Spatial-temporal variation of Aureococcus anophagefferens blooms in relation to environmental factors in the coastal waters of Qinhuangdao, China

Harmful Algae 86 (2019) 106–118

Contents lists available at ScienceDirect

Harmful Algae journal homepage: www.elsevier.com/locate/hal

Spatial-temporal variation of Aureococcus anophagefferens blooms in relation to environmental factors in the coastal waters of Qinhuangdao, China

T

Peng Yaoa,b, , Lei Leia,c, Bin Zhaoa,c, Jinpeng Wanga,c, Lin Chena,c ⁎

a

Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education/Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China b Laboratory of Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China c College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China

ARTICLE INFO

ABSTRACT

Keywords: Qinhuangdao Brown tide Aureococcus anophagefferens 19′-Butanoyloxyfucoxanthin Hydrological variables Minutocellus polymorphus

The brown tides occurring in the coastal scallop cultivation area of Qinhuangdao, China, in recent years are caused by Aureococcus anophagefferens and significantly impact the scallop industry and the marine ecosystem in this region. Long-term investigations of phytoplankton and hydrological variables in the Qinhuangdao sea area were conducted in this study to understand the spatial-temporal variations of A. anophagefferens in relation to environmental factors. Samples were collected during twelve cruises from July 2011 to December 2013 and were analyzed for the temperature, salinity, dissolved oxygen (DO), nutrients and phytoplankton pigments. All diagnostic pigments of A. anophagefferens, such as chlorophyll c3 (Chl c3), Chl c2, 19′-butanoyloxyfucoxanthin (Butfuco), fucoxanthin (Fuco), and diadinoxanthin (Diad), were detected in the surface water by using high-performance liquid chromatography (HPLC). The highest concentrations of But-fuco (5.64 μg L−1), Fuco (37.94 μg L−1) and chlorophyll a (Chl a, 17.25 μg L−1) occurred in different seasons and sampling sites. The A. anophagefferens bloom (as indicated by But-fuco) usually expanded from the south to the north of the Qinhuangdao sea area, close to scallop-culturing regions. The bloom unusually starts in May, reaches its peak in June and almost disappears in August, with the temperature ranging from ca. 19 °C to 23 °C. The redundancy analysis (RDA) indicated that relatively high salinity (> 29) and low inorganic nutrients were suitable for the development of the A. anophagefferens bloom. The ratios of diagnostic pigments to Chl a were not constant during different cruises and generally obeyed two different linear relationships, thus indicating the co-occurrence of the blooms of A. anophagefferens and other species, such as Minutocellus polymorphus. In summary, our work reports the long-term variation of A. anophagefferens blooms based on diagnostic pigments and environmental controls, which may provide more insights into the formation mechanisms of the brown tide in this region.

1. Introduction Brown tides caused by the pelagophyte Aureococcus anophagefferens have received considerable attention for several decades since their first occurrence in the 1980s along the eastern coast of the USA (Gobler et al., 2005, and references therein). Since then, it has bloomed frequently in estuaries of Long Island and has shown apparent expansion along the eastern coast of the USA (e.g., Anderson et al., 1993; Mulholland et al., 2004) and even South Africa (Probyn et al., 2001, 2010). More recently, in 2009, massive A. anophagefferens blooms began to appear in coastal waters near the scallop cultivation area of Qinhuangdao, China (e.g., Kong et al., 2012; Zhang et al., 2012; Zhen

et al., 2016; Qiao et al., 2017). Blooms of A. anophagefferens can cause reproductive failure and mortality in suspension-feeding animals, such as scallops, hard clams, oysters and mussels, along with concomitant brown discoloration of the seawater (Sieburth et al., 1988; Wazniak and Glibert, 2004). Therefore, ecological studies of A. anophagefferens are essential to better understand the formation mechanism, harmful pattern and environmental effects of the brown tide. Aureococcus anophagefferens is a tiny, spherical, and nonmotile picophytoplankton species (cell size 2∼3 μm) with no flagella (DeYoe et al., 1997). Recent studies have revealed that brown tides of A. anophagefferens often occur under low inorganic nutrient conditions (Cosper et al., 1989; Keller and Rice, 1989; LaRoche et al., 1997), which

⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education/Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (P. Yao).

https://doi.org/10.1016/j.hal.2019.05.011 Received 15 November 2017; Received in revised form 22 May 2019; Accepted 23 May 2019 Available online 31 May 2019 1568-9883/ © 2019 Elsevier B.V. All rights reserved.

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 1. Locations of the sampling sites in the coastal area of Qinhuangdao, China, during the twelve cruises from July 2011 to December 2013. Arrows indicate the direction of the currents (from Bian et al., 2016). YSWC: Yellow Sea Warm Current; LDCC: Liaodong Coastal Current (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

may be largely attributed to its ability to obtain nutrients from dissolved organic compounds, such as urea, proteins, amino acids, chitobiose and acetamide (Berg et al., 1997, 2002; Mulholland et al., 2002, 2009; Dzurica et al., 1989). Environmental factors, such as the temperature, salinity and light, also play significant roles in the growth of A. anophagefferens (Cosper et al., 1989; Popels et al., 2007). For example, it has been shown that coastal waters with relatively high salinities (≥24) (Cosper et al., 1989; LaRoche et al., 1997), low-level light intensities (Popels et al., 2007) and temperatures retained at approximately 20 °C (Cosper et al., 1989; Gobler et al., 2002) were the appropriate conditions for their growth. To monitor the development of A. anophagefferens blooms, a simple, effective, quick and reproducible analytical method for identification and quantification is definitely needed. The identification of A. anophagefferens by traditional microscopic observation is quite difficult because their tiny, indistinguishable cells are not easily recognized. Characteristic pigments, however, can serve as chemotaxonomic markers for identifying phytoplankton groups, through which method the phytoplankton community structure and abundance can be determined rapidly and accurately (Andersen et al., 1996; Ansotegui et al., 2001). For A. anophagefferens, the pigment suites of 19′-butanoyloxyfucoxanthin (But-fuco), fucoxanthin, chlorophyll c3, chlorophyll c2, diadinoxanthin and chlorophyll a can be used as diagnostic tools (Jeffrey and Vesk, 1997; Bidigare, 1989; Alami et al., 2012). For example, in the early 1990s, concentrations of But-fuco in seawater and the regressions of the But-fuco with direct cell counts were successfully used to track the presence of A. anophagefferens in the coastal bays of Maryland and Virginia (Trice et al., 2004). It took several years to confirm the causative species of the brown tides in Qinhuangdao, China, by means of microscopy, pigment chemotaxonomy and genetic analyses. Through high-performance liquid chromatography (HPLC) pigment analysis, Kong et al. (2012) found high concentrations of But-fuco and other pigments in their samples, thus narrowing down the possible causative species to half a dozen species that belong to pelagophytes, haptophytes or silicoflagellates. Clone libraries were then constructed using eukaryote-specific 18S rDNA by Zhang et al. (2012), who found that all 8 pelagophyte clones from the Qinhuangdao blooming area were 99.7–100% similar to A. anophagefferens, the species that caused brown tides along the coasts of the eastern Unites States and South Africa. Furthermore, clone libraries and a real-time polymerase chain reaction (RT-PCR) were employed to analyze the spatial variation of the microalgae community structure

and confirmed that the causative species of brown tide was A. anophagefferens (Gu et al., 2015; Guo et al., 2015). Moreover, Dong et al. (2014) used RDA-seq technology to profile the transcriptome of a Chinese strain of A. anophagefferens grown on urea, nitrate, and a mixture of urea and nitrate under N-replete, limited and recovery conditions and found that A. anophagefferens primarily uses urea rather than nitrate, which was similar to findings reported by Berg et al. (1997) and Lomas et al. (1996) using 15N tracer methods. Similarly, Zhen et al. (2016) found that A. anophagefferens was the dominant species in the local eukaryotic microalgae community during the brown tide in June 2012 in the Qinhuangdao scallop culture area based on an 18S rDNA sequence analysis, and they also found that decreasing concentrations of dissolved inorganic nitrogen (DIN), increasing amounts of human aquaculture activities and the suitability of temperatures were the main reasons for the occurrence of brown tides in Qinhuangdao. Despite such efforts, the distribution of A. anophagefferens, and most importantly, the effects of environmental factors on the development of the brown tide in the Qinhuangdao sea area are still poorly understood. In this study, the pigment concentrations and some environmental parameters, such as the temperature, salinity, dissolved oxygen (DO), and dissolved inorganic nutrients in seawater samples collected from the coastal scallop cultivation area of Qinhuangdao from July 2011 to December 2013 were examined. The primary objective of this study was to use But-fuco and other diagnostic pigments as proxies to reconstruct the spatial-temporal variation of A. anophagefferens in this region and to determine linkages between the brown tide and environmental factors. It is worth noting that due to certain condition limitations, no organic nutrients were analyzed in this study. The discussion on the effects of environmental factors on the evolution of A. anophagefferens brown tide was mainly based on current data, while the discussion related to dissolved organic nitrogen (DON) was based on the literature results. Nevertheless, this study still helps to obtain a better understanding of the occurrence and development of the A. anophagefferens brown tide and its controls in the coastal waters of Qinhuangdao and provides comparable data for future studies in other coastal waters.

107

Harmful Algae 86 (2019) 106–118 2.22 ± 1.03 0.25 ± 0.17

2. Materials and methods 2.1. Study area and sample collection

0.09 ± 0.04 0.45 ± 5.34

17.01 ± 7.05 3.74 ± 5.41

0.60 ± 0.14 0.40 ± 0.14

0.33 ± 0.13 0.49 ± 0.15

107 ± 60 28 ± 9

The study area was located between 39.0 °N-40.2 °N latitude and 118.8 °E-120.0 °E longitude in the coastal area of Qinhuangdao in the Bohai Sea, with an average depth of 20 m (Fig. 1). Luanhe River, in the southwestern part of Qinhuangdao, together with other small rivers, drains into the Bohai Sea (Fig. 1). The oceanographic regime of the study area is generally governed by southward currents, including the remnant of the Yellow Sea Warm Current (YSWC) and Liaodong Coastal Current (LDCC) (Bian et al., 2016) (Fig. 1). The currents bring warm and high salinity water to the study area, which makes Qinhuangdao an ice-free harbor. Qinhuangdao is a well-known breeding base of scallops, with the shellfish industry being the leading aquaculture industry (Liu et al., 2011). Twelve cruises were conducted from July 2011 to December 2013 (Table 1), and sampling was performed onboard local fishing boats (Fig. 1). At each station, environmental parameters, including the salinity, temperature, and DO of the surface water, were determined and recorded directly by a multiparameter water quality analyzer (HQ40d, Hach, USA). The surface water was then collected into a prewashed plastic bottle and was stored under a cool and low-light environment before being transported to a land-based laboratory after each cruise for further treatment. For nutrient analysis, subsamples were filtered through 0.45-μm pore size acetate cellulose filters (Xidoumen, Hangzhou, China) that had been presoaked in diluted hydrochloric acid (approx. pH = 2) overnight and rinsed with Milli-Q water (Millipore Corporation, USA) (Yao et al., 2010). The filtrates were poisoned by the addition of chloroform (approx. 1.0 × 10−3 v/v). For each sample, half of the filtrates were stored at -20 °C pending NH4+, NO3-, NO2- and PO43- measurements, and the other half were stored at room temperature until SiO32- analysis. For the pigment analysis, 500-1000-mL subsamples were prefiltered over a 200-μm silk sieve to remove larger zooplankton and other debris and then were filtered through GF/F filters (0.7 μm, Whatman, Maidstone, UK) under gentle vacuum pressure (less than 0.03 MPa) and dim light. The filters were wrapped in aluminum foil and stored at −80 °C prior to the HPLC analysis.

0.58 ± 0.41 1.28 ± 0.26 4.73 ± 1.41 3.91 ± 1.05 28.48 ± 0.47 27.77 ± 0.79 24.37 ± 0.48 6.17 ± 1.37

7.99 ± 0.44 11.99 ± 0.75

2.93 ± 1.94 6.19 ± 0.67

8.24 ± 3.15 11.39 ± 6.78

1.10 ± 0.36 88 ± 37 0.31 ± 0.09 0.67 ± 0.10 8.65 ± 3.99 0.09 ± 0.02 0.21 ± 0.07 5.22 ± 1.78 29.62 ± 0.08 20.95 ± 1.95

9.43 ± 0.55

2.29 ± 0.62

7.73 ± 1.73

0.31 ± 0.19 158 ± 86 0.25 ± 0.10 0.72 ± 0.10 3.64 ± 2.18 0.10 ± 0.06 0.30 ± 0.14 9.54 ± 6.20 30.00 ± 0.33 18.77 ± 2.26

8.64 ± 0.53

3.46 ± 2.30

13.30 ± 8.23

0.29 ± 0.22 110 ± 98 0.51 ± 0.19 0.42 ± 0.19 2.44 ± 2.40 0.08 ± 0.03 0.38 ± 0.13 2.85 ± 2.13 29.71 ± 0.36 2.93 ± 0.33

12.63 ± 0.85

4.32 ± 5.75

7.55 ± 6.82

0.91 ± 0.87 46 ± 44 0.61 ± 0.16 0.37 ± 0.15 5.89 ± 4.24 0.25 ± 0.05 0.12 ± 0.16 3.09 ± 1.53 26.17 ± 0.90 2.70 ± 0.44

11.88 ± 0.23

6.91 ± 7.04

10.12 ± 8.19

3.63 ± 5.38 56 ± 16 0.53 ± 0.08 0.45 ± 0.08 17.29 ± 21.3 0.08 ± 0.01 0.06 ± 0.03 1.96 ± 0.46 33.48 ± 0.12 20.68 ± 0.98

9.39 ± 0.43

2.36 ± 0.78

4.38 ± 1.10

0.58 ± 0.21 268 ± 452 0.70 ± 0.19 0.28 ± 0.17 8.44 ± 11.7 0.08 ± 0.05 0.17 ± 0.06 1.76 ± 0.69 / /

/

14.9 ± 26.3

16.81 ± 26.2

0.28 ± 0.15 126 ± 64 0.23 ± 0.10 0.75 ± 0.10 1.04 ± 0.46 0.04 ± 0.03 0.07 ± 0.03 / /

/

2.97 ± 0.81

0.96 ± 0.60

4.00 ± 1.16

1.66 ± 0.41 13 ± 3 0.50 ± 0.06 0.45 ± 0.07 13.75 ± 1.53 0.69 ± 0.08 0.49 ± 0.20 / /

/

3.92 ± 1.29

4.28 ± 1.19

8.69 ± 2.32

/ 0.87 ± 0.38 759 ± 701 805 ± 682 0.01 ± 0.01 0.14 ± 0.10 0.98 ± 0.01 0.81 ± 0.14 / 20.32 ± 2.97 0.07 ± 0.08 0.07 ± 0.06 0.08 ± 0.04 1.23 ± 1.11 19.55 ± 14.5 25.37 ± 16.9 30.97 ± 0.21 29.23 ± 0.35 22.76 ± 1.06 25.84 ± 0.58

July 6, 2011 Aug. 22, 2011 Dec. 5, 2011 Apr. 13, 2012 May 15, 2012 Jun. 19, 2012 Dec. 4, 2012 Mar. 27, 2013 Jun. 3, 2013 Jun. 27, 2013 Sep. 1, 2013 Dec. 3, 2013

2.28 ± 0.11 7.81 ± 0.18

0.23 ± 0.13 3.80 ± 2.48

19.86 ± 14.5 30.40 ± 16.4

NH4+/DIN Silicate (μmol/L) Phosphate (μmol/L) DIN (μmol/L) Nitrite (μmol/L) Nitrate (μmol/L) Ammonium (μmol/L) DO (mg/L) Salinity Temperature (°C) Cruises

Table 1 Monthly average temperature, salinity, DO and nutrients of surface samples from the coastal scallop cultivation area of Qinhuangdao from July 2011 to December 2013.

NO3−/DIN

N/P

Si/N

P. Yao, et al.

2.2. Nutrient analysis The dissolved inorganic nutrients in surface waters were determined colorimetrically on a nutrient autoanalyzer (AA3, Seal, UK). The detection limits for NO3−, NO2−, NH4+, PO43- and SiO32- were 0.02, 0.01, 0.04, 0.02 and 0.03 μmol L-1, respectively. The analytical uncertainty for all the dissolved nutrients in the replicate samples was better than 3%. 2.3. Pigment extraction and HPLC analysis Pigments were extracted in acetone and were ultrasonicated in an ice bath (59 KHz, 250 W, Kudos, China) for 5 min after the frozen filters were thawed and cut into small pieces. Extracts were separated from the debris by centrifugation. The supernatant was decanted into a testtube, and the extraction procedure was repeated until the filtrates turned colorless. The filtrates were then mixed and blown to dryness with nitrogen, redissolved with 200 μL of 95% methanol (in Milli-Q water), and filtered through a syringe filter (0.45-μm PTFE, Whatman, UK) before injection. Sample volumes of 30∼60 μL were then injected automatically into the HPLC system. The whole extraction procedure was performed under dim light. Pigment analysis was performed by HPLC following the method of Zapata et al. (2000). The Waters Alliance 2695 separation module equipped with two detectors, namely, a Waters 2996 photodiode array detector (PAD; 1.2 nm optical resolution) and a Waters 2475 multi λ 108

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

values of 9.54 μmol L−1 and 5.22 μmol L−1, respectively. In September and December 2013, the NH4+ concentrations decreased gradually to a similar level as in December 2011. The highest average value of NO3− concentrations (14.88 μmol L-1) was found in May 2012, with the highest level (68.37 μmol L-1) found at the Funing site (F01). In June 2012 and late-June 2013, the NO3− concentrations remained at a low level (approximately 2.30 μmol L-1). In winter seasons (e.g., December of all three investigated years), the NO3- concentrations were all at relatively high levels (> 4.00 μmol L-1) (Table 1, Fig. 2). NO2− was the least abundant nitrogenous nutrient during the sampling periods at different sites. From April to June 2012, the NO2− concentrations were at very low levels (∼0.10 μmol L-1), especially in June, which was similar to the NH4+. From March to lateJune 2013, NO2- concentrations decreased gradually from ∼0.38 μmol L-1 to ∼0.21 μmol L-1, but the values in late-June 2013 were still slightly higher than that in June 2012 (Table 1, Fig. 2). The sum of three dissolved inorganic nitrogen (DIN) nutrients varied in a wide range from 2.29 to 70.05 μmol L−1, with high values found in July 2011, August 2011 and May 2012 (Table 1). During the brown tide period in June 2012, the DIN was relatively low (4.38 ± 1.10 μmol L−1), but during the bloom in late June 2013, it was not very low, because the concentrations of NH4+ and NO3- increased at each station, especially NH4+. High NH4+/DIN values (> 0.50) were mainly observed in spring and summer seasons (e.g., July and August 2011, April 2012 and early-June 2013), whereas low ratios of NH4+/DIN (< 0.50) were found mainly in December. The ratios of NO3-/DIN were high in the three December cruises (avg. 0.50, 0.61 and 0.49, respectively) and in May and June 2012 (avg. 0.70 and 0.53, respectively) (Table 1). In June 2013, the ratios of NO3-/DIN were lower than those in June 2012, since NH4+ contributed to a greater proportion of DIN than NO3- in 2013 (Table 1). Compared with other nutrients, the concentrations of PO43− showed a different spatial-temporal distribution pattern (Fig. 2). For most of the sampling periods (both bloom periods and nonbloom periods), the average values of the PO43− concentrations were below 0.10 μmol L-1. Relatively high values (> 0.50 μmol L-1) occurred only in December cruises (Table 1, Fig. 2). The concentrations of SiO32- fluctuated in a wide range from 0.18 to 57.10 μmol L-1, with an average value of 3.74 μmol L-1 (Table 1). The highest value (57.05 μmol L-1) of SiO32- was found in June 2012 at site F02, which was much higher than that of other stations in June 2012. The concentrations of SiO32- showed increasing trends from April 2012 to June 2012 and from March 2013 to September 2013 but decreased from summer to winter cruises. Relatively high levels of SiO32- were found in August 2011, June 2012 and September 2013 (avg. 20.32 μmol L-1, 17.29 μmol L-1 and 17.01 μmol L1 , respectively) (Table 1). Unlike nitrogenous nutrients and PO43−, SiO32- was at relatively high levels during the brown tide bloom periods (e.g., on June 19, 2012, and June 27, 2013). The ratios of N/P also changed in a wide range from 8.50 to 1891, with an average value of 27.50 (Table 1). The N/P ratios were high (> 100) during most of the investigated periods because of the low concentrations of PO43−. Relatively low levels of N/P (< 100) were found in December cruises due to the increased concentration of PO43− and in the bloom periods since DIN decreased at that time (Table 1). The ratios of Si/N fluctuated from 0.05 to 14.40, with an average value of 0.25 (Table 1). During most periods, the ratios of Si/N were below 1.00, and relatively high values were found in December 2011, June 2012, June 27, 2013, and September 2013 (avg. 1.66, 3.63, 1.10 and 2.22, respectively) (Table 1).

fluorescence detector, was employed. A reverse-phase column (Waters Symmetry C8, 150 × 4.6 mm, 3.5-μm particle size, 100 Å pore size) was used as the stationary phase. For the mobile phase, eluent A was a mixture of methanol: acetonitrile: aqueous pyridine (50:25:25, v/v/v) and eluent B was a methanol: acetonitrile: acetone (20:60:20, v/v/v) solution. The mobile phases were prepared using all HPLC-grade organic solvents (Merck, Germany) and Milli-Q water. A linear elution gradient procedure was used. The mobile phase was pumped from 0% to 40% of eluent B in 22 min, then was increased to 95% at 28 min and was maintained at 95% for a further 10 min. The initial conditions were restored by a reversed linear gradient within 2 min. After each run, the sampler was flushed with a loop volume with 30% methanol (in water). The flow rate was 1 mL min−1. The pigments were identified by comparison of their retention time and absorption spectra with authentic standards and literature values (Jeffrey and Vesk, 1997). The quantification of pigments was based on the external standard method, and the pigment standards included: chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll c3 (Chl c3), chlorophyll c2 (Chl c2), diadinoxanthin (Diad), diatoxanthin (Diat), fucoxanthin (Fuco), alloxanthin (Allo), prasinoxanthin (Pras), peridinin (Peri), 19′-butanoyloxyfucoxanthin (But-fuco), 19′-hexanoyloxyfucoxanthin (Hex-fuco), zeaxanthin (Zea), lutein (Lut), and β, β-carotene (βcaro), which were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA) and 14C Agency (DHI, Denmark). The detection limit of Chl a was 0.02 μg L−1 (for a sample volume of 1000 mL and an extraction volume of 1.5 mL), and the difference between two replicate measurements was usually < 1% (Yao et al., 2010). 2.4. Statistical analysis A redundancy analysis (RDA) with a Monte-Carlo permutation test was performed to reveal the correlations between the significant pigment abundance of field samples and environmental factors, following the results of pretested, detrended correspondence analysis (DCA) using Canoco (version 5). The program SPSS was used to run a one-way analysis of variance (ANOVA) to examine the significant differences in the data between two or more groups. Surfer 11 and Origin 8.5 were used for graphics. 3. Results 3.1. Temperature, salinity and DO Temperatures in the surface waters fluctuated from 2.40 °C to 26.61 °C, with the highest value in August 2011 (25.84 ± 0.58 °C) and the lowest value in December 2012 (2.70 ± 0.44 °C) (Table 1). The salinity varied from 25.20 to 33.60 throughout the study period, with the highest level in June 2012 (33.48 ± 0.12) and the lowest value in December 2012 (26.17 ± 0.90) (Table 1). The DO changed from 2.12 to 13.90 mg L−1, with high levels found in winter cruises (December 2012 and December 2013) and in spring cruises (March 2013) and the lowest levels found in the summer cruise (July 2011) (Table 1). 3.2. Dissolved inorganic nutrients The concentrations of NH4+ varied in a wide range during the investigation (Table 1, Fig. 2). The summer cruises (July and August) in 2011 showed high levels of NH4+, averaging 19.55 and 25.37 μmol L−1, respectively. The highest NH4+ concentrations were both found at Shanhaiguan sites in these two cruises (57.42 μmol L−1 and 63.90 μmol L−1, respectively). In December 2011 and April 2012, NH4+ decreased to a low level, and during the brown tide periods in mid-May and late-June 2012, the concentrations of NH4+ were even lower, with average values of 1.76 μmol L−1 and 1.96 μmol L−1, respectively. In early June and late-June of 2013, the NH4+ concentrations were higher than those in the same period in 2012, with average

3.3. Composition and abundance of major pigments In addition to Chl a, the major pigments that were detected in most field samples were Chl c3, Chl c2, But-fuco, Fuco and Diad (Table 2). Other accessory pigments, including Peri, Allo, Zea, Chl b and β-caro, were also found in some samples. Chl c3 was found in the samples from 109

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 2. Variation of NH4+, NO3−, NO2−, SiO32- and PO43- in the surface waters at selected stations S01 (a), S02 (b), F01 (c) and F02 (d) in the coastal area of Qinhuangdao, China, from July 2011 to December 2013.

most periods, except in December 2011, with a mean value of 2.16 μg L−1 during the sampling periods (Table 2). The highest concentration of Chl c3 was found in June 2012, especially at the sites of F02 (23.73 μg L−1) and S02 (22.51 μg L−1), with a mean value of 18.32 μg L-1 (Table 2, Figs. 3b and 5 a). During the bloom in June 2013, the mean level of Chl c3 was 1.31 μg L−1, which was much lower than that in June 2012. Chl c2 had a similar temporal distribution with Chl c3, but the concentrations of Chl c2 were much higher, with a mean value of 9.49 μg L-1 for all samples (Table 2, Figs. 3 and 5b). During the bloom period in June 2012, the mean concentrations of Chl c2 reached 53.08 μg L-1 (Table 2), and the highest value was found in the coastal water of Funing (69.55 μg L−1) (Fig. 3e). Similarly, the concentration of Chl c2 on June 27, 2013 (5.83 μg L−1), was also lower than that in June 2012 (Table 2). Concentrations of But-fuco in the coastal waters of Qinhuangdao changed markedly during the investigation periods. On July 6, 2011, But-fuco was detected in most sites, except F01, F02 and X02, and relatively high concentrations of But-fuco were found in S01 (2.27 μg L−1) and S02 (1.74 μg L−1) (Fig. 4a). In contrast, in August 2011, But-fuco was barely detected at almost all sites, and there was a

sharp decreasing trend from July to August (Fig. 5c). In 2012, no Butfuco was detected in April, but a low concentration of But-fuco began to appear in May, ranging from 0.10 to 0.49 μg L−1. In June 2012, when the brown tide concentration increased, But-fuco concentrations ranged from 2.03 to 5.64 μg L−1, and peak values were found at the sites of F01 (4.93 μg L−1) and F02 (5.64 μg L−1) (Fig. 4b). A similar trend was observed in the summer of 2013. On June 3, 2013, almost no But-fuco was detected in the study area, but after June 27, 2013, peak values of Butfuco increased to 5.56 μg L−1, with the highest value found in the coastal water near Changli (C01) (Fig. 4c). In addition, low levels of But-fuco were also detected in September 2013, in the coastal waters near Shanhaiguan and Funing (∼0.03 μg L−1 and ∼0.04 μg L−1, respectively). Almost no But-fuco was detected in seawater samples in December cruises during the investigation (Fig. 5c). Concentrations of Fuco varied from 0.04 to 37.94 μg L−1, with a mean value of 2.90 μg L-1 (Table 2). Similar to But-fuco, Fuco was abundant in samples from summer cruises. On July 6, 2011, concentrations of Fuco ranged from 0.72 to 8.51 μg L−1, with a mean value of 4.32 μg L−1, while on June 19, 2012, the average concentration of Fuco reached 10.90 μg L-1 (Table 2), and high concentrations were

Table 2 Monthly average concentrations of major pigments of surface samples from the coastal scallop cultivation area of Qinhuangdao from July 2011 to December 2013. ND = not detected, NA = not available. Cruises

Chl c3 (μg L−1)

Chl c2 (μg L−1)

But-fuco (μg L−1)

Fuco (μg L−1)

Diad (μg L−1)

Chl a (μg L−1)

Jul. 6, 2011 Aug. 22, 2011 Dec. 5, 2011 Apr. 13, 2012 May 15, 2012 Jun. 19, 2012 Dec. 4, 2012 Mar. 27, 2013 Jun. 3, 2013 Jun. 27, 2013 Sep. 1, 2013 Dec. 3, 2013 Average

4.38 ± 5.70 0.44 ± 0.35 ND 0.10 ± 0.26 1.44 ± 1.10 18.32 ± 4.91 0.13 ± 0.10 0.08 ± 0.02 0.20 ± 0.21 1.31 ± 1.18 0.06 ± 0.06 0.12 ± 0.15 2.16 ± 5.33

24.3 ± 12.1 7.45 ± 3.09 1.11 ± 0.69 ND 4.95 ± 2.55 53.08 ± 12.1 7.17 ± 5.89 1.72 ± 0.57 3.06 ± 2.48 5.83 ± 6.20 1.05 ± 0.41 1.53 ± 1.55 9.49 ± 15.4

0.73 ± 0.87 0.02 ± 0.05 ND ND 0.25 ± 0.22 3.60 ± 1.47 0.02 ± 0.03 ND 0.02 ± 0.06 1.66 ± 2.27 0.02 ± 0.02 0.004 ± 0.01 0.49 ± 1.22

4.32 ± 2.16 1.95 ± 0.94 0.26 ± 0.07 0.70 ± 0.85 0.81 ± 0.41 10.90 ± 2.34 0.91 ± 0.60 0.84 ± 0.47 0.70 ± 0.79 10.85 ± 15.63 1.53 ± 1.79 2.91 ± 2.13 2.90 ± 5.11

0.66 0.38 0.08 0.17 0.15 1.22 0.24 0.09 0.07 0.58 0.13 0.14 0.33

7.22 7.99 1.63 1.88 2.18 7.70 2.09 1.40 1.13 4.44 2.44 2.45 3.84

110

± ± ± ± ± ± ± ± ± ± ± ± ±

0.48 0.20 0.06 0.13 0.11 0.61 0.16 0.06 0.09 0.56 0.10 0.16 0.42

± ± ± ± ± ± ± ± ± ± ± ± ±

4.58 3.50 1.35 1.59 0.66 1.05 1.06 1.01 1.05 2.81 1.67 2.64 3.49

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 3. Spatial distributions of Chl c3 (a–c), Chl c2 (d–f) and Fuco (g–i) during the investigated periods of July 6, 2011, June 19, 2012, and June 27, 2013, respectively.

found in the coastal waters near Funing and Shanhaiguan (13.59 μg L−1 and 12.50 μg L−1, respectively) (Figs. 3e and 5 d). On June 27, 2013, Fuco increased from north to south, with a mean value of 10.85 μg L-1 (Table 2), and the highest concentration (37.94 μg L−1) of Fuco was found near Changli (C01) (Figs. 3f and 5 d). During other cruises, Fuco was at a relatively low level (avg. < 3.00 μg L−1) (Table 2, Fig. 5d). The concentrations of Diad were widely distributed at different sites in the sampling periods. The concentrations of Diad fell in the range of 0.01–2.09 μg L−1, with a mean value of 0.33 μg L−1 (Table 2). The highest concentration of Diad was found near Funing during the brown tide period on June 19, 2012 (2.09 μg L−1) (Fig. 4e). On June 27, 2013, the concentrations of Diad (avg. 0.58 μg L−1) were also high but were lower than those on July 6, 2011 (avg. 0.66 μg L−1), and June 19, 2012 (avg. 1.22 μg L−1) (Table 2, Fig. 5e). The concentrations of Chl a varied in a wide range of 0.21 to 17.25 μg L−1, with a mean value of 3.84 μg L-1 (Table 2). In the two summer cruises of 2011, the concentrations of Chl a were high, with a mean value of 7.22 μg L−1 on July 6 and 7.99 μg L−1 on August 22 (Fig. 4d and 5f). During the bloom period on June 19, 2012, the concentrations of Chl a were also at a high level (avg. 7.70 μg L−1) and

were distributed homogeneously in space (Figs. 4e and 5 f). In late June 2013, Chl a was at a relatively low level and increased from north to the south, with an average value of 4.44 μg L−1 (Table 2) and the highest value of 9.12 μg L−1 in the coastal waters near Changli (C01) area (Figs. 4f and 5 f). During the winter cruises on December 5, 2011, and December 4, 2012, and the spring cruise on March 27, 2013, the concentrations of Chl a were all at low levels (avg. < 2.10 μg L−1). Interestingly, the lowest Chl a concentration was not found in these cruises but was found on June 3, 2013, especially near Funing (F01 and F02 stations) (Table 2). The scatter plots between the biomarker pigments and Chl a showed that all the samples roughly obeyed two different linear relationships (Fig. 6). Samples from the bloom period (e.g., July 2011, June 2012 and June 2013) approximately obeyed the line with greater slopes, with the ratios of Chl c3, Chl c2, But-fuco, Fuco and Diad to Chl a being 3.66, 10.45, 0.68, 1.87 and 0.26, respectively. Samples collected in August 2011 obeyed the line with lower slopes, with ratios of Chl c2, Fuco and Diad to Chl a being 0.85, 0.31, and 0.03, respectively. The concentrations of But-fuco and Chl c3 in samples from August 2011 were very low and showed no increase with Chl a. Due to the low concentrations, no 111

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 4. Spatial distributions of But-fuco (a–c), Diad (d–f) and Chl a (g–i) during the investigated periods of July 6, 2011, June 19, 2012, and June 27, 2013, respectively.

SiO32- (0.44) were not significant (Table S1). The second axis had a positive correlation with N/P (0.68), a relatively weak correlation with DO (0.49) and a weak correlation with PO43- (-0.48) (Table S1). Moreover, samples from different cruises were divided into distinct groups through RDA (Fig. 6). The community of sites (especially S01, S02 and F02) from June 19, 2012, were positively related to the salinity, Si/N and temperature but were negatively related to NH4+, NO3−, NO2− and DIN. Samples from June 3, 2013, were divided into two communities, with the community (S02, F01, F02 and C02) positively correlated with NH4+, NO3−, NO2− and DIN and negatively correlated with the salinity, Si/N and temperature; and another community (S01 and C01) was positively related to PO43- and negatively related to DO. Samples from June 27, 2013, were divided into three groups, of which C02 was included in one of the communities of June 3, 2013 (S02, F01, F02 and C02), S01 and S02 were included with the communities of June 3, 2013 (S01 and C01), and the F01 and C01 sites were included in the communities of June 19, 2012.

significant relationships between these pigments and Chl a were observed in other sampling periods. 3.4. Correlation of A. anophagefferens blooms with environmental variables A redundancy analysis (RDA) was conducted to identify the determinant environmental factors that contributed to the distribution of major pigments and the abundance of A. anophagefferens. The first RDA axis explained 87.2% of the total variation, while the second RDA axis explained only 2.74% (Fig. 7). Of all the environmental factors analyzed, salinity (p = 0.014), Si/N (p = 0.006), NH4+ (p = 0.014), NO2− (p = 0.006), DIN (p = 0.080) and N/P (p = 0.068) were shown to be significant in driving the distribution of pigments and abundance of A. anophagefferens, and these factors together provided 53.0% of the total RDA explanatory power. The first axis had significant positive correlations with salinity (0.64), Si/N (0.60) and significant negative correlations with NO2− (-0.63), NH4+ (-0.62) and DIN (-0.61), while the correlations between the first axis and the temperature (0.48) and

112

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 5. Temporal variations of (a) Chl c3, (b) Chl c2, (c) But-fuco, (d) Fuco, (e) Diad and (f) Chl a (μg L−1) at selected sampling sites during investigation.

4. Discussion

et al., 2015; Yu et al., 2015). In this study, the pigment composition of phytoplankton samples was characterized using Chl c3, Chl c2, But-fuco, Fuco, Diad and Chl a and was consistent with the pigment component of A. anophagefferens in a previous study (Bidigare, 1989). The temporal distribution of these pigments showed obvious seasonal variation, especially But-fuco. In the coastal waters of Qinhuangdao, the concentrations of But-fuco usually increased in May or early-June each year and reached a peak value in mid- to late-June, and soon afterwards

4.1. Spatial-temporal distribution of But-fuco and A. anophagefferens Brown tides in the coastal waters of Qinhuangdao have been reported previously. These works have provided results based on pigment analysis or molecular biology and confirmed that the causative organism was A. anophagefferens (Kong et al., 2012; Zhang et al., 2012; Gu

Fig. 6. Relationships between Chl a and (a) Chl c3, (b) Chl c2, (c) But-fuco, (d) Fuco, and (e) Diad during the investigation. Different symbols represent different cruises. 113

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 7. RDA ordination plots for the relationship between the significant pigment abundance of field samples and environmental factors in the coastal waters of Qinhuagdao, China, during three cruises.

decreased and almost disappeared in August (Fig. 5c). This result was similar to the survey made along the U.S. eastern coast of Maryland and Virginia, where the concentrations of But-fuco were found to increase in May or June of each year and rapidly disappeared in July (Trice et al., 2004). This variation pattern was also consistent with the results of a 15-year observational study on the brown tides of Long Island (Gobler et al., 2004). The concentrations of But-fuco in the coastal waters of Qinhuangdao differed from year to year (Fig. 5c), which indicates that the intensity of the brown tide changed annually. During the two bloom periods in this study, the peak values of But-fuco on June 19, 2012, and June 27, 2013, were 5.64 and 5.56 μg/L, respectively, both of which can be specified as category 3 blooms (> 3.71 μg/L; ≥2 × 105 cells/ mL, Gastrich and Wazniak, 2002; Trice et al., 2004). The mean values of But-fuco during these two cruises were 3.60 and 1.66 μg/L, respectively, which suggests that the bloom in 2012 in Qinghuangdao was more intense than that in 2013. In Mid Chincoteague of the coastal bays of Maryland and Virginia, a very high concentration of But-fuco (6.85 μg/L) during brown tide was observed in 2002 (Trice et al., 2004), which was 20% higher than the highest result in this study (Table 3). At the same time, in other regions of the coastal bays of Maryland and Virginia, the concentrations of But-fuco varied significantly across regions with time (Table 3) (Trice et al., 2004) but showed no significant increases or decreases in any bay segment (Glibert et al., 2014). Kong et al. (2012) made a survey in the coastal waters of Qinhuangdao in 2011, and their results showed that the highest concentration of But-fuco was 6.16 μg/L at the bottom and 5.74 μg/L at the surface of the blooming area (Table 3), which was very close to the results of this study. These results indicated that the bloom intensities were quite different in distinct regions and sampling periods, which may be associated with diverse environmental factors, such as temperature, salinity, and nutrients.

Fig. 8. Relationships between But-fuco and (a) temperature, (b) salinity and (c) DO during the investigation.

4.2. Response of A. anophagefferens to environmental forces The occurrence and abundance of A. anophagefferens in coastal waters has been commonly attributed to nutrient loadings and physical factors, such as temperature, salinity and light (e.g., Berg et al., 1997; Cosper et al., 1989; LaRoche et al., 1997; Gobler et al., 2002; Lomas et al., 1996, 2001), which is more complicated than simple eutrophication-related blooms (Sunda et al., 2006). Similar to previous findings (Nuzzi and Waters, 2004), A.

Table 3 Maximum concentrations of But-fuco during brown tide in the coastal waters of Qinhuangdao scallop culture area and other regions (μg L−1). Area

1995

1999

2000

2001

2002

Mid Chincoteague Newport Bay Sinepuxent Bay Lower Chincoteagure Qinhuangdao (bottom) Qinhuangdao (surface) Qinhuangdao (surface)

4.70 6.04 2.43 0.27

2.44 1.51 0.27 0.88

3.10 1.49 1.09 0.23

6.71 5.37 4.75 0.06

6.85 5.89 2.72 0.18

114

2011

6.16 5.54

2012

5.64

2013

Reference

5.56

Trice et al., Trice et al., Trice et al., Trice et al., Kong et al., Kong et al., This study

2004 2004 2004 2004 2012 2012

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

anophagefferens in the Qinhuangdao area was well adapted to temperatures from 19 °C to 23 °C (Fig. 8a). The average temperatures during the two bloom periods in June 2012 and late-June 2013 were 20.68 °C and 20.95 °C (Table 1), respectively, both of which were close to the optimal temperature (∼20 °C) for the growth of A. anophagefferens. The positive correlations between But-fuco and temperature, however, were not that significant, as was revealed by RDA (Fig. 7), since the temperatures of three cruises were close, thus obscuring the impact of the temperature to some degree. The sea water temperature may play an important role in the spatial-temporal variation of brown tide in the Qinhuangdao area. The bloom of A. anophagefferens first occurred in the middle-south region of Qinhuangdao and then extended to the northern areas (Fig. 5). During the growing period of the brown tide in May 2012, almost no But-fuco was found in the northern region of Qinhuangdao, but it was detected at each station in the middle-south region. Temperatures in the middlesouth area were higher than those in the north during this season and were close to the temperature that is suitable for the growth of A. anophagefferens (Gobler et al., 2002). Salinity is another significant factor that impacts the growth of A. anophagefferens. The results showed that But-fuco could be found when the salinity was above 29.50 (Fig. 8b). The RDA also revealed significant positive correlations between the salinity and But-fuco (Fig. 7, Table S1), which was consistent with previous findings that A. anophagefferens grows optimally at salinities above 24 and is more likely to occur during dry years with higher salinities (Cosper et al., 1989; LaRoche et al., 1997). Based on the RDA results, the abundance of But-fuco and Fuco was positively related to DO but not significantly (Fig. 7, Table S1). High concentrations of But-fuco were detected when DO varied from ca. 8.80 mg/L to 10.00 mg/L (Fig. 8c). Gu et al. (2015) also demonstrated that DO may play an important role in causing the change in the eukaryotic microalgal community structure and diversity in Qinhuangdao. It is important to note that the DO in sea waters may vary significantly even on the diel time scale. Further studies are needed to clearly identify the relationships between the brown tide (as detected using the pigment But-fuco) and DO. Nutrients were crucial factors for the growth of A. anophagefferens, and their impacts on the initiation and duration of the brown tide have been documented in many coastal areas (Cosper et al., 1989; LaRoche et al., 1997; Glibert et al., 2007, 2014; Gobler et al., 2004, 2011). The negative correlation between inorganic nitrogen (including NH4+, NO3, NO2- and DIN) and the abundance of But-fuco in Qinhuangdao indicated that A. anophagefferens was adapted to low concentrations of inorganic nitrogen conditions (Figs. 9a-d), which was also observed in other estuaries, e.g., the east coast of the U.S. (Cosper et al., 1989; LaRoche et al., 1997). This phenomenon may be partly attributed to the ability of A. anophagefferens to utilize organic compounds, such as urea, amino acids, proteins and glucose, which has been confirmed both in culture and in the field (e.g., Dzurica et al., 1989; Berg et al., 1997, 2002; Lomas et al., 1996, 2001, 2004; Glibert et al., 2007; Mulholland et al., 2002, 2009). A laboratory study indicated that the Chinese strain of A. anophagefferens collected from the Qinhuangdao sea area has an uptake preference and a strong capability for photosynthesis on urea (Ou et al., 2018a). More recently, an examination of the activities of extracellular enzymes suggested that the hydrolysis of dissolved organic matter, in particular dissolved organic phosphorus, might significantly contribute to the occurrences of brown tides in the coastal waters of Qinhuangdao (Ou et al., 2018b). The accumulation of dissolved organic nutrients may provide a competitive advantage to A. anophagefferens over other picoalgal species that result in significant blooms (Lomas et al., 2004; Ou et al., 2018a). Unfortunately, dissolved organic nutrients were not included in this study due to certain condition limitations. Further studies are needed to elucidate the relationships between dissolved organic nutrients and the brown tide based on long-term field investigations.

In addition, during the brown tide period in June 2012 and June 2013, peak values of But-fuco were found on the Funing and Changli coast, respectively, both of which were important scallop cultivation areas of Qinhuangdao. Using samples collected simultaneously with ours, Zhen et al. (2016) reported that in waters approximately three nautical miles off Changli coast, the 18S rDNA copies were the highest, which indicated that the Changli coast was the most severe region that was afflicted with brown tide in Qinhuangdao. Similarly, based on the results of real-time fluorescent quantitative PCR, Qiao et al. (2017) also found that A. anophagefferens blooms occurred in the coastal waters of Funing and Changli. The frequent aquacultural activity can cause disturbance of sediments, thus increasing the nutrient inputs from the bottom that can be utilized by A. anophagefferens (Berg et al., 2002; Mulholland et al., 2002; Wazniak and Glibert, 2004). Additionally, the disturbance of water and the feeding activity would increase the concentrations of suspended particulate matter in seawater and decrease the light transmittance, thereby resulting in a low-light condition that is beneficial for the growth of A. anophagefferens (Probyn et al., 2010). 4.3. Variation of pigment ratios during blooms: implications for the cooccurrence of the blooms of A. anophagefferens and other species Relationships between pigments and Chl a in Qinhuangdao during blooms may indicate the co-occurrence of the bloom of A. anophagefferens and other taxa. The ratios of But-fuco, Fuco and Diad to Chl a in the bloom periods were very close to the results of a previous study in the same area (Kong et al., 2012) (Table S2), but were higher than the values of pure strains of A. anophagefferens (Alami et al., 2012; Bidigare, 1989), which suggested the presence of other But-fuco-containing and/ or Fuco-containing phytoplankton, such as haptophytes (type 8), chrysophytes and diatoms (Jeffrey and Vesk, 1997; Zapata et al., 2004; Llewellyn and Gibb, 2000). Furthermore, the ratios of Chl c3 and Chl c2 to Chl a were also higher than the reference values of A. anophagefferens, which also indicated the presence of haptophytes and chrysophytes (Zapata et al., 2004). In particular, as shown in Fig. 6, there were increases in Chl c2, Fuco and Diad with Chl a but basically no changes in But-fuco and Chl c3 in August 2011, right after the A. anophagefferens bloom of that year, which possibly indicated that there was a bloom of other species rather than that of A. anophagefferens. Diatoms are the most likely species, since Chl c2, Fuco and Diad are their signature pigments (Llewellyn and Gibb, 2000). In fact, based on molecular phylogenetic analysis, it recently has been found that the A. anophagefferens brown tide event was not monospecific; a diatom species, Minutocellus polymorphus also bloomed during the brown tide in the coastal waters of Qinhuangdao (Yu et al., 2015; Qiao et al., 2017; Xu et al., 2017), which was also recorded during the brown tide of 1985 in Narragansett Bay (USA) (Smayda and Villareal, 1989). In addition, Qiao et al. (2017) found the highest abundance of M. polymorphus in September 2013, along with the decrease in A. anophagefferens, which was attributed to the persistence of this diatom species and different nutrient utilization strategies for these two microalgae. Interestingly, besides M. polymorphus, other small algae species (pico- and/or nanosized) were also overserved during brown tide events in both Qinhuangdao and other sea areas. For example, the 1985 “brown-tide” of A. anophagefferens in Narragansett Bay co-occurred not only with M. polymorphus but also with Skeletonema costatum, Thalassiosira pseudonana, and a dinoflagellate species Prorocentrum minimum (Smayda and Villareal, 1989). Similarly, Qiao et al. (2017) also found significant growth of P. minimum along with M. polymorphus during an A. anophagefferens bloom in July 2013, in Qinhuangdao. Moreover, besides those small algae taxa that co-occurred with A. anophagefferens in Narragansett Bay, Xu et al. (2017) also detected a markedly higher abundance of Bathycoccus in Qinhuangdao. More recently, a study on the phytoplankton community succession in the Qinhuangdao coastal areas showed that another diatom species, 115

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

Fig. 9. Relationships between But-fuco and (a) ammonium, (b) nitrate, (c) nitrite, (d) DIN, (e) phosphate, and (f) silicate during the investigation.

Chaetoceros decipiens may serve as a potential species for the breakout of brown tides in the area by RDA ordination (Cui et al., 2018). There have been few attempts to discuss the co-occurrence mechanisms of A. anophagefferens blooms with other species, but none have come to any uniform conclusions. For example, experiments using various levels of A. anophagefferens and M. polymorphus or P. minimum did not show an apparent inhibition of the growth of these potentially co-occurring species by A. anophagefferens (Cosper et al., 1989). Recently, laboratory experiments showed that A. anophagefferens and M. polymorphus shared a similar allelopathic effect, with significant growth suppression by each other (Chen et al., 2018). Nevertheless, current evidence supports the existence of close interactions between small algae species and A. anophagefferens. Small-algae dominance in the phytoplankton community may provide a basis for the formation of A. anophagefferens brown tides (Xu et al., 2017).

region were highest in the near-shore scallop aquaculture regions. The A. anophagefferens blooms usually occur in June. In addition to the appropriate temperature, relatively high salinity and low inorganic nutrients were also potential factors that drive the brown tides. The highest concentrations of major pigments (such as But-fuco, Fuco and chlorophyll a) occurred in different seasons and sampling sites, thus suggesting an apparent decoupling of the biological origins of these pigments. The variation of pigment ratios further indicated the co-occurrence of A. anophagefferens and other species, such as M. polymorphus. Further studies, such as broader spatial-temporal scale investigations and in situ mesocosm experiments, are needed in Qinhuangdao coastal areas to better constrain the formation mechanisms and controlling factors of the brown tides and the role of A. anophagefferens in phytoplankton community succession. Acknowledgments

5. Conclusions

This study was supported by the Ocean Public Welfare Scientific Research Project (No. 201205031) and the Creative Team Project of the Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology (LMEES-CTSP-

According to the results of pigment analysis, the causative species of brown tides in the Qinhuangdao sea area was further identified as A. anophagefferens. Most of the pigments and hydrological variables in this 116

Harmful Algae 86 (2019) 106–118

P. Yao, et al.

2018-2). We thank Dong Li, Yu Zhen, Bin Gu, and Lingling Zhang for the sampling assistance. Guodong Chen is appreciated for analytical support. Finally, this manuscript was greatly improved, thanks to the editor’s and the anonymous reviewers’ constructive suggestions and insightful comments. This is MCTL contribution No. 66.[CG]

polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao. Acta Oceanolog. Sin. 34 (2), 132–136. Jeffrey, S.W., Vesk, M., 1997. Introduction to marine phytoplankton and their pigment signatures. In: Jeffrey, S.W. (Ed.), Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods. UNESCO Publishing, Paris, pp. 37–85. Keller, A.A., Rice, R.L., 1989. Effects of nutrient enrichment on natural populations of the brown tide phytoplankton Aureococcus anophagefferens (Chrysophyceae). J. Phycol. 25 (4), 636–646. Kong, F.Z., Yu, R.C., Zhang, Q.C., Yan, T., Zhou, M.J., 2012. Pigment characterization for the 2011 bloom in Qinhuangdao implicated “brown tide’’ events in China. Chin. J. Oceanol. Limnol. 30 (3), 361–370. LaRoche, J., Nuzzi, R., Waters, R., Wyman, K., Falkowski, P.G., Wallace, D.W.R., 1997. Brown tide blooms in Long Island’s coastal waters linked to interannual variability in groundwater flow. Glob. Change Biol. Bioenergy 3 (5), 397–410. Liu, W.C., Zeng, Z.C., Zhang, R.Q., Li, H., 2011. Investigation report on Qinhuangdao shellfish industry development. Modern Fish. Info. 26 (6), 10–14. Llewellyn, C.A., Gibb, S.W., 2000. Intra-class variability in the carbon, pigment and biomineral content of prymnesiophytes and diatoms. Mar. Ecol. Prog. Ser. 193, 33–44. Lomas, M.W., Glibert, P.M., Berg, G.M., 1996. Characterization of nitrogen uptake by natural populations of Aureococcus anophagefferens (Chrysophyceae) as a function of incubation duration, substrate concentration, light and temperature. J. Phycol. 32, 907–916. Lomas, M.W., Glibert, P.M., Clougherty, D.A., Huber, D.E., Jones, J., Alexander, J., Haramoto, E., 2001. Elevated organic nutrient ratios associated with brown tide blooms of Aureococcus anophagefferens (Pelagophyceae). J. Plank. Res. 23, 1339–1344. Lomas, M.W., Kana, T.M., MacIntyre, H.L., Cornwell, J.C., Nuzzi, R., Waters, R., 2004. Interannual variability of Aureococcus anophagefferens in Quantuck Bay, Long Island: natural test of the DON hypothesis. Harmful Algae 3 (4), 389–402. Mulholland, M.R., Gobler, C.J., Lee, C., 2002. Peptide hydrolysis, amino acid oxidation, and nitrogen uptake in communities seasonally dominated by Aureococcus anophagefferens. Limnol. Oceanogr. 47 (4), 1094–1108. Mulholland, M.R., Boneillo, G.E., Minor, E., 2004. A comparison of N and C uptake during brown tide (Aureococcus anophagefferens) blooms from two coastal bays on the east coast of the USA. Harmful Algae 3 (4), 361–376. Mulholland, M.R., Boneillo, G.E., Bernhardt, P.W., Minor, E.C., 2009. Comparison of nutrient and microbial dynamics over a seasonal cycle in a mid-atlantic coastal lagoon prone to Aureococcus anophagefferens (Brown Tide) Blooms Estuar. Coast 32 (6), 1176–1194. Nuzzi, R., Waters, R.A., 2004. Long-term perspective on the dynamics of brown tide blooms in Long Island coastal bays. Harmful Algae 3 (4), 279–293. Ou, L.J., Cai, Y.Y., Jin, W.Y., Wang, Z., Lu, S.H., 2018a. Understanding the nitrogen uptake and assimilation of the Chinese strain of Aureococcus anophagefferens (Pelagophyceae). Algal Res. 34, 182–190. Ou, L.J., Liu, X.H., Li, J.J., Qin, X.L., Cui, L., Lu, S.H., 2018b. Significant activities of extracellular enzymes from a brown tide in the coastal waters of Qinhuangdao, China. Harmful Algae 74, 1–9. Popels, L.C., MacIntyre, H.L., Warner, M.E., Zhang, Y.H., Hutchins, D.A., 2007. Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae). J. Phycol. 43 (1), 32–42. Probyn, T., Pitcher, G., Pienaar, R., Nuzzi, R., 2001. Brown tides and mariculture in Saldanha Bay, South Africa. Mar. Pollut. Bull. 42 (5), 405–408. Probyn, T.A., Bernard, S., Pitcher, G.C., Pienaar, R.N., 2010. Ecophysiological studies on Aureococcus anophagefferens blooms in Saldanha Bay, South Africa. Harmful Algae 9 (2), 123–133. Qiao, L., Chen, Y.Y., Mi, T.Z., Zhen, Y., Gao, Y.H., Yu, Z.G., 2017. Quantitative PCR analysis of the spatiotemporal dynamics of Aureococcus anophagefferens and Minutocellus polymorphus and the relationship between brown tides and nutrients in the coastal waters of Qinhuangdao, China. J. Appl. Phycol. 29 (1), 297–308. Sieburth, J.M., Johnson, P.W., Hargraves, P.E., 1988. Ultrastructure and ecology of Aureococcus anophagefferens gen. et sp.nov. (Chrysophyceae): the dominant picoplankter during a bloom in Narragansett Bay, Rhode Island, summer 1985. J. Phycol. 24 (3) 426–425. Smayda, T.J., Villareal, T.A., 1989. The 1985 ‘brown-tide’and the open phytoplankton niche in Narragansett Bay during summer. In: Cosper, E.M. (Ed.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer, Berlin, pp. 159–188. Sunda, W.G., Graneli, E., Gobler, C.J., 2006. Positive feedback and the development and persistence of ecosystem disruptive algal blooms. J. Phycol. 42 (5), 963–974. Trice, T.M., Glibert, P.M., Lea, C., Van Heukelem, L., 2004. HPLC pigment records provide evidence of past blooms of Aureococcus anophagefferens in the Coastal Bays of Maryland and Virginia, USA. Harmful Algae 3 (4), 295–304. Wazniak, C.E., Glibert, P.M., 2004. Potential impacts of brown tide, Aureococcus anophagefferens, on juvenile hard clams, Mercenaria mercenaria, in the Coastal Bays of Maryland, USA. Harmful Algae 3, 321–329. Xu, X., Yu, Z.M., Cheng, F.J., He, L.Y., Cao, X.H., Song, X.X., 2017. Molecular diversity and ecological characteristics of the eukaryotic phytoplankton community in the coastal waters of the Bohai Sea, China. Harmful Algae 61, 13–22. Yao, P., Yu, Z.G., Deng, C.M., Liu, S.X., Zhen, Y., 2010. Spatial-temporal distribution phytoplankton pigments in relation to nutrient status in Jiaozhou Bay, China. Estuar. Coast. Shelf Sci. 89 (3), 234–244. Yu, J., Zhang, L.L., Sun, Y., Zhang, L., Jiao, W.Q., Dou, H.Q., Guo, H.B., Bao, Z.M., 2015. Diversity of nanoplankton during the brown tide in the Bohai Sea. Period. Ocean Univ. China 45 (3), 73–78 (in Chinese, with English abstract).

References Alami, M., Lazar, D., Green, B.R., 2012. The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities. Biochim. Biophys. Acta Bioenergy 1817 (9), 1557–1564. Anderson, D.M., Keafer, B.A., Kulis, D.M., Waters, R.M., Nuzzi, R., 1993. An immunofluorescent survey of the brown tide chrysophyte Aureococcus anophagefferens along the northeast coast of the United States. J. Plankton Res. 15, 563–580. Andersen, R.A., Bidigare, R.R., Keller, M.D., Latasa, M., 1996. A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans. Deep Sea Res. Part II Top. Stud. Oceanogr. 43 (2), 517–537. Ansotegui, A., Trigueros, J.M., Orive, E., 2001. The use of pigment signatures to assess phytoplankton assemblage structure in estuarine waters. Estuar. Coast. Shelf Sci. 52 (6), 689–703. Berg, G.M., Glibert, P.M., Lomas, M.W., Burford, M.A., 1997. Organic nitrogen uptake and growth by the Chrysophyte Aureococcus anophagefferens during a brown tide event. Mar. Biol. 129, 377–387. Berg, G.M., Repeta, D.J., Laroche, J., 2002. Dissolved organic nitrogen hydrolysis rates in axenic cultures of Aureococcus anophagefferens (Pelagophyceae): comparison with heterotrophic bacteria. Appl. Environ. Microb. 68 (1), 401–404. Bian, C., Jiang, W., Pohlmann, T., Sündermann, J., 2016. Hydrography-physical description of the Bohai Sea. J. Coastal. Res. 74, 1–12. Bidigare, R.R., 1989. Photosynthetic pigment composition of the brown tide alga: unique chlorophyll and carotenoid derivatives. In: Cosper, E.M. (Ed.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer, New York, pp. 57–76. Chen, Y.Y., Gao, Y.H., Chen, C.P., Li, X.S., Liang, J.R., Sun, L., Wang, Y.Y., 2018. Interactions between the brown tide-causing microalga Aureococcus anophagefferens and the small diatom Minutocellus polymorphus under laboratory culture. J. Appl. Phycol. https://doi.org/10.1007/s10811-018-1704-y. Cosper, E.M., Dennison, W., Milligan, A., Carpenter, E.J., Lee, C., Holzapfel, J., Milanese, L., 1989. An examination of the environmental factors important to initiating and sustaining “brown tide” blooms. In: Cosper, E.M. (Ed.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer, Berlin, pp. 317–340. Cui, L., Lu, X.X., Dong, Y.L., Cen, J.Y., Cao, R.B., Pan, L., Lu, S.H., Ou, L.J., 2018. Relationship between phytoplankton community succession and environmental parameters in Qinhuangdao coastal areas, China: a region with recurrent brown tide outbreaks. Ecotox. Environ. Safe 159, 85–93. DeYoe, H.R., Stockwell, D.A., Biolagare, R.R., Latasa, M., Johnson, P.W., Hargraves, P.E., Suttle, C.A., 1997. Description and characterization of the algal species Aureoumbra lagunensisgen. et sp. nov. and referral of Aureoumbra and Aureococcus to the Pelagophyceae. J. Phycol. 33 (6), 1042–1048. Dong, H.P., Huang, K.X., Wang, H.L., Lu, S.H., Cen, J.Y., Dong, Y.L., 2014. Understanding strategy of nitrate and urea assimilation in a Chinese strain of Aureococcus anophagefferens through RNA-Seq analysis. PLoS One 9 (10), e111069. Dzurica, S., Lee, C., Cosper, E.M., Carpenter, E.J., 1989. Role of environmental variables, specifically organic compounds and nutrients, in the growth of the chrysophyte Aureococcus anophagefferens. In: Cosper, E.M. (Ed.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer, New York, pp. 229–252. Gastrich, M., Wazniak, C.E., 2002. A brown tide bloom index based on the potential harmful effects of the brown tide alga, Aureococcus anophagefferens. Aquat. Ecosyst. Health Manage. 5 (4), 435–441. Glibert, P.M., Wazniak, C.E., Hall, M.R., Sturgis, B., 2007. Seasonal and interannual trends in nitrogen in Maryland’s Coastal Bays and relationships with brown tide. Ecol. Appl. 17 (5), S79–S87. Glibert, P.M., Hinkle, D.C., Sturgis, B., Jesien, R.V., 2014. Eutrophication of a Maryland/ Virginia coastal lagoon: a tipping point, ecosystem changes, and potential causes. Estuar. Coast 37, S128–S146. Gobler, C.J., Renaghan, M.J., Buck, N.J., 2002. Impacts of nutrients and grazing mortality on the abundance of Aureococcus anophagefferens during a New York brown tide bloom. Limnol. Oceanogr. 47 (1), 129–141. Gobler, C.J., Boneillo, G.E., Debenham, C., Caron, D.A., 2004. Nutrient limitation, organic matter cycling, and plankton dynamics during an Aureococcus anophagefferens bloom in Great South Bay. NY. Aquat. Microb. Ecol. 35 (1), 31–43. Gobler, C.J., Lonsdale, D.J., Boyer, G.L., 2005. A review of the causes, effects, and potential management of harmful brown tide blooms caused by Aureococcus anophagefferens (Hargraves et Sieburth). Estuaries 28 (5), 726–749. Gobler, C.J., et al., 2011. Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics. PNAS 108 (11), 4352–4357. Gu, B., Zhen, Y., Mi, T.Z., 2015. Characterization of phytoplankton community in the coastal waters of Qinhuangdao during brown tide. Period. Ocean Univ. China 45 (7), 64–72. Guo, H., Liu, Y.J., Zhang, Y.X., Zhang, W.W., Zhang, Z.F., 2015. A quantitative

117

Harmful Algae 86 (2019) 106–118

P. Yao, et al. Zapata, M., Rodriguez, F., Garrido, J.L., 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar. Ecol. Prog. Ser. 195, 29–45. Zapata, M., Jeffrey, S.W., Wright, S.W., Rodriguez, F., Garrido, J.L., Clementson, L., 2004. Photosynthetic pigments in 37 species (65 strains) of Haptophyta: implications for oceanography and chemotaxonomy. Mar. Ecol. Prog. Ser. 270, 83–102.

Zhang, Q.C., Qiu, L.M., Yu, R.C., Kong, F.Z., Wang, Y.F., Yan, T., Gobler, C.J., Zhou, M.J., 2012. Emergence of brown tide caused by Aureococcus anophagefferens Hargraves et Sieburth in China. Harmful Algae 19 (9), 117–124. Zhen, Y., Qiao, L.L., Gu, B., Mi, T.Z., 2016. Characteristics of eukaryotic microalgal community and its abiotic influencing factors during brown tide blooms near Qinhuangdao, China. Harmful Algae 57, 1–12.

118