Environ Sci Pollut Res (2012) 19:540549 DOI 10.

1007/s11356-011-0578-1

RESEARCH ARTICLE

Influence of sampling sufficiency on biodiversity analysis of microperiphyton communities for marine bioassessment
Henglong Xu & Wei Zhang & Yong Jiang & Mingzhuang Zhu & Khaled A. S. Al-Rasheid

Received: 27 April 2011 / Accepted: 26 July 2011 / Published online: 13 August 2011 # Springer-Verlag 2011

Abstract Introduction With quick responses to environmental changes, easy sampling, relative immobility, increasing availability of easily used taxonomic references, and allowing standardization for temporal and spatial comparisons, the biodiversity measures of microperiphyton communities have widely been accepted as useful indicators to evaluate environmental stress and anthropogenic impact. Materials and methods The influence of sampling sufficiency for biodiversity analysis of microperiphyton communities was studied using a range of statistical methods in coastal waters of the Yellow Sea, northern China, from May to June 2010. Samples were collected from two depths using an artificial substrate. Results Sampling sizes represented a significant influence on biodiversity analysis of microperiphyton communities, e.g., 20 slide replicates (350 cm2) were sufficient for the microperiphyton communities at both depths, while 10 slide replicates (175 cm2) could meet the sampling strategy only for the samples with colonization times of 10 days or more at a depth of 1 m for recovering 90% species during the study period. Otherwise, more slide replicates were required with the increase of water depths and shortening colonization times for recovering microperiphyton species, e.g., for recovering 90% species of a mature microResponsible editor: Philippe Garrigues H. Xu (*) : W. Zhang : Y. Jiang : M. Zhu Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China e-mail: henglongxu@126.com K. A. S. Al-Rasheid Zoology Department, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia

periphyton community (>10 days), 10 slide replicates (175 cm2) were sufficient at a depth of 1 m, while for the young samples (>10 days) much more (1530) slide replicates were required at both depths in this study. Furthermore, to achieve <10% standard errors, six (105 cm2) and nine (160 cm2) slide replicates were required for biodiversity analysis of the microperiphyton communities with various colonization times at depths of 1 and 3 m during the summer season, respectively. Conclusion These results suggest that sampling sizes represented a significant influence on biodiversity analysis of microperiphyton communities for monitoring programs and ecological conservation researches in marine ecosystems. Keywords Microperiphyton . Biodiversity . Artificial substrate . Species accumulation curve . Marine bioassessment

1 Introduction As the primary components of periphyton communities, microperiphytons play a crucial role for energy flux and element cycling in many aquatic ecosystems (Fischer et al. 2002; Debenest et al. 2009; Kathol et al. 2009). Autotrophic microalgae that are predominated by benthic diatoms are responsible for the bulk of primary production (Debenest et al. 2009; Duong et al. 2010), while protozoan consumers mediate the flow of both matter and energy from plankton to benthos in most aquatic ecosystems (Kathol et al. 2009; Norf et al. 2009; Xu et al. 2011). With their rapid responses to environmental stress, easy sampling, and allowing standardization for spatiotemporal discriminations, microperiphytons have widely been used as helpful bioindicators to assess water quality in aquatic environ-

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2010; Tan et al. 2010; Xu et al. 2011). So far, a number of approaches based on a range of sample sizes and the exposure depths for collecting microperiphyton communities using artificial substrates have been reported (Strder-Kypke 1999; Burkovskii and Mazei 2001; Nayar et al. 2005; Kralj et al. 2006; Mieczan 2010). However, the influence of sample sizes on analyzing ecological patterns of microperiphyton communities has been the subject of few specific attentions (Dorigo et al. 2009, 2010; Xu et al. 2009a, b, 2011). In this study, microperiphyton communities with different colonization ages were investigated using the glass slide method in coastal waters of the Yellow Sea, near Qingdao, northern China, from May to June 2010. It should be noted, however, that because of constraints of the methods used, only the data mainly for periphytic ciliates, sarcodines, and diatoms are available. Our aims were: (1) to reveal the influence of sampling size on analyzing ecological features of microperiphyton communities with different colonization times and exposure water depths and (2) to determine an optimal sampling strategy of large spatial/temporal scale monitoring programs and ecological conservation researches in marine ecosystems using microperiphyton.
Fig. 1 Sampling station located at coastal waters of the Yellow Sea, near Qingdao, northern China

2 Materials and methods ments (Gold et al. 2002; Khatoon et al. 2007; Risse-Buhl and Ksel 2009; Morin et al. 2010). The biodiversity measures (e.g., richness, diversity, and evenness of species) of a community are commonly used to evaluate water quality in many aquatic ecosystems based on both traditional microscopy and molecular methods (Burkovskii and Mazei 2001; Gong et al. 2005; Kralj et al. 2006; Dorigo et al. 2009, 2010). However, they are problematic due to their dependence on sample sizes (Warwick and Clarke 2001; Dorigo et al. 2.1 Sampling station and strategy This survey was carried out in coastal waters of the Yellow Sea, near Qingdao, northern China, from May 18 to June 16, 2010 (Fig. 1). This coastal area is 8 m deep with a transparency of 3 m with such a water condition status: water temperature, 1419C; pH 8; salinity, 3031 psu; dissolved oxygen, 67 mg l1 during the study period. The glass slide systems were designed, deployed, anchored, and sampled as described by Xu et al. (2009a, b).

Table 1 Species numbers of microperiphyton observed in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples with 20 slide replicates at depths of 1 and 3 m in coastal waters of the Yellow Sea, near Qingdao, northern China, during the study period

Taxa 1-m samples Ciliate Sarcodine Diatom Total 3-m samples Ciliate Sarcodine Diatom Total

1 day

3 days

7 days

10 days

14 days

21 days

28 days

6 0 14 20 3 0 12 15

20 1 12 33 14 1 13 28

32 1 14 47 26 1 14 41

43 2 13 58 35 2 11 48

44 2 12 58 44 2 11 57

47 0 11 58 41 0 9 50

50 0 12 62 45 0 9 54

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Fig. 2 Species numbers of microperiphyton in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples with 120 slide replicates (a, b) and coefficients of variation in species number of the samples with 120, 220, 320,

1920 slide replicates (c, d) at depths of 1 (a, c) and 3 m (b, d) in coastal waters of the Yellow Sea during the study period

A total of 280 glass slides (17.5 cm2 for each) were used as artificial substrates for collecting microperiphyton communities at depths of 1 and 3 m below the water surface. For each depth, a total of seven polyvinyl chloride (PVC) frames were used to hold a total of 140 slides, 20 of which were randomly collected from each PVC frame at time intervals of 1, 3, 7, 10, 14, 21, and 28 days, during the study period. From both depths, samples were collected simultaneously.

2.2 Identification and enumeration Identification and enumeration of microperiphyton species were carried out following the methods outlined by Xu et al. (2009a, b). Taxonomic classification of microalgae, ciliates, and sarcodines was based on published keys and guides, such as Steidinger and Tangen (1997), Song et al. (2009), Fan et al. (2010), Jiang et al. (2010), and Pan et al. (2010).

Table 2 Parameters of SACs of microperiphyton communities with various colonization times at depths of 1 and 3 m in coastal waters of the Yellow Sea during the study period Smax predicted maximum accumulative species number, Kn slide replicate number for recovering 50% of the predicted maximum number of species, R2 regression coefficient

Parameters 1-m samples Smax Kn R2 3-m samples Smax Kn R2

1 day

3 days

7 days

10 days

14 days

21 days

28 days

22.9 2.5 0.96 17.3 3.3 0.98

36.1 1.8 0.98 31.3 3.6 0.88

48.5 1.6 0.92 45.0 2.5 0.98

58.4 0.7 0.85 49.2 2.0 0.94

59.4 0.9 0.96 58.4 1.3 0.97

57.0 0.8 0.89 51.4 1.0 0.86

62.8 1.3 0.95 54.3 1.5 0.90

Environ Sci Pollut Res (2012) 19:540549 Table 3 Estimated slide replicate numbers required to recover 7595% species in microperiphyton samples with various colonization times at depths of 1 and 3 m in coastal waters of the Yellow Sea during the study period rs (%) 1 day 3 days 7 days 10 days 14 days 21 days

543 28 days

rs recovery rate of species

1-m samples 75 7.5 90 22.5 95 47.5 3-m samples 75 9.9 90 29.7 95 62.7

5.4 16.2 34.2 10.8 32.4 68.4

4.8 14.4 30.4 7.5 22.5 47.5

2.1 6.3 13.3 6.0 18.0 38.0

2.7 8.1 17.1 3.9 11.7 24.7

2.4 7.2 15.2 3.0 9.0 19.0

3.9 11.7 24.7 4.5 13.5 28.5

The enumeration of microperiphyton was carried out in vivo at a 100-fold magnification under an inverted microscope as soon as possible after sampling (generally within 24 h; Xu et al. 2009a, b). For recovering all species colonizing the glass slides, one surface of an entire slide (17.5 cm2) from a total of 40 slide replicates was examined at each colonization period using bright field illumination and occurrences were recorded. For the enumeration of individual abundances, one entire slide surface was examined at each colonization period of <14 days, whereas for colonization periods of 14 days or more, 10 randomly chosen fields of view per slide were examined and the dominant ciliates were enumerated. For each of 20 sample sizes with 120 slide replicates, the cell numbers were calculated to confirm the average abundance of microperiphyton individuals (individuals per square centimeter).
Table 4 Estimated recovering rates of microperiphyton species in the samples with 150 slide replicates during various colonization periods at depths of 1 and 3 m in coastal waters of the Yellow Sea during the study period Slide replicate 1-m sample 1 2 3 5 10 15 20 30 40 50 3-m sample 1 2 3 5 10 15 20 30 40 50 1 day

2.3 Data analyses The species accumulation curves (SACs) can be fitted by the MichaelisMenten equation: Sn Smax =1 Kn =n 1

where Sn is the accumulative species number at nth slide replicate; Smax is the predicted maximum accumulative species number; n is the replicate of glass slides; and Kn is a constant, i.e., the slide replicate number when the accumulative species number reach 50% of the predicted maximum accumulative species number (Flather 1996). Two parameters (Smax and Kn) of SACs were estimated using the SIGMAPLOT. We define the percentage of maximum accumulative species number as the recovering rate of
7 days 10 days 14 days 21 days 28 days

3 days

29 44 55 67 80 86 89 92 94 95 23 38 48 60 75 82 86 90 92 94

36 53 63 74 85 89 92 94 96 97 22 36 45 58 74 81 85 89 92 93

38 56 65 76 86 90 93 95 96 97 29 44 55 67 80 86 89 92 94 95

59 74 81 88 93 96 97 98 98 99 33 50 60 71 83 88 91 94 95 96

53 69 77 85 92 94 96 97 98 98 43 61 70 79 88 92 94 96 97 97

56 71 79 86 93 95 96 97 98 98 50 67 75 83 91 94 95 97 98 98

43 61 70 79 88 92 94 96 97 97 40 57 67 77 87 91 93 95 96 97

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Fig. 3 Abundances (individuals per square centimeter) of microperiphyton in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples of 120 slide replicates (a, b) and coefficients of variation in individual

abundance of the samples with a range of 120, 220, 320, 1920 slide replicates (c, d) at depths of 1 (a, c) and 3 m (b, d) in coastal waters of the Yellow Sea during the study period

species (rs), i.e., rs =100 Smax/Sn. Thus, the rs value can be calculated following the model: rs 100=1 Kn =n 2

The standard errors of the microperiphyton community parameters among different sample sizes were summarized using the coefficient of variation (CV). For visualizing these CV values, the line plots with scale values were used. The nonparametric KolmogorovSmirnov test was used to evaluate the differences in the parameters of SACs (e.g., Smax) based on different sample sizes between depths of 1 and 3 m at the 0.05 level.

3 Results 3.1 Species accumulation curves and required slide replicates The species numbers of the microperiphyton in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples with 20 slide replicates at depths of 1 and 3 m in coastal waters of the Yellow Sea

during the study period are summarized in Table 1. The ciliates and diatoms are the primary contributors to the microperiphyton communities with all 20 sample sizes at both depths. However, it should be noted that the ciliate assemblages became the most important component in taxonomic composition with an increase of colonization periods, especially in the samples of more than 10 days. The SACs for each sample with a colonization period of 128 days were summarized in Fig. 2a, b. Regression analyses showed that these SACs were well fitted by the model (Eq. 1; R2 0.90, P <0.05). Although no significant differences were found (P <0.05), the Smax values were higher at a depth of 1 m than at 3 m, whereas the Kn values were in a reverse case, i.e., more replicates of glass slides were required to recover 50% of the maximum number of species for 3-m samples than the samples at a depth of 1 m (Table 2). The required numbers of slide replicates to recover 75 95% species of microperiphyton varied according to different colonization periods (Table 3). It was shown that more slide replicates were required with the increase of

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Fig. 4 Species richness (a, b) of microperiphyton communities in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples of 120 slide replicates (a, b) and coefficients of variation in species richness index (D) of the

samples with a range of 120, 220, 320, 1920 slide replicates (c, d) at depths of 1 (a, c) and 3 m (b, d) in coastal waters of the Yellow Sea during the study period

water depths and shortening colonization times for recovering microperiphyton species (Table 3). For example, for recovering 90% species of a mature microperiphyton community with a colonization period of 10 days or more, 10 slide replicates (175 cm2) were sufficient at a depth of 1 m, while for the young samples with colonization times of fewer than 10 days, much more (1530) slide replicates were required at both depths. It should be addressed that 10 slide replicates were sufficient to recover 75% microperiphyton species in all samples. However, to recover 95% species, 20 slide replicates could meet the sampling strategy of the mature samples at a depth of 1 m, whereas larger sample sizes were needed in the other samples, especially for the young samples at both depths (Table 3). The recovery rates of species for the different sampling regimes (150 slide replicates) were estimated using the statistical model (Eq. 2; Table 4). It should noted that: (1) 10 slide replicates were sufficient to recover 80% and 90% species in the mature and young samples and (2) 20 slide replicates were adequate to record 90% species

for the mature and young microperiphyton communities at both depths (Table 4). 3.2 Standard errors for analyses of species number The CVs in species number of the microperiphyton samples with a range of 120, 220, 320, 1920 slide replicates are summarized in Fig. 2c, d. The CV values in terms of species number showed a clear decreasing trend with the increase of slide replicates and colonization periods (Fig. 2c, d). It is should be addressed that the six and nine slide replicates were required to achieve <10% standard errors for recovering species in the 1- and 3-m samples within all exposure periods and that the young communities generally needed more slide replicates than the mature ones for achieving a sampling effort at both depths (Fig. 2c, d). For example, two and three slide replicates were sufficient to achieve <10% standard errors for the 14-day samples, while more (approximately six) slide replicates were needed for the 7-day communities at depths of 1 and 3 m, respectively (Fig. 2c, d).

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Fig. 5 Species diversity (a, b) of microperiphyton communities in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples of 120 slide replicates (a, b) and coefficients of variation in species diversity index (H) of the

samples with a range of 120, 220, 320, 1920 slide replicates (c, d) at depths of 1 (a, c) and 3 m (b, d) in coastal waters of the Yellow Sea during the study period

3.3 Standard errors for analyses of individual abundance The individual abundances of the microperiphyton in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples with a range of 120 slide replicates at two depths are summarized in Fig. 3a, b. The abundances of microperiphyton showed an increasing trend with the increase of slide replicates from 1 to 10 and leveled off at the replicates of more than 10 glass slides (Fig. 3a, b). The analyses of standard errors revealed that the CV values in abundance decreased with the increase of slide replicate numbers (Fig. 3c, d). For achieving <10% standard errors approximately eight slide replicates were sufficient to measure individual abundance for all microperiphyton samples. It should be noted that more slide replicates were required for the young communities compared with the mature ones (Fig. 3c, d). For example, less (one to two) slide replicates were adequate to achieve <10% standard errors for 14-day communities compared with 7-day samples (six slide replicates; Fig. 3c, d).

3.4 Standard errors for analyses of diversity indices Species richness, species diversity, and species evenness of the microperiphyton samples of different sample sizes represented an increasing trend with shortening colonization period in both 1- and 3-m samples (Figs. 4a, b, 5a, b, and 6a, b). The coefficients of variation in all three indices of ciliate communities with a range of 120, 220, 320, 1920 slide replicates were shown in Figs. 4c, d, 5c, d, and 6c, d. It was shown that six and nine slide replicates are sufficient to archive <10% coefficients of variation in species richness (Fig. 4a, b), while 7 and 10 slides are the least replicates needed to reach <5% coefficients of variation in species diversity and evenness (Figs. 5c, d and 6c, d) for 1- and 3-m samples, respectively.

4 Discussion A number of investigations have demonstrated that SACs may provide information on the relationship between

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Fig. 6 Species evenness (a, b) of microperiphyton communities in 1-, 3-, 7-, 10-, 14-, 21-, and 28-day samples of 120 slide replicates (a, b) and coefficients of variation in species evenness index (J) of the

samples with a range of 120, 220, 320, 1920 slide replicates (c, d) at depths of 1 m (a, c) and 3 m (b, d) in coastal waters of the Yellow Sea during the study period

sampling effort and number of recovering species (Thompson et al. 2003; Ugland et al. 2003; Colwell et al. 2004; Izsk and Papp 2011). In the present study, the species accumulation curves of microperiphyton communities with various colonization times were found to be significantly fitted to the model (Eq. 1) used. Based on our data, it was demonstrated that the required samples size varied with both colonization times and exposure water depths. Our data suggest that (1) 10 slide replicates were sufficient to recover 80% and 90% species in the mature and young samples at a depth of 1 m and (2) 20 slide replicates were adequate to record 90% species for the mature and young microperiphyton communities at both depths, respectively. This implies that a surface of 350 cm2 is sufficient to recover 90% species in the microperiphyton samples within colonization times of 128 days at both depths during the study period. According to these results, analyzing 300 cm2 (17 slide replicates) or more for each sample in some previous studies on mature microperiphytic communities would recover >90% species (Nayar et al. 2005; Dorigo et al.

2010; Morin et al. 2010). Thus, fewer than five slide replicates, which is the commonly used sampling effort strategy in some previous investigations, might achieve <85% of species (Coppellotti and Matarazzo 2000; Gong et al. 2005). However, just two glass slides (35 cm2) for 14-day samples in some previous studies on periphytic communities would result in <75% recovery rates of the species expected (Railkin 1998; Strder-Kypke 1999; Kralj et al. 2006; Xu et al. 2009a, b). The analyses of standard errors showed that six and nine slide replicates were sufficient to achieve <10% standard errors for measuring species number, individual abundance, and three diversity indices of the microperiphyton communities with a range of colonization times at depths of 1 and 3 m, respectively. It should be noted, however, that fewer slide replicates were required for analyzing both community patterns of microperiphyton at a depth of 1 m compared with cases at the deeper layer. This may be mainly due to the weak sunlight conditions and less food supply and at a depth of 3 m in the water columns with a transparency of 3 m. This may also be the reason for both accumulative

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Environ Sci Pollut Res (2012) 19:540549 Acknowledgments This work was supported by The Natural Science Foundation of China (project no. 41076089), and funded by Research Group (project no. RGP-VPP-083), King Saud University Deanship of Scientific Research. Special thanks are due to Prof. Weibo Song, Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean University of China, for helpful discussions in the preparation of the manuscript.

species number and individual abundance being higher at a depth of 1 m than at 3 m during the study period. It should be noted that the present study has traditionally been based on the microscopy method, i.e., identification of microperiphyton taxa at the lowest possible taxonomic level. However, this method requires skilled taxonomic expertise and is time-consuming and expensive, thus limiting the use of microperiphytons for monitoring water quality, in particular by the environmental agencies that often deal with large-scale sampling in a limited time span. The molecular methods, such as denaturing gradient gel electrophoresis, which have previously been used for analyzing prokaryote and eukaryote community richness and diversity in river systems, and HPLC pigment analysis to assess the global taxonomic composition of the photoautotrophic communities have been proven to be effective techniques for reducing the costs of ecological monitoring researches by identifying taxa to the highest possible category without losing information (Dorigo et al. 2009, 2010). Several researches on benthic communities have suggested that these methods make it easy to determine and compare the species composition of periphytic communities (Amann et al. 1995; Szabo et al. 2008; Dorigo et al. 2009, 2010). Thus, the molecular methods could be employed in order to determine the sample size for the analysis of biodiversity of microperiphyton communities in the future. In summary, the sampling size represented a significant influence on biodiversity analysis of microperiphyton communities, e.g., 20 slide replicates (350 cm2) were sufficient for the microperiphyton communities at both depths, while 10 slide replicates (175 cm2) could meet the sampling strategy only for the samples with colonization times of 10 days or more at a depth of 1 m for recovering 90% species during the study period. Otherwise, more slide replicates were required with the increase of water depths and shortening colonization times for recovering microperiphyton species, e.g., for recovering 90% species of a mature microperiphyton community (>10 days), 10 slide replicates (175 cm2) were sufficient at a depth of 1 m, while for the young samples (>10 days), much more (1530) slide replicates were required at both depths in this study. Furthermore, to achieve <10% standard errors, six (105 cm2) and nine (160 cm2) slide replicates were required for biodiversity analysis of the microperiphyton communities with various colonization times at depths of 1 and 3 m during the summer season, respectively. These results suggest that sampling sizes represented a significant influence on biodiversity analysis of microperiphyton communities for monitoring programs and ecological conservation researches in marine ecosystems. Further studies, however, on a range of marine waters and over extended time periods using both microscopy and molecular methods are needed in order to verify this conclusion.

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