of the three dominant species of Qinhuangdao green tides

Sample numbers	Primes	Molecular identification	Accession numbers
K-1; K-2; K-3; K-4; K-5	ITS	Ulva australis	ON263399; ON263400; ON263401; ON263402; ON263403
Y-1; Y-2; Y-3; Y-4; Y-5	ITS	Bryopsis plumosa	ON263404; ON263405; ON263406; ON263407; ON263408
H-1; H-2; H-3; H-4; H-5	ITS	Ulva linza/prolifera	ON263394; ON263395; ON263396; ON263397; ON263398
H-1; H-2; H-3; H-4; H-5	5S	Ulva prolifera	ON286724; ON286725; ON286726; ON286727; ON286728
Note: ITS is the abbreviation of internal transcribed spacer.

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3.2 Effects of temperature and light on the growth of dominant green tide species

Within the temperature and light ranges of the experimental setup, the optimum combination of temperature and light for U. australis was 10℃ and 150 μmol/(m²·s), resulting in a growth rate of about 12% per day (Fig. 3). The RGR of U. australis decreased with increasing temperature in the range of 15–25℃. The optimum temperature and light conditions for the growth of B. plumosa were 15℃ and 150 μmol/(m²·s), obtaining RGR of 10.2% per day (Fig. 4). The temperature range of 20–25℃ was most suitable for the growth of U. prolifera. The optimum temperature and light conditions forU. prolifera were 20℃ and 200 μmol/(m²·s), resulting in RGR of 18.6% per day (Fig. 5).

Figure  3.  Effects of temperature and light on the growth of Ulva australis. RGR: relative growth rate.

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ANOVA was performed to test the significant effects of light and temperature on the growth of U. prolifera,U. australis, and B. plumosa. The results showed that temperature had a significant effect on the growth of the three algaes (P<0.01), while light had no significant effect on the growth of the three seaweeds relative to temperature (P>0.05). So, temperature exerted a stronger influence on the RGR of all the three species. For example, at a light condition of 150 μmol/(m²·s), the RGR of U. prolifera varied from 4.2% to 16.8% at a temperature of 10−25℃. In comparison, the variation of RGR at different light levels was relatively small at a certain temperature. RGR ofU. australis, B. plumosa, andU. prolifera were generally highest at 15℃, 20℃, and 25℃, respectively, and decreased towards both lower and higher temperatures.

All co-culture experiments were conducted under a light range of 50−200 μmol/(m²·s). At the temperature of 10℃, the growth rate of U. australis was significantly higher than that of B. plumosa (P<0.05). The growth rate ofB. plumosa was significantly higher than that ofU. australis at the temperature of 15℃ (P<0.05). At 20℃ and 25℃, the growth rate ofU. prolifera was significantly higher than that of B. plumosa (P<0.05) (Fig. 6).

Figure  4.  Effects of temperature and light on the growth of Bryopsis plumose. RGR: relative growth rate.

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Figure  5.  Effects of temperature and light on the growth of Ulva prolifera. RGR: relative growth rate.

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Figure  6.  The relative growth rate (RGR) of co-culture two algae under different temperature and light conditions.

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3.3 Effect of nutrients on the RGR of dominant green tide species

At the same concentrations of ${{\rm {NO}}_3^-} $ , ${{\rm {NH}}_4^+} $ , or ${{\rm {PO}}_4^{3-}} $ , the growth rate of U. prolifera was significantly higher than those of B. plumosa and U. australis (P<0.05). Compared with ${{\rm {NO}}_3^-} $ and ${{\rm {NH}}_4^+} $ , changes in ${{\rm {PO}}_4^{3-}} $ concentration had no significant effect on the growth rates of all three species (P>0.05) (Figs 7−9).

Figure  7.  The relative growth rate (RGR) of three species of green tide algae at different nitrate concentrations.

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Figure  8.  The relative growth rate (RGR) of three species of green tide algae at different ammonium salt concentrations.

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Figure  9.  The relative growth rate (RGR) of three species of green tide algae at different phosphate concentrations.

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3.4 Nutrient uptake by dominant green tide species

The uptake rates of ${{\rm {NO}}_3^-} $ , ${{\rm {NH}}_4^+} $ , and ${{\rm {PO}}_4^{3-}} $ were accelerated with increasing nutrient levels. In the 4-h experiment, the nutrient uptake rates of the three species decreased gradually over time. In the same period, U. prolifera had stronger uptake rates of ${{\rm {NO}}_3^-} $ , ${{\rm {NH}}_4^+} $ , and ${{\rm {PO}}_4^{3-}} $ than B. plumosa and U. australis (P<0.05) (P>0.05) (Figs 10−12).

Figure  10.  Comparison of the nitrate uptake capacity of Ulva australis, Bryopsis plumosa, and Ulva prolifera.

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Figure  11.  Comparison of the ammonium uptake capacity of Ulva australis, Bryopsis plumosa, and Ulva prolifera.

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Figure  12.  Comparison of the phosphate uptake capacity of Ulva australis, Bryopsis plumosa, and Ulva prolifera.

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Bryopsis plumosa,U. australis, and U. prolifera showed saturated ${{\rm {NO}}_3^-} $ , ${{\rm {NH}}_4^+} $ , and ${{\rm {PO}}_4^{3-}} $ uptake kinetics. In the 4-h experiment, The R_max and R_max/K_s values of U. prolifera were higher compared with those of U. australis and B. plumosa. Compared with the uptake of ${{\rm {NO}}_3^-} $ , the uptake of ${{\rm {NH}}_4^+} $ resulted in higher R_max values for all species. Compared with phosphate uptake, the three species showed higher R_max values for nitrogen uptake ( ${{\rm {NO}}_3^-} $ and ${{\rm {NH}}_4^+} $ ) (Tables 2-4).

Table  2.  Kinetic parameters of nutrient uptake at different concentrations forUlva prolifera

Nutrition	Time	Ulva prolifera
		Rmax/(μmol·g−1·h−1)	Ks/(μmol·L−1)	Rmax/Ks	r2
${{\rm {NO}}_3^-} $	0−1 h	104.31	8.19	12.74	0.98
	1−2 h	81.84	16.69	4.90	0.98
	2−3 h	65.34	19.48	3.35	0.98
	3−4 h	50.31	24.48	2.06	0.97
${{\rm {NH}}_4^+} $	0−1 h	157.35	12.68	12.41	0.99
	1−2 h	118.62	18.80	6.31	0.99
	2−3 h	85.41	19.71	4.33	0.98
	3−4 h	66.70	22.20	3.00	0.98
${{\rm {PO}}_4^{3-}} $	0−1 h	48.41	16.72	3.90	0.98
	1−2 h	44.25	19.27	2.30	0.94
	2−3 h	23.32	9.98	2.34	0.98
	3−4 h	19.57	14.53	1.35	0.99

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Table  3.  Kinetic parameters of nutrient uptake at different concentrations for Bryopsis plumosa

Nutrition	Time	Bryopsis plumosa
		Rmax/(μmol·g−1·h−1)	Ks/(μmol·L−1)	Rmax/Ks	r2
${{\rm {NO}}_3^-} $	0−1 h	79.43	13.4	5.93	0.97
	1−2 h	49.48	13.92	3.55	0.96
	2−3 h	29.83	12.68	2.35	0.95
	3−4 h	25.72	14.58	1.76	0.93
${{\rm {NH}}_4^+} $	0−1 h	117.59	21.81	5.39	0.96
	1−2 h	89.83	20.68	4.34	0.98
	2−3 h	52.12	18.76	2.78	0.99
	3−4 h	45.47	33.69	1.35	0.97
${{\rm {PO}}_4^{3-}} $	0−1 h	22.48	7.21	3.12	0.92
	1−2 h	20.79	14.99	1.39	0.98
	2−3 h	10.17	5.73	1.77	0.98
	3−4 h	11.89	18.56	0.64	0.93

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Table  4.  Kinetic parameters of nutrient uptake at different concentrations for Ulva australis

Nutrition	Time	Ulva australis
		Rmax/(μmol·g−1·h−1)	Ks/(μmol·L−1)	Rmax/Ks	r2
${{\rm {NO}}_3^-} $	0−1 h	83.42	9.46	8.82	0.98
	1−2 h	71.31	17.82	4.00	0.97
	2−3 h	52.96	21.82	2.43	0.98
	3−4 h	33.56	20.75	1.82	0.98
${{\rm {NH}}_4^+} $	0−1 h	138.38	17.36	7.97	0.99
	1−2 h	107.06	22.10	4.84	0.98
	2−3 h	65.80	18.01	3.65	0.97
	3−4 h	51.45	25.58	2.01	0.97
${{\rm {PO}}_4^{3-}} $	0−1 h	21.82	12.63	1.73	0.99
	1−2 h	19.16	21.69	0.88	0.97
	2−3 h	8.75	6.04	1.45	0.84
	3−4 h	7.01	12.17	0.58	0.99

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4. Discussion

4.1 Temperature as a key environmental factor for the succession of dominant species of Qinhuangdao green tides

Eutrophication, especially the increase in inorganic nitrogen, largely drives macroalgae blooms (Fong et al., 1996). Studies have shown that river pollution in Qinhuangdao is becoming more and more serious, and the eutrophication of coastal areas in Qinhuangdao has shown an increasing trend in recent years (Wang et al., 2013). Fong et al. (1996) assume that other factors (temperature and light) also play an important role in the growth of macroalgae when the nutrient supply is sufficient. Fan et al. (2015) showed that temperature is a key environmental factor for the succession of dominant species of raft-fouling green macroalgae. In addition, the phenomenon of species succession during macroalgae blooms in other coastal waters was also controlled by temperature. Yoshida et al. (2015) showed that in the Hiroshima Bay green tide, Japan, Ulva spp. is the dominant species in summer and autumn, whereas in winter, U. australis, a cold-water species, becomes the dominant species.

The strain of Ulva prolifera in the Bohai Sea green tides is not the same as that in the Yellow Sea green tides. The green tide in Qinhuangdao is not correlated with the green tide Yellow Sea and has a different origin and development mode (Han et al., 2022a). In previous study, the relationship between green tides in Qinhuangdao and the Yellow Sea (Rudong and Qingdao) was evaluated by genetic analyses of U. prolifera based on SNP markers. In total, 12945 SNP markers were obtained for all samples. The U. prolifera populations in Rudong and Qingdao were closely clustered, while samples from Qinhuangdao clustered separately. These results showed genetic differentiation was lower among floating U. prolifera populations in Rudong and Qingdao than in Qinhuangdao. Physiological experiments showed that the net buoyancy of floating U. prolifera were higher in Qingdao and Rudong compare to Qinhuangdao under the same light conditions (Han et al., 2022a). Previous studies have shown that the attached macroalgae on seaweed beds in the coastal waters of Jinmenghaiwan Bathing Beach are the major original source of the green tide blooms in the Bohai Sea (Song et al., 2019).

We found that U. australis, B. plumosa, and U. prolifera could tolerate a wide range of temperature and light levels and maintained growth rate of 3.4%–12.2% per day, 4.6%–14.2% per day, and 3.2%–18.6% per day, respectively, at the tested temperature and light ranges. The high RGR indicated the great ecological advance for this species under the favored environmental conditions (Xiao et al., 2016). Compared to the relatively smaller variation as a result of the light conditions, the growth rates of these three algae were more significantly influenced by temperature. According to the results, within the temperature and light range of the experimental setup, the optimum growth temperatures of U. australis, B. plumosa, and U. prolifera were 10℃, 15℃, and 20–25℃, respectively. According to the data released by the Department of Natural Resources of Hebei Province on the seawater temperature of Jinmenghaiwan Bathing Beach from April to September 2018–2020 (Fig. 13), the sea water temperature is low in April (about 10℃), which is conducive to the growth of U. australis, making it the dominant species in the first stage. In May, the sea water temperature is about 15℃, which is conducive to the growth of B. plumosa. In the co-culture experiment with B. plumosa and U. australis, B. plumosa gained competitive advantages due to its high growth rate and became the dominant species in the second stage. The indoor experiment showed that the growth rate ofU. prolifera was highest at 20–25℃. After June, the sea water temperature exceeds 20℃, which is suitable for the growth of U. prolifera. This species also showed obvious growth advantages in the co-culture withB. plumosa and became the dominant species in the third stage. Therefore, we believe that the sea water temperature is the key environmental factor for the succession of dominant species of green tides in the Qinhuangdao coastal area.

Figure  13.  Seawater temperature of Jinmenghaiwan Bathing Beach from April to September 2018–2020 ( http://zrzy.hebei.gov.cn/heb/gongk/gkml/yjgl/hyzh/).

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4.2 Growth and nutrient uptake capacity of the three green tide algae

According to the previous study, the content of NO₃-N in the water column of Bohai Bay accounted for more than half of the total nitrogen, 53.08%, while the content of NH₄-N also accounted for 23.57%, and these two forms of nitrogen accounted for 76.65% of the total nitrogen, indicating that the nitrogen nutrients in the water column mainly existed in the form of inorganic nitrogen. Both PO₄-P are the main forms of phosphorus, with PO₄-P content in the water column accounting for 76.46% of the total phosphorus (Song et al., 2019). This region is phosphate limited with nitrogen to phosphorus ratio above 20 (Zhang et al., 2018). The main occurrence area of green tide in Qinhuangdao is the sea area adjacent to Jinmenghaiwan Bathing Beach (Han et al., 2022b), which is adjacent to Tang River Estuary and rich in nutrient. According to the preliminary field survey, the approximate ranges of nitrate, ammonium and phosphate in the green tide occurrence area from April to September were 4.08–9.20, 2.15–4.47 and 0.12–0.47, respectively (unpublished data).

In this study, compared with U. australis and B. plumosa, U. prolifera had higher growth and nutrient uptake capacities. The growth rate is mainly determined by the ability of nutrient uptake from adjacent waters (Taylor and Rees, 1999; Ale et al., 2011), indicating that U. prolifera can use and store nutrients more effectively for rapid growth. At high nitrogen concentrations, species with higher nutrient uptake rates have an advantage over species with lower rates (Pedersen and Borum, 1997). The Jinmenghaiwan Bathing Beach is the main area affected by Qinhuangdao green tides, which is adjacent to the mouth of the Tang River and rich in nutrient salts (Wu et al., 2015). For nitrate, ammonium, and phosphate uptake, U. prolifera had higher R_max values than U. australis and B. plumosa, indicating that this species has a higher uptake rate at the same nutrient concentration. The R_max/K_s ratio describes the relationship between uptake rate and substrate concentration, with higher values indicating competitive advantages and higher affinity. The significant difference in the R_max/K_s values of the three dominant species of Qinhuangdao green tides indicates that U. prolifera had obvious competitive advantages in taking up and storing nitrogen and phosphate from neighboring waters compared with U. australis and B. plumosa. A high nutrient absorption ability greatly contributes to the rapid growth rate and ecological competitive advantage of this species over the other co-occurring species, especially in eutrophic waters (Luo et al., 2012; Wang et al., 2012). This study also explains why the biomass of the third stage of the green tide, with U. prolifera as the dominant species, was the highest, with the greatest impact on the local ecological environment.

The maximum absorption rate of the three algae for ${{\rm {NH}}_4^+} $ was higher than that to ${{\rm {NO}}_3^-} $ . For example, in the interval of 0–1 h, the R_max values of U. prolifera,U. australis, and B. plumosa of ${{\rm {NO}}_3^-} $ were 104.31 μmol/(g·h), 83.42 μmol/(g·h), and 79.43 μmol/(g·h), respectively, whereas those of ${{\rm {NH}}_4^+} $ were 157.35 μmol/(g·h), 138.38 μmol/(g·h), and 117.59 μmol/(g·h), respectively. This is because the algae absorb ammonia nitrogen and convert it directly into glutamate, which is used for algal growth. After the uptake of nitrate salts, they must be reduced to ammonia nitrogen or low-valence ammonium salt before being absorbed and used by the algae. Since ${{\rm {NO}}_3^-} $ is reduced to ${{\rm {NH}}_4^+} $ by nitrate enzyme before assimilation (Hurd et al., 1995; Abreu et al., 2011), the higher affinity to ${{\rm {NH}}_4^+} $ might be related to the less energy required for the assimilation of this nutrient.

In conclusion, U. australis, B. plumosa, and U. prolifera, the three dominant species of the green tide in Qinhuangdao, showed significant interspecific differences in physiological characteristics such as growth and nutrient uptake capacities. Temperature has a significant effect on the growth of the three species and is the key environmental factor for the dominant species succession of green tides in Qinhuangdao. Ulva prolifera has a higher growth rate than U. australis and B. plumosa at the same nitrate, ammonium, and phosphate concentrations, with obvious competitive advantages in nutrient uptake capacity. Therefore, the strong growth and nutrient uptake capacities of U. prolifera might be the main reason why U. prolifera becomes the dominant species, with the highest biomass and the greatest influence on the local ecological environment.