Variability in the Structure of Planktonic Microalgae Assemblages in Water Column Associated with Posidonia oceanica (L.) Bed in Tunisia

Patterns of phytoplankton in areas with seagrass (Posidonia oceanica) and areas without seagrass were quantified in the coast of Chebba (East of Tunisia). Replicate samples were collected at August 2011, from four stations (separated by 500 of metres). The diversity of phytoplankton was high around P. oceanica meadows compared to area without seagrass. A possible explanation to this finding is that water motion and hydrodynamics forces cause leaves agitation allowing the passage of epiphytic species in the water column. Our results also show an increase of abundance of potentially toxic dinoflagellates around Posidonia bed such as Alexandrium minitum, Amphidinium carterae, Karenia selliformis, Coolia monatis, Karlodinium veneficum, Ostreopsis ovata, Prorocentrum concavum, P. minimum, P. rathymum, and P. lima. Installation of fish farms on Posidonia beds should be avoided, not only to preserve this vulnerable habitat, but also to avoid fish contamination by toxic species derived from the resuspension of epiphytic community on seagrass substrata to the water column.


Introduction
Marine phytoplankton constitutes the base of the pelagic food-web and is responsible for about half of the net annual primary production on Earth [1].These range from diatoms and dinoflagellates to cyanobacteria.They are a direct as well as indirect food source for many animal species [2].Leaves of seagrass systems offer substrata suitable for settlement and growth of a number of microcolonists characterized by a high diversity of species [3].Among the epiphytic community, a large number of potentially toxic species of dinoflagellates (Alexandrium minutum, Amphidinium carterae, Karenia selliformis, Coolia monatis, Karlodinium veneficum, Ostreopsis siamensis, Prorocentrum concavum, Prorocentrum minimum, Prorocentrum rathymum, and Prorocentrum lima) were found in Tunisia [4,5] and in the north of the Mediterranean Sea [6].Some of them are potential toxin producers [7].
Although a good number of surveys have focused on the phytoplankton community in the north basin of the Mediterranean Sea, the south part of the basin has received little attention.Many authors have bemoaned the lack of data in some Mediterranean regions, particularly North Africa [8].Few studies analyzing changes in phytoplankton assemblages in the north of Tunisia have been published [4,9] and to our knowledge no studies on plankton assemblages in the east coast have been conducted up to now.This region, with other neighbouring areas, contains 20% of the potential yield of small pelagic fish stocks of the country.Moreover, several fish farms have recently been installed in this area.Knowledge of potentially toxic species of microalgae is necessary to assess the influence of those species on fisheries exploitation and human health.
At Chebba, east of Tunisia, we analyzed the phytoplankton community structure around different habitats.We hypothesized that phytoplankton communities' abundances are higher around Posidonia beds.
We thus compared microalgae assemblages around three kinds of Posidonia oceanica beds and around unvegetated habitats and examined their variability.

Materials and Methods
This study was conducted in the locality of Oued Lafrann (35 ∘ 15  18  N, 11 ∘ 07  28  E) in the region of Chebba (east of Tunisia).The climate is semiarid(average precipitation: 350 mm year −1 ) and sunny with strong northerly winds.This locality is characterized by a good seagrass status with barrier and fringing reefs in addition to an extensive plain meadow that has the highest cover and shoot densities in the east of Tunisia [10].In this region, we found a tiger meadow [10] with tiger-shaped cords of seagrass from 10 to 30 m long and spaced about 1-1.5 m with dead matte [11].This area is under consideration as a marine protected area (submitted in Italo-Tunisian project iTunES) because of its biocenotic richness and its distinctive ecosystems.Prospecting and sampling were conducted during August 2012 in four stations (i) a fringing reef meadow, (ii) tiger meadow, (iii) plain meadow, and (iv) unvegetated habitat (Figure 1).At each station, twelve replicate water samples (about 10 m apart) were selected.Water column samples, using 1-litre glass bottle, were conducted by SCUBA diving above canopy for vegetated stations or on the bottom for unvegetated station.All samplings were collected about noon.
Samples were fixed with Lugol's solution and finally preserved in 5% formalin.All sampling water was kept in the dark at ambient temperature until their microscopic observation.Settling long glass tubes used for sedimentation procedure were 2 cm wide by 21 cm long and have a base plate that contains a coverslip onto which the algae settle.To mix the sample, the bottle was gently tilted back and forth 10 times before pouring.A 50 mL subsample was poured into the settling chamber and left to settle for 24 h.Subsamples were examined in an inverted microscope at medium (×200) magnification by scanning the entire surface of the settling chamber to enumerate epiphytic microalgae [12].The total number of microalgae individuals () contained in 1 litre [expressed as number of individual per litre] is obtained by the following conversion:  = ( × 1000)/V; with  = number of individuals counted and V = volume of the sedimentation chamber (50 mL).The identified taxa were divided into groups (diatoms, dinoflagellates, cyanobacteria).
2.1.Data Analysis.Data were tested for normality using the Kolmogorov-Smirnov test [13] and for heteroscedasticity using Cochran's  test, and transformed if necessary [14].
Analyses of similarity (ANOSIM) randomization tests (with Log( + 1)-transformed data) were used to test for differences in species abundance between stations [15].Differences found using ANOSIM were followed up using the SIMPER analysis to identify which species primarily accounted for the observed differences between stations.SIMPER generates a ranking of the species responsible for the significant differences.These analyses used a matrix composed of Bray-Curtis similarity coefficient generated with Log( + 1)-transformed species abundance data.
A one-way Analysis of variance (ANOVA) was used to test the hypothesis that the abundance of each of the most abundant taxa differed between stations.The Tukey's HSD (Honestly Significant Difference) test was applied for multiple comparisons of means.

Results
The highest mean value of dinoflagellates abundance was recorded in the fringing reef meadow, whereas the lowest mean value has been detected in the tiger meadow.The lowest mean value of diatoms abundance was recorded in the unvegetated station, whereas the highest mean value was detected in fringing reef meadow.Cyanobacteria abundance was very low in all stations (Figures 2(a For microalgae, 61 species were counted including 60 species around P. oceanica bed stations and 34 at the unvegetated station (see the appendix).Analysis of similarity (ANOSIM) of epiphytic microalgae species abundances showed significant differences between stations (Table 1).
Abundances of microalgae and cyanobacteria were included in the univariate analyses of variances.Dinoflagellates differed significantly between stations with high abundance in fringing reef meadow (Tukey test).Significant differences were detected for diatoms with high abundance in the plain meadow (Tukey test).When the abundance of toxic dinoflagellates and cyanobacteria are grouped, significant differences were detected between stations with high abundance in vegetated habitats (ANOVA, Table 2).Analyses of similarity percentage (SIMPER) showed that the average dissimilarity between vegetated and unvegetated stations is high (89.15%).This procedure also allowed determining the species that contribute to this dissimilarity, they are Pleurosigma sp.Nitzschia fontifugua, Guinardia sp., Ostreopsis ovata, Licmophora sp., Coolia monotis, Polykrikos sp., Prorocentrum micans, and Coscinodiscus sp. that are absent at unvegetated stations and the cyanobacteria Spirulina subsalsa that was absent around Posidonia beds.Species Navicula sp., Peridinium sp., and Prorocentrum lima have unequal abundance between the groups of stations (Table 3).

Discussion
Our study shows two key results: (1) a high abundance of phytoplankton in the water column around P. oceanica beds and (2) high number and abundance of potentially toxic dinoflagellates species around vegetated habitats.
The diversity of phytoplankton was high around P. oceanica meadows.A possible explanation to this finding is that water motion in our study site causes blade agitation (and other substratum) allowing the passage of epiphytic (and benthic) species into the water column as found by other previous studies [4,6,16].For example, positive and significant correlations between epiphytic and planktonic Ostreopsis cell abundances have been found previously [6,16] which supports the hypothesis of cell resuspension from the macrophyte surfaces to the water column.
Vegetated ecosystems are ideal habitats for benthic diatoms and other epiphytes [17] since seagrass leaves and algal thalli may represent an order of magnitude greater surface area for the colonization and growth of diatoms [18].These high abundances in the presence of macrophytes may be due to an increase in (i) the amount of physical structure (usable as living space), (ii) the number of microhabitats, (iii) sediment deposition and stabilization, and (iv) food resources and (v) protection from predators, and also to (vi) a reduction of hydrodynamic forces [19].Vila et al. [20] suggest that benthic dinoflagellates are particularly affected by hydrodynamics, as the mat is only loosely attached, and they are easily resuspended in the water column.Brahim et al. [21] have found that abundances of micro-and macroorganisms were higher in the P. oceanica leaf habitat than in the water column.This may be explained by the very high predatory pressure exerted by young and planktivorous (larvae, juveniles, and adults) fish [22] on animals remaining in the water column.The blades of P. oceanica furnish these animals with refuge and protection from predatory fish [19].A similar observation was made by Mabrouk et al. [5] in the eastern coast of Tunisia (Mahdia), where a significant correlation between the leaf area index and the abundance of epiphytic species indicates that the phenological parameters of the host plant influence the abundance of epiphytic microorganisms.
It seems that benthic dinoflagellates have no preference for specific macrophytes, but they prefer three-dimensional, flexible, and high surface area as proposed by Vila et al. [20].On the contrary, Turki [9] had found that epiphytic dinoflagellates displayed host substratum preference on Cymodocea nodosa where the cell abundance was higher than on P. oceanica.An important variability in the abundance of Ostreopsis Seagrass Posidonia beds concentrate a wide variety of organism that feed on their epiphytes [2].Grazing has a negative effect on the abundance of phytoplankton in the water column around the seagrass beds [23].Despite this effect, the abundance of phytoplankton in vegetated areas remains higher than in the unvegetated station.
The effect of other environmental parameters should be mentioned, such as wave activity [24], nutrient availability, salinity, and light intensity [25].Nutrient concentration is higher around seagrass [26] and as a consequence the plankton population is more diversified around Posidonia bed.
Our results also show an increase of abundance of potentially toxic dinoflagellates around Posidonia beds such as Alexandrium minutum, Amphidinium carterae, Karenia selliformis, Coolia monatis, Karlodinium veneficum, Ostreopsis ovata, Prorocentrum concavum, P. minimum, P. rathymum, and P. lima.Prorocentrum species are mainly associated with okadaic acid and the production of analogues [27].Moreover, Marr et al. [28] concluded that the underestimation of toxic dinoflagellates associated with a toxic event might be due, in part, to the lack of sampling of the benthic and epiphytic communities.
No evidence of pollution at our studied area since we did not detect nutrient enrichment and the phenological parameters of P. oceanica are good [29].Moreover, this station is quite far from any anthropogenic disturbance.

Conclusion
The results of this survey highlight the increases of toxic dinoflagellates around vegetated area.We, thus, believe that our data may be, on a North African scale, not only worthwhile but also useful in initiating other investigations.Finally, a study by FAO [30] recommends that aquaculture installation on Posidonia beds should be avoided whenever possible and our data support this recommendation, not only to preserve this vulnerable habitat [31], but also to avoid fish contamination by toxic species derived from the passage of epiphytic community on seagrass substrata to the water column.

Figure 1 :
Figure 1: Map of the study area, showing the sampling station.

Figure 2 :
Figure 2: Average abundance of phytoplankton in water column in prospected stations.

Table 1 :
Results of analysis of similarity ANOSIM of phytoplankton abundances in water column according to habitat.Transformation log( + 1).

Table 2 :
Analysis of variance ANOVA of the abundances of major groups of microalgae and cyanobacteria in prospected stations.

Table 3 :
Results of SIMPER procedure showing distribution of discriminating species between stations around Posidonia bed and unvegetated habitat.

Table 4 :
Species list of microalgae and cyanobacteria identified on water column around P. oceanica bed and in unvegetated station.