Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea

Harmful Algae 55 (2016) 179–190

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Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea Stefano Accoroni *, Tiziana Romagnoli, Salvatore Pichierri, Cecilia Totti Dipartimento di Scienze della Vita e dell’Ambiente, Universita` Politecnica delle Marche, via Brecce Bianche, Ancona 60131 Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 August 2015 Received in revised form 3 March 2016 Accepted 4 March 2016 Available online

Composition and temporal variation of the microphytobenthos communities of the Conero Riviera (northern Adriatic Sea) were investigated in the course of an annual cycle, focusing on their relationships with blooms of the benthic dinoflagellate Ostreopsis cf. ovata. Sampling was carried out from March 2009 to March 2010 on undisturbed benthic substrata (macroalgae and pebbles). Samples for the study of microphytobenthos were collected with a monthly frequency, while those for the study of Ostreopsis bloom weekly. Benthic diatoms dominated the microphytobenthos communities for most of the annual cycle (except the summer), both in terms of abundance and biomass. In summer, cyanobacteria were dominant (54.04  9.18 and 24.29  11.11% of total abundance and biomass, respectively), while benthic dinoflagellates were an important component of the community in terms of biomass only at the peak of the Ostreopsis bloom in late summer (up to 91% of the total biomass). Among diatoms, the most abundant forms throughout the year were motile species (77.5  3.71% of the population), while erect diatoms formed the majority of the biomass in winter and spring (48.66  16.66 and 48.05  5.56% of total population, respectively). Diatoms were mainly affected by DIN availability, while the patterns of biomass of O. cf. ovata and cyanobacteria were related to salinity and temperature. The biomass of Ostreopsis was also affected by the availability of phosphorus. The results of this study suggest that the proliferation of Ostreopsis affected the structure of the benthic diatom community: motile diatoms were significantly more abundant during the Ostreopsis bloom peak than during the rest of summer, probably because they benefited from the abundant mucilaginous mat covering the benthic substrata. In the course of the O. cf. ovata bloom the diversity of the microphytobenthos was significantly lower than during the rest of the year, suggesting an influence of both the shading produced by the mucous mat and allelopathic compounds possibly produced by O. cf. ovata. ß 2016 Elsevier B.V. All rights reserved.

Keywords: Adriatic Sea Microphytobenthos community Benthic dinoflagellates Ostreopsis cf. ovata Diatom growth forms Mucilaginous mat

1. Introduction Microphytobenthos communities consist of aquatic microalgae associated with benthic substrata, although these assemblages often include settled cells or colonies of phytoplanktonic species. All microalgal groups (mainly cyanobacteria, diatoms, dinoflagellates and cryptophytes) are represented in the microphytobenthos, but diatoms and cyanobacteria are typically the dominant groups (MacIntyre et al., 1996). Diatoms are usually the main component of microphytobenthos communities in temperate regions (Facca et al., 2002; Welker et al., 2002; Totti, 2003; Blasutto et al., 2005; Munda, 2005; Cibic et al., 2007; Totti et al., 2007a), while the

* Corresponding author. Tel.: +39 071 2204919; fax: +39 071 2204650. E-mail address: [email protected] (S. Accoroni). http://dx.doi.org/10.1016/j.hal.2016.03.003 1568-9883/ß 2016 Elsevier B.V. All rights reserved.

importance of benthic cyanobacteria generally increases at lower latitudes (MacIntyre et al., 1996; Ortega-Morales et al., 2005). Studies on the microphytobenthos of the northern Adriatic Sea have focused primarily on epipelon (i.e., microphytobenthos associated to soft bottoms) (Facca et al., 2002; Welker et al., 2002; Totti, 2003; Blasutto et al., 2005; Cibic et al., 2007), whereas the epilithon has been scarcely investigated (Munda, 2005; Totti et al., 2007a) and, to the best of our knowledge, there are no studies concerning the microepiphytic communities. Dinoflagellates are a major component of microphytobenthos communities particularly in tropical areas, where they are known for the production of toxins responsible for fish toxicity and human intoxications (Skinner et al., 2011; Chan, 2014). Recently, benthic dinoflagellate blooms have also become a common occurrence in temperate regions (Rhodes, 2011; Parsons et al., 2012), with significant negative impacts on human health and activities. In

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particular, in the last decade intense blooms of the benthic dinoflagellate Ostreopsis cf. ovata have regularly occurred in the Mediterranean Sea (Vila et al., 2001; Turki, 2005; Aligizaki and Nikolaidis, 2006; Mangialajo et al., 2011; Illoul et al., 2012; Ismael and Halim, 2012; Pfannkuchen et al., 2012), as well as in other temperate regions (Chang et al., 2000; Rhodes et al., 2000; Pearce et al., 2001; Taniyama et al., 2003; Shears and Ross, 2009; Selina et al., 2014). In these regions, O. cf. ovata produces palytoxin-like compounds, i.e., isobaric palytoxin and ovatoxin-a, b, c, d, e, f, g and h and mascarenotoxin-a and c (Scalco et al., 2012; Uchida et al., 2013; Garcı´a-Altares et al., 2014; Brissard et al., 2015) that cause negative effects on human health (Gallitelli et al., 2005; Kermarec et al., 2008; Tichadou et al., 2010; Del Favero et al., 2012) and benthic marine organisms (Gorbi et al., 2012, 2013; Pagliara and Caroppo, 2012; Carella et al., 2015). Numerous studies have assessed the relationships between bloom dynamics and environmental parameters, mainly hydrodynamics, water temperature and nutrients (e.g., Vila et al., 2001; Shears and Ross, 2009; Totti et al., 2010; Mabrouk et al., 2011; Mangialajo et al., 2011; Accoroni et al., 2012b, 2015a; Cohu et al., 2013; Selina et al., 2014), whereas the interactions between Ostreopsis cf. ovata and the other components of microphytobenthos communities are almost unknown. Several studies highlighted the capacity of many dinoflagellates to produce allelochemicals affecting other co-occurring microalgae under unfavorable environmental conditions (Tillmann and John, 2002; Fistarol et al., 2003; Grane´li and Johansson, 2003; Fistarol et al., 2004; Suikkanen et al., 2004; Grane´li and Hansen, 2006; Prince et al., 2008; Hakanen et al., 2014). Monti and Cecchin (2012) documented a weak allelopathic effect of O. cf. ovata in culture on other benthic dinoflagellates that often co-occur with this alga in the field (i.e., Prorocentrum minimum and Coolia monotis), but further studies are required to assess similar interactions with the other species of the microphytobenthos community. The aim of this study was to investigate the abundance, biomass and community structure of epilithic and epiphytic microphytobenthos communities in relation to environmental parameters and to the 2009 bloom of Ostreopsis cf. ovata in the Conero Riviera (northern Adriatic Sea), an area where Ostreopsis blooms are among the largest in the Mediterranean basin (Mangialajo et al., 2011). 2. Materials and methods 2.1. Sampling and sample treatment The study was carried out in Portonovo, Conero Riviera (northern Adriatic Sea: 438330 4100 N 138360 0600 E, Fig. 1), a coastal site characterized by a shallow rocky bottom and directly exposed to wave action. This bay is a popular site for summer holidays and therefore is moderately affected by human impact during the summer season. Sampling was carried out from March to November 2009, and subsequently in March 2010. Two sets of samples were collected for different purposes. Samples for a quantitative and qualitative analysis of the microphytobenthos communities were collected with monthly frequency. Samples for a study of the Ostreopsis bloom were collected fortnightly until the appearance of Ostreopsis cells and weekly throughout the period of the bloom. Macroalgae, i.e., Ulva rigida (Ulvophyceae) and Dictyota dichotoma (Phaeophyceae) and benthic hard substrata (pebbles) were collected in three replicates at approximately 0.5 m depth. Undisturbed samples were collected following the protocol recommended by Totti et al. (2010). Surface temperature and salinity were measured with a CTD, Model 30 Handheld Salinity, Conductivity and Temperature System, YSI (Yellow Spring, OH USA) and wave height was recorded according to the Douglas scale.

Fig. 1. Map of the study area showing the location of the sampling station (Portonovo) in the Conero Riviera (N Adriatic Sea).

Water samples for nutrient analysis were collected in polyethylene bottles (50 ml) near the sampled substrata, avoiding resuspension phenomena, immediately filtered using GF/F Whatman filters (25 mm) and stored in triplicates in 4 ml polyethylene bottles at 22 8C until the analysis. Macroalgae and pebbles were treated following the method of Totti et al. (2010), modifying the detachment procedure (in seaweed samples), in order to remove all benthic diatoms, including forms strongly attached to the substrata (i.e., Cocconeis), as follows: plastic bottles with seaweed thalli and their storage water were immersed in an ultrasonic bath to detach epiphytic microalgae: samples were sonicated for 10 min several times (at least 6), with 5 min intervals between successive treatments in order to avoid excessive heating. Then the thalli were rinsed with filtered seawater until epiphytic cells were completely removed. The seaweed thalli were weighed to determine fresh (g fw) and dry (g dw) weight and their surface areas were calculated following the procedure described in Accoroni et al. (2011), i.e., using a conversion factor obtained from the ratio of ‘fresh weight’/‘dry weight’/‘surface’ utilized to estimate the thallus area measuring the wet or dry weight. Pebbles were scraped with a blade and rinsed with filtered seawater. Then the area of the scraped surface was measured using a measuring tape. Finally, the water samples were fixed with 0.8% neutralized formaldehyde (Throndsen, 1978) and stored at 4 8C in darkness until the analyses. 2.2. Nutrient analysis The analyses of N–NO3, N–NO2, N–NH4, and P–PO4 and Si– Si(OH)4 were performed following the colorimetric method by Strickland and Parsons (1972), using an Autoanalyzer QuAAtro Axflow. Detection limits were 0.02 mmol l1 for N–NO3, N–NO2, N– NH4 and Si–Si(OH)4 and 0.03 mmol l1 for P–PO34 . 2.3. Microscope analysis Samples were analyzed with an inverted microscope (Zeiss Axiovert 135) equipped with phase contrast, at 400 and 200 magnification for identification and counting of benthic microalgae.

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Sub-samples (5–25 ml) were settled in counting chambers after homogenization, according to the Utermo¨hl’s sedimentation method (Edler and Elbra¨chter, 2010). Counting was performed in different ways. For Ostreopsis cf. ovata it was performed at 200 on a known area (10–30 random fields, 1–2 transects or the whole sedimentation chamber), in order to count a representative cell number. The microphytobenthos community was examined at 400 magnification on 30 random fields; then a counting at 200 on the whole sedimentation chamber was performed in order to obtain a correct evaluation of uncommon taxa. During the counting, cells were measured to calculate cell biovolumes, which allowed to estimate biomass following Menden-Deuer and Lessard (2000). Abundance and biomass values were expressed as cells cm2 and mg C cm2 of substrata for both macroalgae and pebbles. Microalgae taxa were identified following Hasle and Syvertsen (1997), Hustedt (1930, 1985), Peragallo and Peragallo (1897– 1908), Steidinger and Tangen (1997) and Van Heurck (1880–1885). The identification of Ostreopsis cf. ovata was confirmed by molecular analysis (Accoroni et al., 2011; Perini et al., 2011). Epiphytic and epilithic diatoms were subdivided into the growth forms identified by Round (1981): erect (i.e., species attached to the substratum by mucus pads or peduncles), adnate (species lying on the substratum through the valve face, with limited motility), motile (biraphid diatoms capable to move freely on the substratum), tube-dwelling (i.e., naviculoid and nitzschioid diatoms living in mucilage tubes) and planktonic (i.e., true plankton species that settle on the substratum remaining healthy). 2.4. Scanning electron microscope analysis For the observation of the microphytobenthos community, some preserved fragments of macroalgal thalli were dehydrated by immersion in ethanol at increasing gradations (10, 30, 50, 70, 80, 90, 95 and 100%), and treated in a Critical Point Dryer (Polaron CPD 7501). Then, each fragment was placed on a biadhesive tape mounted on a stub. The identification of diatoms at the lowest taxonomical level possible has been made through SEM analysis, after removal of organic matter: subsamples were cleaned with sulphuric and nitric acids following the von Stosch method (Hasle and Syvertsen,

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1997). Then, one or more drops of cleaned material were poured on Nucleopore polycarbonate filters fixed on stubs and air-dried completely. The stubs were sputtered with gold-palladium in a Sputter Coater (Polaron CPD7501) for observation under a Philips XL20 scanning electron microscope. 2.5. Statistical analysis Differences in (i) absolute and percent abundance and biomass of each major group of microphytobenthos (Bacillariophyceae, Dinophyceae and Cyanophyceae) in the whole study period and in each season, (ii) absolute and percent abundance and biomass of diatom growth forms (adnate, erect, motile, tube-dwelling and planktonic) in the whole the study period and in each season (including Ostreopsis bloom period) and (iii) Shannon diversity index (H0 ) among seasons (including Ostreopsis bloom period) and substrata were assessed using one-way analyses of variance (ANOVAs). When significant differences for the main effect were detected (p < 0.05), a post-hoc Tukey’s pairwise test was also performed. Pearson correlation matrices with environmental parameters, i.e., water temperature, salinity and nutrient concentrations, were built up and significant correlations (p < 0.05) were extracted. Moreover, all environmental parameters were tested for significant correlations (Pearson) with abundances of each microphytobenthos group, diatom growth forms and main taxa. Principal Component Analysis (PCA) based on r algorithm (correlation coefficient) was performed with a matrix of variables containing environmental parameters and biomass values of Ostreopsis, cyanobacteria and diatom growth forms. The statistical analyses were performed using the Statistica 10.0 (StatSoft Inc., Tulsa, OK, USA) software. 3. Results 3.1. Environmental parameters In the course of the study, surface temperature ranged from a minimum value of 10.4 8C in March to a maximum of 27.3 8C in August (Table 1). The temperature decreased from August until late October, when it became lower than 16 8C and remained around this value until the end of the study period.

Table 1 Temperature (8C), salinity, nutrient concentrations (mmol l1) and their molar ratios in water column. Sampling

Temperature

Salinity

N–NH4

N–NO2

N–NO3

DIN

P–PO4

Si–Si(OH)4

N/P

Si/N

25/03/2009 07/04/2009 07/05/2009 18/05/2009 03/06/2009 15/06/2009 03/07/2009 17/07/2009 31/07/2009a 06/08/2009a 11/08/2009a 21/08/2009a 27/08/2009a 03/09/2009a 09/09/2009a 15/09/2009a 23/09/2009a 29/09/2009a 06/10/2009a 21/10/2009a 28/10/2009a 11/19/2009

10.4 13.9 17.2 20.9 18.7 22.4 24.3 25.9 27.3 25.9 26.2 27.3 26.6 26.4 24.9 22.6 23 22.8 21.8 15.2 13.7 15

32.5 29.7 29.1 30.2 30.4 32.6 33.2 33.4 33.8 33 33.2 33.2 35.1 36 34.2 35 35.6 34.7 32.9 34.6 33.4 35.3

7.861 3.289 9.482 6.822 2.67 1.437 7.091 1.823 3.72 1.308 3.463 1.297 2.187 1.645 6.951 0.638 0.811 2.185 2.098 2.673 0.744 n.d.

0.804 0.535 0.520 0.355 0.157 0.020 0.124 0.024 0.097 0.015 0.072 0.169 0.020 0.031 0.234 0.064 0.215 0.020 0.020 0.530 1.085 n.d.

19.037 16.469 17.629 14.03 9.609 1.955 3.232 0.969 2.151 1.406 1.496 1.219 1.373 1.636 3.395 1.689 1.874 1.401 1.985 4.049 15.509 n.d.

27.702 20.293 27.631 21.207 12.436 3.412 10.447 2.816 5.968 2.729 5.031 2.685 3.580 3.312 10.580 2.391 2.900 3.606 4.103 7.252 17.338 n.d.

0.020 0.066 0.215 0.056 0.020 0.020 0.225 0.106 0.111 0.152 0.324 0.250 0.190 0.150 0.139 0.188 0.135 0.018 0.008 0.164 0.176 n.d.

7.738 43.044 17.283 88.996 12.867 4.471 20.171 13.775 5.146 10.556 24.658 8.597 62.427 21.454 88.628 34.433 17.935 10.358 20.766 127.494 16.737 n.d.

1385 307 129 379 622 171 46 27 54 18 16 11 19 22 76 13 21 200 513 44 99 n.d.

0.28 2.12 0.63 4.20 1.03 1.31 1.93 4.89 0.86 3.87 4.90 3.20 17.44 6.48 8.38 14.40 6.18 2.87 5.06 17.58 0.97 n.d.

a

Days when Ostreopsis cf. ovata has been detected.

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Salinity ranged from 29.1 to 35.6 (Table 1), without large variation throughout the annual cycle, with the highest values in late summer-autumn and the lowest values in spring. Dissolved inorganic nitrogen (DIN), obtained as the sum of nitrate (NO3), nitrite (NO2) and ammonium (NH4), ranged from 2.391 to 27.702 mmol l1 (Table 1). The concentration of DIN was highest in early spring (27.702–27.631 mmol l1) and began to decrease at the end of May, with values ranging between 2.391 and 17.338 mmol l1 during the Ostreopsis cf. ovata bloom. In the course of the study NO3 ranged from 0.969 to 19.037 mmol l1 and was the main nitrogen source from March to June, although NH4 (0.638–9.482 mmol l1) represented an important additional source, particularly from June to mid-October. The overall contribution of NO2 as nitrogen source was minor. PO4 ranged from 0.008 to 0.324 mmol l1, without a clear seasonal trend and with the highest values (>0.200 mmol l1) in early May, early July and mid-August. A N:P ratio around Redfield values (16) was recorded only in August and mid-September, in correspondence with the start of the bloom and a strong increase of the abundance of Ostreopsis cf. ovata, respectively. The values of Si(OH)4 oscillated between 4.471 and 127.494 mmol l1, and did not show a seasonal trend. Salinity was positively correlated with temperature (r = 0.528, n = 35, p < 0.01), and negatively correlated with DIN (r = 0.862, n = 35, p < 0.001) and Si(OH)4 (r = 0.418, n = 35, p < 0.05). No correlation between salinity and PO4 was observed. 3.2. Microphytobenthos community A diverse microphytobenthos community was observed on all benthic substrata during the study period (Fig. 2). The taxa recorded are listed in Table 2.

The microphytobenthos was dominated for most of the year by diatoms, which showed significantly higher percent abundances (p < 0.001) and percent biomass (p < 0.001, Table 3) than cyanobacteria and dinoflagellates. Abundance and biomass of diatoms ranged from 2  103 to 3.6  106 cells cm2 and 0.14 to 38.86 mg C cm2 respectively, with the highest values recorded in spring (Figs. 3 and 4). Cyanobacteria ranged from 0 to 1.7  106 cells cm2 and 7.04 mg C cm2 in abundance and biomass respectively, and represented the main group in summer (Figs. 3 and 4). This group consisted mainly of filamentous non-heterocytic forms, tentatively attributed to Oscillatoriales, and an unidentified species of Spirulina. Dinoflagellates were the least represented group in the microphytobenthos community, showing significantly lower percent abundances than both diatoms and cyanobacteria (p < 0.001, Table 3). Their abundance and biomass ranged from 0 to 15.7  103 cells cm2 and 36.67 mg C cm2, respectively; in terms of biomass dinoflagellates were the dominant group during the bloom of Ostreopsis cf. ovata (see below). The community structure was characterized by a marked seasonal variation (Fig. 3B and D): in winter diatoms showed higher percent abundance and biomass than in other seasons, whereas in summer their percent abundance and biomass were significantly lower than in winter (Table 4). Conversely, cyanobacteria were not recorded until May but prevailed in summer, when they showed higher (although not significantly) percent abundance and biomass values than in the rest of the year (Table 4). Benthic dinoflagellates reached their annual maximum during the Ostreopsis cf. ovata bloom peak in September (26.1  103 cells cm2 and 57.59 mg C cm2 for abundance and biomass, respectively, Figs. 3 and 4). Even at this stage, however, the abundance of dinoflagellates did not exceed the 7% of the entire microphytobenthos community (Fig. 3B and D), while the

Fig. 2. Epiphytic community on natural samples of macrophytes. (A) Epiphytic Ostreopsis cf. ovata on a seaweed thallus: it is evident that sediment and mucilaginous mat covers the surface. (B) Licmophora abbreviata attached with mucilaginous stalk on a macroalga. Several pennate diatoms and fungal hyphae are visible. (C) Several Achnanthes spp. attached with mucilaginous stalk arising from the spines of the seaweed thallus. (D) Two cells of Striatella unipunctata attached to the substrata with mucilaginous stalk. Fungal hyphae are visible on the macroalgal surface. (E) A dense covering of Cocconeis spp. on the macroalgal surface. (F) Epiphytic Proschkinia sp. on a seaweed thallus. Scale bars = 50 mm (A, B, C and E); 20 mm (D); 10 mm (F).

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Table 2 List of microalgal taxa recorded on benthic substrata during the study period. Bacillariophyceae Achnanthes brevipes Agardh Achnanthes longipes Agardh Amphora spp. Asterionellopsis glacialis (Castracane) Round Bacillaria paxillifera (O.F. Mu¨ller) Hendey Caloneis cf. alpestris (Grunow) Cleve Campylodiscus sp. Cerataulina pelagica (Cleve) Hendey Chaetoceros danicus Cleve Chaetoceros lorenzianus Grunow Chaetoceros tenuissimus Meunier Chaetoceros spp. Cocconeis scutellum Ehrenberg Cocconeis spp. Cyclotella spp. Cylindrotheca closterium (Ehrenberg) Lewin and Reimann Cymbella spp. Dactyliosolen fragilissimus (Bergon) Hasle Diploneis cf. schmidti Cleve Diploneis spp. Entomoneis ornata (J.W. Bailey) Reimer Entomoneis spp. Fragilaria spp. Grammatophora marina (Lyngbye) Ku¨tzing Guinardia flaccida (Castracane) H. Peragallo Guinardia striata (Stolterfoth) Hasle Licmophora cf. abbreviata Agardh Licmophora cf. flabellata (Greville) C. Agardh Licmophora spp. Melosira spp. Navicula spp. Nitzschia cf. sigma (Ku¨tzing) W. Smith Nitzschia longissima (Bre´bisson in Ku¨tzing) Ralfs in Pritchard Nitzschia spp. Plagiotropis spp. Pleurosigma spp. Psammodictyon spp. Rhabdonema adriaticum Kutzing

contribution to the total biomass was very high (up to 91%, Fig. 4B and D), to the point that in this period benthic dinoflagellates had significantly higher absolute and percent biomass (24.24  20.05 mg C cm2 and 89.45  1.37%) than both diatoms (3.04  2.63 mg C cm2 and 10.15  1.14%, p < 0.01) and cyanobacteria (0.17  0.16 mg C cm2 and 0.41  0.24%, p < 0.001). Statistical analyses did not reveal any correlation between composition and abundance of the whole microphytobenthos community and temperature, salinity and nutrient concentrations. A significant positive correlation was only observed between diatom abundances and DIN (r = 0.574, n = 35, p < 0.001), and a significant negative correlation was found between diatom abundances and salinity (r = 0.675, n = 35, p < 0.001). No significant differences were found either in terms of abundance or biomass of the total microphytobenthos community between pebbles (186,634  35,691 cells cm2 and 11.93  4.38 mg C cm2,

Rhoicosphenia spp. Skeletonema marinoi Sarno and Zingone Striatella unipunctata (Lyngbye) C.A. Agardh Synedra spp. Tabularia spp. Thalassionema frauenfeldii (Grunow) Hallegraeff Thalassionema nitzschioides (Grunow) Mereschkowsky Thalassiosira spp. Tryblionella spp. Und. tube-dwelling diatoms Und. centric diatoms Und. pennate diatoms Dinophyceae Alexandrium spp. Amphidinium cf. carterae Hulburt Coolia monotis Meunier Diplopsalis lenticula Bergh Ostreopsis cf. ovata Fukuyo Prorocentrum lima (Ehrenberg) Dodge Prorocentrum micans Ehrenberg Prorocentrum minimum (Pavillard) Schiller Protoperidinium longispinum Kofoid Und. naked dinoflagellates Und. thecate dinoflagellates Cryptophyceae Und. Cryptophyceae Prasinophyceae Und. Prasinophyceae Others flagellates Und. phytoflagellates <10 mm Cyanophyceae Spirulina sp. Und. Oscillatoriales

respectively) and macroalgae (348,286  118,555 cells cm2 and 6.66  2.15 mg C cm2, respectively). The Shannon diversity index varied in relation to seasonality, with maxima in spring and autumn (Fig. 5) even if, considering the average values between all substrata, no significant differences among seasons were detected (1.24  0.22, 1.26  0.15, 1.09  0.13 and 1.40  0.20 in winter, spring, summer and autumn, respectively). The Shannon diversity index was significantly higher for pebbles (1.38  0.12) than for macroalgae (1.06  0.10, p < 0.05). The results of the PCA are shown in Fig. 6. The percentages of explained variance of the first two components (PCA1 and PCA2) were 34.47 and 19.60%, respectively. The first component (PCA1) shows the DIN gradient, which is opposite to that of salinity and temperature, highlighting that high diatom biomass values were associated with high DIN availability. Cyanobacterial biomass was related to high temperature values. The second axis (PCA2) shows

Table 3 Results of ANOVA and Tukey’s tests about both absolute and percent abundance and biomass of benthic diatoms, cyanobacteria and dinoflagellates in the study period.

Abundance cells cmS2 %

Diatoms

Cyanobacteria

Dinoflagellates

Avg  SE

Avg  SE

Avg  SE

96911  20740 67.18  6.69

118308  60309 32.26  6.69

571  523 0.56  0.36

p-Level

Tukey test

***

Diato > dino, cyano Cyano > dino

***

Biomass mg C cmS2 %

6.34  1.52 76.69  6.74

Mean values (Avg)  standard error (SE). Diato = diatoms; cyano = cyanobacteria; dino = dinoflagellates. * p < 0.05. *** p < 0.001.

0.56  0.26 11.58  4.58

2.26  2.01 11.73  5.81

* ***

Diato > cyano Diato > dino, cyano

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Fig. 3. Trend in abundance and percent abundance of microphytobenthos community. Abundance (cells cm2, A, C) of benthic diatoms (^, dotted lines) (left y-axis) cyanobacteria (~, dotted lines) (left y-axis) and dinoflagellates (&, bold lines) (right y-axis), on seaweeds (A) and pebbles (C). Error bars indicate standard deviation. Percent abundances of benthic diatoms (grey), dinoflagellates (black) and cyanobacteria (crossed), on seaweeds (B) and pebbles (D). The trend of benthic dinoflagellates coincided with that of Ostreopsis cf. ovata representing in average the 96.2% of the dinoflagellate abundance).

Fig. 4. Trend in biomass and percent biomass of microphytobenthos community. Biomass (mg C cm2, A, C) of benthic diatoms (^, dotted lines) (left y-axis) cyanobacteria (~, dotted lines) (left y-axis) and dinoflagellates (&, bold lines) (right y-axis), on seaweeds (A) and pebbles (C). Error bars indicate standard deviation. Percent biomass of benthic diatoms (grey), dinoflagellates (black) and cyanobacteria (crossed), on seaweeds (B) and pebbles (D). Benthic dinoflagellates were represented mainly by Ostreopsis cf. ovata (on average 98.0% of the dinoflagellate biomass).

Table 4 Results of ANOVA and Tukey’s tests about percent abundance and biomass of benthic diatoms, cyanobacteria and dinoflagellates in each season.

% Abundance

% Biomass

Winter

Spring

Summer

Autumn

Microalga group

Avg  SE

Avg  SE

Avg  SE

Avg  SE

p-Level

Tukey test

Diatoms Dinoflagellates Cyanobacteria Diatoms

89.65  10.35 0.00  0.00 10.35  10.35 98.42  1.49

80.83  14.63 0.00  0.00 19.16  14.63 94.45  4.89

44.43  8.58 1.53  0.91 54.04  9.18 46.49  12.14

69.71  11.27 0.02  0.02 30.27  11.27 88.73  5.19

*

Summer < winter

**

Summer < winter, spring Summer < autumn

*

Dinoflagellates Cyanobacteria

0.10  0.06 1.48  1.48

0.63  0.31 4.91  4.59

29.22  14.29 24.29  11.11

5.03  4.39 6.24  2.13

Mean values (Avg)  standard error (SE). * p < 0.05. ** p < 0.01.

the phosphorus gradient and highlights that not only diatoms but also Ostreopsis, was positively related to P availability. 3.3. Benthic diatoms Among diatoms, the most represented taxon was unidentified pennate species (66.0%, mainly belonging to the size fraction

<20 mm), followed by species of Licmophora (8.6%), Navicula (6.6%) and Amphora (3.1%). In terms of biomass Licmophora was the dominant taxon (48.6%), followed by unidentified pennate diatoms (19.1%), Navicula (6.8%), Grammatophora (3.5%) and Cocconeis spp. (3.0%). In terms of growth forms (adnate, erect, motile, tube-dwelling and planktonic), motile forms (mainly naviculoid diatoms)

S. Accoroni et al. / Harmful Algae 55 (2016) 179–190

Fig. 5. Temporal trend of Shannon diversity index (H0 ) of microphytobenthos community analyzed on macroalgae (bold line) and pebbles (dotted line). Vertical dotted bars indicate the period of Ostreopsis cf. ovata bloom.

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was detected (Table 5). Adnate (mainly Amphora spp. and Cocconeis spp.), tube-dwelling and planktonic diatoms occurred with substantially lower abundance and biomass values than motile and erect forms (Table 5). All growth forms exhibited the highest values in spring. Motile, erect and adnate diatoms increased slightly again in autumn (Fig. 7). The contribution of the planktonic species to diatom abundance and biomass was significantly higher in winter than in the rest of year (Table 6), due to an intense winter bloom of Skeletonema marinoi (data not shown), whose cells settled on benthic substrata. Motile diatoms were the most abundant throughout the annual cycle, while in terms of biomass these forms were dominant only in summer and autumn. The erect forms were the main group in winter and spring (Table 6). A significant negative correlation was observed between the abundances of Synedra and water temperature (r = 0.479, n = 22, p < 0.05), while a significant positive correlation was found between DIN values and Pleurosigma (r = 0.492, n = 18, p < 0.05) and Synedra (r = 0.914, n = 18, p < 0.001); the latter genus showed also a significant negative correlation with PO4 (r = 0.480, n = 18, p < 0.05). 3.4. Ostreopsis cf. ovata and other benthic dinoflagellates

Fig. 6. Principal component analysis (PCA), based on r algorithm of environmental parameters (T = temperature, S = salinity, DIN = dissolved inorganic nitrogen, P = phosphorous, Si = silica) and biomass values of (Ostr) Ostreopsis, (Cyano) cyanobacteria and diatom growth forms: Ere = erect, Adn = adnate, Mot = motile, Tub = tube-dwelling and Pla = planktonic.

represented the most abundant fraction, showing significantly higher values than other growth forms (p < 0.001, Table 5). Erect forms (mainly Licmophora cf. abbreviata followed by Tabularia spp. and Synedra spp.) showed significantly higher absolute and percent biomass than adnate, tube-dwelling and planktonic diatoms (p < 0.001), while no significant difference from the motile forms

Ostreopsis cf. ovata represented the main taxon among the benthic dinoflagellates, accounting on average for the 96.2% of the total abundance and 98.0% of the biomass of this group, followed by unidentified naked dinoflagellates. Differently from O. cf. ovata, which bloomed in late summer-early autumn, no clear seasonal pattern was observed for the other benthic dinoflagellates recorded in the microphytobenthos community. Cells of O. cf. ovata were first recorded on 31 July 2009, when it occurred with low abundance (Fig. 8). The peak of the bloom took place in late summer (end of September/early October) and coincided with temperature values around 23 8C, while the bloom declined in late October/early November in correspondence with prolonged conditions of stormy sea. The bloom of O. cf. ovata on benthic substrata showed a similar temporal pattern, with higher values on pebbles (2467  788 cells cm2) than on macroalgae (540  144 cells cm2, p < 0.01). The highest abundances were recorded on 23 September 2009, with 26.1  103 and 8.7  103 cells cm2 on rocks and macroalgae, respectively (Fig. 8). During the peak of the bloom, the characteristic brownish spotty network-shaped mat of O. cf. ovata covered all the benthic substrata. Among benthic dinoflagellates, other species of potentially toxic microalgae were recorded during the study: Prorocentrum lima and Amphidinum cf. carterae were found on all substrata with mean abundances two orders of magnitude lower than those of O. cf. ovata (3  1 and 1  1 cells cm2, respectively). Coolia monotis was also

Table 5 Results of ANOVA and Tukey’s tests about both absolute and percent abundance and biomass of diatom growth forms (adnate, erect, motile, tube-dwelling and planktonic) in the all study period.

Abundance cells cmS2 %

Adnate

Erect

Motile

Tube-dwelling

Planktonic

Avg  SE

Avg  SE

Avg  SE

Avg  SE

Avg  SE

p-Level

Tukey test

4287  1785 4.77  0.90

11,568  3765 10.46  2.49

74,507  17,159 77.55  3.71

1978  1655 0.72  0.44

4571  2561 6.50  3.07

***

Motile > adnate, erect, tube-dwelling, planktonic Motile > adnate, erect, tube-dwelling, planktonic Erect > tube-dwelling

*** *

Biomass mg C cmS2 %

0.36  0.10 9.80  2.26

3.78  1.19 38.99  3.87

2.03  0.48 45.37  3.79

0.04  0.03 0.26  0.11

0.13  0.08 5.58  3.79

*** *** ***

Mean values (Avg)  standard error (SE). * p < 0.05. ** p < 0.01. *** p < 0.001.

Erect > adnate, tube-dwelling, planktonic Erect > adnate, tube-dwelling, planktonic Motile > adnate, tube-dwelling, planktonic

S. Accoroni et al. / Harmful Algae 55 (2016) 179–190

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Fig. 7. Trend in abundance (cells cm2, A, C) and biomass (mg C cm2, B, D) of benthic diatom growth forms (adnate, erect, motile, tube-dwelling and planktonic diatoms) on seaweeds (A, B) and pebbles (C, D).

Table 6 Results of ANOVA and Tukey’s tests about percent abundance and biomass of diatom growth forms (adnate, erect, motile, tube-dwelling and planktonic) in each season.

Season % Abundance

Winter

Adnate

Erect

Motile

Tube-dwelling

Planktonic

Avg  SE

Avg  SE

Avg  SE

Avg  SE

Avg  SE

p-Level

Tukey test

24.53  13.18

42.56  14.51

0.13  0.08

28.69  17.24

***

Motile > adnate, tube-dwelling PLanktonic > tube-dwelling Motile > adnate, erect, tube-dwelling, planktonic Motile > adnate, erect, tube-dwelling, planktonic Motile > adnate, erect, tube-dwelling, planktonic

4.10  1.61

**

% Biomass

Spring Summer Autumn

5.41  1.92 2.18  0.49 9.60  3.01

7.96  2.10 8.70  2.45 5.41  1.91

83.24  4.29 86.02  3.42 82.70  2.72

1.88  1.53 0.26  0.19 0.39  0.27

1.51  0.75 2.82  1.16 1.90  0.93

Winter

7.87  3.99

40.66  16.66

22.26  10.42

0.18  0.15

29.03  16.41

*** *** *** * ***

Spring

8.98  2.20

48.05  5.56

42.02  5.87

0.52  0.36

0.44  0.17

Summer

3.32  0.63

36.54  5.46

58.97  5.46

0.08  0.04

1.06  0.32

*** *** *** *** *

Autumn

25.68  9.52

28.82  6.36

43.42  7.08

0.28  0.19

1.79  0.85

***

Erect > adnate Erect > tube-dwelling Erect > adnate, tube-dwelling, planktonic Motile > adnate, tube-dwelling, planktonic Erect > adnate, tube-dwelling, planktonic Motile > adnate, tube-dwelling, planktonic Motile > erect Motile > tube-dwelling, planktonic

Mean values (Avg)  standard error (SE). * p < 0.05. ** p < 0.01. *** p < 0.001.

recorded (2  2 cells cm2), although this species was recently removed from the list of harmful algae (www.marinespecies.org/ hab/index.php). A significant positive correlation was observed between Prorocentrum lima abundances and temperature (r = 0.470, n = 22, p < 0.05). 3.5. Microphytobenthos community structure during the Ostreopsis cf. ovata bloom Throughout the period of occurrence of Ostreopsis cf. ovata (31 July 2009 to 28 October 2009), diatoms were significantly less abundant than in the rest of the year, both in terms of absolute (39  103  14  103 and 130  103  28  103 cells cm2, respectively, p < 0.05) and percent values (47.73  9.16 and 78.29  7.85%, respectively, p < 0.05). Diatom biomass was significantly lower when O. cf. ovata was present than in the rest of the year (2.14  0.94 and 8.73  2.10 mg C cm2 respectively, p < 0.05), as well as in terms of the percent values (47.81  12.53 and 93.20  3.04%, respectively, p < 0.001). The percent abundances of cyanobacteria were significantly higher at this time (50.73  9.65%) than in rest of the year (21.71  7.85%, p < 0.05); the same pattern was found for the

dinoflagellates (1.54  0.91% and 0.0028  0.0008%, respectively, p < 0.05). Among the diatom growth forms, in summertime motile diatoms showed higher percent abundances at the time of the bloom peak on both benthic substrata (91.22  2.82% and 98.34  0.89% respectively for rocks and macroalgae) than in the rest of the summer (73.24  6.17% and 92.95  0.85%, respectively for rocks and macroalgae), although this difference was significant only on macroalgae (p < 0.01). The Shannon diversity index of the microphytobenthos community was significantly lower when Ostreopsis cf. ovata was recorded (0.99  0.13) than in the rest of the year (1.35  0.08, p < 0.05). 4. Discussion The relationships of Ostreopsis cf. ovata blooms with temperature, nutrients and hydrodynamics in the Conero Riviera have been widely discussed in previous papers (Totti et al., 2010; Accoroni et al., 2011, 2012a,b, 2015a). While the onset of the bloom occurs at temperature values higher than 25 8C related to cyst germination (Accoroni et al., 2014), the peaks typically occur in late summer, in correspondence with decreasing temperatures values. Significantly

S. Accoroni et al. / Harmful Algae 55 (2016) 179–190

Fig. 8. Trend in Ostreopsis cf. ovata abundance on benthic substrata (macroalgae and rocks) (cells cm2, left y-axis) and water temperature (8C, right y-axis) (modified from Accoroni et al., 2011). Error bars indicate standard deviations.

higher abundances are reported from pebbles than from macroalgae, suggesting that living substrata support lower concentration of epibionts, probably due to the production of some allelopathic compounds (Accoroni et al., 2015b). Microphytobenthos communities of the northern Adriatic Sea have been investigated both on soft (Facca et al., 2002; Welker et al., 2002; Totti, 2003; Blasutto et al., 2005; Cibic et al., 2007) and hard substrata (Munda, 2005; Totti et al., 2007a), indicating that diatoms are the major, in some cases exclusive (Welker et al., 2002), component of these communities. In this study, the microphytobenthos community of natural substrata (macroalgae and rocks) of the Conero Riviera (northern Adriatic Sea) has been investigated for the first time. Diatoms were the dominant microalgal group for the whole year except summer, when an increase of filamentous cyanobacteria was observed. Dinoflagellates occurred with very low abundances except during the bloom of Ostreopsis, which dominated the benthic dinoflagellate community. Even in this period, however, dinoflagellates represented only 7% of microphytobenthos community in terms of abundance, while they were major contributors in terms of biomass, accounting for more than 90% of the total microphytobenthos biomass. Diatoms were most abundant in spring and autumn, with a marked decrease in winter and summer, similarly to what observed in other Mediterranean (Barranguet, 1997; Romagnoli et al., 2007) and northern European (Stief et al., 2013) areas. On the contrary, in the Gulf of Trieste (N Adriatic Sea), Welker et al. (2002) and Cibic et al. (2007, 2012) found a different pattern, with two peaks generally in May and August, and a marked interannual variability. In summer, a significant increase of cyanobacteria was observed, as already reported in the same area on soft bottoms (Totti, 2003), as well as on epizoic communities in the Ligurian Sea (Romagnoli et al., 2007). Cyanobacteria are recognized as being dependent on high temperatures (Paerl and Otten, 2013). Nevertheless, a clear relationship between cyanobacteria and water temperature was not observed in this study, as other factors not considered in this study (e.g., light irradiance and daylength) may explain this seasonal pattern in cyanobacteria abundances (Berg and Sutula, 2015). Motile forms represented the main fraction in terms of diatom abundance, as already reported in this area (Totti et al., 2007a) and in Gulf of Trieste (Welker et al., 2002). In particular, in this study the dominance of motile forms throughout all the year was highlighted. In terms of biomass, however, erect diatoms became the dominant forms in winter and in spring. The decrease of erect forms in summer was already observed by Totti et al. (2007a) and may be explained considering that in summer the grazing pressure is higher than in other seasons, and the erect diatoms are the most exposed to grazing among all growth forms (Hillebrand et al., 2000; Wellnitz and Ward, 2000). A high contribution of

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phytoplankton cells has been recorded in benthic communities in winter, in relation to the settlement of a species (Skeletonema marinoi) commonly blooming in winter in the northern Adriatic Sea (Totti et al., 2000; Bernardi-Aubry et al., 2004; Totti et al., 2005). The present study showed that the microphytobenthos community diversity was significantly lower on macroalgae than on pebbles, while no significant differences were observed in terms of either abundance or biomass. This may result from the possible inhibitory activity of seaweed species toward microalgal species (Jeong et al., 2000; Jin and Dong, 2003; Nagayama et al., 2003; Nan et al., 2004, 2008; Jin et al., 2005; Wang et al., 2007a,b; Tang and Gobler, 2011; Ye and Zhang, 2013; Accoroni et al., 2015b). Moreover, although no measurements of light intensity were taken, there is reason to hypothesize that these results may be also related to the lower light intensity present in the macroalgae microenvironment than on exposed pebbles (Hauxwell et al., 2001; Sundba¨ck and McGlathery, 2005). In this regards, Facca et al. (2002) observed in the Venice lagoon (N Adriatic Sea) that the microphytobenthos diversity index (H’) displayed a positive significant correlation with the underwater light transmission. During the Ostreopsis cf. ovata bloom, dinoflagellates reached significantly higher abundances than during the rest of the year. Contrarily to previous observations for other common benthic dinoflagellates in this area (e.g., Prorocentrum lima, Totti, 2003), O. cf. ovata shows a strong seasonal behavior since it was first documented in 2006 (Totti et al., 2007b). In the northern Adriatic Sea, it typically thrives in late summer (end of September), differently from what observed in other Mediterranean areas (Mangialajo et al., 2011). Nutrient budgets in the northern Adriatic basin are strongly affected by river inputs. Riverine waters flowing in the northern Adriatic Sea have phosphorus content markedly lower compared to that of nitrogen (Tedesco et al., 2007; Cozzi and Giani, 2011), resulting in a high N:P ratio. Nutrient availability strongly affects microphytobenthos communities in terms of both abundance and community structure (Totti, 2003). Indeed, the abundances of benthic diatoms (and particularly of genera Pleurosigma and Synedra) along the Conero Riviera showed a significant positive correlation with total inorganic nitrogen, in agreement with what observed in the Venice lagoon (Facca et al., 2002). Diatoms are highly productive in NO3-enriched environments, while cyanobacteria (especially picocyanobacteria) and many chlorophytes and dinoflagellates, may be better adapted to use of NH4 (Glibert et al., 2016). These preferences were recognizable in the seasonal pattern of diatoms, cyanobacteria and dinoflagellates shown in this study: the main nitrogen source for the main part of the year was NO3 when diatoms were the dominant microalgal group, except that in summer when NH4 supplied an important contribution and an increase of filamentous cyanobacteria and dinoflagellates was observed. Nutrient availability (especially of P) affected also the Ostreopsis bloom development. The effect of N:P ratio, in synergy with local hydrodynamic conditions and temperature, has been proposed to explain the bloom dynamics in the northern Adriatic Sea (Accoroni et al., 2015a), as optimal N:P ratio for Ostreopsis growth is around Redfield value (Vanucci et al., 2012; Vidyarathna and Grane´li, 2013) and in the sampling period of this study, it is possible to note an increase of PO4 concentrations (resulting in a N:P ratio around Redfield values) only in August even when the Ostreopsis cf. ovata bloom started, and in mid-September, corresponding to a marked increase of O. cf. ovata abundances. Conversely, cyanobacteria did not show any relationship with nutrient concentrations. In this regard, although several studies showed that some cyanobacterial blooms, mainly in freshwater environments, are associated with eutrophication, several marine species bloom when concentrations

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S. Accoroni et al. / Harmful Algae 55 (2016) 179–190

of inorganic N and P are low, highlighting the importance of organic N and P as nutrient sources as well as their possibile ability to fix nitrogen (O’Neil et al., 2012). In this study, although diatoms showed abundances at least one order of magnitude higher than those of benthic dinoflagellates, during the Ostreopsis cf. ovata bloom they showed a general decrease in both percent and absolute abundances. This decrease is probably linked to the typical annual cycle of the benthic diatoms in this area, which is characterized by a general decrease of abundances in summertime (Totti et al., 2007a) driven mainly by nutrient availability and water temperature rather than to a direct influence of the presence of O. cf. ovata within the microphytobenthos community. Nevertheless, an influence of Ostreopsis cf. ovata presence could be recognized considering the composition of the diatom community in terms of growth forms: in fact, during the Ostreopsis bloom peak, motile diatoms showed higher percent abundances than during the rest of the summer. This could be explained considering that during the Ostreopsis bloom the benthic microenvironment changed due to the development of a copious biofilm (named ‘mat’) characterized by the presence of a network of several trichocyst filaments in the mucous matrix (Honsell et al., 2013). Inside the mucus, the motile life strategy is the most successful (Hudon and Legendre, 1987; De Nicola and McIntire, 1990) as these biraphid taxa are not only able to avoid mucus shading, but also would benefit from that tridimensional microenvironment rather than a smooth substratum. Moreover, with regard to the toxin effects, although the presence of palytoxins inside the mucus filaments has been documented (Giussani et al., 2015), diatoms seem to be not affected by such compounds (Pichierri et al., 2014). The microphytobenthos community showed a significant lower value of Shannon diversity index during the presence of Ostreopsis cf. ovata than in the rest of the year. Considering that there were not significant differences in terms of diversity among the four seasons, this result would lead to hypothesize an influence of Ostreopsis presence due to both the shadowing produced by mucous mat and the possible allelopathic compounds produced by O. cf. ovata. In this respect, it is well-known that several toxic dinoflagellates can produce allelochemicals in order to compete with other co-occurring algae especially under unfavorable environmental conditions (Tillmann and John, 2002; Fistarol et al., 2003, 2004; Grane´li and Johansson, 2003; Suikkanen et al., 2004; Grane´li and Hansen, 2006; Prince et al., 2008). A weak allelopathic activity have been observed in O. cf. ovata on the growth of Coolia monotis and Prorocentrum minimum (Monti and Cecchin, 2012). This study highlighted that the relationships between O. cf. ovata and the other benthic microalgae are complex, and possibly involve a number of factors, such as allelochemicals and mucus production, competition for nutrients and substrata. In this regards, further studies are required to clarify these aspects. Acknowledgments This research was funded by the ISPRA-Italian Ministry of the Environment and MURST (PRIN 2007). We are grateful to Fabio Rindi for the English revision. The authors would like to thanks the anonymous reviewers for their valuable comments and suggestions to improve the quality of the manuscript.[SS] References Accoroni, S., Romagnoli, T., Colombo, F., Pennesi, C., Di Camillo, C.G., Marini, M., Battocchi, C., Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Tartaglione, L., Penna, A., Totti, C., 2011. Ostreopsis cf. ovata bloom in the

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