Benthic ciliate and meiofaunal communities in two contrasting habitats of an intertidal estuarine wetland

Benthic ciliate and meiofaunal communities in two contrasting habitats of an intertidal estuarine wetland

Journal of Sea Research 70 (2012) 50–63 Contents lists available at SciVerse ScienceDirect Journal of Sea Research journal homepage: www.elsevier.co...

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Journal of Sea Research 70 (2012) 50–63

Contents lists available at SciVerse ScienceDirect

Journal of Sea Research journal homepage: www.elsevier.com/locate/seares

Benthic ciliate and meiofaunal communities in two contrasting habitats of an intertidal estuarine wetland Yongfen Du a, b, Kuidong Xu b,⁎, Alan Warren c, Yanli Lei b, Renhai Dai b a b c

The Key Laboratory of Coast and Island Development of Ministry of Education, Nanjing University, Nanjing 210093, China Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK

a r t i c l e

i n f o

Article history: Received 16 September 2011 Received in revised form 7 March 2012 Accepted 13 March 2012 Available online 28 March 2012 Keywords: Ciliates Community composition Meiofauna Seagrass Tidal flats Jiaozhou Bay, China

a b s t r a c t Annual variations in benthic meiofaunal and ciliated protozoan communities were investigated using monthly samplings from June 2006 to May 2007 in two habitats characterized by different vegetal coverage in an estuarine intertidal wetland of Qingdao Jiaozhou Bay, China. The sediment composition was stable at each site: sediments densely covered with seagrass (Suaeda glauca) in the lower estuarine site (Station S) were finer, with higher content of organic matter, phaeopigments and water than sediments at the upper estuarine site (Station S-P) which was unvegetated other than for patches of S. glauca and common reed (Phragmites australis). Chlorophyll a exhibited a similar distribution in the two habitats. A total of 14 meiofaunal groups, and 249 species of ciliates belonging to 37 genera, 28 families and 16 orders, were isolated from the two sites. Univariate and multivariate measures of the communities were significantly different between the two habitats. There were higher abundances of ciliates and meiofauna, and a greater diversity of ciliates, at Station S than Station S-P (223 vs. 61 species). Herbivorous ciliates were numerically predominant in ciliate communities at both sites. The representative ciliates at Station S-P belonged to the Cyrtophorida and appeared to be a reduced subset of the assemblage at Station S, which was characterized by members of the Prostomatida, Cyrtophorida, Hypotrichida and Scuticociliatida. More than 96% of the total meiofauna were nematodes, accounting for 93% of the differences in the abundance compositions of the meiofaunal communities between habitats. The average individual weights of nematodes were nearly 3 times greater at Station S than Station S-P, indicating a distinctive species composition at each site. Temperature, salinity and food availability were key factors that regulated the ciliate and meiofaunal community structure. Nematodes were the dominant group in terms of the combined abundance, biomass and benthic metabolism of ciliates and meiofauna. With respect to the dominance of herbivorous ciliates and epistrate-feeder nematodes in seagrass sediment, predator–prey relationships and competition for food resources between nematodes and ciliates are likely to be important factors in controlling the abundances of these groups. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Estuaries are widely recognized as highly specialized types of ecosystem with unique marine and brackish water communities of organisms (Levin et al., 2001; McLusky and Elliott, 2004). The benthic realm forms an integral and important component of estuarine ecosystems, with the benthos encompassing a large variety of fauna whose body sizes span more than nine orders of magnitude (Schmid et al., 2000). The ciliated protozoa (ciliates), which are members of the microfauna, and the microscopic metazoans, which constitute the meiofauna, are considered to be important elements in the benthic microbial food web. They are characterized by their small size, short life span, high turnover rate and complicated trophic structure

⁎ Corresponding author. Fax: + 532 82898776. E-mail address: [email protected] (K. Xu). 1385-1101/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2012.03.004

(Coull, 1999; Epstein, 1997a,b; Kuipers et al., 1981). Additionally, their high sensitivity to anthropogenic inputs makes them excellent sentinels of estuarine pollution (Grego et al., 2009; Irizuki et al., 2011; Moreno et al., 2011; Sutherland et al., 2007). In comparison to the intensively studied meiofauna, the distribution and ecological importance of ciliates in marine sediments have received little attention. This is largely due to the methodological difficulties involved in extracting these fragile microorganisms from sediments for the purpose of qualitative and quantitative analyses. In recent years, however, the application of silica sol density gradient centrifugation in combination with the quantitative protargol stain (QPS) has enabled quantitative investigations of the benthic ciliate assemblages to be carried out with high taxonomic resolution (Hamels et al., 2004, 2005; Wickham et al., 2000; Xu et al., 2010). Jiaozhou Bay is located on the southern coast of the Shandong Peninsula in eastern China (Fig. 1). It is a semi-enclosed bay of the Yellow Sea that has undergone extensive environmental changes in

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

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Fig. 1. Map showing the intertidal sampling sites (●) at the Dagu River estuary discharging into Jiaozhou Bay (36°14′N, 120°06′E). Station S was covered by Suaeda glauca and station S-P was covered by S. glauca and Phragmites australis during monthly samplings.

recent years, due both to natural factors (including sediment supplies) and human activities such as farming and construction. For example, the world's longest bridge over water crosses Jiaozhou Bay. Nevertheless, the perimeter region of Jiaozhou Bay is one of only seven estuarine wetland ecosystems listed in China Biodiversity Conservation Strategy and Action Plan as requiring priority conservation attention. The estuarine wetland of the River Dagu, the largest river discharging into Jiaozhou Bay, supports about 75% of the total vegetation in the region. Thus it is one of its most important ecosystems and has led to its establishment of a nature reserve (Ministry of Environmental Protection of the People's Republic of China, 2010). However, for a number of historical and methodological reasons, the micro- and meiofauna of the River Dagu estuary has received far less attention than other biotopes in Jiaozhou Bay such as the pelagium and macrofauna (Yuan et al., 2007; Zhang et al., 2001). Furthermore, there is a paucity of knowledge of the fauna in unvegetated patches within seagrass-dominated estuaries worldwide despite the ecological importance of seagrass biotopes (Boström et al., 2006). We investigated the benthic ciliate and meiofauna communities in the River Dagu estuary using the recently developed Ludox-QPS method (Du et al., 2009; Xu et al., 2010). We compared the environmental characteristics, and the communities of ciliates and meiofauna (i.e. species composition, diversity, abundance, biomass, spatial and seasonal distributions) in seagrass sediments in the lower part of the estuary with those in sediments with a patchy distribution of halophytes, or which are otherwise unvegetated, in the upper part of the estuary. The main aims are to determine: (1) whether the seagrass habitat supports ciliate and meiofaunal communities that are as distinctive as that of the macrofauna; (2) the most important factors regulating the ciliate/meiofaunal variation spatially and seasonally, and; (3) the relative importance of ciliates and nematodes in terms of their abundance, biomass and metabolism in the benthic ecosystem. Potential trophic interactions between ciliates and nematodes are also discussed. 2. Materials and methods 2.1. Study sites This investigation was undertaken in the tidal estuary of the River Dagu which is located in the northwestern part of Jiaozhou Bay (Fig. 1). The shoreline of the estuary consists mainly of muddy sediments and is extensively covered with halophytes (Ma et al., 2006). Much of the area of sediment deposition has been reclaimed for shrimp farming or salt production (Song et al., 2008). A number of jetties for fishing boats have also been constructed along the estuary.

Two sites representing two distinct habitats were selected for the investigation. One was located in the lower section of the estuary and was exposed to a strong tidal flow of marine waters. This area of the estuary is covered by a dense canopy of the seagrass (also known as or seepweed) Suaeda glauca (Bunge). The other site was located in the upper section of the estuary and was more protected from strong tidal flows of marine waters. In this area of the estuary most of the sediment was unvegetated and subaerially exposed over much of the ebb tidal phase, although S. glauca and the common reed, Phragmites australis, occurred in patches. The two habitats are designated Stations S and Station S-P respectively, based on the type and extent of plant cover. 2.2. Sample collection and measurement of environmental factors Monthly sampling was carried out during ebb tide from June 2006 to May 2007. Eight randomly selected replicate samples were collected at each site by coring sediments to a depth of 8 cm using a modified syringe with an inner diameter of 23 mm. The sediment was extruded carefully through the bottom of the cores and sectioned immediately into 0–0.5 cm, 0.5–2 cm, 2–4 cm and 4–8 cm depth layers. For the analysis of benthic organisms, each of the four samples was initially diluted in-situ with filtered water, fixed with an equal volume of ice-cold glutaraldehyde (2% final concentration) and stored at 4 °C in the dark until being processed. The corresponding layers of the other four replicate cores were pooled and stored in a frozen state for later environmental analyses. On each sampling occasion, the in situ temperature was measured using a thermometer with the bulb immersed in the sediment to a depth of 5 cm. Seepage water from the uppermost 10 cm layer of the sediment was collected for salinity measurement using a refractometer. The water content of the sediment was determined as the percentage of weight loss after drying the sediment at 60 °C for 72 h. The organic matter (OM) content of the sediment was measured using the K2Cr2O7–H2SO4 oxidization method (Gaudette et al., 1974; Nelson and Sommers, 1982) The concentrations of sediment chlorophyll a (chl-a) and total phaeopigments (pheo) were determined via spectrophotofluorimetry (Lorenzen and Jeffrey, 1980; MacIntyre and Cullen, 1995) using the corrected formula of Wang (1986) for the calculation of pheo. Surface sediment samples were used for determination of grain size distribution using a Laser Diffraction Particle Size Analyzer (Cilas 940L). 2.3. Extraction, enumeration and identification of organisms The Ludox-QPS method, which combines Ludox density gradient centrifugation (Xu et al., 2010) and quantitative protargol staining

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(QPS) (Montagnes and Lynn, 1993), was used for the enumeration and identification of benthic ciliates and meiofauna. Prior to Ludox centrifugation, sample elutriation and salt reduction were performed, during which a 500 μm mesh was used for the removal of macrofauna. A subsample of 3–5 ml was used for the extraction of ciliates and meiofauna. The extracted organisms were then concentrated using vacuum equipment (Sartorius Stedium, Germany) onto a 25 mm diameter, 5 μm pore-sized cellulose nitrate filter membrane. Finally, the filter was stained according to the modified QPS protocol of Skibbe (1994). The QPS-prepared slides were examined under a Leica 4500B microscope. Ciliates and meiofauna were enumerated at a 200× magnification, while ciliate identification was undertaken at 400× and 1000× magnifications with the aid of relevant publications (e.g., Carey, 1992; Lynn and Small, 2002). The classification of the ciliate groups mainly follows Corliss (1979). Meiofauna organisms were sorted to group level according to Higgins and Thiel (1988) and Giere (2009). In each sample, the abundance and biomass of ciliates and meiofauna were analyzed. The biovolumes of ciliates were calculated using cell size measurements obtained from the protargol preparations according to the commonly used geometric equations (Hillebrand et al., 1999). Ciliate biomasses were calculated using the conversion factor 0.19 pg C μm − 3 of biovolume (Putt and Stoecker, 1989) and multiplied by their respective abundances. Ciliates were classified into four functional groups according to their trophic status, i.e. herbivores, omnivores, bacterivores and carnivores. Trophic status was determined from observations of food vacuole contents and data published in the scientific literature (e.g., Fenchel, 1969; Song et al., 2009). Taxa that primarily feed on benthic micro-photoautotrophs were designated herbivores; those feeding on both benthic microphotoautotrophs and bacteria were considered to be omnivores;

and those whose trophic status could not be determined were assigned to the ‘unknown’ category. The wet weight (WW) of meiofauna was calculated according to the empirical equation WW = CLW2 1.13, where C is a dimensionless conversion factor specific to each taxon, L is the total individual length, W is the maximum width of an individual, and 1.13 is the specific gravity (Warwick and Price, 1979; Wieser, 1960). The organic carbon content was estimated by assuming a dry matter/weight ratio of 0.25 (Wieser, 1960) and a conversion factor of 0.45 for the carbon content, although for ostracods and bivalves these factors were 0.28 and 0.43, respectively (Feller and Warwick, 1988; Widbom, 1984). 2.4. Data analysis Univariate and multivariate statistical methods were used to test the null hypothesis that there were no differences between sites in terms of ciliate and meiofauna assemblages at any given season or sediment layer. The following parameters were used as univariate descriptors of the ciliate and meiofauna assemblages: ciliate abundance and biomass (CA and CB); ciliated species number and Shannon– Wiener diversity index (CN and CH); meiofauna abundance and biomass (MA and MB); nematode abundance and biomass (NA and NB). A two/three-way permutation analysis of variance (PERMANOVA; Anderson, 2001) was applied. All PERMANOVA tests were conducted on Euclidean-distance similarity matrices using 9999 random permutations and the residuals were permuted under a reduced model. Pearson correlation analyses were used to estimate possible relationships between environmental factors and faunal descriptor parameters using SPSS 19.0 software package. Abundance and biomass were analyzed from ln (x+ 1)-transformed data. Depth-integrated faunal descriptors were used for determining correlations with temperature, salinity,

Table 1 PERMANOVA results from univariate analysis of the benthic environments. Twelve sampling dates were combined into four groups, representing the four seasons, spring (March, April and May), summer (June, July and August), autumn (September, October and November) and winter (December, January and February), and for sediment composition analysis, the four groups are spring (April and May), summer (July and August), autumn (October and November) and winter (December, January) to set a balance for the available data. Bold values indicate significant differences (p b 0.05). Chl-a: chlorophyll a; Pheo: phaeopigments; OM: organic matter; Mgs: medium grain size. Source Stations (Sta.) Seasons (Sea.) Depths (Dep.) Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual Sta. Sea. Sta. × Sea. Residual Sta. Sea. Sta × Sea Residual Stations(Sta.) Seasons (Sea.) Sta × Sea Residual

Degrees of freedom Chl-a 1 3 3 3 3 9 9 64 OM 1 3 1 3 1 3 3 32 Salinity 1 3 3 16 Mgs 1 3 3 8 Silt content 1 3 3 8

Mean squares

Pseudo-F

Significance level (p)

0.0002 2.076 4.747 0.211 0.230 0.612 0.091 0.228

0.0009 90.124 20.858 0.928 1.010 2.691 0.388

0.9787 0.0001 0.0001 0.4457 0.4170 0.0086 0.9451

14.963 0.104 0.304 0.182 0.166 0.078 0.084 0.073

204.446 1.425 4.153 2.489 2.264 1.060 1.153

0.0001 0.2464 0.0501 0.0758 0.1392 0.3768 0.3468

776.344 79.760 2.399 13.667

58.806 5.836 0.176

0.0001 0.0076 0.9100

2545.833 332.386 437.645 117.473

21.672 2.829 3.726

0.0027 0.1091 0.0393

49.281 174.703 48.016 98.376

0.501 1.776 0.488

0.5207 0.2152 0.7534

Degrees of freedom Pheo. 1 3 3 3 3 9 9 64 Temp. 1 3 3

16 Water content 1 3 3 16 Sand content 1 3 3 8 Clay content 1 3 3 8

Mean squares

Pseudo-F

Significance level (p)

5.344 0.355 0.142 0.221 0.221 0.122 0.098 0.072

75.127 4.971 1.996 3.109 3.114 1.720 1.371

0.0001 0.0036 0.1236 0.0331 0.0310 0.1051 0.2158

3.375 496.723

0.303 44.534

0.5761 0.0001

2.518

0.226

0.8766

1624.109 18.263 46.020 11.328

143.376 1.612 4.063

0.0001 0.2217 0.0199

2199.141 276.053 316.910 101.668

21.631 2.715 3.117

0.0027 0.1094 0.0677

1297.080 12.707 98.153 18.442

70.332 0.689 5.322

0.0003 0.5851 0.0296

11.154

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because not all parameters were measured at all four sediment depth layers. The PRIMER 6.0 computer program (Clarke and Gorley, 2006; Clarke and Warwick, 1994) was used in the SIMPER and Bio-Env analyses.

water content and sediment composition, although in the case of the latter only seven months of data were used. A two/three-way PERMANOVA was used for the analysis of the multi-variate structure of the meiofauna and nematode communities. All data were ln (x + 1) transformed to valorize the contribution of the rare species and diminish the relative importance of dominant species. For all the data sets the analysis was conducted on a Bray− Curtis dissimilarity matrix (McArdle and Anderson, 2001). All univariate and multivariate analyses were performed using PERMANOVA.exe (Anderson, 2005). When the number of possible permutable units was not sufficient to perform a reasonable test via permutation, a P-value was obtained using a random Monte Carlo sample from the asymptotic permutation distribution. The twelve sets of samples were divided into the following four groups, representing four seasons according to the climate characteristics of the north temperate zone: spring (March, April and May); summer (June, July and August); autumn (September, October and November); and winter (December, January and February). Analysis of similarity percentages (SIMPER) was conducted to identify which meiofaunal taxa or ciliate species were the most important in characterizing the differences between habitats and among months/seasons. By linking of multivariate biotic patterns to a suite of environmental variables, Bio-Env analysis was applied to establish the best subset of factors determining the biotic communities. Depth-integrated descriptors were used in the Bio-Env analysis

a

3.1. Environmental variables PERMANOVA analysis revealed that the two sampling stations differed significantly in a range of environmental parameters, the exceptions being temperature, silt content and concentrations of chl-a and pheo (Table 1). Particle size distribution remained relatively constant at Station S, the medium grain size (mgs) ranging from 5 to 11 μm (6.5–7.5 ϕ) and the sediment composition being also constant (Fig. 2a, b). By contrast, the mgs at Station S-P was much higher and displayed much greater variation, ranging from 12.8 to 79.7 μm (3.6–6.3 ϕ). The sediment temperature presented typical seasonality for a northern temperate zone, with a warming start in spring and the maximum temperature occurring at the end of July to early August, followed by a cooling period, with a seasonal minimum being observed on January (Fig. 2c). The interstitial water salinity was in the range of 21–38‰ at Station S and 13–24‰ at Station S-P (Fig. 2d), with each site spanning two of the three distinctive salinity classes, i.e., mesohaline 5–18‰, polyhaline 18–30‰ and euhaline

S

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Percentage (%)

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3. Results

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3 2 1 0 06- 06- 06- 06- 06- 06- 06- 07- 07- 07- 07- 07-

6

7

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9 10 11 12 1

2

3

4

5

Fig. 2. Variations of sediment composition (a, b) temperature (c), salinity (d), water content (e), organic matter content (f), concentrations of chlorophyll a (g) and phaeopigments (h) at stations S and S-P during monthly samplings from June, 2006 to April, 2007. Nine months data of sediment composition were available from April, 2006 to January, 2007.

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g 7

0-0.5 cm 0.5-2 cm

Chlorophyll a (µg/g)

6

2-4 cm 4-8 cm

5 4 3 2 1 0 8 7 6 5 4 3 2

Station S

07-5

07-4

07-3

07-2

07-1

06-12

06-11

06-9

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Phaeopigments (µg/g)

h

Station S-P Fig. 2 (continued).

>30‰ (Anonymous, 1959). In addition to salinity, the water content, organic matter content and pheo concentration were, when significantly different, higher at Station S than at Station S-P (Fig. 2e, f, h). However, no difference in chl-a content was found between the two sites, i.e. 0.28–3.18 μg/g at Station S and 0.30 to 4.05 μg/g at Station S-P (Fig. 2e). 3.2. Ciliate community 3.2.1. Total abundance and biomass Except in January at Station S-P, ciliates were observed in each month, with mean abundances of 0–496 cells/ml being detected at Station S and of 0 to 2183 cells/ml at Station S-P, and the highest mean numbers appearing in the top 0–0.5 cm of sediment in May (Fig. 3a). Generally, 22.4–87.8% of the ciliates at Station S, and 13.6– 94.4% of the ciliates at Station S-P, inhabited the top 0–0.5 cm layer, although the proportion of the ciliate communities in this layer was lower in late autumn and winter. Approximately 34–97% of the ciliates at Station S, and over 88% of ciliates at Station S-P, inhabited in the top 0–2 cm layer. The results from a three-way PERMANONA of ciliate abundance showed significant differences between the stations, seasons and depths (Table 2). Pair-wise comparisons revealed that the ciliate abundance: (1) was, when significantly different, higher at Station S than at Station S-P; (2) showed seasonality in the order spring ≥ autumn = summer > winter in the top 0–2 cm layers at Station S, and spring ≥ autumn > summer = winter in the top of 0–0.5 cm layer at Station S-P; and (3) decreased significantly in the top 4 cm of sediment with increasing sediment depth in summer at Station S and in spring at Station S-P; it also decreased significantly in the top 2 cm of sediment in spring, autumn and winter at Station S and in summer and autumn at Station S-P. Pearson

correlation analysis showed that ciliate abundance was positively correlated with temperature and chl-a at Station S, and with temperature, chl-a and salinity at Station S-P (Table 3). The mean biomass varied from 0 to 4.79 μg C/ml at station S and 0 to 11.12 μg C/ml at Station S-P, with the highest mean numbers appearing in the top layer of 0–0.5 cm in May (Fig. 3b). The percentage of ciliate biomass in the 0–0.5 cm layer ranged from 21.4 to 75.6% at Station S and from 25.7 to 45.9% at Station S-P, whereas in the top 0–2 cm layer it was 50.7–98.7% at Station S and 31.7–100% at Station S-P. The three-way PERMANONA showed that ciliate biomass was significantly influenced by the sampling station, season and depth (Table 1). Pair-wise comparisons revealed that the average ciliate biomass: (1) was higher in the 0–0.5 cm layer in summer at Station S than at Station S-P; (2) showed seasonal variation in the order autumn ≥ spring ≥ summer > winter at Station S and spring ≥ autumn ≥ summer > winter at Station S-P only in the 0–0.5 cm layer; and (3) decreased conspicuously in the top two layers with increasing sediment depth in summer and winter at Station S and in spring at Station S-P. Pearson correlation analysis showed that ciliate biomass was positively correlated with temperature at Station S, but with salinity at Station S-P (Table 2).

3.2.2. Ciliate community composition and diversity A total of 249 ciliate species belonging to 37 genera, 28 families and 14 orders were identified. Among these, 188 ciliate species were found exclusively at Station S, only 35 exclusively at Station S-P, and 26 at both sites. Among the 14 ciliate orders identified, 8 were common to both stations, 4 were found only at Station S and 2 were observed only at Station S-P. The dominant ciliate species in each sample, together with their feeding ecology, are presented in

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

Abu. of ciliate (inds. ×103/ml)

a

b

Station S

0.5

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0-0.5cm 0.5-2cm 2-4cm 4-8cm

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Bio. of ciliate (µg C/ml)

0-0.5cm 0.5-2cm 2-4cm 4-8cm

0.5 0.0

d 5.0

12.0

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Station S-P

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0-0.5cm 0.5-2cm 2-4cm 4-8cm

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f 6.0

1.0

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0-0.5cm 0.5-2cm 2-4cm 4-8cm

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h 150

10 8

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0-0.5cm 0.5-2cm 2-4cm 4-8cm 06- 06- 06- 06-06- 06- 06-07- 07- 07-07- 076 7 8 9 10 11 12 1 2 3 4 5

0-0.5cm 0.5-2cm 2-4cm 4-8cm

2 0

06- 06-06- 06- 06- 06-06- 07- 07- 07- 07-076 7 8 9 10 11 12 1 2 3 4 5

Fig. 3. Variations of abundance (Abu.) and biomass (Bio.) of ciliates and meiofauna (Meio.) at Stations S and S-P during the monthly sampling campaigns.

Appendix 1. The percentage composition of the ciliates according to their abundances, partitioned at the order level and based on feeding types, are shown in Fig. 4. The orders represented by the greatest numbers of species were Prostomatida (49 species at Station S and nine at Station S-P), Haptorida (29 species at Station S and 13 at Station S-P), Cyrtophorida (27 species at Station S and five at Station S-P), Hypotrichida (17 species at Station S and six at Station S-P) and Scuticociliatida (10 species at Station S and 11 at Station S-P). As for

the functional groups, at Station S herbivores were the numerically predominant feeding type from the end of winter to mid-autumn, whereas bacterivores dominated from late-autumn until the end of winter, with carnivorous ciliates sometimes showing high species richness but low abundance (e. g. July and December). By contrast, herbivores were the dominant feeding type throughout the year at Station S-P with bacterivores dominating intermittently for short time periods (e.g. early winter and early summer).

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Table 2 PERMANOVA results for the uni- and multivariate descriptors of the ciliate and meiofaunal communities. CA: ciliate abundance; CB: ciliate biomass; CN: number of ciliate species; CH: Shannon–Wiener index of ciliates; MA: meiofaunal abundance; MB; meiofaunal biomass; NA: nematode abundance; NB: nematode biomass. Bold values indicate significant differences (p b 0.05). Ln (x + 1)-transformed data were used for the abundance and biomass analysis. Source

Degrees of freedom

Mean squares

Pseudo-F

Significance level (p)

1 3 3 3 3 9 9 64

6.914 13.972 59.057 0.100 1.104 2.428 1.886 0.832

8.309 16.792 70.956 0.120 1.327 2.918 2.267

0.0064 0.0001 0.0001 0.9462 0.2772 0.0068 0.0327

605.010 180.843 408.677 51.236 58.677 39.899 9.936 18.472

32.740 9.786 22.116 2.773 3.175 2.159 0.538

0.0001 0.0002 0.0001 0.0477 0.0318 0.0344 0.8503

6.147 0.283 36.496 2.780 0.311 0.230 0.243 0.3168

19.454 0.892 115.201 8.838 0.983 0.725 0.766

0.0001 0.4505 0.0001 0.0001 0.4136 0.6756 0.6459

5.716 0.250 35.095 2.705 0.245 0.260 0.235 0.322

17.780 0.778 109.085 8.407 0.762 0.809 0.732

0.0001 0.5067 0.0001 0.0001 0.5264 0.6011 0.6745

25468.361 8134.653 9759.730 5166.445 4768.920 4491.696 3785.757 3620.801

7.0339 2.2466 2.6955 1.4269 1.3171 1.2405 1.0456

0.0001 0.0004 0.0001 0.0373 0.0779 0.0403 0.3384

6211.142 460.322 2792.161 797.942 1508.590 407.014 371.261 391.986

15.845 1.174 7.123 2.036 3.859 1.038 0.947

0.0001 0.3096 0.0001 0.0230 0.0002 0.4206 0.5537

Degrees of freedom

Mean squares

Pseudo-F

Significance level (p)

0.006 0.527 2.147 0.226 0.023 0.347 0.205 0.078

0.082 6.767 27.572 2.890 0.299 4.455 2.632

0.7782 0.0005 0.0001 0.0346 0.8400 0.0002 0.0107

19.938 2.461 3.555 1.358 1.176 0.253 0.352 0.166

58.132 7.176 10366 3.959 3.427 0.738 0.484

0.0001 0.0007 0.0001 0.0130 0.0214 0.0681 0.8769

25.193 0.534 20.188 0.685 3.965 0.067 0.528 0.185

136.137 2.888 109.091 3.702 21.427 0.363 2.851

0.0001 0.0463 0.0001 0.0168 0.0001 0.9516 0.0082

11.126 0.057 12.497 0.684 1.584 0.201 0.248 0.163

68.350 0.347 76.771 4.201 9.745 1.233 1.525

0.0001 0.7947 0.0001 0.0075 0.0001 0.2786 0.1612

1 3 3 3 3 9 9 64

22449.142 7574.507 9505.728 6131.729 4683.633 4806.150 4449.922 3781.158

5.937 2.003 2.514 1.622 1.239 1.271 1.170

0.0001 0.0008 0.0001 0.0058 0.0987 0.0164 0.0601

1 3 3 3 3 9 9 64

29845.641 1326.328 3366.742 1467.855 3540.236 1181.081 744.277 1049.588

24.436 1.264 3.208 1.399 3.373 1.125 0.709

0.0001 0.2358 0.0010 0.1707 0.0009 0.2962 0.8708

Univariate analyses CA Stations (Sta.) Seasons (Sea.) Depths (Dep.) Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual

CB

CN Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep Sta. × Sea. × Dep. Residual

1 3 3 3 3 9 9 64 MA 1 3 3 3 3 9 9 64 NA 1 3 3 3 3 9 9 64

1 3 3 3 3 9 9 64 CH′ 1 3 3 3 3 9 9 64 MB 1 3 3 3 3 9 9 64 NB 1 3 3 3 3 9 9 64

Multivariate analyses CA Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual Sta. Sea. Dep. Sta. × Sea. Sta. × Dep. Sea. × Dep. Sta. × Sea. × Dep. Residual

1 3 3 3 3 9 9 64 MA 1 3 3 3 3 9 9 64

CB

MB

The detected spatio-temporal variation in ciliate diversity in terms of both species number and the Shannon–Wienner diversity index is given in Fig. 5a, b. Three-way PERMANONA analysis for ciliate diversity showed that: (1) when there were significant differences, ciliate diversity was higher at Station S than at Station S-P; (2) there was a seasonal variation in the order autumn ≈ spring ≥ summer > winter at both stations; and (3) there was a decrease in the number of species in the top two layers with increasing sediment depth in summer and spring at both stations and likewise in the Shannon–Wienner index except during winter at Station S. Multivariate analyses of the ciliate assemblages also showed significant differences between sites, seasons and depths

(Table 2). Based on SIMPER analysis, 14 species accounted for approximately 60% of the differences in ciliate abundance composition between the two sites. Prorodon sp. 2, Spirodyseria cf. ganghwaensis, Chlamydodon triquetrus, scuticociliate sp. 1, Euplotes sp. 1, Prorodon sp. 4, Prorodon sp. 9 and Prorodon sp. 1 occurred primarily at Station S, and Pseudochilodonopsis sp., Chilodontopsis sp., scuticociliate. sp. 4, Metacystis sp. 8, Uronema cf. minima and Euplotes sp. 2 were more abundant at Station S-P. Bio-Env analysis indicates that the combinations of parameters that best explain differences observed in ciliate communities were temperature and pheo concentration at Station S (r = 0.425) and temperature, salinity, organic matter and water content at Station S-P (r = 0.534).

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

57

Table 3 Pearson correlation coefficients among the abiotic and biotic parameters.

Stations S and S-P Medium grain size Silt content Clay content Sand content Temperature Salinity Water content Organic matter Chlorophyll a Phaeopigments Ciliate abundance (CA) Ciliate biomass (CB) Ciliate number (CN) Ciliate diversity (CH′) Meiofauna abundance (MA) Meiofauna biomass (MB) Nematode abundance (NA) Nematode biomass (NB) Station S Medium grain size Silt content Clay content Sand content Temperature Salinity Water content Organic matter Chlorophyll a Phaeopigments Ciliate abundance (CA) Ciliate biomass (CB) Ciliate number (CN) Ciliate diversity (CH′) Meiofauna abundance (MA) Meiofauna biomass (MB) Nematode abundance (NA) Nematode biomass (NB) Station S-P Medium grain size Silt content Clay content Sand content Temperature Salinity Water content Organic matter Chlorophyll a Phaeopigments Ciliate abundance (CA) Ciliate biomass (CB) Ciliate number (CN) Ciliate diversity (CH′) Meiofauna abundance (MA) Meiofauna biomass (MB) Nematode abundance (NA) Nematode biomass (NB)

CA

CB

CN

CH′

MA

MB

NA

NB

− 0.312 0.207 0.259 − 0.327 0.475⁎ 0.415⁎

− 0.397 0.199 0.353 − 0.393 0.292 0.0290 − 0.070 − 0.043 0.238⁎ − 0.064 – – 0.676⁎⁎ 0.287⁎ 0.447⁎⁎ 0.382⁎⁎ 0.448⁎⁎ 0.378⁎⁎

− 0.453 0.134 0.372 − 0.473 0.457⁎ 0.692⁎⁎ 0.543⁎⁎ 0.369⁎⁎ 0.231⁎

− 0.527 0.068 0.448 − 0.557⁎ 0.270 0.716⁎⁎ 0.725⁎⁎ 0.600⁎⁎

− 0.233 − 0.167 0.125 − 0.305 0.074 0.342 0.524⁎⁎ 0.389⁎⁎ 0.511⁎⁎

− 0.618⁎ − 0.003 0.492 − 0.679⁎⁎ − 0.051 0.684⁎⁎ 0.786⁎⁎ 0.686⁎⁎ 0.477⁎⁎ 0.221⁎

− 0.245 − 0.102 0.150 − 0.311 0.084 0.328 0.512⁎⁎ 0.376⁎⁎ 0.504⁎⁎

− 0.476 − 0.061 0.345 − 0.543⁎⁎ − 0.060 0.575⁎⁎ 0.796⁎⁎ 0.557⁎⁎ 0.411⁎⁎ 0.250⁎

0.001 0.039 0.473⁎⁎ − 0.016 – 0.800⁎⁎ 0.764⁎⁎ 0.539⁎⁎ 0.663⁎⁎ 0.577⁎⁎ 0.662⁎⁎ 0.550⁎⁎

0.149 0.164 − 0.188 0.207 0.718⁎⁎ 0.222 0.168 − 0.188 0.520⁎⁎ − 0.185 – 0.784⁎⁎ 0.866⁎⁎ 0.778⁎⁎ 0.699⁎⁎ 0.737⁎⁎ 0.691⁎⁎ 0.652⁎⁎

− 0.107 0.131 0.033 − 0.181 0.589⁎⁎ 0.655⁎⁎ − 0.197 − 0.125 0.454⁎⁎ − 0.094 – 0.819⁎⁎ 0.877⁎⁎ 0.351⁎⁎ 0.640⁎⁎ 0.509⁎⁎ 0.643⁎⁎ 0.499⁎⁎

0.196 – – – 0.811⁎⁎ 0.538⁎⁎ 0.609⁎⁎ 0.535⁎⁎ 0.565⁎⁎

0.169 0.349⁎⁎ – – – – 0.464⁎⁎ 0.597⁎⁎ 0.471⁎⁎ 0.529⁎⁎

0.133 – – – – – 0.846⁎⁎ 0.999⁎⁎ 0.894⁎⁎

– – – – – – 0.834⁎⁎ 0.929⁎⁎

0.130 – – – – – – – 0.891⁎⁎

0.095 0.056 − 0.139 0.267 0.690⁎ 0.273 0.392 − 0.068 0.277 0.093

0.104 0.064 − 0.157 0.317 0.666⁎ 0.373 0.413 − 0.085 0.341⁎

0.080 0.016 − 0.123 0.329 0.675⁎ 0.462 0.327 − 0.110 0.345⁎

− 0.162 − 0.161 0.168 − 0.242 − 0.432 0.305 0.184 0.225 0.643⁎⁎

− 0.054 − 0.113 0.050 0.051 − 0.255 0.507 0.356 0.262 0.741⁎⁎

− 0.096 − 0.091 0.101 − 0.195 − 0.419 0.295 0.178 0.220 0.627⁎⁎

0.086 0.046 − 0.101 0.112 − 0.461 0.164 0.520 0.197 0.591⁎⁎

− 0.012

− 0.058

− 0.149

− 0.248

− 0.135

− 0.098

– 0.877⁎⁎ 0.593⁎⁎ 0.504⁎⁎ 0.555⁎⁎ 0.500⁎⁎ 0.546⁎⁎

– – 0.844⁎⁎ 0.518⁎⁎ 0.575⁎⁎ 0.514⁎⁎ 0.532⁎⁎

– – – 0.532⁎⁎ 0.586⁎⁎ 0.528⁎⁎ 0.516⁎⁎

– – – – 0.915⁎⁎ 0.999⁎⁎ 0.957⁎⁎

– – – – – 0.903⁎⁎ 0.906⁎⁎

– – – – – – 0.958⁎⁎

– – – – – – –

− 0.053 0.159 0.059 − 0.066 0.384 0.635⁎ − 0.367 − 0.146 0.217 − 0.187

0.113 − 0.047 − 0.148 0.060 0.565 0.649⁎ − 0.052 − 0.276 0.195 − 0.097

0.309 − 0.231 − 0.259 0.0295 0.330 0.329 0.215 − 0.051 0.060 0.172

0.327 − 0.321 − 0.387 − 0.234 0.733⁎⁎ − 0.214 0.597⁎ 0.332 0.444⁎⁎

0.423 − 0.435 − 0.482 0.352 0.309⁎ 0.011 0.132 0.340 0.404⁎⁎

0.261 − 0.245 − 0.314 0.172 0.737⁎⁎ − 0.220 0.591⁎ 0.332 0.443⁎⁎

0.397 − 0.418 − 0.477 0.316 0.540⁎ − 0.213 0.233 0.200 0.357⁎

0.148

0.133

0.148

0.154

– 0.750⁎⁎ 0.072 0.434⁎⁎ 0.363⁎ 0.437⁎ 0.313⁎⁎

– – 0.573⁎⁎ 0.557⁎⁎ 0.338⁎ 0.562⁎ 0.341⁎

– – – 0.291⁎⁎ 0.081 0.292⁎

– – – – 0.848⁎⁎ 0.999⁎⁎ 0.847⁎

– – – – – 0.841⁎⁎ 0.961⁎⁎

– – – – – – 0.841⁎⁎

– – – – – – –

0.108

*Correlated (0.01 b p b 0.05), **significantly correlated (p b 0.01), Bold values indicate correlated or significant correlated; values were determined at four depths in each sampling to allow correlation of ciliate abundance (CA), biomass(CB), species number (CN)and the Shannon–Wienner index (CH), meiofaunal abundance and biomass (MA and MB), nematode abundance and biomass (NA and NB), chlorophyll a and phaeopigments; the top two layers were analyzed for organic matter content and bioparameters; Integration over the four sediment depth layers for the bioparameters was performed for temperature, salinity, water content and sediment composition (medium grain size, content of silt, clay and sand), and seven months of samples was analyzed to determine the sediment composition.

3.3. Meiofauna community 3.3.1. Total abundance and biomass The mean abundance varied from 13 to 5526 inds./ml at Station S, with the highest value being observed in the top layer of 0–0.5 cm in February and lowest being found in the 4–8 cm layer in August. At Station S-P, the mean abundance ranged from 8 to 1035 inds./ml,

with the highest level detected in the top 0–0.5 cm layer in August and the lowest in the 4–8 cm layer in May (Fig. 3e, f). Generally, 25.3 to 83.4% of the meiofauna at Station S and 17.1 to 63.2% of those at Station S-P were distributed in the top 0–0.5 cm layer, and approximately 40–95% of meiofauna at Station S and 41–78% of those at Station S-P inhabited the top 0–2 cm layer. Three-way PERMANONA analysis reveals that there were significantly differences between

58

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63 Haptorida Heterotrichida Karyorelictida Prostomatida others

Peritrichida Hymenostomatida Nassulida Scuticociliatida

Cyrtophorida Hypotrichida Pleurostomatida Synhymeniida

Haptorida

Cyrtophorida

Hypotrichida

Karyorelictida

Nassulida

Oligotrichida

Primociliatida

Prostomatida

Scuticociliatida

Synhymeniida

others

Bacterivory Omnivory

Carnivory Unknow

100% 80% 60% 40% 20% 0% Bacterivory Omnivory

Carnivory Unknow

herbivory

herbivory

100% 80% 60% 40% 20% 0% 06- 06- 06- 06- 06- 06- 06- 07- 07- 07- 07- 07-

6

7

8

9 10 11 12 1

2

3

4

5

Station S

06- 06- 06- 06- 06- 06- 06- 07- 07- 07- 07- 07-

6

7

8

9 10 11 12 1

2

3

4

5

Station S-P

Fig. 4. Percent composition of ciliate abundance partitioned at orders level and feeding type (no ciliates were found in January at Station S-P).

the sampling stations, depth, and the interaction effect of station and season (Table 2). Pair-wise comparisons revealed that meiofaunal abundances: (1) were higher at Station S than at Station S-P; (2) showed seasonal variation only at Station S-P in the order summer ≥ autumn > spring≈ winter; (3) decreased drastically below the 2–4 cm layer at both sampling stations. Pearson analysis shows that meiofauna abundance was positively correlated with chl-a at Station S, and with chl-a, temperature and water content at Station S-P (Table 3). The mean meiofaunal biomass varied from 0.13 to 140.17 μg C/ml at Station S, with the highest value occurring in the 0–0.5 cm layer in March, whereas it ranged from 0.01 to 10.85 μg C/ml at Station S-P, with the highest value in the 0–0.5 cm layer in August (Fig. 3g, h). Meiofaunal biomass in the 0–0.5 cm layer accounted for 28.3–77.9% of the total biomass at Station S and 8.0–87.2% of that at Station S-P., and in the upper 2 cm layer it accounted for 56.1–98.7% at Station S and 13.4% to 91.7% at Station S-P. Three-way PERMANONA analysis showed that meiofaunal biomass was significantly influenced by the sampling station, season and depth (Table 2). Pair-wise comparisons revealed that meiofaunal biomass: (1) was, higher at Station S than at Station S-P when the two values were significant different; (2) showed a seasonal pattern only in the top 0–0.5 cm at Station S in the order spring > winter ≥ autumn ≈ summer; and (3) decreased conspicuously in the top 2 cm layer with increasing sediment depth, except in winter and spring at Station S-P. Pearson analysis showed that meiofaunal biomass was positively correlated with chl-a at Station S, and with chl-a and temperature at Station S-P (Table 3). 3.3.2. Meiofauna composition A total of 14 higher meiofaunal taxa were identified. Among these, nematodes were the predominant group in terms both of abundance and biomass at both stations, while the average weight of individual

nematodes was nearly three times higher at Station S (0.04 ± 0.011 μg) than at Station S-P (0.14 ± 0.007 μg). At Station S, nematodes accounted for 96 ± 4.8% of the total abundance, followed by crustacean nauplii (2.5%), Copepoda (1.3%), Turbellaria (0.2%) and Polychaeta (0.1%). Also at Station S, nematodes accounted for 44.7 ± 21.6% of total meiofaunal biomass, followed by Copepoda (43.3%), crustacean nauplii (6.3%), Polychaeta (4.9%) and Turbellaria (0.5%). At Station S-P, nematodes accounted for 98.5 ± 1.7% of the total meiofaunal abundance, followed by Crustacean nauplii (0.27%), and 72.1± 21.6% of the total meiofaunal biomass, followed by Polychaeta (13.7%). Three-way univariate PERMANONA analysis of nematode abundance revealed similar results as those for the total meiofauna (Table 2). Significant correlations between the total meiofauna and nematodes were found in terms of abundance and biomass, and the correlation between environmental parameters and nematode abundance and biomass were also similar to that for the total meiofauna (Table 3). The results from multivariate PERMANONA of the structure of higher meiofaunal taxa showed significant differences between sites and sediment depths and a significant effect of interaction between the two (Table 2). Based on SIMPER analysis, nematodes accounted for 92.9% of the difference in terms of meiofaunal abundance composition, followed by crustacean nauplii (3.3%) and copepods (2.1%); in terms of biomass composition, nematodes accounted for 51.1% of the difference, followed by copepods (29.7%), Polychaeta (7.8%) and crustacean nauplii (4.5%). Each of the four groups mentioned above was more abundant at Station S than at Station S-P. Low correlation coefficients were observed between the stations and meiofaunal community structure in terms of abundance composition based on Bio-Env analyses, with a value of 0.151 for the best combination at Station S, i.e. OM, chl-a concentration, pheo concentration and water

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

70

0-0.5 cm

0.5-2 cm

2-4 cm

59

4-8 cm

0-8 cm

Species number ( N )

60

50

40

30

20

10

0

Shannon-Wiener index ( H’ )

10

8

6

4

Station S

07-5

07-4

07-3

07-2

07-1

06-12

06-11

06-9

06-10

06-8

06-7

06-6

07-5

07-3

07-4

07-2

07-1

06-12

06-11

06-10

06-9

06-8

06-7

0

06-6

2

Station S-P

Fig. 5. Diversity of ciliates at stations S and S-P during monthly samplings. Diversity was measured by species number (N) and the index of Shannon–Wienner (H’).

content, and a value of only 0.099 for the best combination at Station S-P, i.e. chl-a and pheo concentration.

4. Discussion 4.1. Benthic environments Both sampling stations were well sheltered from the actions of waves and currents. This was reflected in the stable sediment size distributions at both sites, there being no visible signs around our site markers indicating the occurrence of erosion or deposition between sampling campaigns. Sediments densely covered with halophytes were finer, and had a higher organic matter content, than those that were unvegetated or with halophytes distributed in patches. These findings are consistent with those of other studies (Danovaro and Gambi, 2002; Fonseca et al., 1983, 2011) and may be explained by the trapping of fine-grained sediments by the halophytes (Fonseca and Bell, 1998; Wang et al., 2006). Furthermore, primary production

cycles of halophytes were responsible for a large amount of sediment organic carbon (Danovaro, 1996). The chl-a concentration, which is commonly used to estimate benthic microalgal biomass (Grinham et al., 2007; Underwood and Kromkamp, 1999), showed a great deal of complexity because of biotic and abiotic effects (Blanchard et al., 2001; Epstein, 1997a, 1997b). Some results observed in this investigation, e.g. higher concentrations in the top layer, a rapid decrease with depth and an increase concentration from late winter and early spring, are consistent with previous findings (e.g., Koh et al., 2007). On the other hand, the present observation that chl-a concentrations at the two sites were comparable throughout the period of sampling differed from that reported by Li and Ding (2007) in tidal flats of Yangtze River Estuary, China, where chl-a concentration was higher in the unvegetated sediments than vegetated areas in the bulrush transition zone colonized by Scirpus mariqueter. This was probably due to differences in the morphological characteristics of the halophytes in the two estuaries, the effects of S. glauca on chl-a concentrations in River Dagu estuary being due to their shorter stems.

60

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

4.2. Communities of ciliates and meiofauna Seagrass is typically considered to act as an ecosystem engineer by playing an important role in structuring benthic assemblages, serving to reduce the physical stress and protecting smaller invertebrates from predators thereby enhancing food availability (Bos et al., 2007; Boström et al., 2006; Orth et al., 1984). Nevertheless, those reports related to the high abundance, biomass and diversity of fauna in seagrass biotopes focus mainly on macrofauna (e.g., Hirst and Atrill, 2008; Lee et al., 2001). In the present investigation, uni- and multivariate analyses both indicated significant differences in the communities of meiofauna and ciliates between that two sampling sites. There were higher abundances of ciliates and meiofauna, and a higher diversity of ciliates, in the sediments of the S. glauca marsh (Station S) than in the unvegetated sediments where seagrass and reeds were distributed in patches (Station S-P). The ciliate species number (223) is much higher than that reported for other intertidal sediments (Hamels et al., 2004, 2005; Meng et al., 2011). This can be attributed not only to the highly productive nature of the estuary wetland, but also to the methods used for the isolation and identification of ciliates, and the intensive sampling regime, employed in the present study. Nevertheless, this is still likely to be a conservative estimate of species diversity because of the limitations of all direct observation techniques in recovering cryptic species. In the case of free-living protists, for example, recent studies using molecular methods indicate that even the highest estimates of species diversity are likely to be at least an order of magnitude too low, mainly due to the difficulty in detecting less abundant taxa in environmental samples (Bass and Boenigk, 2011). The majority of ciliate species in the S. glauca marsh (Station S) are typical marine benthic ciliates (Carey, 1992; Song et al., 2009). At the two sites 75.5% of the species isolated were restricted to the seagrass sediment (Station S) and 10.4% were restricted to the unvegetated sediment interspersed with patches of S. glauca and P. australis (Station S-P). The dominant ciliates species at Station S-P were a reduced subset of the assemblage inhabiting Station S. This case is in contrast to other findings of communities of macrofauna and nematodes inhabited in seagrass and unvegetated sediments. e.g., Van Houte-Howes et al. (2004) found that in three New Zealand estuaries, there were relative few macrofauna associated exclusively with either seagrass beds or unvegetated sediments, and the majority of species were common to the both habitats. However, differences in the relative abundances at the two habitats were significant. By contrast, Fonseca et al. (2011) observed that in coastal habitats of New South Wales, Australia, 32% of nematodes species were restricted to seagrass sediments and 25% were restricted to unvegetated sediments, indicating that each habitat has a distinctive faunal assemblage. These findings suggest that microfauna respond to environmental changes created by seagrass in different ways to the macro- and meiofauna. It is also noteworthy that ciliate diversity at these sites conforms to the paradigm that marine habitats support greater species diversity than brackish water habitats (Adăo et al., 2009; Hourston, et al., 2011; Sanders, 1968). Hence, whether or not there are distinctly different ciliate communities in the seagrass sediments vs. the unvegetated sediments will require further studies as there is a paucity of information regarding the importance of this matrix (Boström et al., 2006). Differences in the meiofaunal/nematode composition between vegetated and unvegetated sediments, or along salinity gradients in estuaries, have been reported previously (e.g., Adăo et al., 2009; Fonseca et al., 2011; Hourston, et al., 2011; Soetaert et al., 1995). Likewise, significant differences in the species composition and abundances of the meiofauna at the two sampling sites were also observed in the present study. For example, a higher abundance of meiofauna, together with contrasting seasonal dynamics of these organisms, was found in the S. glauca salt marsh in the lower estuary (Station S) compared to that in unvegetated tidal flats with patches of halophytes in

the upper estuary (Station S-P). Furthermore nematodes, which was the predominant meiofaunal group at both sites in terms of abundance and biomass and accounted for 93% of the differences in the meiofaunal communities, exhibited a nearly 3 times higher average individual weight in the S. glauca salt marsh sediments (Station S) than in the unvegetated sediments at Station S-P. This is significant because body size represents an important attribute in terms of morphological and functional diversity within food webs (Perkins and Reiss, 2010; Platt and Warwick, 1983; Reiss et al., 2009; Schratzberger et al., 2007; Yamamuro, 2000). Although many studies have been reported on feeding strategies of ciliates, the quantitative importance of the different functional groups within specific ecosystem remains poorly understood. Epstein (1997b) revealed a seasonal pattern for microbial assemblages characterized by two distinct phases: a herbivore-dominated phase in the spring period and a bacterivore-dominated phase in autumn. Likewise, herbivores and bacterivores were also found to be dominant ciliate groups in the muddy sediments of the River Dagu estuary in the present study. This contrasts with findings for the offshore seabed of the Yellow Sea where carnivorous ciliates were found to be the dominant group (Meng et al., 2011). These contrasting findings are comparable to that for functional groups of nematodes, e.g., epistrate-feeding nematodes that graze on the microphytobenthos, being the dominant group in sediments associated with seagrass in shallow waters and estuaries (Danovaro and Gambi, 2002; Fonseca et al., 2011; Hourston et al., 2011) whereas in the deep sea, where refractory detritus may be the basal food resource for nematodes providing up to 99% of their carbon requirements, non-selective deposit feeders are dominant (Gontikakia et al., 2011; Ingels, et al., 2011). The transition among functional groups observed for the ciliates and nematodes probably reflects the different carbon fluxes and energy flows in the microbial food web among the different habitats. 4.3. Factors regulating the communities of ciliates and meiofauna Factors known to play a role in the regulation of ciliate and meiofaunal communities, e.g., food availability, sediment grain size, temperature, salinity and organic matter content (Dietrich and Arndt, 2000; Epstein, 1997b; Fenchel, 1969; Giere, 2009; Hamels, et al., 2005), were investigated in the present study (Table 3). It is noteworthy that there were significant correlations between the ciliates and the meiofauna in univariate analyses indicating a potential biotic interaction between these groups of organisms. The importance of temperature and salinity for the ciliate community was revealed by Bio-Env analyses. By contrast, the low Bio-Env coefficients for the meiofaunal communities indicate that factors other than those investigated here, e.g., bacterial production alone or in combination with temperature etc., may be of greater importance for the distribution of these organisms. The seasonal dynamics of chl-a concentration in the present investigation (Fig. 2e) closely parallel those of benthic diatoms, as reported by Epstein (1997b) who found that during a herbivore-dominated phase, the abundance of benthic diatoms was high at the end of the winter, and decreased gradually due to grazing by herbivores resulting in a collapse of diatoms abundance at mid-summer. This pronounced dynamic of chl-a concentration could, to some extent, be linked to the population dynamics of herbivorous ciliates at both sites (Fig. 5) as well as the meiofauna at Station S. This implies that food availability is a major controlling factor for the ciliate and meiofaunal communities. Although specific feeding groups of the main meiofauna, namely nematodes, was not investigated in this study, the dominance of epistrate-feedering nematodes, which graze on the microphytobenthos, has been reported more than once in seagrass-dominated and other estuarine habitats (Danovaro and Gambi, 2002; Fonseca et al., 2011 and references therein; Hourston et al., 2011). Likewise, Fonseca et al. (2011) also found that higher abundances of non-selective deposit

Y. Du et al. / Journal of Sea Research 70 (2012) 50–63

feeders and omnivorous/predatory nematodes were present in the unvegetated sediments compared to the vegetated sediments. Therefore, in addition to diatoms, other food source organisms such as bacteria could also be important regulators of meiofaunal populations in the unvegetated sediments. Another feature that might influence the population dynamics of benthic communities is the root-rhizome of S. glauca. Root residues were often observed in sediment samples from Station S whereas they were rarely observed in the unvegetated sediments at Station S-P. It was noted that ciliate numbers declined much less abruptly with depth in the former than the latter, the ratio of ciliates at the four different depth layers (i.e. 0–0.5 cm, 0.5–2 cm, 2–4 cm and 4–8 cm) being 8:3:1:1 at Station S vs. 170:14:1:2 at station S-P. Commonly, sediments with a median grain size of 120–250 μm harbor the highest abundance of microorganisms (Fenchel, 1969). By contrast, the small sediment grain size (7–80 μm) in the present study is probably not conducive to the movement of organisms. On the other hand, the interstitial space available for microorganisms was probably enhanced by the growth of seagrass roots and rhizomes, though the dense root-rhizome mat may inhibit the activities of some macrofauna, e.g., deep-burrowing polychaetes (Van Houte-Howes et al., 2004). Furthermore, the detrital material of the seagrass probably constituted an important food source for many benthic organisms (Currin et al., 1995). Taking these findings together, we are in agreement with Coull (1999) who noted that the distribution patterns of meiofauna are ultimately linked to temperature, either directly or indirectly as a result of temperature-induced control of food abundance and type and anoxic depth levels. Furthermore it has frequently been demonstrated that salinity plays an important role in the distribution of organisms in estuaries (Hourston et al., 2011; Kchaou et al., 2009; Sanders, 1968; Soetaert et al., 1995). Given the sensitivity of microorganisms to environmental change, it is likely that such controls apply even more strongly to ciliates. Therefore these factors, along with the presence of seagrass, resulted in contrasting spatio-temporal variations in the ciliate and meiofauna communities in the present investigation. Temperature, salinity and food availability thus appear to be the key factors that regulate ciliate and meiofaunal benthic assemblages in the River Dagu estuary. 4.4. Possible relationships between ciliates and meiofauna Previous studies have shown that heterotrophic protists dominate estuarine benthic habitats in terms of abundance, whereas the meiofauna contribute most of the biomass (Garstecki et al., 2000; Hamels et al., 2004). However, in the present study, the meiofauna organisms (mainly nematodes) were dominant in terms of both abundance and biomass, the contribution of benthic ciliates surpassing that of the meiofauna only in spring at Station S-P, mainly because of a bloom of Pseudochilodonopsis sp. Additionally, the contribution of the protists to the benthic ecosystem is disproportionate to their biomass because of their high turnover rate. According to the formula R = a W − 0.249 (Fenchel, 1974; Hamels et al., 2004), where the weight-specific metabolic rate (R) is related to body weight (W), and ‘a’ is a constant with a value of 10 − 1.94 for ciliates and 10 − 1.64 for meiofauna, the mean individual body weight (roughly total biomass/total abundance) and biomass were estimated for each group, and relative metabolic rates were calculated for the ciliates, nematodes, copepods and the other meiofauna for each sample. This calculation also shows that the meiofauna was the most important component, accounting for approximately 86% (20–100%) of the estimated combined metabolic rate of the ciliates and meiofauna, with nematodes alone accounting for about 61% (19–100%). By contrast, ciliates played the role of the key consumers only in April– May at Station S-P, where they contributed up to 58–80% of the total metabolism.

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There are currently few available studies addressing the interactions between ciliates and nematodes in the benthos. Most of available reports on trophic relationships for these groups are limited to those between nematodes/ciliates and diatoms/bacteria (e.g., Epstein, 1997a, b; Epstein et al., 1992; Franco, et al., 2008; Pascal et al., 2008). Hamels et al. (2001a, 2001b) provided the first quantitative data on the predation of ciliates by nematodes and found that ciliates were an important carbon source for nematode, and the main ecological importance of ciliates was their role as a link between primary producers and bacteria on the one hand and nematodes on the other, in the microbial loop. However, in addition to predator–prey relationships, competition between herbivorous nematodes and ciliates is also likely to be important, at least in seagrass sediments, because of their common preference for the microphytobenthos as a food source. Whether nematodes and ciliates select a specific diatom, and thus whether competition between these groups exists, will require further investigation. The application of stable isotope technique (Maria et al., 2011), could provide deeper insights regarding these trophic interactions. Supplementary data to this article can be found online at doi:10. 1016/j.seares.2012.03.004. Acknowledgements The study was supported by the Knowledge Innovation Program of Chinese Academy of Sciences (no. KZCX2-YW-417) and the National Natural Science Foundation of China (nos. 40706047; 40906066). Thanks are due to Zifeng Zhan, Zhaocui Meng and Jiadong Wang for their assistance in collecting field samples, and Shichao Yang for her assistance with the measurement of organic matter content. We also thank Prof. Michael Collins, Bernd Wuennemann, and Burghard Flemming, for their helpful comments on the manuscript. We are thankful to two anonymous reviewers for their timely and constructive comments. References Adăo, H., Alves, A.S., Patríciob, J., Neto, J.M., Costa, M.J., Marques, J.C., 2009. Spatial distribution of subtidal Nematoda communities along the salinity gradient in southern European estuaries. Acta Oecologia 35, 287–300. Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32–46. Anderson, M.J., 2005. PERMANOVA: a FORTRAN computer program for permutational multivariate analysis of variance. Department of Statistics, University of Auckland, New Zealand. Anonymous, 1959. Symposium on the classification of brackish waters, Venice, 8–14 April, 1958. Archivio di Oceanografia e Limnologia, 11 (Suppl.). Bass, D., Boenigk, J., 2011. Everything is everywhere: a twenty-first century de-/ reconstruction with respect to protists. In: Fontaneto, D. (Ed.), Biogeography of Microscopic Organisms. : Systematics Association Special Volume Series, No. 79. Cambridge University Press, Cambridge, UK, pp. 88–110. Blanchard, G.F., Guarini, J.-M., Orvain, F., Sauriau, P.-G., 2001. Dynamic behaviour of benthic microalgal biomass in intertidal mudflats. Journal of Experimental Marine Biology and Ecology 264, 85–100. Bos, A., Bouma, T., Dekort, G., Vankatwijk, M., 2007. Ecosystem engineering by annual intertidal seagrass beds: sediment accretion and modification. Estuarine, Coastal and Shelf Science 74, 344–348. Boström, C., Jackson, E.L., Simenstad, C.A., 2006. Seagrass landscapes and their effects on associated fauna: A review. Estuarine, Coastal and Shelf Science 68, 383–403. Carey, P.G., 1992. Marine Interstitial Ciliates. An Illustrated Key. Chapman and Hall, London. 351 pp. Clarke, K.R., Gorley, R.N., 2006. PRIMER v 6: User Manual/Tutorial. PRIMER-E, Plymouth. 190 pp. Clarke, K.R., Warwick, R.M., 1994. Changes in Marine Communities: an Approach to Statistical Analysis and Interpretation. Natural Environment Research Council, UK. 144 pp. Corliss, J.O., 1979. The ciliate protozoa. Characterization, Classification and Guide to the Literature. Pergamon Press, Oxford. 455 pp. Coull, B.C., 1999. Role of meiofauna in estuarine soft-bottom habitats. Australian Journal of Ecology 24, 327–343. Currin, C.A., Newell, S.Y., Paerl, H.W., 1995. The role of standing dead Spartina alterniflora and benthic microalgae in salt marsh food web: considerations based on multiple stable isotope analysis. Marine Ecology Progress Series 121, 99–116. Danovaro, R., 1996. Detritus–bacteria–meiofauna interactions in a seagrass bed (Posidonia oceanica) of the NW Mediterranean. Marine Biology 127, 1–13.

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