INTRODUCTIONTop

Diverse and highly productive epiphytic assemblages composed mainly of microscopic algae are attached to the seagrass and macroalgae leave and benefit from this relationship by gaining a structure on which to grow and by consuming nutrients that the vegetation releases (Hauxwell et al. 2001Hauxwell J., Cebrian J., Furlong C., et al. 2001. Macroalgal canopies contribute to eelgrass (Zostera marina) decline in temperate estuarine ecosystems. Ecology 82: 1007-1022., Perez et al. 2008Perez M., Garcia T., Invers O., et al. 2008. Physiological responses of the seagrass Posidonia oceanica as indicators of fish farm impact. Mar. Pollut. Bull. 56: 869-879.).

The host plants play a key role in shaping the composition of the epiphytic community structure (Johnson et al. 2005Johnson M.P., Edwards M., Bunker F., et al. 2005. Algal epiphytes of Zostera marina: Variation in assemblage structure from individual leaves to regional scale. Aquat. Bot. 82: 12-26.), since the phenological parameters such as leaf length, leaf area and leaf area index of some magnoliophytes increase during the warm seasons (Mabrouk et al. 2009Mabrouk L., Hamza A., Sahraoui H., et al. 2009. Caractéristique et phénologie de l’herbier de Posidonia oceanica (L.) Delile sur les cotes de Mahdia (région est de la Tunisie). Bull. Inst. Nat. Sci. Tech. Océan. Pêche Salammbô. 36: 139-148.). The structure of epiphytic communities is also influenced by factors such as the age of the leaf (Mazzella et al. 1994Mazzella L., Buia M.C., Spinoccia L. 1994. Biodiversity of epiphytic diatom community on leaves of Posidonia oceanica. In: Marino D. and Montresor M. (eds), Proceedings of the 13th Diatom Symposium, Biopress, Bristol, UK.), the seasonal cycle of macroalgae (Gambi et al. 1992Gambi M.C., Lorenti M., Russo G.F., et al. 1992. Depth and seasonal distribution of some groups of the vagile fauna of the Posidonia oceanica leaf stratum: structural and trophic analyses. PSZN I. Mar. Ecol. 13: 17-39.) and grazing (Mirella et al. 2012Mirella P.C.V., Brendan P.K., Melanie J.B., et al. 2012. Epiphyte grazing enhances productivity of remnant seagrass patches. Aust. Ecol. 37: 885-892.).

The composition and abundance of epiphyte communities can also be influenced by abiotic factors such as irradiance, temperature, salinity and inorganic nutrients. Temperature contributes significantly to the temporal variation of diatom epiphytes (Johnson et al. 2005Johnson M.P., Edwards M., Bunker F., et al. 2005. Algal epiphytes of Zostera marina: Variation in assemblage structure from individual leaves to regional scale. Aquat. Bot. 82: 12-26.) and epiphytic dinoflagellates (Armi et al. 2010Armi Z., Turki S., Trabelsi E., et al. 2010. First recorded proliferation of Coolia monotis (Meunier, 1919) in the North Lake of Tunis (Tunisia) correlation with environmental factors. Environ. Monit. Assess. 164: 423-433.), and salinity is an important factor in the distribution of the microepiphyte community (Johnson et al. 2005Johnson M.P., Edwards M., Bunker F., et al. 2005. Algal epiphytes of Zostera marina: Variation in assemblage structure from individual leaves to regional scale. Aquat. Bot. 82: 12-26.). The influence of other factors such as hydrodynamics and light intensity on the development of epiphytes has also been documented (Nesti et al. 2009Nesti U., Piazzi L., Balata D. 2009. Variability in the structure of epiphytic assemblages of the Mediterranean seagrass Posidonia oceanica in relation to depth. Mar. Ecol. 30: 276-287.).

Epiphytic microalgae may include many toxic species that can damage fisheries and cause human health hazards. In recent years, the proliferation of toxic epibenthic species appears to be expanding on a global scale, probably due to either global climate change (Hallegraeff 2010Hallegraeff G.M. 2010. Ocean climate change, phytoplankton community responses, and harmful algal blooms: a formidable predictive challenge. J. Phycol. 46: 220-235.) or anthropogenic impacts such as eutrophication and transfer of ballast water (Hallegraeff et al. 2010Hallegraeff G.M., Bolch C.J.S., Huisman J.M., et al. 2010. Planktonic dinoflagellates. Algae of Australia phytoplankton of temperate coastal waters. CSIRO Publishing/ABRS. Melbourne, 145-212.).The increase in studies in various ecosystems all around the world over the past few decades could also explain their apparent global proliferation (Van Dolah 2000Van Dolah F. 2000. Marine algal toxins: origins, health effects, and their increased occurrence. Env. Health Persp. 108 (Suppl. 1): 133-141., Maso and Garcés 2006Maso M., Garcés E. 2006. Harmful microalgae blooms (HAB); problematic and conditions that induce them. Mar. Pollut. Bull. 53: 620-630.).Few studies (Bomber et al. 1989Bomber J.W., Rubio M.G., Norris D.R. 1989. Epiphytism of dinoflagellates associated with the disease ciguatera: substrate specificity and nutrition. Phycologia 28: 360-368.) have focused on the effect of the substrata on the growth of toxic epiphytic species, and their relationships with their hosts. Although the distribution of epiphytes has been shown to depend largely on their host (Cohu et al. 2013Cohu S., Mangialajo L., Thibaut T., et al. 2013. Proliferations of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in North Western Mediterranean Sea. Harmful Algae 24: 32-44., Accoroni et al. 2016aAccoroni S., Romagnoli T., Pichierri S., et al. 2016a. Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea. Harmful Algae 55: 179-190.), the question that still needs to be addressed is whether there is an affinity between a toxic species and a given substrate. Furthermore, Aligizaki and Nikolaidis (2006)Aligizaki K., Nikolaidis G. 2006. The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece. Harmful Algae 5: 717-730. highlighted correlations between the abundance of these toxic species in the water column and their abundance on macrophytes. Since most environmental monitoring programmes have focused on water column concentration of toxic species, understanding the distribution patterns of epiphytic species on macrophytes should be particularly useful for the design of monitoring programmes.

This study aims to characterize the temporal variability of epiphytic microalgae on different substrates (magnoliophytes and macroalgae) and in the water column, with a special focus on epiphytic toxic dinoflagellates. We particularly wish to examine the following hypotheses:

1) Do the diversity and abundance of epiphytic microalgae vary between substrata and environmental conditions?
2) Is there a relationship between the concentrations of the toxic species present on specific substrata and in the water column?
3) Do toxic epiphytic species show different distribution patterns on the leaves of Posidonia oceanica?

MATERIALS AND METHODSTop

Study area

The study area was in the locality of Oued Lafrann (35°15′18″N, 11°07′28″E) in the region of Chebba (north of the Gulf of Gabès in Tunisia) (Fig. 1). The climate is semiarid and sunny with strong northward winds. This region is not subjected to a major human impact. It has clear water in which artisanal and selective fisheries are very active.

The study area is colonized by many macrophytes. Cymodocea nodosa is present in shallow water (from 0.5 m to 18 m deep). P. oceanica beds, with foliage density exceeding 455.25 shoots m^–2, follow Cymodocea and reach up to 20 m depth. The new invasive magnoliophyte Halophila stipulacea (Sghaier et al. 2011Sghaier Y., Zakhama-Sraieb R., Benamer I., et al. 2011. Occurrence of the seagrass Halophila stipulacea (Hydrocharitaceae) in the southern Mediterranean Sea. Botanica Marina 54: 575-582.) with scattered tufts (2-3 m²) has also been recorded in this area. Chlorophyta Penicillus capitatus (Lamarck), with a density exceeding 1000 ind m^–2, is intermixed with Cymodocea. Zostera noltii is identified in scattered tufts in shallow muddy hollows and is sometimes associated with the seagrass C. nodosa (Caye and Meinesz 1985Caye G., Meinesz A. 1985. Observations on the vegetative development, flowering and seeding of Cymodocea nodosa (Ucria) Ascherson on the Mediterranean coasts of France. Aquat. Bot. 22: 277-289.). Photophylic algae generally colonize rocks, and tough substrates between 0.5 and 3 m depth, such as Cystoseira, represented by Cystoseira amentacea, Cystoseira stricta, Cystoseira compressa, Cystoseira barbata, occupy sandy bottoms.

Data sampling and processing

The sampling was conducted in a small creek covering a coastline of about 500 m, where diverse substrates (rocky blocks and sandy surfaces with dense vegetation) were present. The sampling, performed monthly from March 2013 to March 2014 in the same creek, was conducted by diving from 0.5 to 2 m depth. The study area was well covered by different types of marine vegetation. Ten substrata were investigated: four magnoliophytes (Posidonia oceanica, Zostera noltii, Cymodocea nodosa and Halophila stipulacea) and six macroalgae (Padina pavonica, Cystoseira mediterranea, Dictyota dichotoma, Dictyopteris membranacea, Penicilus capitatus, Asparagopsis armata). Most of the sampled vegetation was not permanent during the sampling period. Some types of vegetation, such as Posidonia oceanica, were present throughout the year; others such as Padina pavonica, Cystoseira mediterranea and Halophila stipulacea, appeared for a few months. P. oceanica, a perennial species, showed a rather good vitality in the study area, as was confirmed by several previous studies (Mabrouk et al. 2009Mabrouk L., Hamza A., Sahraoui H., et al. 2009. Caractéristique et phénologie de l’herbier de Posidonia oceanica (L.) Delile sur les cotes de Mahdia (région est de la Tunisie). Bull. Inst. Nat. Sci. Tech. Océan. Pêche Salammbô. 36: 139-148., 2011Mabrouk L., Hamza A., Ben Brahim M., et al. 2011. Temporal and depth distribution of microepiphytes on Posidonia oceanica (L.) Delile leaves in a meadow off Tunisia. Mar. Ecol. 32: 148-161.).

Macrophytes and seawater samples were collected in triplicate following the protocol agreed by a consortium of experts in the framework of the ENPI-CBCMED project M3-HABs (http://m3-habs.net) and recently published in Accoroni et al. (2016b)Accoroni S., Romagnoli T., Penna A., et al. 2016b. Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea. J. Phycol. 52: 1064-1084.. Before collection of the benthic substrata and in order to avoid resuspension, we sampled 1.5L of seawater at about 30 cm from the macrophyte in plastic bottles for nutrient analysis (500 ml) and planktonic identification (1L). The leaf beam of the magnoliophytes and total macroalgal thalli were then covered with a plastic bag (two different sizes, 50/30 cm and 40/20 cm, depending on the size of the vegetation) and gently detached from their substrate. The plant samples within the storage water were shaken vigorously to dislodge the epiphytic cells. They were then re-rinsed with filtered sea water (FSW) (2x 100 ml). The total retrieved volume was noted. It generally ranged between 400 and 1000 ml.

The macrophyte was then weighed to determine the fresh weight. All collected samples were preserved in a seawater formalin (3‰) solution and kept in the dark at ambient temperature until transfer to the laboratory.

For each retrieved sample, three subsamples (10 mL) were counted by means of an inverted microscope according to Utermohl’s sedimentation method (Utermohl 1958Utermohl H. 1958. Zur Vervollkommung der quantitativen Phytomicroorganisms-Methodik. Mitt. Int. Ver. Theor. Angew. Limnol. 9: 1-38.). The number of epiphyte species and their abundance, expressed as number of individuals per g of fresh weight of macrophyte (FW), was determined for each sampling period and depth.

Some of the recorded dinoflagellates, namely Ostreopsis cf. ovata and P. lima, were reported to be toxic and others, such as C. monotis, to be potentially toxic (Calabretti et al. 2017Calabretti C., Citterio S., Delaria M.A., et al. 2017. First record of two potentially toxic dinoflagellates in tide pools along the Sardinian coast. Biodiversity 18: 2-7., David et al. 2017David H., Kromkamp J.C., Orive E. 2017. Relationship between strains of Coolia monotis (Dinophyceae) from the Atlantic Iberian Peninsula and their sampling sites. J. Exp. Mar. Biol. Ecol. 487: 59-67.). C. monotis strains collected in the Gulf of Gabès were shown to be toxic to mice after intra-peritoneal injection (3.6 10⁷ cells ml^–1), causing loss of coordination, hind limb paralysis and respiratory difficulty (Abdennadher 2014Abdennadher M. 2014. Étude taxonomique et écophysiologique des dinoflagellés toxiques du Golfe de Gabès: Alexandrium minutum, Prorocentrum lima, Coolia spp. and Ostreopsis ovata. Ph.D. thesis. Univ. Science. Sfax, Tunisia.).

During the period of confirmed high abundance of epiphytic toxic (Ostreopsis cf. ovata, Prorocentrum lima) and potentially toxic (Coolia monotis) dinoflagellates, generally occurring in September (Mabrouk et al. 2014Mabrouk L., Ben Brahim M., Hamza A., et al. 2014. Temporal and spatial zonation of macroepiphytes on Posidonia oceanica (L.) Delile leaves in a meadow off Tunisia. Mar. Ecol. 36: 77-92.), a triplicate of shoots (that totalized 15 leaf bundles) of P. oceanica were prospected in the densest meadows (2 m depths). The different sections of the leaf (apical, middle and basal) were separated in the field and each part was gently covered with a plastic bag. In the laboratory, two persons held the sectioned parts horizontally by two clamps on each side, and the inner and outer sides of the leaf were gently scraped with a lamella. The scrapings were immersed in 10 ml of filtered sea water formalin (3‰). The abundance of microepiphyte was expressed by cells g^–1 FW on each part of the leaf.

Water column temperature was measured in situ using a multi-parameter type 340i / SET. Inorganic nutrients (NO₂⁻, NO₃⁻, NH₄⁺, PO₄³⁻, Si(OH)₄), total-nitrogen (TN) and total phosphate (TP) were analysed with a BRAN and LUEBBE type 3 autoanalyser, and concentrations were determined colorimetrically using a UV-visible (6400/6405) spectrophotometer (APHA 1992APHA. 1992. Standard methods for examination of water and waste water. APHA, AWWA. Washington, DC., USA.).

Data analysis

The Shannon index (Gray et al. 1992Gray C.A., Otway N.M., Laurenson F.A., et al. 1992. Distribution and abundance of marine fish larvae in relation to effluent plumes from sewage outfalls and depth of water. Mar. Biol. 113: 549-559.) was calculated to express diversity taking into account the number of species and abundance of individuals within each species. It is given by the following formula:

$$H^{\prime }=-{\displaystyle {\sum }_{i=1}^S{p}_i}\log p_i$$

(1)

where p_i is the proportional abundance or percentage of the species importance: p_i = n_i/N; S is the total number of species; n_i is the number of individuals of a species in the sample; N is the total number of individuals of all species in the sample.

To examine the relationships between the abundance of toxic and potentially toxic epiphytic dinoflagellates on macrophyte leaves and in the water column, the bivariate Pearson correlation test was used (SPSS software).

One-way ANOVA analyses were conducted to test the difference in toxic epiphyte concentrations between the studied substrata and to compare the abundances of epiphytic toxic dinoflagellates on the different parts of P. oceanica leaves. The Student-Newman-Keuls (SNK) post hoc test was used for post hoc multiple comparisons of means (Underwood 1997Underwood A.J. 1997. Experiments in ecology. Their logical design and interpretation using analysis of variance. Cambridge University Press, Cambridge. 504 pp.). Cochran’s C test was used before each analysis to check the homogeneity variance and data were log (x+1) transformed when necessary (Underwood 1997Underwood A.J. 1997. Experiments in ecology. Their logical design and interpretation using analysis of variance. Cambridge University Press, Cambridge. 504 pp.).

The similarity in epiphytic composition and abundance between the studied substrates was analysed by means of cluster analyses. We conducted a hierarchical agglomerative clustering analysis (Clarke and Warwick 2001Clarke K.R., Warwick R.M. 2001. Change in Marine Communities: An Approach to Statistical Analysis and Interpretation. 2nd Edition, PRIMER-E, Plymouth, 172 pp.) using the routine “CLUSTER” of the PAST software to depict the relative differences in epiphytic substrata.

A co-inertia analysis (Dolédec and Chessel 1994Dolédec S., Chessel D. 1994. Co-inertia analysis: an alternative method for studying species: environment relationships. Freshw. Biol. 31: 277-293.), which is a direct extension of multiple regressions to the modelling of a multivariate response matrix (Legendre and Legendre 1998Legendre P., Legendre L. 1998. Numerical ecology (2nd English Edn). Elsevier Science B.V., Amsterdam. 853 pp.), was conducted to examine the correlation between an array of response variables (in this case the ten substrates) and of independent explanatory variables (epibenthic abundance) conditional to a third matrix (here environmental parameters), keeping the environmental effect constant. A simple log (x+1) transformation was applied to the data to stabilize variance (Frontier 1973Frontier S. 1973. Etude statistique de la dispersion du zooplancton. J. Exp. Mar. Biol. Ecol. 12: 229-262.). Computing and graphical displays were performed with R-2.4.0 software (R-Development Core Team 2006R-Development Core Team. 2006. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) using the packages ade4 1.4.2 (Chessel et al. 2012Chessel D., Dufour A.B., Dray S., et al. 2012. Analysis of ecological data: Exploratory and euclidean methods in environmental sciences. R package version 1.5-1.).

RESULTSTop

Detailed nutrient mean concentrations are reported in Table 1. These mean concentrations were calculated taking into account the number of samples of each substrate during the study period. The results showed that nutrient concentrations varied from one substrate to another. Zostera noltii and Asparagopsis armata exhibited more variability than the other substrates.

The concentrations of nitrite and nitrate were high in magnoliophytes, with a maximum recorded for P. oceanica, whereas in macroalgae the maximum was recorded for Cystoseira mediterranea. Ammonium concentrations showed a different trend, with the highest values being recorded in macroalgae (Table 1). The highest concentrations of phosphorus and silicate were recorded for Dictyopteris membranacea (Table 1).

During the study period, a total of 31 microalgal taxa were identified (Table 2). Three algal groups were represented in our study area: namely, Baccillariophyceae (19 species), Dinophyceae (9 species) and Cyanophyceae (3 species). The species number differed according to the host species (Table 2). The highest species diversity was recorded on P. oceanica and A. armata. Only a few species were recorded on macrophytes (Table 2).

On magnoliophytes, the highest abundances of diatoms were recorded on P. oceanica leaves during spring. This class was also rather important in spring and summer, with abundance exceeding 60% and reaching over 96%of total epiphyte microalgae on macroalgae, especially on Asparagopsis armata (Table 3).

Dinoflagellates accounted for the highest abundances on P. oceanica in winter. This class also showed high abundances on Cymodocea nodosa (Table 3). On macroalgae, Cystoseira mediterraneas howed the highest abundances of dinoflagellates. Comparing to diatoms, dinoflagellates showed a low abundance on Padina pavonica and this group was almost absent on Asparagopsis armata.

The diversity index (H′) of epibenthic species was very high on P. oceanica, P. pavonica, H. stipulacea and D. dichotoma (Table 3). The highest diversity index (H’) was recorded on P. oceanica for magnoliophyte (H’=3.656) and on D. dichotoma for macroalgae (H′=3.835) during spring, and the lowest was recorded on Z. noltii during the same season (H′=1.825) (Table 3).

The co-inertia plot (Fig. 2A) illustrated close relationships between the composition of phytoplankton communities and the water properties above the ten sampling substrates. The overall model explained 33% of the total variation (permutation test, p=0.02, 1000 replicates). This variation was due to microphytoplankton taxa (20%) and to physical and chemical variability (18.52%) (Fig. 2B). Posidonia, Cystoseira and Halophila substrates showed close links between nitrite and nitrate and the phytoplankton species, as was illustrated by the position of Ostreopsis, P. lima and total dinoflagellates (Fig. 2A). In contrast, Dictyota dichotoma, Zostera noltii and Asparagopsis armata substrates were surrounded by the numerically dominant Coolia monotis, total phytoplankton and diatoms (Fig. 2A).

The toxic and potentially toxic dinoflagellates were mostly concentrated on P. oceanica, where they represented about 65% of the total epiphyte microalgae, followed by Cystoseira mediterranea and Cymodocea nodosa.

On P. oceanica, the occurrence frequency of toxic and potentially toxic dinoflagellates was high both on substrate and in the water column (Table 4). The highest occurrence of toxic dinoflagellates was observed for P. lima, with 66.83% on macrophytes and 54.26% in the water column (Table 4). The frequency was low for Ostreopsis, with only 1.84% on macrophytes and 9.30% in the water column (Table 4). C. monotis did not exceed 10% in the water column and was about 2.76% on macrophytes (Table 4). The other epiphytic species were barely observed within the water column and on macrophyte leaves, except for the epiphytic Polykrikos kofoidii (27.76%) (Table 4).

The abundance of the epiphytic dinoflagellates Ostreopsis cf. ovata was higher on magnoliophytes than on macroalgae, especially for Cymodocea nodosa, on which it reached 22.73% of the total dinoflagellates in autumn (Table 5). This toxic species did not show a significant difference in concentrations between the studied substrata, although the concentrations reached 0.5 10³ cells g^–1 FW on Cymodocea nodosa, 10³ cells g^–1 FW on Posidonia leaves in February and September (Fig. 3A), and relatively high abundances on Padina pavonica in September and November (Fig. 3C). A significant positive correlation (P<0.05, R²=0.30) was observed between the species concentrations on Padina pavonica and in the water column above this macroalga (Fig. 3C, D).

The abundance of P. lima on Posidonia leaves accounted for 62.59% of the total dinoflagellates (Table 5). During the sampling period, P. lima was the most dominant and frequent species on magnoliophytes as well as on macroalgae. This species significantly accumulated on P. oceanica (P<0.05), with the highest concentrations, exceeding 10⁴ cells g^–1 FW, being observed from February to May (Fig. 3A). On macroalgae, this species did not show significant variations between substrata. It exceeded 8.3 10³ cells g^–1 FW during March on Cystoseira mediterranea (Fig. 3E), whereas on Padina pavonica and on Halophila stipulacea, concentrations did not exceed 2 10³ cells g^–1 FW (Fig. 3C, G). In contrast to other toxic species, P. lima showed significant variations in its abundance on P. oceanica over time (P<0.05). This species was also present in the water column above the P. oceanica bed, with a concentration reaching over 10³ cells L^–1 (Fig. 3B). A significant correlation (P<0.005, R²=0.80) was pointed out between P. lima concentrations on different substrata and in the water column.

C. monotis was also present on different substrates, with a maximum of 37.5% recorded on Dictyota dichotoma (Table 5). The monthly abundance of C. monotis showed no significant difference between the studied substrata (P>0.05) (Fig. 3). This species showed generally low concentrations in the water column sampled near Posidonia, Cystoseira and Halophila (Fig. 3F, H). P. micans showed the highest concentrations on P. oceanica but its abundances were rather low on other substrates (Table 5). Other substrata were barely observed in our study area. P. lima and C. monotis were the main species present on these substrates, where the maximum concentration was approximately 1.7 10³ cells g^–1 FW on Asparagopsis armata during April.

In spring, when the maximum of substrates were available, the clustering analysis of epiphytic species similarity between different substrates showed four groups (Fig. 4). The first cluster was composed of C. mediterranea, which hosted C. monotis, P. lima and P. micans (Fig. 4). The second cluster was composed of Z. noltii, H. stipulacea, A. armata and D. dichotoma, which hosted only two species, P. lima and C. monotis (Fig. 4). The third cluster was composed of P. pavonica, hosting mainly P. lima and C. monotis (Fig. 4). Finally, the last cluster was composed of P. oceanica, which showed high dissimilarity to the other substrates, hosting the different epiphytic dinoflagellates with a dominance of P. lima (Fig. 4).

According to the SNK test results, the distribution of Ostreopsis cf. ovata on Posidonia leaf revealed three groups (a homogeneous subset) (Table 6). The highest abundance was marked on the inner face of the apical and the middle parts of the leaf. This toxic species was also present with a relatively high abundance on the inner face of the basal part. On the other hand, it was particularly absent on the outer face of Posidonia leaf (Fig. 5). As regards P. lima, there were only two identified groups of the distribution of this species on the leaf of P. oceanica (Table 6). The first group was formed on the inner face of the middle part and the outer and inner face of the apical part, where the abundance was the highest (Fig. 5). The second group formed the outer face of the middle and basal parts of the leaf, where the abundance was lower (Fig. 5).

The results of the ANOVA showed that the abundance of Ostreopsis cf. ovata and P. lima showed variability on the different parts of the Posidonia leaf (apical, middle and basal) (Fig. 5) and according to their position on the inner and outer faces of the leaves (P_{Ostreopsis cf. ovata}<0.0001; P_P.lima<0,005) (Table 7). Coolia monotis was only represented in a single subset that was recorded in low abundances and only on inner faces of Posidonia leaf (Fig. 5). The distribution showed no differences according to the face or part of the leaf (P_C.monotis>0.05) (Table 7).

DISCUSSIONTop

Posidonia seagrass beds, in contrast to macrophytes, which are generally rather scattered with a high inter-annual variability, cover large areas of the Gulf of Gabès and are structured in valleys (Mabrouk et al. 2009Mabrouk L., Hamza A., Sahraoui H., et al. 2009. Caractéristique et phénologie de l’herbier de Posidonia oceanica (L.) Delile sur les cotes de Mahdia (région est de la Tunisie). Bull. Inst. Nat. Sci. Tech. Océan. Pêche Salammbô. 36: 139-148., 2011Mabrouk L., Hamza A., Ben Brahim M., et al. 2011. Temporal and depth distribution of microepiphytes on Posidonia oceanica (L.) Delile leaves in a meadow off Tunisia. Mar. Ecol. 32: 148-161., Ben Brahim 2013Ben Brahim M., Hamza A., Ben Ismail S., et al. 2013. What factors drive seasonal variation of phytoplankton, protozoans and metazoans on leaves of Posidonia oceanica and in the water column along the coast of the Kerkennah Islands, Tunisia? Mar. Pollut. Bull. 71: 286-298.). During the year, Posidonia was by far the substrate hosting the greatest biomass and diversity of epiphytes (Table 3). This result could be explained by the diverse conditions that Posidonia offers for the success of epiphytic species: (i) the amount of physical structure usable as living space, as Posidonia provides both a shading effect and high microhabitat diversity because of its large leaf areas (Kikuchi and Pérès 1977Kikuchi T., Peres J.M. 1977. Consumer ecology of seagrass beds. In: McRoy C.P., Helfferich C. (ed.) Seagrass ecosystems: a scientific perspective. Marcel Dekker, New York. pp. 147-194.); (ii) coexistence of Posidonia seagrass material, dead or alive, suspended particulate organic matter and leaf epiphytes as potential food sources within the ecosystem (Dauby 1989Dauby P. 1989. The stable carbon isotope ratios in benthic food webs of the gulf of Calvi, Corsica. Contin. Shelf. Res. 9: 181-195.); (iii) protection from predators thanks to a dense rhizome mat; and (iv) the reduction of hydrodynamic forces (Lewis 1984Lewis F.G. 1984. Distribution of macrobenthic crustaceans associated with Thalassia, Halodule, and bare sand substrata. Mar. Ecol. Prog. Ser. 19: 101-113.). The P. oceanica canopy tends to mitigate currents and waves, thereby reducing the forces exerted on individual shoots (Koch et al. 2006Koch E.W., Ackerman J.D., Verduin J., et al. 2006. Fluid dynamics in seagrass ecology, In: Larkum A.W.D., Orth R.J., Duarte C.M. (eds), Seagrasses: Biology, Ecology and Conservation, Springer, Amsterdam, The Netherlands, pp. 193-225.).

Posidonia offers a greater surface for epiphytes than macroalgae such as Padina pavonica and Cystoseira mediterranea. However, the latter two host a relatively abundant population of epiphytes. Though they do not have the highest biomass of epiphytic species, marine macroalgae hosted the highest species diversity (Table 3), probably because they showed spatial complexity and could modulate the availability of resources, therefore affecting assemblages of associated epibiota (Gestoso et al. 2010Gestoso I., Olabarria C., Troncoso J.S. 2010. Variability of epifaunal assemblages associated with native and invasive macroalgae. Mar. Freshw. Res. 61: 724-731.). In particular, host algae with a branched structure like Cystoseira mediterranea or with a filamentous structure like Dictyota dichotoma usually have a high degree of structural complexity, which may make them more suitable as habitats for epibiota (Totti et al. 2009Totti C., Poulin M., Romagnoli T., et al. 2009. Epiphytic diatom communities on intertidal seaweeds from Iceland. Polar Biol. 32: 1681-1691.).

Diatoms were the dominant group and prevailed throughout the sampling period. This dominance could be attributed to their successful behaviour in attaching to the algae and establishing a mutualistic relationship with their host (Romagnoli et al. 2007Romagnoli T., Bavestrello G., Cucchiari E., et al. 2007. Microalgal communities epibiontic on the marine hydroid Eudendrium racemosum in the Ligurian Sea, during an annual cycle. Mar. Biol. 151: 537-552.). Indeed, pennate diatoms have the ability to cling to seaweeds by mucilage stalks and sheaths or gelatinous pads or by the attachment of the cell along its entire valve face. The centric forms are often trapped by the thallus of seaweeds or held in the tangle of attached forms (Totti et al. 2009Totti C., Poulin M., Romagnoli T., et al. 2009. Epiphytic diatom communities on intertidal seaweeds from Iceland. Polar Biol. 32: 1681-1691.).

A high diversity and abundance of confirmed toxic and potentially toxic dinoflagellate species hosted in vegetated habitats were recorded, especially on P. oceanica leaves (Table 3). Particularly P. lima, the most abundant species (Fig. 3), seems to affect P. oceanica leaves. This species has been reported as a widespread dinoflagellate in many coastal waters and estuaries around the world, generally in summer and autumn (Levasseur et al. 2003Levasseur M., Couture J.Y., Weise A.M., et al. 2003. Pelagic and epiphytic summer distributions of Prorocentrum lima and P. mexicanum at two mussel farms in the Gulf of St. Lawrence, Canada. Aquat. Microb. Ecol. 30: 283-293.), in the Fleet lagoon in the UK (Foden et al. 2005Foden J., Purdie D.A., Morris S., et al. 2005. Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK. Harmful Algae 4: 1063-1074.), in Greek coastal waters (Aligizaki et al. 2009Aligizaki K., Nikolaidis G., Katikou P., et al. 2009. Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters. Harmful Algae 8: 299-311.), along the coast and inside the harbours of the Abruzzo region in the Adriatic Sea (Ingarao et al. 2009Ingarao C., Lanciani G., Verri C., et al. 2009. First record of Prorocentrum lima (Dinophyceae) inside harbor areas and along the Abruzzo region coast, W Adriatic. Mar. Poll. Bull. 58: 596-600.), and on the northern coasts of Tunisia (Aissaoui et al. 2014Aissaoui A., Amri Z., Akrout F., et al. 2014. Environmental factors and seasonal dynamics of Prorocentrum lima population in coastal waters of the Gulf of Tunis, South Mediterranean. Water. Environ. Res. 86: 2256-2270.). In the study area, it reached about 25000 cells g^–1 FW on Posidonia leaves, which is higher than the 70 cells g^–1 FW found in the same area by Mabrouk et al. (2011)Mabrouk L., Hamza A., Ben Brahim M., et al. 2011. Temporal and depth distribution of microepiphytes on Posidonia oceanica (L.) Delile leaves in a meadow off Tunisia. Mar. Ecol. 32: 148-161.. However, these concentrations were lower than those reported for Cymodocea nodosa in Greece, where the abundance reached 133000 cells g^–1 FW (Aligizaki et al. 2009Aligizaki K., Nikolaidis G., Katikou P., et al. 2009. Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters. Harmful Algae 8: 299-311.). The high concentrations of P. lima on Posidonia raise the problem of its sampling representativeness, since most monitoring programmes focused on the water column, which might lead to an underestimation of the species abundance (Marr et al. 1992Marr J.C., Jackson A.E., McLachlan J.L. 1992. Occurrence of Prorocentrum lima, a DSP toxin-producing species from the Atlantic coast of Canada. J. Appl. Phycol. 4: 17-24.). P. lima concentrations showed a significant relationship between P. oceanica and the water column (R²=0.79), suggesting that the species, being a weekly swimming dinoflagellate that can even be affected by low water motion conditions (Richlen and Lobel 2011Richlen M.L., Lobel P.S. 2011. Effects of depth, habitat, and water motion on the abundance and distribution of ciguatera dinoflagellates at Johnston Atoll, Pacific Ocean. Mar. Ecol. Prog. Ser. 421: 51-66.), might move from one compartment to another. The establishment of a direct relationship between the concentration of this species in the water column and on macrophytes allows us to assess the concentration in one compartment by referring to the concentration in the other one. Moreover, to the best of our knowledge, the toxicity threshold used for this species in the monitoring programmes was only established for the water column (Abdennadher 2014Abdennadher M. 2014. Étude taxonomique et écophysiologique des dinoflagellés toxiques du Golfe de Gabès: Alexandrium minutum, Prorocentrum lima, Coolia spp. and Ostreopsis ovata. Ph.D. thesis. Univ. Science. Sfax, Tunisia.), so the use of this relationship to extrapolate to the substrata needs to be further investigated.

Ostreopsis cf. ovata had no preference for a given substratum, as indicated by the absence of a significant difference in concentrations between the substrata studied. The significant relationship found between the species on Padina and in the water column above this alga could be explained by the fact that this species is loosely attached to hard substrates and seaweeds with mucilaginous strands (Tindall and Morton 1998Tindall D.R., Morton S.L. 1998. Community dynamics and physiology of epiphytic/benthic dinoflagellates associated with ciguatera. In: Anderson D.M., Cembella A.D., Hallegraeff G.M. (eds), Physiological Ecology of Harmful Algal Blooms, NATO ASI Series G41. Springer-Verlag, Berlin. pp. 293-313.). Water motion could cause leaf agitation, allowing the shift of epiphytic species into the water column. A particularly low abundance, with a maximum of 1.85 10³ cells g^–1 FW recorded on Posidonia leaves, was observed during this survey compared with the high species abundances observed in the western Mediterranean, where 7.2 10⁶ cells g^–1 FW was reported in Catalonia (Mangialajo et al. 2011Mangialajo L., Ganzin N., Accoroni S., et al. 2011. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 57: 408-420.), 2.5 10⁶ cells g^–1 FW on the Genoa coasts (Mangialajo et al. 2008Mangialajo L., Bertolotto R., Cattaneo-Vietti R., et al. 2008. The toxic benthic dinoflagellate Ostreopsis ovata: quantification of proliferation along the coastline of Genoa, Italy. Mar. Poll. Bull. 56: 1209-1214.) and 1.7 10⁶ cells g^–1 FW in the Adriatic Sea (Totti et al. 2010Totti C., Accoroni S., Cerino F., et al. 2010. Ostreopsis ovata bloom along the Conero Riviera (northern Adriatic Sea): relationships with environmental conditions and substrata. Harmful Algae 9: 233-239.). In the eastern Mediterranean (Greece), a maximum abundance of 0.41 10⁶ cells g^–1 FW was observed (Aligizaki and Nikolaidis 2006Aligizaki K., Nikolaidis G. 2006. The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece. Harmful Algae 5: 717-730.). These findings suggest that the study area might have some constraints preventing the accumulation of this toxic species, known to cause serious health concerns in other ecosystems and particularly in the Mediterranean (Totti et al. 2010Totti C., Accoroni S., Cerino F., et al. 2010. Ostreopsis ovata bloom along the Conero Riviera (northern Adriatic Sea): relationships with environmental conditions and substrata. Harmful Algae 9: 233-239., Cohu et al. 2011Cohu S., Thibaut T., Mangialajo L., et al. 2011. Occurrences of the toxic dinoflagellate Ostreopsis cf. ovata in relation with environmental factors in Monaco (NW Mediterranean). Mar. Pollut. Bull. 62: 2681-2691.).

Leaves of the seagrass P. oceanica hosted the highest population, especially of P. lima, whereas Ostreopsis cf. ovata and other species were very scarce or planktonic (Fig. 3). This opposing pattern between Ostreopsis cf. ovata and P. lima was also illustrated in the divergence of these species in the cluster analysis (Fig. 4). A habitat separation between Ostreopsis spp. and Prorocentrum spp. has already been reported in the Pacific Ocean (Richlen and Lobel 2011Richlen M.L., Lobel P.S. 2011. Effects of depth, habitat, and water motion on the abundance and distribution of ciguatera dinoflagellates at Johnston Atoll, Pacific Ocean. Mar. Ecol. Prog. Ser. 421: 51-66.). This behaviour could be attributed to allelopathic effects between dinoflagellates leading to possible niche separation. Indeed, some phytoplankton species, including P. lima (Sugg and VanDolah 1999Sugg L.M., VanDolah F.M. 1999. No evidence for an allelopathic role of okadaic acid among ciguatera-associated dinoflagellates. J. Phycol. 35: 93-103.), produce and release secondary metabolites that negatively affect the growth of other organisms (Rizvi and Rizvi 1992Rizvi S.J.H., Rizvi V. 1992. Allelopathy: basic and applied aspect. Chapman & Hall, London, 480 pp.). These species quickly cause cell lyses of most competitors within minutes, when the latter are exposed to either certain amounts of the allelochemicals or to certain cell densities of the allelopathic algae. Such allelopathy is thought to reduce competition for nutrients, vitamins, etc. (Fistarol et al. 2004Fistarol G.O., Legrand C., Selander E., et al. 2004. Allelopathy in Alexandrium spp.: effect on a natural plankton community and on algal monocultures. Aquat. Microb. Ecol. 35: 45-56.). Indeed, the co-inertia plot showed that the distribution of Ostreopsis cf. ovata, and to a lesser degree P. lima, was explained by nitrogen, mainly nitrate and nitrite, which might suggest competition between these species for nitrogen availability. Both species were documented to be positively correlated with nutrient availability (nitrate, nitrite, phosphate, and silicate) concentrations in the waters surrounding Hawaii (Parsons and Preskitt 2007Parsons M.L., Preskitt L.B. 2007. A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawaii. Harmful Algae 6: 658-669.). Cohu et al. (2013)Cohu S., Mangialajo L., Thibaut T., et al. 2013. Proliferations of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in North Western Mediterranean Sea. Harmful Algae 24: 32-44. reported that phosphate concentration, rather than nitrogen or silicate concentration, was positively associated with Ostreopsis cf. ovata abundances in the north western Mediterranean Sea. Furthermore, many studies have shown that nutrient limitation decreases Ostreopsis cf. ovata growth, an effect that is more accentuated under N-limitation (Accoroni et al. 2014Accoroni S., Romagnoli T., Pichierri S., et al. 2014. New insights on the life cycle stages of the toxic benthic dinoflagellate Ostreopsis cf. ovata. Harmful Algae 34: 7-16.).

For P. oceanica, there is an increase in the cover of most epiphytic species in the apical and middle regions of the leaves (Fig. 5). This result had already been reported in previous studies (Alcoverro et al. 2004Alcoverro T., Perez M., Romero J. 2004. Importance of within-shoot epiphyte distribution for the carbon budget of seagrasses: the example of Posidonia oceanica. Bot. Mar. 47: 307-312.) and explained by the fact that the apical part of the leaves, and to a lesser degree the middle part, expose their epiphytes to high light intensities and water movement. This would promote photosynthetic organisms such as epiphytic macroalgae, which increase the nutrient intake from water and remove inhibitory substances (Trautman and Borowitzka 1999Trautman D.A., Borowitzka M.A. 1999. Distribution of the epiphytic organisms on Posidonia australis and P. sinuosa, two seagrasses with differing leaf morphology. Mar. Ecol. Prog. Ser. 179: 215-229). Moreover, the epiphytic species zonation on leaves of Posidonia was reported to be related to the concentration of phenolic compounds produced in abundant quantities, depending on the state of stress caused by environmental conditions (Dumay et al. 2004Dumay O., Costa J., Desjobert J.M., et al. 2004. Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition. Phytochemistry 65: 3211-3220.). However, the use of artificial leaves made of plastic tape showed the same apico-basal distribution of epiphytic algae (Trautman and Borowitzka 1999Trautman D.A., Borowitzka M.A. 1999. Distribution of the epiphytic organisms on Posidonia australis and P. sinuosa, two seagrasses with differing leaf morphology. Mar. Ecol. Prog. Ser. 179: 215-229), supporting the hypothesis that epiphyte settlement was unlikely to be the result of changes in the surface chemistry of the leaves (Borowitzka et al. 2006Borowitzka M.A., Lavery P., Keulen M. 2006. Epiphytes of seagrasses. In: Larkum A.W.D., Orth R.J., Duarte C.M. (eds), Seagrasses: Biology, Ecology and Conservation. Springer, Dordrecht, pp. 441-461.). These variations were likely due to differences in hydrodynamic or light intensity related to the shape and orientation of the leaves. The inner surface of adult and intermediate leaves seemed to be the most exposed (Borowitzka and Lethbridge 1989Borowitzka M.A., Lethbridge R.C. 1989. Seagrass epiphytes. In: Larkum A.W.D., McComb A.J., Shepherd S.A. (eds), Biology of Seagrasses. Elsevier, Amsterdam, pp. 304-345.).

The concentration of P. lima on the outer surface of the Posidonia leaf, explained by the behaviour it uses to escape predators (Ben Brahim et al. 2010Ben Brahim M., Hamza A., Hannachi I., et al. 2010. Variability in the structure of epiphytic assemblages of Posidonia oceanica in relation to human interferences in the Gulf of Gabes, Tunisia. Mar. Environ. Res. 70: 411-421.), is in opposition to the general behaviour of other epiphytic species, particularly Ostreopsis cf. ovata, which has been shown to prefer the inner face of Posidonia leaves (Alcoverro et al. 2004Alcoverro T., Perez M., Romero J. 2004. Importance of within-shoot epiphyte distribution for the carbon budget of seagrasses: the example of Posidonia oceanica. Bot. Mar. 47: 307-312., Peirano et al. 2011Peirano A., Cocito S., Banfi V., et al. 2011. Phenology of the Mediterranean seagrass Posidonia oceanica (L.) Delile: medium and long-term cycles and climate inferences. Aquat. Bot. 94: 77-92.). This would suggest competition for space between Ostreopsis cf. ovata and P. lima, and might support their apparently opposed distribution pattern.

CONCLUSIONSTop

This study has highlighted the diversity of epiphytic microorganisms on vegetated ecosystems, particularly on macroalgae, and has confirmed the previous finding on the potential of P. oceanica to accumulate epiphytic biomass. This finding suggests that more attention should be paid to the protection of the P. oceanica meadows and their associated epiphytes.

P. lima, by far the most abundant epiphytic toxic species on all vegetated substrates, showed a preference for P. oceanica. A significant correlation was found between the species concentration on that substrate and in the water column. More effort should be made to accurately determine this relationship under different hydrological conditions. One of the practical implications of this result is the recommendation to include the sampling of P. lima on Posidonia leaves in HAB monitoring programme and to set up the toxicity threshold of this species on P. oceanica leaves.

P. lima showed an opposed distribution pattern to that of Ostreopsis cf. ovata on Posidonia leaves, suggesting that competition for space and nutrient between the two species is likely. This hypothesis needs to be investigated in order to assess and apprehend the proliferation mechanisms of the two species.

ACKNOWLEDGEMENTSTop

We wish to thank Mr. Jamil JAOUA, founder and former head of the English Teaching Unit at the Sfax Faculty of Science, for proofreading our paper.

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