Phytoplankton Assemblage Structure Shaped by Key Environmental Variables in the Pearl River Estuary,South China

ZHANG Xia,ZHANG Jingping,HUANG Xiaoping,and HUANG Liangmin

Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou 510301,P. R.China

1 Introduction

Estuaries are transition zones linking freshwater and marine systems,and are therefore characterized by gradients of chemical,physical and biological components in the water column.These gradients strongly influence the spatial and temporal distribution,species composition and abundance of phytoplankton in estuaries (Quinlan and Phlips,2007).Hence,the phytoplankton community will be composed of a series of assemblages along the freshwater to marine continuum as a result of physical tolerances and resource competition (Attrill and Rundle,2002).

Phytoplankton dynamics are mainly forced by bottom-up and top-down controls,and these interactions are more complicate in estuaries,because of the seasonal variations of the river discharge besides wind and tidal energy (Pennock and Sharp,1994; Cloern,1996).In many estuaries,salinity appears to be the main factor determining the shifts in many phytoplankton taxa,and other factors (i.e.,temperature,light and nutrient availability) have coupling effects with salinity to explain the spatial and temporal alteration of the phytoplankton composition (Quinlan and Philips,2007).Phytoplankton in the Suwannee Rive estuary progressively changes from a freshwater to marine dominated community along a salinity gradient (Quinlan and Philips,2007).

It is common accepted that salinity and nutrient concentrations are not necessarily independent in estuaries,as both are a function of the riverine discharge.It has been approved that most of dissolved inorganic nitrogen in the PRE is from the freshwater discharges and its concentration declines along the salinity axis resulting from dilution and biological uptake (Linet al.,2004).Hence nitrate shows the conservative behaviour (Huanget al.,2003; Yinet al.,2000).However,there is no linear relationship between the phosphate and salinity,suggesting that the main source of phosphate not depends on the freshwater input.Huanget al.(2003) indicated that land-based sources outside the estuary brought by coastal current and flood tide current should be the main sources of the phosphate in the PRE.

Phosphorus is considered as a frequent limiting nutrient to phytoplankton growth in the PRE and other estuaries (Smith,2006; Yin and Harrison,2008).In addition,low light availability has been proposed as another main reason for lower yields of phytoplankton (Cloern,1987;Fisheret al.,1988; Kocumet al.,2002) and much of the temporal variability of phytoplankton biomass in estuaries is related to variations in light availability.Consequently,some phytoplankton species serve as seed populations downstream and start to bloom when light penetration or other ambient conditions are improved in the PRE (Yinet al.,1995).Huanget al.(2004) observed that distribution of the dominant phytoplankton species varies with salinity of seawater,and their abundances correlate negatively with nutrients and zooplankton.In addition,Yinet al.(2004) sketched a typical spatial variability of phytoplankton biomass in the PRE: low biomass and productivity due to rapid dilution and light limitation in turbid estuarine waters; a regional maximum of biomass and productivity under intermediate salinities in the coastal plume and low biomass due to nutrient limitation in oceanic waters.

Phytoplankton diversity and distribution,seasonal and spatial variations of biomass and bloom events,and their linear correlations with nutrient have been widely investigated in the PRE (Huanget al.,2004; Yinet al.,2000,2001,2004).However,most of these studies only included one or two distinct seasons and little explored the relationship between dominant phytoplankton species and key environmental factors.In this study,the spatial and temporal composition and abundance of phytoplankton were examined in relation to physico-chemical and zooplankton variables in three seasons under distinct hydrodynamic conditions.To achieve these goals a series of exploratory statistical methods have been employed in the analysis of the combined data sets.

2 Materials and Methods

2.1 Study Area

The Pearl River Estuary (PRE),being located in southern Guangdong Province,is formed by inflows of the Pearl River to the South China Sea (SCS) through 8 entrances,4 of which enter the Ling Ding Yang estuary,a sub-estuary of the Pearl River Estuary.Geographically,the Pearl River has a subtropical climate with a long summer and a short winter; the annual average temperature is 21–22℃ and total rainfall is 1600–2000 mm.The annually averaged river discharge is 10524 m3s?1,with 20% of it occurring during the dry season from October to March and 80% during the wet season from April to September (Zhao,1990).In recent decades,the Pearl River Delta area has been under rapid socio-economic changes,including the population growth,industrialization,and urbanization.A large amount of domestic,industrial,and agricultural effluent is discharged into the river system,causing deterioration of the aquatic environment in this estuary (Huanget al.,2003; Qiuet al.,2010).Then the harmful algal blooms (HAB) happened more frequently in recent years in the PRE (Yinet al.,2001).

2.2 Sampling and Analysis

Three surveys were conducted at 16 stations respecttively in July 2006 (high river flow season,summer),March 2007 (low river flow season,spring),and September 2007 (normal river flow season,autumn) in the PRE(Fig.1).The whole study area is within the sub-tidal zone with strong fluvial runoffs and marine water inter-reactions.Temperature and salinity were measured using YSI6600.Integrated water samples were collected with 5 L Niskin bottles through the full depth of the water column.

Fig.1 Sampling stations of the Pearl River Estuary.

Suspended solid content (SSC) samples were acquired through pre-weighed Whatman GF/F fiber filters (25 mm).The filters were dried and weighed to determine the amount in mg L?1of sample.Dissolved oxygen was determined according to Dickson (1995).Total phosphorus(TP) concentration was measured by colorimetry after digestion of the unfiltered samples with K2S2O8to orthophosphate (Ebinaet al.,1983).Total nitrogen (TN) was digested simultaneously with TP.After digestion,TN was measured as nitrate and absorbance was measured at 543 nm.Nitrate (plus nitrite) and ammonium were determined manually following the procedures of Woodet al.(1967)and Slawyk and MacIsaac (1972),respectively.DIN was the sum of nitrate,nitrite and ammonia.DIP concentration was measured based on the methods by Armstronget al.(1967).

A plankton net (77 μm mesh size) was employed in collecting phytoplankton and samples were stored with Lugol’s solution and identified according to Jinet al.(1965).Zooplankton was sampled using a modified WP-2 net (mouth size,0.5 m2; mesh size,505 μm) by towing vertically from 1 m above the bottom to the surface and the filtered water volume was determined from the rope length multiplied by mouth size (Zhang,1993).The samples collected were preserved immediately in 5% formaldehyde for further identification of species under microscope (Chen and Zhang,1974; Chen and Shen,1974).

2.3 Data Analysis

Non-metric multidimensional scaling (NMDS) was designed to distinguish distinct regions within the estuary based on the phytoplankton assemblages.These analyses were undertaken using PRIMER (v.6) software (Plymouth Routines in Multivariate Ecological Research,Plymouth Marine Laboratory,Plymouth,UK; Clarke and Gorley,2006).On the basis of Bray–Curtis similarities,the similarity percentages analysis (SIMPER) was applied to phytoplankton species abundance,in order to allow the separation of every two groups of stations according to different phytoplankton community structure.We intended to analyze the dominant phytoplankton density as dependent factors in a linear regression analysis with water temperature,salinity,SSC,NO3-N,NH4-N,DIN,TN,DIP,TP concentrations and copepod density as the independent factors.Owing to the dominance of the copepods in zooplankton community,we considered copepods as main efficient consumers.Since several of the variables obviously were correlated,we performed a principal component regression (PCR),i.e.,principal component scores from a principal component analysis (PCA) instead of the actual x-variables in the regression analysis (Quinn and Keough,2002).Statistical analysis was performed using SPSS 17.0 for Windows.

3 Results

3.1 Environmental Variables

In our surveys,the average of water temperature was higher in summer (29.3℃) and autumn (27.6℃) than that in spring (19.9℃).The average of salinity was higher in spring (20.4) than those in autumn (10.9) and summer(9.4).In summer,a stratified layer and salinity gradient were observed in the middle reaches of the estuary resulting from large river discharge and salt wedge intrusion (Fig.2a).A well developed salt wedge could extend to station S8.In spring,salinity difference existing between the surface and bottom layer was much smaller.Salinity was roughly less than 25 in the upper reaches(S1?S5) or on the western side (S9,S10 and S12) of the PRE,while it varied from 30 to 35 in the middle and lower reaches (S8,S11 and S13?S16).About 95% of the sediment load is delivered during the wet season (Harrisonet al.,2008).SSC was higher in high flow season(41.65 mg L?1) than in spring (25.34 mg L?1) and exhibited the peak values near the river mouth in both seasons(Fig.2b).

Fig.2 Variations of salinity in surface and bottom layers (a)and SSC (b) in the Pearl River Estuary.

Generally,the distribution patterns of nutrient were similar to that of SSC and the concentration decreased as the riverine water mixed with the shelf water (Fig.3).DIN mainly came from the runoff of Pearl River and the concentration in the northern area was generally higher than that in the southern part.The maximum DIP concentration was observed at coastal stations S4 and S7 (near Shenzhen Bay) in two seasons of 2007 (Fig.3) which were under intense anthropogenic pressure and implicated that land based pollutants near Shenzhen Bay contributed to phosphate load greatly (Huanget al.,2003).Meanwhile,the lowest DIP values in summer,spring and autumn were 0.28 μmol L?1,0.43 μmol L?1and 0.06 μmol L?1,respectively,which were then observed in the outer estuary.

Fig.3 Spatial variation of phosphorus and nitrogen concentrations in three seasons.

3.2 Abundance and Community Structure of Phytoplankton and Zooplankton

Average density of phytoplankton in summer,spring and autumn was 4.0×105,1.8×105and 2.3×105cells L?1,respectively (Table 1).In these three seasons,the higher abundance occurred in the lower section of the PRE,and the maximum value appeared at the station 15,which was situated in the southern waters of Hong Kong.In the upstream and middle PRE (S1 to S10),phytoplankton abundance was several orders of magnitude lower than those detected in the downstream.Phytoplankton abundance of the three seasons was negatively related with SSC,DIN and DIP while positively with salinity (Fig.4).151 species were identified in the three surveys,belonging to 42 genera of Bacillariophyta (105 species),10 genera of Pyrrhophyta (31 species),4 genera of Chrysophyta(8 species) and 5 genera of Cyanobacteria (7 species).Diatoms almost dominated the community in the three cruises except at some stations in the autumn cruise(Fig.5). The most important diatom species (average abundance >5% of the total) wereCoscinodiscus jonesianus,Skeletonema costatum,Thalassiosira subtilis,Biddulphia sinensis,Pseudonitzschia pungens,P.delicatissima,Aulacoseira granulata,A.granulata v.angustissima,Chaetocerospseudocurvisetus,C.curvisetusandC.lorenzianus.

The NMDS ordination of stations generated by phytoplankton abundance is illustrated in Fig.6.Stress values associated with this two-dimensional plot was 0.04,0.14 and 0.07 for the three seasons,respectively,revealing that this representation of stations was sound (Clarke and Gorley,2006).The successive position of the stations revealed algal community differences along the estuary.

In the summer,stations located in the upper estuary (S1 to S7) were classified as group A characterized by the prevalence of freshwater phytoplankton species (A.granulataandA.granulatav.angustissima).S16 located in the outer estuary,formed group C.Group B (S8 to S15)individualized dominance of coastal and estuarine species(S.costatumandP.delicatissima) (Fig.5,Fig.6a).

Table 1 Mean and ranges of the physico-chemical parameters,phytoplankton and zooplankton abundance of three surveys in the Pearl River Estuary

Fig.4 Linear regression of phytoplankton abundance (log10-transformed) and SSC (a),salinity (b),DIN (c),DIP (d)in the PRE.

Fig.5 Spatial variation of dominant phytoplankton species in three studied seasons.

Fig.6 NMDS analyses of the phytoplankton community structure in three studied seasons.

In the spring,Stations S1 to S14 and S16 were categorized into one group (group A),while S15 constituted another one (group B) (Fig.6b).Components of group A were characterized by dominance of marine species (e.g.Thalassiosira subtilisandBiddulphia sinensis) and freshwater species only made a minor contribution in the community: group B was dominated byC.pseudocurvisetusand total phytoplankton abundance was much larger than that in group A (Fig.5).

In the early autumn cruise,16 stations could be separated into three groups according to phytoplankton structure (Fig.6c).Group A consisted of S1 to S10 with an overwhelming speciesMicrocystisspp.Stations in the inner estuary (S11 to S14) were clustered as group B.Outer stations S15 and S16 were labeled as group C withP.pungens,SkeletonemaandChaetocerosbeing dominant species (Fig.5).

The average of zooplankton abundance was highest in summer (778 ind.m?3),followed by those in autumn (734 ind.m?3) and spring (351 ind.m?3).Average copepod densities were 594,294 and 407 ind.m?3in summer,spring and autumn,respectively.The copepod assemblages were dominated byPseudodiaptomus poplesia,Sinocalanus solstitialis,S.laevidactylus,Acarfia spinicaudaandPavocalanus crassirostrisin the three seasons (Fig.7).

Fig.7 Spatial variation of total zooplankton,dominant copepods abundance in three studied seasons.

3.3 Environmental Variables Related with Phytoplankton Community Explored by PCR

The ten abiotic and biotic variables that potentially explained the horizontal heterogeneity of phytoplankton community structure in the summer and spring were interpreted in terms of principal components (Table 2).In both seasons,SSC and nutrient species were positively associated with each other and both of them were negatively associated with salinity.In the summer of 2006,the first principal component (PC1),which explained 60.07%of the variation of the environmental data,was positively correlated with nutrient species (NO3-N,NH4-N,DIN,TN,DIP and TP),SSC and water temperature were negatively correlated with salinity.The second component(PC2),which explained 11.49% of the variation of the environmental data,was correlated with copepod density.In the spring,PC1 explained 57.89% of the variation of the environmental data,was positively correlated with SSC,salinity,nutrient species and copepods.The second component (PC2) was positively related with temperature and explained 19.95% of the variation of the environmental data.The linear regression models including principal components and dominant phytoplankton species are shown in Table 3.In the summer,PC1 was negatively related withS.costatum,P.delicatissimaand positively related withA.granulataandA.granulatav.angustissima.In the spring,T.subtilisandB.sinensiswere negatively related with PC1.

Table 2 Results of principal component analysis (PCA) describing the major environmental gradients of variation in two seasons

Table 3 Regression analysis data of the principal components (PC) as dependent factors for the dominant phytoplankton species in two seasons

4 Discussion

4.1 Key Environmental Factors Regulating Phytoplankton Abundance

In turbid estuaries like the Schelde Estuary,phytoplankton biomass is mainly regulated by concentrations of suspended matter (Kromkamp and Peene,1995;Kocumet al.,2002).Although suspended sediment concentration in the PRE is lower than the Yangtze River estuary and Yellow River estuary,Yin and Harrison(2008) pointed out low chlorophylladuring summer in the PRE mainly resulted from high turbidity due to high freshwater discharge.The 10 mg L?1has been regarded as a threshold concentration of SSC for phytoplankton inhibition (DeMasteret al.,1996; Ragueneauet al.,2002).In our summer and spring surveys,suspended solid content was much higher than this threshold value except at the outer stations S15 and S16.According to Cloern (1987),average euphotic depth in the inner estuary (S1 to S14) in summer was only 1.43 m,while at the outer sites S15 and S16 such average depths were 3.18 and 3.90 m,respectively.Hence,phytoplankton growth was light-limited and phytoplankton could not accumulate in the inner estuary in spite of the high nutrients (Yinet al.,2000; Yinet al.,2004).In the Chesapeake and Delaware Bays,a chlorophyll maximum occurred downstream of the turbidity maximum (Fisheret al.,1988).Turbidity was considered as the main limiting factor for phytoplankton abundance in the PRE and exhibited an inversed relationship.

It has been widely accepted that the Ks (half-saturation constant) is associated with nutrient limitation for algal uptake (Aksnes and Egge,1991).Typical Ks values of nitrate and phosphate for natural phytoplankton populations are ca.1?2 μmol L?1and 0.1?0.5 μmolL?1,respectively (Fisheret al.,1988).As for phosphate,its concentration was always below 0.5 μmol L?1in outer stations,suggesting a potential P-limitation.In the present study,NO3?concentrations at the outer stations S15 and S16 were both higher than 2 μmol L?1in the three seasons,indicating that N was abundant.Yin and Harrison (2008)figured in the region of the chl.amaximum,phosphorus was rapidly depleted by phytoplankton and excess of N was left in the water.In the present study,phytoplankton abundance was negatively related with DIP in the three surveys.The maxima of phytoplankton density occurred in the outer estuary (S15 and S16) where phosphate was reduced to extremely low level.Therefore,phosphate may be another factor regulating phytoplankton abundance in the PRE,which was also suggested by Huanget al.(2003).

4.2 Environmental Factors Shaping Horizontal Phytoplankton Community Structure

Along the estuary axis,phytoplankton composition shifted from dominant freshwater species to salinitytolerant species,and finally to the increasing dominance of estuarine forms (Wong and Townsend,1999).It is obvious that the different dominating species of diatoms at different sites are expected to use distinct strategies in order to adapt to the diverse trophic conditions and mixing levels (Reynoldset al.,2002; Alves-de-souzaet al.,2008).In our case,configuration explored by NMDS gave a good representation of the distance matrix,allowing an easier identification of the dominant species assemblage at the salinity gradient.A.granulataandA.granulatavar.angustissimahad the highest abundance near Humen mouth (S1) with a surface water salinity of 0.9 in the summer,and then decreased with increasing salinity along the estuarine axis.This distribution pattern was very similar to those reported by Huanget al.(2004).A.granulataandA.granulatavar.angustissimaare typical riverine phytoplankton in large rivers of the world(Rojoet al.,1994; Lewiset al.,1995).With a limited salinity tolerance,these two freshwater species were restricted to the head of the estuary in the summer and exhibited a positive relationship with PC1,which reflected the freshwater inflow effects.

On the contrary,other dominant diatoms such asS.costatum,P.delicatissima,T.subtilisandB.sinensiswere negatively associated with PC1,indicating that their development was restricted by turbid freshwater inflow.S.costatumis a pollution-tolerant and indicator species of eutrophication (Nassar,2000).In the competition experiments with two marine diatoms,S.costatumsupplantedPhaeodactylum tricornutumat high N: P ratios with the medium (Pauw and Naessens-Foucquaert,1991).S.costatumis well adapted to higher N: P conditions andhigher N: P ratio is one of a characteristic of the nutrient regime in the PRE.The optimum temperature and salinity ranges forS.costatumwere 20–26℃ and 25–30,respectively (Jin,1965).Chenet al. (2005) found that the growth ofS.costatumwas little affected by a salinity shift from 18 to 35.7.Another cosmopolitan genus,Pseudonitzschiais well known for its adaption to great ranges in salinity,15–40 for optimal growth in some species(Thessenet al.,2005).In spring,eurythermal speciesT.subtilisandB.sinensisalways appear in coastal waters and estuaries which are generally characterized by high nutrient concentrations (Qasimet al.,1973).Sudden changes in salinity (15?30) and temperature (12?20℃)almost did not influence growth ofB.sinensis(Jahnke and Baumann,1983).In the present study,two independent variables,salinity and turbidity,played important roles in the distribution of the major phytoplankton species.

The hydrographic conditions of the PRE and other estuaries (e.g.,Swan,Yura and Pearl Estuary) drastically change between wet and dry periods (Thompson,1998;Harrisonet al.,2008).In the spring survey,probably because of the decrease of the freshwater flushes from the Pearl River,the inner estuary was relatively homogenous.Hence,sampling stations of the inner estuary were classified as one group.Some estuarine and marine species were detected at the head of the estuary.

In the autumn,an enhanced proliferation of potentially toxicMicrocystiswas detected at the lower section of the PRE.Microcystisblooms have often been associated with varying concentrations of the microcystin in the surrounding water.However,during our study,Microcystisabundance was below a blooming level (105cells L?1),which probably imposed limited impairment to environment.The higher temperatures along with water column stability may have supported cyanobacteria,as have been observed in several estuaries and coastal waters,including the Chesapeake Bay (Marshall and Nesius,1996),the Florida Bay (Phlipset al.,1999),the Neuse River estuary(Valdes-Weaveret al.,2006),the San Francisco Bay(Ninget al.,2000),and the York River estuary (Sinet al.,2000).Cyanobacteria abundance in these ecosystems varies considerably,but in general warmer estuaries tend to be associated with higher abundance of cyanobacteria(Murrel and Lores,2004).During the dominance ofM.aeruginosa,water temperatures were from 24.7 to 33.9℃in the eutrophic Furuike Pond,Japan (Imaiet al.,2009).Furthermore,Christianet al.(1986) found the optimum water temperature for this species to be about 28℃.Our investigation is consistent with these results.

The growth ofM.aeruginosawas restricted in the brackish region and negatively impacted by the salinity in Patos Lagoon (Yuneset al.,1997).In addition,M.aeruginosagrew optimally at salinities up to 4,above which growth rate declined to zero at salinity about 25 in laboratory experiments (Robson and Hamilton,2003).In the present study,the salinity was beyond its tolerance threshold in the middle of the estuary.It is probable that colonies ofMicrocystisdeveloped in this area have been seeded from upstream and transported by river discharge and the wind-induced advection,which has also been observed in San Francisco Bay Estuary (Lehmanet al.,2005) and a Western Australia estuary (Robson and Hamilton,2003).

The absence of significant relationship between phytoplankton and zooplankton density was observed in the present study.Low levels of phytoplankton in the inner estuary was probably not related with top-down force as zooplankton (copepods) abundance was very low and restricted only at given regions of the estuary (Liet al.,2004).Aggregations of copepods and other zooplankton tended to occur at boundaries or fronts in the vertical or horizontal planes (Petipa,1985).Tanet al.(2004) observed that a high value of Chl.ausually coincided with a low ingestion rate in the PRE.Hence,phytoplankton abundance was more likely controlled by abiotic factors than by biotic mechanisms in this estuary.

Acknowledgements

This study was supported by the National Natural Sciences Foundation of China (Nos.31000185,41076069),the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No.XDA11020205).

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