A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas

Harmful Algae 4 (2005) 673–695 www.elsevier.com/locate/hal A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas Magda Vilaa,*, Maria Grazia Giacobbeb, Mercedes Masóa, Ester Gangemib, Antonella Pennac, Nagore Sampedroa, Filippo Azzarob, Jordi Campa, Luca Galluzzic a Institut de Ciències del Mar, Passeig Marı́tim de la Barceloneta, 37-49, Barcelona 08003, Spain b Istituto per l’Ambiente Marino Costiero, CNR, Spianata S. Raineri 86, Messina 98122, Italy c Centro Biologia Ambientale, University of Urbino, Vle Trieste 296, Pesaro 61100, Italy Received 22 June 2004; accepted 14 July 2004 Abstract Alexandrium minutum is a toxic dinoflagellate widespread along the Mediterranean coasts. This species is frequently detected year-round at low concentrations within the Mediterranean basin. However, it only proliferates recurrently in some localities. Two affected areas are the Catalan and Sicilian coasts. In order to identify the factors determining the A. minutum blooms in the Mediterranean Sea, we compare the bloom conditions in two harbours: Arenys de Mar (Catalan coast, Spain) and Syracuse (Sicily, Italy), during 2002–2003. Arenys de Mar harbour is a fishing and leisure harbour and receives an input of freshwater rich in nutrients. Likewise, the Syracuse harbour – located on the Ionian coast of Sicily – is subject to freshwater inputs. Some points of this site are used for productive activities such as shellfish farming. A. minutum from the two areas studied were morphologically and genetically identical. In both sites, recurrent blooms take place from winter to spring. Surface water temperatures and salinities during A. minutum bloom events were 12–14.5 8C and 32–38, and 16–24 8C and 32–37.7 for Arenys and Syracuse, respectively. During the blooms, the spatial distribution of A. minutum in the two harbours, the physicochemical characteristics and the phytoplankton community were studied. Similarities in composition of the phytoplankton community were evidenced, with a clear dominance of dinoflagellates over the other taxa. In Arenys, the second dominant species was Prorocentrum micans followed by Scrippsiella spp. and Dinophysis sacculus. The same species were found in Syracuse although P. triestinum, and alternatively Lingulodinium polyedrum, reached cell densities much higher than the other dinoflagellates giving marked water discolourations. # 2004 Elsevier B.V. All rights reserved. Keywords: Alexandrium minutum; HAB; Mediterranean Sea; Phytoplankton assemblages; Toxic dinoflagellates * Corresponding author. Tel.: +34 932309500; fax: +34 932309555. E-mail address: magda@icm.csic.es (M. Vila). 1568-9883/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2004.07.006 674 M. Vila et al. / Harmful Algae 4 (2005) 673–695 1. Introduction Once the increment in harmful algae blooms (HAB) has been accepted as a common phenomenon in coastal waters (Anderson, 1989; Hallegraeff, 1993), we should aim at investigating the causes of such an increase. Comparative studies will allow us to identify critical processes controlling HAB occurrence and consequently to develop prediction capacities (GEOHAB science plan, Glibert and Pitcher, 2001). For such a type of study, it is important to take advantage of recurrent blooms since they never occur there by chance. The genus Alexandrium includes about 30 species and at least nine of them produce a number of neurotoxins that can lead to paralytic shellfish poisoning (PSP) events. A. minutum, which is one of the toxic species, is a small armored dinoflagellate originally described from a red tide in the Alexandria harbour (Egypt, SE Mediterranean Sea; Halim, 1960). This species has been reported over a number of geographical areas and in a wide range of coastal hydrographic regimes (i.e. Hallegraef et al., 1988; Tahri-Joutei et al., 2000; Maguer et al., 2000; Yoshida et al., 2000; Daly Yahia Kefi et al., 2001a,b; Usup et al., 2002). A. minutum is an unchained species that is well characterized morphologically (see Balech, 1995). However, some specimens identified as A. minutum differ morphologically from the species redescription (Balech, 1989), especially concerning their plate ornamentation (Montresor et al., 1990), and the lack of a ventral pore (Danish specimens in Hansen et al., 2003). This suggests that more than a single species could be included under the name of A. minutum (Moestrup et al., 2002). A. lusitanicum was described as a distinct species based on the different shape of the s.a. plate (Balech, 1995). However, Franco et al. (1995) suggested that A. lusitanicum and A. minutum are nonspecific in view of the apparent variability of this feature. A. minutum is widely distributed in the Mediterranean Sea and events of paralytic shellfish poisoning (PSP) have been frequently associated with this species in different basins such as the Northern Adriatic Sea (Honsell et al., 1996), Eastern Aegean (Koray and Buyukisik, 1988), Tyrrhenian Sea (Giacobbe et al., 2003b), and Catalan-Balearic Basin (Delgado et al., 1990; Forteza et al., 1998). A. minutum blooms in the Mediterranean seem to be restricted to coastal enriched sites, particularly harbours, estuaries or lagoons (Belin, 1993; Giacobbe and Maimone, 1994; Vila et al., 2001). The inoculum for bloom initiation in semi-enclosed areas could be the result of offshore advection of cells or excystment of local benthic cysts. Resting cysts in the local sediment of Arenys Harbour (Catalan Sea) have been detected recently (Garcés et al., 2004). These authors highlighted the potential main role of cyst beds in the outbreaks of A. minutum in water bodies with restricted water exchange such as harbours. A lot of effort has been made to understand the ecological preferences and the adaptive strategies of A. minutum under laboratory conditions, i.e. nutritional preferences and toxin production in relationship with grazers (Bagøien et al., 1996; Frangópulos et al., 2000; Guisande et al., 2002; Lippemeier et al., 2003), or competition and allelopathy (Cannon, 1996; Arzul et al., 1999; Tillmann and John, 2002; Fistarol et al., 2004). However, there is a gap in comparative field studies in areas which are affected by blooms of the same species. Those are essential for a better understanding of the A. minutum bloom conditions. In this study, we analyze the occurrence of A. minutum in two human impacted areas of the Mediterranean: the Catalan coast (North-western Mediterranean) and the Eastern coast of Sicily (Eastern Mediterranean). It is known from the last decade that blooms of A. minutum are recurrent in harbours of these two regions. The main objective of the present study is to compare these systems in order to provide further insights into the conditions that make a certain locality susceptible to blooms of this species. First of all, we provide evidence that we are dealing with the same species based on morphological and genetic information of the organism. To identify the factors related to A. minutum blooms, we examined the A. minutum distribution and related parameters at different scales in both areas. First, we focus on the A. minutum distribution and inorganic nutrients concentration on a regional scale during the period 2002–2003. Two target sites were intensively studied: Arenys de Mar harbour (Catalonia) and Syracuse bay (Sicily), where A. minutum blooms have been described since 1996 and 2001, respectively. We also studied the small-scale spatial M. Vila et al. / Harmful Algae 4 (2005) 673–695 distribution of A. minutum, environmental variables, species competitiveness (phytoplankton community) and potential grazing impact during bloom events at those localities. 2. Methods 2.1. Extensive monitoring A number of stations were examined along the Catalan coast of Spain (24) and Ionian (14) and Tyrrhenian (11) littoral of Sicily (Italy) to establish the distribution of A. minutum in both areas (Table 1, Fig. 1). The stations included different systems (harbours, bays, beaches and lagoons). Temperature and salinity were measured and water samples collected at the surface for phytoplankton, chlorophyll-a and inorganic nutrient analyses. In Catalonia, sampling in harbours was carried out on 2–4 occasions per month from March to September and once to twice a month for the rest of the year. Catalan beaches were sampled once a week in summer. Monitorings along the coast of Sicily were conducted on various occasions in 2002 and 2003 (Table 1). 2.2. Study areas Arenys de Mar harbour is situated on the Catalan coast (NE of Spain) at 418340 N and 28330 E. Its extension is approximately 30 ha – total surface – of which 17 ha is water. It is a shallow (0.5–6 m depth), fishing and leisure harbour receiving an input of freshwater rich in nutrients. The Syracuse harbour (37830 N, 158170 E), located on the Ionian coast of Sicily (Syracuse bay, extension about 700 ha) is also subject to freshwater inputs (riverine and spring waters). Some spots of this bay (depth: 0.5–8 m at the sampling area; 25–30 m at the entrance) are also used for productive activities such as shellfish farming. Both harbours were intensively monitored over time (2002–2003) and space during A. minutum blooms. 2.3. Sampling in Arenys de Mar harbour Two stations (st. R and T, Fig. 1C) were examined during 2002 and 2003. Station R was sampled all year 675 long and st. T during the A. minutum blooms (since mid-January to mid-March). Both st. R and T were sampled twice or even three times a week during that period. Temperature and salinity were measured directly by microprocessor conductivity meter WTW Model LF197. Two temperature sensors that store data every 15 min were placed (st. T) at the surface and bottom (2 m). Surface water samples were collected for phytoplankton, chlorophyll-a and nutrient analysis. Hundred and fifty milliliters was preserved with Lugol’s iodine solution for microscopic analysis. Sixty milliliters was filtered on Whatman GF/F glass fibre filters, and frozen at 20 8C for chlorophyll analysis, and 60 ml was frozen immediately (20 8C) for nutrient analyses. In 2002, a cruise was performed during the maintenance phase of the A. minutum bloom (18 February 02), just after it had reached its maximum concentration, to analyze the horizontal variability inside the harbour (30 stations). The phytoplankton and microzooplankton community were analyzed as well as environmental parameters. A vertical transect was repeated at four stations at midday (12–13 h) and in the afternoon (17–18 h, local time) with the aim of analyzing vertical variability. The Pearson correlation coefficient was calculated after logarithmic transformation (log + 1) when necessary (i.e. phytoplankton cell numbers). In 2003, a different sampling scheme was applied: a total of five stations inside the harbour were monitored during the bloom development (same parameters like those of 2002 were analyzed). 2.4. Sampling in Syracuse Four stations (st. 1–3 inside the harbour; st. 4 in the mussel area, Fig. 1C) were sampled from March to October (2002 and 2003) with a usual sampling frequency of 2–4 times per month. The parameters considered and procedures were the same as indicated for Arenys. Temperature and salinity were measured using a mobile probe (Multiline F/SET-3 WTW). On 11 May 2002 and 2 April 2003, surveys to study the horizontal distribution and composition of phytoplankton inside the area were carried out. Sampling points were increased to 14, including a maximum bloom site that was examined vertically from surface to bottom (0–5 m). Zone Code Station Latitude (N) Longitude Type (E) Catalan coast – Catalanobalear Basin North Ca1 Roses 428150 000 38100 000 North Ca2 Empuriabrava 428140 3300 38 80 600 North Ca3 Estartit 42830 000 38120 1200 Sampled Max. Conc. period (month) 2002 2003 Chl-a Month > 104 Mean S.D. n 38110 5600 Beach 5–9 418590 1800 418580 2700 418530 4100 418510 4800 418510 2900 418500 3000 418470 3000 418400 1800 418340 1800 38120 1900 38120 4500 38110 4500 38100 1700 3880 4000 3870 600 3830 1400 28470 4800 28330 1800 Beach Beach Beach Beach Beach Harbour Beach Harbour Harbour 5–9 5–9 5–9 5–9 5–9 1–12 5–9 1–12 1–12 n.d. n.d. <103 n.d. n.d. <103 n.d. 103 107 Center Center Center Center Center Ca14 Ca15 Ca16 Ca17 Ca18 Premià Olı́mpic Barcelona Castelldefels Vilanova 418290 600 418230 1200 418200 600 418150 5200 418120 1800 28200 5400 28120 600 28100 1200 18570 1100 18430 4200 Harbour Harbour Harbour Beach Harbour 1–12 1–12 1–12 5–9 1–12 103 104 <103 <103 104 South South South South South South Ca19 Ca20 Ca21 Ca22 Ca23 Ca24 Torredembarra Tarragona Cambrils L’Ametlla L’Ampolla St. Carles de la Ràpita 41870 3000 41850 000 41830 4200 408520 4800 408480 3600 408360 3600 18240 000 18120 5400 1830 4800 08480 1200 08420 4800 08360 2400 Harbour Harbour Harbour Harbour Harbour Harbour 1–12 1–12 1–12 1–12 1–12 1–12 103 <103 105 103 <103 103 North Ca4 Sicilian coast – Tyrrhenian Sea Salina Si1 Salina Rinella Lipari Si2 Marina di Porto Salvo Vulcano Si3 Three stations Milazzo Si4 Marina di Nettuno Marinello Si5 Lago Verde 388320 5300 148490 4500 388280 3000 148570 3000 Harbour 8 Harbour 6 388250 148570 388120 5800 158150 700 Beach 5–10 Harbour 3 38880 3500 Lagoon 15820 4500 3–6 n.d. n.d. <103 <103 n.d. <103 n.d. <103 106 February, March 103 <103 July 103 <103 105 March, April <103 103 105 May, May <103 <103 104 April Mean S.D. 2.6 5.7 3.1 2.1 4.5 2.6 57 39 36 135 69 56 3.8 6.7 23 2.3 1.0 0.4 1.6 1.7 0.7 7.4 1.2 4.7 13 2.0 12 26 0.8 21 23 0.2 26 28 1.4 17 13 1.1 3 11 0.4 35 25 13.6 18 21 0.9 36 76 3.7 147 302 36 328 47 23 68 21 27 12 57 61 n Si/DIN Si/PO4 Mean S.D. n Mean S.D. n 50 57 0.9 199 36 2.4 175 69 2.0 0.8 3.5 2.9 57 34 36 232 69 77 60 460 182 57 36 69 20 24 2.1 2.1 24 16 16 24 23 25 41 22 11 18 30 121 367 19 21 27 17 30 36 19 36 68 1.4 1.1 1.2 1.8 1.8 0.6 1.2 0.5 0.4 0.7 1.2 0.6 1.3 2.0 0.7 0.7 0.4 0.2 19 21 27 17 30 36 19 36 68 33 19 40 13 20 13 22 26 96 30 21 58 16 52 19 46 21 124 19 21 27 17 30 36 19 36 68 727 17 27 10 154 36 48 68 28 57 0.4 0.8 0.6 2.0 0.6 0.3 0.8 0.6 3.7 1.0 36 48 68 28 57 76 16 11 19 26 158 18 19 22 61 36 48 68 28 57 39 157 1328 379 556 347 36 67 36 57 36 69 0.8 0.9 0.5 0.4 0.8 2.0 0.6 1.0 0.2 0.2 0.3 4.7 36 15 67 58 36 204 57 85 36 236 69 141 17 129 670 249 421 240 36 67 36 57 36 69 6.8 0.0 42 2 23 12 14 2 42 2 1 17 4.1 3.1 3.9 3.5 4.9 4.4 2.1 5.3 2.5 4.5 6.4 15.9 2.1 3.6 2.0 10.3 15.2 50.9 1.3 7.5 1.9 24.0 36 67 36 57 36 69 27 85 417 199 304 167 12.6 33.3 42 6 33 6 42 6.2 6 2 0.4 6 1 3.0 n.d. 103 n.d. n.d. <103 March 105 April 1 M. Vila et al. / Harmful Algae 4 (2005) 673–695 42820 4500 North North North North North North North Center Center Platja de l’Estartit Ca5 Pals Ca6 Sa Riera Ca7 Llafranc Ca8 Castell Ca9 Fosca Ca10 Palamós Ca11 St. Pol Ca12 Blanes st. R Arenys de Mar 103 <103 <103 105 April 104 104 September, July <103 <103 Harbour 1–12 Harbour 1–12 Harbour 1–12 DIN/PO4 676 Table 1 A. minutum spatial distribution (maxima cell concentration), chlorophyll-a and nutrient relationships (mean, standard deviation and n) over the 2002–2003 period along the Catalan and Sicilian coasts Portorosa Si6 Four stations 15860 Harbour 2–6 104 March, April 378510 600 158180 1700 Beach 5–6 <103 May 378140 1600 158120 1000 Harbour 2–3 n.d. 378120 5300 158110 000 Harbour 2–9 103 n.d. 37890 2000 37890 600 158120 1500 158130 4600 Harbour Beach 2–4, 6, 9 2–9 <103 <103 <103 n.d. 37880 3300 158130 2000 Beach 5–9 n.d. 37880 2800 158130 1300 Beach 2–9 n.d. 37840 500 37830 5100 158170 5500 158170 100 Harbour Harbour 4–10 3–10 10 37830 3200 37830 2500 158160 3900 158170 4000 Harbour Bay 4 3–10 103 106 104 37830 2400 158160 2200 Harbour 3–10 105 105 37820 1600 158170 2300 Bay 3–10 104 104 368480 800 15850 2900 Lagoon 3 5 164 317 33 0.3 0.1 13 55 62 13 16 10 2 1.1 0.3 2 16 5 2 242 40 2 0.3 0.1 2 69 27 2 0.8 1.2 11 37 43 13 1.1 0.9 13 27 25 13 June 0.4 0.5 0.5 0.5 10 22 22 58 16 93 11 24 0.8 1.5 0.8 3.3 11 24 11 53 7 98 11 24 <103 July 0.5 0.3 22 61 113 23 0.8 0.5 23 26 30 23 103 July 1.1 1.4 22 39 51 24 0.7 0.8 24 17 14 24 105 105 April June, March April April 2.2 27.6 1.8 80.6 8 53 28 22 19 29 7 26 1.0 1.1 1.0 1.0 7 26 16 14 9 11 7 26 1.1 1.2 31 16 34 8 31 4 32 0.8 1.8 0.6 2.1 4 32 11 28 9 19 4 32 April, March April, April 5.7 7.5 52 21 22 30 0.8 0.7 30 14 24 30 2.3 1.8 23 40 44 27 0.5 0.4 27 16 16 27 n.d. M. Vila et al. / Harmful Algae 4 (2005) 673–695 Sicilian coast – Ionian Sea Taormina Si7 Lido Mendolia Augusta Si8 Foce fiume Roadstead Mulinello Augusta Si9 Marcellino Roadstead Mouth Priolo Si10 IAS S. Panagia Si11 Magnisi Bay, Priolo peninsula S. Panagia Si12 Lidi Bay, Priolo S. Panagia Si13 ENEL Bay, Priolo Power Station Siracusa Si14 Marmoreo Siracusa st. 1 Sanità Marittima Siracusa st. 8 st. 8 Siracusa st. 2 Fonte Aretusa Siracusa st. 3 Foce AnapoCiane Siracusa st. 4 Campo Mitili Noto Si30 Stagni di Vendicari 38870 Months with A. minutum concentrations above 104 cells l1 are indicated. Sampling stations are ordered from north to south. The zone, geographical coordinates, station type and sampled months are indicated. Codes are used as in Fig. 4. 677 678 M. Vila et al. / Harmful Algae 4 (2005) 673–695 Fig. 1. (A) Study area. (B) Geographical distribution of A. minutum along the Catalan and Sicilian coasts (Mediterranean Sea). Presence is indicated by black triangles (~) and absence by open triangles (~). Further information in Table 1. (C) Sampling points in the Arenys de Mar harbour (Catalan coast) and Syracuse bay (Sicilian coast). Open triangles (~) refer to stations sampled during the 2002 cruise, black triangles 679 M. Vila et al. / Harmful Algae 4 (2005) 673–695 Table 2 List of A. minutum strains, sample location and EMBL accession numbers Species Strain Sampling, location and year Accession number A. A. A. A. A. A. CSIC-D1 IEO-AL8C IEO-AL9C CNR-AMIA1 CNR-AMIA4 CNR-AMIA5 Mediterranean, Mediterranean, Mediterranean, Mediterranean, Mediterranean, Mediterranean, AJ312945 AJ532914 AJ621733 AJ621734 AJ318460 AJ532913 minutum minutum minutum minutum minutum minutum Catalan Sea, Arenys, Spain, 1995 Catalan Sea, Arenys, Spain, 2002 Catalan Sea, Arenys, Spain, 2002 Ionian Sea, Syracuse, Italy, 2001 Ionian Sea, Syracuse, Italy, 2001 Ionian Sea, Syracuse, Italy, 2001 CNR, Consiglio Nazionale delle Ricerche, Messina, Italy; CSIC, Institut de Ciències del Mar, Barcelona, Spain; IEO, Instituto Español de Oceanografia, Vigo, Spain. 2.5. Parameters Chlorophyll-a was extracted in 8 ml of 90% acetone overnight at 4 8C, and the concentration measured with a Turner fluorometer (Turner Designs). Analyses of dissolved inorganic nutrients (NO3, NO2, NH4 and PO4) were performed with an Alliance Instruments Evolution II autoanalyzer as described in Grassohoff et al. (1983). Calculations of potential nutrient limitation have been calculated as in Justic et al. (1995). The criteria of probable limitation are as follows: P limitation (P < 0.1 mM; DIN:PO4 > 22; Si:PO4 > 22), N limitation (DIN < 1 mM; DIN:PO4 < 10; Si:DIN > 1), and Si limitation (Si < 2 mM; Si:PO4 < 10; Si:DIN < 1). An aliquot of 10–50 ml of the lugol fixed samples was settled in a counting chamber for 1 day. For phytoplankton enumeration, the appropriate area of the chamber was scanned at 63–400 magnification, depending on the cell density of each species, using a Leica-Leitz DM-Il inverted microscope or a Zeiss Axiovert 200 (Throndsen, 1995). Usually, at least three transects were scanned at 100, one transect at 400 and the complete chamber at 63. Thus, the minimum concentration detected by this method was 20 cells l1. Alexandrium minutum was identified by thecal plate tabulation (Balech, 1995) after adding some drops of the fluorescent dye Calcofluor White M2R (final concentration 10–20 mg ml1; Fritz and Triemer, 1985) to the chamber in order to stain the plates. The chambers were examined under 400 magnification in an inverted microscope with UV excitation fluorescent illumination. The phytoplankton nomenclature is used according to Steidinger and Tangen (1997), Daugbjerg et al. (2000) and Moestrup et al. (2002). For SEM (scanning electron microscopy) cultured cells (strain IEO-AL9C) were fixed with 4% glutaraldehyde, washed in dH2O, dehydrated in a graded ethanol/acetone series, critical point dried and coated with gold. The microscope used was a Hitachi S-350N (Nissei Sangyo Co. Ltd., Tokyo, Japan), operating at 10 kV. 2.6. Culturing and genetic analyses Clonal cultures of A. minutum were established at the Institut de Ciències del Mar (CSIC-D1), Instituto Español de Oceanografia (IEO-AL8C, IEO-AL9C) and Consiglio Nazionale delle Ricerche (CNRAMIA1, CNR-AMIA4, CNR-AMIA5) from water samples taken in 1995, 2001 and 2002 at Arenys de Mar and Syracuse bay (Table 2). All marine cultures were maintained in F/20 and F/2 media (see http:// ccmp.bigelow.org/), at 17  1 8C and a 14:10 h (light:dark) photoperiod. Illumination was provided by a photon irradiance of 100 mmol m2 s1. 2.7. DNA extraction Approximately 5–10 ml of exponentially growing cultures were harvested by centrifugation (4000 rpm) for 10 min at room temperature. The pelleted cells (~) to stations sampled during the 2003 bloom. Those stations sampled both years are indicated by (+). Routinely sampled stations are indicated (st. R and T in Arenys, and st. 1–4 in Syracuse). 680 M. Vila et al. / Harmful Algae 4 (2005) 673–695 were rinsed twice with artificial 0.22 mm sterile seawater. Total DNA was extracted using Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA) or DNeasy Plant Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. 2.8. PCR amplification, cloning and sequencing The 5.8S rDNA and ITS regions (ITS1 and ITS2) were amplified as described by Penna et al. (2005). The PCR products were visualized on 1.8% agarose gel. After electrophoresis analyses, the PCR products were excised from the agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Westburg). Purified PCR fragments were directly sequenced or cloned in the vector pDrive Cloning Vector (Qiagen, Valencia, CA) and sequenced. The nucleotide sequences were performed using the ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Applied Biosystems, Foster City, CA) and the dye terminator method was used according to the manufacturer’s instructions (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer Corp., Foster City, CA). The EMBL accession numbers of the 5.8S rDNA and ITS1-ITS2 regions are indicated in Table 2. 2.9. Sequence analyses Multiple alignments were constructed using the CLUSTAL X program and subsequently rechecked by eye. To determine the ITS and 5.8S rDNA termini for the A. minutum, the rDNA coding region and flanking sequences of ITS1 and ITS2 were aligned with those of Alexandrium species listed in GenBank. 3. Results 3.1. Morphological identification and genetic comparison The identification of A. minutum was based on both microscope observations and genetic analyses. Morphologically, cells are rounded and small-sized, 22.5 mm wide (min.: 14.0 mm; max.: 32.7 mm; n = 112), and 23.2 mm long (min.: 14.2 mm; max.: 33.6 mm; n = 112) on average, although cells in exponentially growing cultures were significantly smaller (Fig. 2). The four main distinctive characters are shown in Fig. 2: direct connection of 10 -Po plates; the presence of a ventral pore (Vp) at the 10 rightanterior side; plate 600 narrow; posterior sulcal plate (Sp) quadrangular, lacking a connecting pore. In the six clonal cultures theca was always smooth, without a reticulation pattern (Fig. 2). The morphological identification was further confirmed by molecular analyses. PCR amplification of the A. minutum isolates produced a single fragment of 520 bp. Sequence alignment of the 5.8S rDNA and ITS regions of A. minutum from Arenys and Syracuse revealed that all six isolate sequences were identical. Selected primers for the conserved 5.8S rDNA and variable ITS-1 region specific for the A. minutum species gave a PCR fragment of 212 bp. Molecular weight of the amplified product was as expected and no other aspecific PCR products were visible when total A. minutum genomic DNA of each isolate was used as template. Speciesspecific primers were tested for cross-reactivity PCR amplification using several genomic DNA from other species (data are shown only for one no-targeted species in Fig. 3). 2.10. Species-specific PCR assay 3.2. Regional scale: A. minutum distribution and inorganic nutrients Species-specific PCR analyses of all six A. minutum isolates were performed by using two species-specific designed primers (Galluzzi et al., submitted). The PCR specificity was tested using a plasmid containing the ITS1-5.8S-ITS2 rDNA sequence of A. minutum. Further, the primer pairs were tested in the PCR assay for possible crossreactivity with no-target DNA by including other species of dinoflagellates and diatoms. Table 1 shows the maximum A. minutum cell concentrations along the Catalan and Sicilian coasts in 2002 and 2003 and the inorganic nutrient ratios. A. minutum cells were widespread along the Catalan and Sicilian coasts (Fig. 1B). They occurred in all the Catalan harbours sampled and in some beach areas. High cell concentrations exceeding 105 cells l1 were detected in four Catalan harbours. In Arenys de Mar harbour, water discolourations coincided with high M. Vila et al. / Harmful Algae 4 (2005) 673–695 681 Fig. 2. SEM micrographs of A. minutum strain IEO-AL9C from Arenys de Mar (Spain). (A) Ventral view of a specimen with ventral pore (Vp) and well-developed intercalary bands. The connection between Po and 10 and the sulcal lists (arrows) can be observed. (B, C) Dorsal view. (D) Epithecal plate pattern. Po = apical pore complex. (E) Structure of the Po plate, f = formamen, c = callus, d = canopy, m = marginal pores. (H) Hypothecal plate pattern. Sp = posterior sulcal plate. Note the small size of these cultured cells. A. minutum concentrations (over 106 cells l1). Various areas of Sicily were also affected by the occurrence of A. minutum, both in Tyrrhenian and Ionian coastal waters (Fig. 1B). The maximum cell densities in Sicily did not exceed 7  103 cells l1, with the exception of a single Ionian zone (Syracuse harbour) and a Tyrrhenian brackish site (Verde Pond) where blooms of this species (>105 cells l1) were observed in spring. The median concentrations of nutrients (nitrate, ammonium and phosphate) over the sampling period in the three areas considered were: 5.2, 2.1 and 0.3 mM for the Catalan coast, 5.5, 1.0 and 0.4 for Sicily (Ionian Sea) and 4.0, 0.4 and 0.34 for Sicily (Tyrrhenian), respectively. The comparison of dissolved inorganic nutrients among different localities of the Catalan and Sicilian coasts during the year 2002–2003 indicated 682 M. Vila et al. / Harmful Algae 4 (2005) 673–695 Fig. 3. A. minutum-specific PCR assay using genomic DNA from six A. minutum isolates, a positive control and Karlodinium sp. (= Gyrodinium corsicum) IRTA-GC5. Target DNA from each isolate and plasmid was used to test species-specific PCR primer for the A. minutum species. The characteristic PCR product sizes generated by species-specific assay are shown by analysis on standard agarose gel. Lane 1, template DNA from the A. minutum CNR-AMIA1; lane 2, A. minutum CNR-AMIA4; lane 3, A. minutum CNR-AMIA5; lane 4, A. minutum CSIC-D1; lane 5, A. minutum IEO-AL8C; lane 6, A. minutum IEO-AL9C; lane 7, template plasmid containing A. minutum ITS1-5.8S-ITS2 rDNA; lane 8, G. corsicum IRTA-GC5. A clean no-template control was included in the PCR assay (not shown on the gel). M, size standards; arrow indicated reactions that produced a single species-specific PCR product. that, irrespective of the strong spatial and temporal variability, Catalan sites in general are characterized by higher amounts of dissolved inorganic nitrogen (DIN) than the Italian region considered (Fig. 4). In Catalonia and some Ionian points of Sicily (Syracuse), the median concentration of DIN usually is higher than 5, and concentrations of PO4–P ranged from 0.1 to 1 mM. Some Spanish harbours, which are located within the south (from Cambrils to St. Carles) and centre (Premià and Arenys de Mar), displayed amounts of DIN 1 or 2 orders of magnitude higher. PO4–P median values above 0.5 mM were only detected in the centre of Catalonia and in some Ionian (the Syracuse urban area) and Tyrrhenian stations (Vulcano). DIN and PO4–P levels were often in the lowest range in beach areas of both regions, with a clearest pattern in Catalonia. Consequently, nutrient ratios showed a high temporal and spatial variability in the three areas (Table 1). Most of the average DIN:PO4 ratios at the coastal sites studied exceeded the Redfield ratio (16:1). The lowest values were found on beach areas, especially in the Tyrrhenian Sea. In almost all the studied coastal sites the average Si:DIN was below the theoretical ratio (1:1), indicating silicate was the most likely inorganic nutrient limiting primary production in these areas. Only the three Tyrrhenian beach areas and some sites in the northern Catalan Sea had ratios higher than 1:1. The average Si:PO4 along both areas exhibited a great spatio-temporal variability, with values as high as 200 to below 16. M. Vila et al. / Harmful Algae 4 (2005) 673–695 Fig. 4. Box plots of median concentrations of inorganic nutrients (DIN and PO4) in Catalan and Sicilian harbours and beaches. Box: 25%, 75%; Whisker: Min, Max. Codes as in Table 1. Beaches are indicated adding B after the code. 683 684 M. Vila et al. / Harmful Algae 4 (2005) 673–695 Fig. 5. Temporal distribution of A. minutum (log cells l1) in Arenys de Mar (st. R) and Syracuse (st. 1 and st. 3) during (A) 2002 and (B) 2003. Temporal variability over an annual cycle (2002) in (C) water temperature, (D) salinity, and inorganic nutrients (in mM) as (E) nitrate, (F) ammonia, (G) phosphate and (H) silicate in Arenys de Mar (st. R) and Syracuse (st. 3). Note that nitrate concentrations are plotted on a logarithmic scale. The temporary pattern of inorganic nutrients over the 2003 cycle is not shown since it shows similar nutrient levels to the presented cycle. Calculations showed that potential limitation of primary production by inorganic nutrient concentrations (DIN, P–PO4, Si) is quite limited at the sites analyzed in this study (20 and 13% of the cases of potential limitation in the Catalan and Sicilian coasts). 3.3. Temporal distribution of A. minutum in two target localities Outbreaks of A. minutum were observed in Arenys de Mar harbour and Syracuse bay (harbour area) M. Vila et al. / Harmful Algae 4 (2005) 673–695 685 Fig. 6. (A) Small-scale temporal evolution of A. minutum in Arenys de Mar harbour during the 2002 bloom event in two stations (st. R and st. T, surface). The difference between water temperature at surface and bottom (st. T) is due to sunny and calm days in front to windy days, indicating water stratification or mixing. Raining events (mm) are indicated by open arrowheads at the bottom. The bloom has been divided into three periods: (1) A. minutum blooming concentrations both at st. R and T; (2) bloom declining at st. R, and still increasing at st. T; (3) bloom finished at both stations. (B–F) Comparison of salinity and dissolved inorganic nutrients (NO3, NH4, PO4 and SiO4) during the three periods at both stations. Error bars: standard error. during the 2 years analyzed (2002 and 2003) (Fig. 5). Surface water temperatures and salinities during A. minutum bloom events were 12–14.5 8C and 32–38, and 16–24 8C and 32–37.7 for the two areas, respectively. Whilst in Arenys A minutum vegetative cells were recorded over all the annual cycle, in Syracuse this species was detected only in spring. The highest densities were found in Arenys with winter to early spring peaks (>106 cells l1) in February 2002 and in March 2003. A more detailed analysis of the temporal evolution of A. minutum densities during the bloom events is presented in Fig. 5. In Syracuse bay, the highest densities occurred in the northern part of the bay (st. 1 and 3, in Fig. 5), being 1–2 orders of magnitude lower than in Arenys de Mar. Both harbours are subject to considerable changes in salinity due to the freshwater inflows supplying a high nutrient load. Concentrations of dissolved inorganic nitrogen (DIN) were much lower in Syracuse than in Arenys (Fig. 5). Nitrate was often the prevalent inorganic nitrogen form, with nitrate peaks (exceeding 200 mM in the Arenys de Mar harbour – st. R), although on some occasions there was a dominance of ammonium–nitrogen in Syracuse, 686 M. Vila et al. / Harmful Algae 4 (2005) 673–695 mostly at the river mouth. Orthophosphates (usually below 1.0 mM in Arenys – st. R) displayed higher values in Syracuse (Fig. 5). Median silicate concentrations were much higher in Arenys than in Syracuse. However, occasional silicate peaks (>30 mM) occurred in both sites (Fig. 5). In both areas, there was no apparent relationship between A. minutum biomass and inorganic nutrient concentrations. However, a common trait in nutrient stoichiometry during the course of blooms suggests that DIN and P–PO4 concentrations were not limiting. In fact, DIN or P–PO4 did not limit in 90% of the cases. In both harbours, chlorophyll concentrations were often lower than 8 mg l1. However, there was an exceptional peak of 380 mg l1 at st. 1 (Sanità Marittima, Syracuse) due to a persistent high biomass bloom of Lingulodinium polyedrum (21 May 2002), with a considerable water discolouration (max. 22  106 cells l1). 3.4. A. minutum bloom in Arenys de Mar (year 2002): small-scale temporal evolution The temporal evolution of the A. minutum bloom in Arenys de Mar harbour is presented (Fig. 6A). Three phases can be distinguished in the 2002 bloom: (1) A. minutum blooming at st. R and T; (2) bloom declining at st. R, and still increasing at st. T; (3) bloom finishing at both stations (Fig. 6A). In the first phase, A. minutum cell concentrations were over 106 cells l1, first at st. R. and after approximately 15 days also at st. T. A parasite infecting A. minutum cells was observed on 26 January at st. R. Infected cells were immobile, dark cysts that sometimes conserved the theca broken around. Unfixed water samples showed that the cysts released a lot of small flagellated cells. The bloom started to decline at st. R after two rain episodes, whereas at st. T it continued to increase until a maximum of 4.7  107 cells l1 was reached on 14 February. The highest concentration at st. T was achieved after 3–4 days of water warming as shown by the difference in temperature between surface (0 m) and bottom (2 m). There was a high variability in salinity and in concentrations of dissolved inorganic nutrients (NO3, NH4, PO4 and SiO4) in Arenys depending on the sampling point (Fig. 6B–F). Salinity was always lower at st. R since it was directly affected by an inflow of freshwater rich in nutrients, especially nitrate and silicate, explaining the big differences between st. R and T. Ammonium and phosphate, although always higher at st. R, were in the same range. The decrease in salinity, observed at st. R and T during the second period, and the overall nutrient increase, were related to the rain events. 3.5. Spatial variability: Arenys de Mar cruise (year 2002) A cruise was carried out in the Arenys de Mar harbour (18 February 2002, Fig. 7) just after the maximum bloom (on 14 February, 2002). 3.5.1. Horizontal distribution A. minutum cells were distributed in the whole harbour, and clearly dominated the planktonic community in all the stations. More than 90% of the phytoplankton cell counts corresponded to A. minutum, except in the harbour’s mouth (60%). On this occasion, maximum concentrations of A. minutum had already been detected in the area of st. T (Fig. 6), from which a dilution gradient was observed towards the harbour mouth. Chl-a followed the same pattern as A. minutum concentrations (n = 57, r = 0.63, p = 0.000). The horizontal distribution of temperature, salinity, DIN and silicate reflects the inflow of freshwater near st. R. Lower salinities were also detected near the northern pier walls and co-occurred with maximum PO4 concentrations (n = 34, r = 0.33, p = 0.038) and high silicate levels, indicating freshwater seepage through the harbour walls. Calculations of potential nutrient limitation in the harbour waters during the cruise suggest no limitation by PO4, only a few cases were limited by DIN (<9%), whereas 47% were limited by SiO4. During the cruise, the values in phosphate and ammonia were in the usual range (mean values 0.40 and 0.62 mM, respectively). In contrast, NO3 levels were very low (<2 mM). The most important co-blooming dinoflagellates were Prorocentrum micans and Dinophysis sacculus. P. micans followed the same pattern as A. minutum (n = 57, r = 0.71, p = 0); maximum D. sacculus concentrations were detected near st. R whereas Scrippsiella spp. were scarce and more homogeneously distributed. High concentrations of microzooplankton were detected (around 104 cells l1). M. Vila et al. / Harmful Algae 4 (2005) 673–695 687 Fig. 7. Surface distribution for the physicochemical parameters and the dominant phyto- and microzooplankton species during the bloom in Arenys (18 February 2002). 688 M. Vila et al. / Harmful Algae 4 (2005) 673–695 Fig. 8. Surface distribution for the physicochemical parameters and the dominant phytoplankton species during the cruises at Syracuse, 2002 and 2003. The main organisms were heterotrophic dinoflagellates such as Polykrikos schwartzii and Gyrodinium sp., loricate ciliates as Tintinnopsis spp. and rotifers (Fig. 5) with Synchaeta triophthalma the most abundant. The microzooplankton species distributions were more heterogeneous in the harbour than for the phytoplankton, probably because their swimming speeds were higher than those of the small dinoflagellates, which gave them the chance to exploit phytoplankton patches less exploited by other micrograzers. S. triophthalma was the dominant grazer at the st. T area, coinciding with the maximum A. minutum concentrations (n = 57, r = 0.50, p = 0). Minimum A. minutum concentrations inside the harbour co-occurred with P. schwartzii and Gyrodinium sp. M. Vila et al. / Harmful Algae 4 (2005) 673–695 3.5.2. Vertical distribution Phytoplankton and microzooplankton species were almost homogeneously distributed along the water column. A. minutum was once again the dominant species at all stations and depths, both at midday and in the afternoon, showing slightly higher cell concentrations at surface. Thus, during the cruise, a thick layer with very high concentrations on A. minutum was widespread in the whole harbour. P. micans achieved relatively high cell concentrations only in the deepest samples at midday (around 40% of total phytoplankton counts). Other significant differences were not found in the vertical pattern between midday and afternoon profiles. 3.6. Spatial variability: Syracuse cruises (2002 and 2003) Two cruises were carried out in the Syracuse harbour (11 May 2002 and 2 April 2003). On both occasions A. minutum cells assembled in the most confined sector of the harbour (northward the river mouth), characterized by a reduced water circulation. The highest cell densities were found in 2003 (1.3  106 l1) at the surface (Fig. 8), with a sharp decrease (one order of magnitude) in cell numbers at 1 m and still lower densities toward the bottom (5 m). Concentrations of nutrients (silicate, DIN and PO4–P) were often maximal near the freshwater inputs with 6.70 mM of inorganic nitrogen (mostly nitrates) and 0.46 mM orthophosphate in the A. minutum patch (cruise 2003, DIN:PO4 = 14.6). In contrast, during the previous cruise in 2002 (Fig. 8), lower cell densities (1.1  103 cells l1) and higher amounts of these nutrients were detected: 20.61 mM DIN – mostly ammonia, 3.21 mM PO4–P, with a DIN:PO4 ratio of 6.4. In 2002, simultaneous blooms of the dinoflagellate Lingulodinium polyedrum took place within the harbour and persisted during the next spring samplings, accounting for the observed high values of chlorophyll a, up to 380 mg l1 on May 21. During the 2003 cruise, the most important, co-blooming dinoflagellate was Prorocentrum triestinum, which reached exceptional concentrations (6  107 cells l1), and was mainly responsible for the water discolouration. Thus, in Syracuse harbour A. minutum was never found as the dominant phytoplankton species. 689 4. Discussion 4.1. Organism identification Cell morphology of A. minutum from the two Mediterranean regions fitted the re-description by Balech (1989), with a ventral pore always present in the material studied and thecal plates lacking ornamentations in the cultured strains. However, on very few occasions, some natural Catalan specimens of A. minutum showed a reticulation in the hypotheca. Our usual morphotype is the most frequent one among the different geographical isolates around the world (see Hansen et al., 2003, Table 5). The toxin composition in strains from Arenys and Syracuse is basically GTX 1-4 (Van Lenning et al., 2004; Giacobbe et al., 2003a,b). ITS15.8S-ITS2 rDNA sequences were identical for the six isolates, confirming the existence of a unique morphotype and genotype in the Mediterranean area. Furthermore, the high specificity of the speciesspecific PCR primers selected for A. minutum was examined against other phytoplankton species and the results obtained allowed detecting the A. minutum species unequivocally. Thus, species-specific PCR assays will be further applied in field phytoplankton populations to discriminate the A. minutum presence. Recently, an A. minutum morphotype, differing from the typical minutum morphotype by the absence of a ventral pore (Vp), has been observed in Denmark (Hansen et al., 2003). Partial large subunit rDNA sequences of Danish specimens clustered together with other European strains of this species having a Vp. Therefore, sequencing of this part of the gene did not resolve intraspecific relationships, as it did not allow the differentiation of populations with or without a ventral pore. These authors suggested that the ventral pore may be a variable feature, making this character questionable. This has important taxonomical implications since it modifies the concept of Alexandrium morphological species, which in this case should be revised. In the near future, atypical A. minutum strains (lacking the ventral pore and with ornamentations) should be analyzed using the high variable ITS sequences in order to possibly discriminate different genetic populations in the Mediterranean Sea. 690 M. Vila et al. / Harmful Algae 4 (2005) 673–695 4.2. Ecological comparison The data obtained from the two Mediterranean regions show the extended distribution of A. minutum in the Mediterranean. However, high-density blooms of this species always occur in confined or semienclosed water areas such as harbours, bays or lagoons. This species was recorded in some beach areas of the Catalan and Sicilian coasts, but densities never exceed 103 cells l1. In both areas, Catalonia and Sicily, A. minutum blooms occur in localities affected by local freshwater inputs that could be related to the supply of macro- and micronutrients. In other reports, riverine inputs of selenium after rainfall have been suggested to be a critical trigger factor for blooms of some dinoflagellates such as A. minutum and Gymnodinium catenatum (Doblin et al., 1999). Studies on batch cultures of A. minutum indicated optimal growth rates at salinities between 20 and 37, with peaks at 25 (0.63  0.07 div day1) (Grzebyk et al., 2003), or even at salinities lower than 15 (Hwang and Lu, 2000). The association between blooms of A. minutum and freshwater outflow could be related to the formation of density gradients acting as a retention mechanism (e.g. fronts) or inducing water stability. In both areas examined, the local outflow of continental waters causes the formation of density fronts that could act as a retention mechanism for the phytoplankton population. Obviously, the direct supply of nutrients flowing in a semi-confined area plays a key role in both areas in sustaining high phytoplankton biomass. Giacobbe et al. (1996) reported that A. minutum proliferations in the Ganzirri lagoon occurred in May, with DIN:PO4 atomic ratios close to 16:1 and a minor competitive pressure from other phytoplankton species. In the present study, A. minutum bloomed in places that differ markedly in inorganic nutrient concentrations and DIN:PO4 ratios (Table 1, Figs. 4 and 5). In fact, Arenys was characterized by high inputs of nitrates and silicates, which are mainly associated with a runoff origin, in contrast to the high concentrations of ammonium and phosphate in Syracuse, which reveal a rather urban origin. However, some common traits can be observed (Table 3): (1) throughout the two Mediterranean regions, Si:DIN ratios tend to be lower than 1 in harbours indicating a potential limitation for diatom growth, and suggesting a possible advantage for dinoflagellate growth (Justic et al., 1995; Masó et al., 2000; Anderson et al., 2002). In contrast, on beach areas, the Si:DIN ratio was much higher than 1, indicating a potential advantage for diatom growth; (2) neither in Arenys nor in Syracuse were there cases of potential nitrogen or phosphate limitation and (3) in both areas nitrates were usually the dominant nitrogen form. Measures of nitrate and ammonium uptake during A. minutum proliferations in Penzé River estuary (NW France) indicated nitrate to be the main source of nitrogen, representing up to 75% of the nitrogen uptake (Maguer et al., 2000). The evident decrease in nitrates during the bloom in Arenys (weeks 3–4, 2002) also supports this observation (Fig. 5). Furthermore, detrimental effects of NH4 at relatively low concentrations were found by Su et al. (1993) in lab conditions, while other authors have not confirmed this effect (Arzul et al., 2001). There are many processes implied in nutrient uptake kinetics, as luxury consumption, local inputs and transient nutrient pulses, or the tight relationship between uptake and external concentration. Therefore, it is not realistic to know if a watermass can support a bloom (Smayda, 1997) because the patterns between a given species or a given community and external nutrient concentration (if there are any) can be interpreted in opposite ways. In fact, in the present study there is not any clear pattern between A. minutum and external inorganic nutrient concentrations. In a single case (Syracuse, 2002), the A. minutum bloom finished simultaneously to an increase in ammonium level. Another example from Arenys suggests that the lack of blooms during the last 6 months of the year could be related to ammonium concentrations higher than 5 mM. On the other hand, a peak of 16 mM NH4 in Syracuse (2003) coincided with an A. minutum peak of 105 cells l1. During the A. minutum blooms in our areas, there was a prevalence of dinoflagellates, despite some slight differences in the phytoplankton species composition and dominance. This observation is consistent with the basic principles of community assembly established by Margalef’s classical Mandala (Margalef, 1978; Margalef et al., 1979). According to the Mandala, red tides would occur if high nutrient concentrations coexisted with relatively low turbulence. Thus, a high nutrient load associated with stratified and/or confined waters favours the M. Vila et al. / Harmful Algae 4 (2005) 673–695 691 Table 3 Similarities and differences between A. minutum blooms in Arenys and Syracuse Biological factors A. minutum maximum density Bloom lifespan Presence of vegetative cells Recurrency Cystbed Dominant phytoplankton group during the bloom Dominant species (life-form types) Bloom decay Physical factors Topography Extension (surface area) Depth Water temperature Salinity Physical structures (fronts/stratification) Chemical factors Freshwater influence DIN/PO4 relationship DIN and PO4 limitations NO3/NH4 ratio PO4 Arenys de Mar Syracuse Over 106 cells l1 Over 105 cells l1; usually 1–2 orders of magnitude lower than in Arenys de Mar One week Spring Known since 2001 Not found Dinoflagellates One month All year long Known since 1996 Present and dense Dinoflagellates A. minutum, P. micans, D. sacculus, Scrippsiella spp. (types I and II) Basically grazed by microzooplankton; possible physical dispersion (after raining); parasitic infection (?) L. polyedrum, P. triestinum (A. minutum) (types I –II and V) Unknown Shallow, semi-confined; fishing and leisure harbour 17 ha 0.5–6 m 12–14.5 (8C) 32–38 Inputs of freshwaters regulate the physical structure Shallow, semi-confined; harbour inside a natural bay; shellfish farming activities 700 ha 0.5–8 m at the sampling area; 25–30 m at the entrance 16–24 (8C) 32–37.7 Inputs of freshwaters regulate the physical structure Continental (nitrates and silicates) Around 300 Not limiting Nitrates as the prevalent inorganic nitrogen form Urban (ammonium and phosphate) 16–40 Not limiting Nitrates as the prevalent inorganic nitrogen form; occasional dominance of ammonium nitrogen near riverine inputs Higher values; around 1.0 mM Usually below 1.0 mM development of red tides. A. minutum dominated in Arenys de Mar harbour, causing water discolouration. The second dominant species was P. micans, followed by D. sacculus and Scrippsiella spp. In contrast, A. minutum never prevailed in Syracuse and P. triestinum and L. polyedrum were the main responsible for water discolourations. P. triestinum is a common species also in Arenys de Mar. However, it seems to show a negative association with A. minutum. In years when P. triestinum was abundant, A. minutum did not reach cell concentrations exceeding 105 cells l1 (Vila et al., in press). A. minutum is described by Smayda and Reynolds (2001) as life-form type I, present in relatively shallow, highly nutrient-enriched habitats, similar to our sites. Also other dinoflagellates such as gymnodinioids and the genera Heterocapsa, Scrippsiella, Prorocentrum are representatives of life-form type I and II. A. minutum and L. polyedrum coexisted in an eutrophicated area on the eastern Adriatic coast with alternating dominance (Marasovic et al., 1995). The last species is, however, adapted to bloom during upwelling relaxations (type V) and therefore able to survive within upwelling habitats (Blasco, 1977). The type V life-form (upwelling relaxation taxa), such as L. polyedrum and G. catenatum (single cells), swim at rates about six times faster than they sink, and can 692 M. Vila et al. / Harmful Algae 4 (2005) 673–695 readily ascend to avoid sinking (Smayda, 2002). A. minutum, L. polyedrum, P. micans and Scrippsiella sp. were the main species of the phytoplankton community in upwelling Atlantic coastal waters of Morocco (Tahri-Joutei et al., 2000). Thus, the lack of L. polyedrum blooms in Catalan waters indicates that Arenys and Syracuse are different habitats. Arenys is a harbour much smaller and shallower than Syracuse, with reduced water mixing and limited cell dispersion. Inorganic nutrients, basically nitrates, are highly available in Arenys. The increased confinement of this site is particularly noticeable because it could play an important role in the maintenance of a huge cyst bed (Garcés et al., 2004). In contrast, A. minutum cysts in sediments from Syracuse have never been found (Bravo, pers. comm.). The bloom decay at both sites occurred quickly (in 6–15 days, Fig. 5), after a week (Syracuse) or a month (Arenys) of sustained high cell concentration (>105 cells l1). It is necessary to highlight that despite the A. minutum parasitic infection at Arenys (st. R), the bloom persisted and some weeks later the whole harbour waters became discoloured. The parasite probably belongs to the new genus Parvilucifera (Apicomplexan) recently reported from Scandinavian waters infecting Dinophysis (Norén et al., 1999), in estuaries of northern Brittany (ErardLeDenn et al., 2002, France) after an A. minutum bloom and, in Tarragona harbour (Spain, Delgado, 1999) after an A. catenella bloom. In laboratory cultures, the parasite is capable of removing a significant fraction of dinoflagellate biomass in a short time (Delgado, 1999; ErardLeDenn et al., 2002). However, the effect of this parasite on natural A. minutum populations did not induce to the bloom decrease (Probert, 1999; this study). In Arenys, the bloom decline at st. R and successive increase at st. T coincided with two rain episodes (Fig. 6A). The increased freshwater input in st. R after the rainfall could have rapidly washed the cells from st. R and those would be accumulated at st. T, as it could be seen in the cruise carried out some days later (Fig. 7). Otherwise, a dense population of rotifers detected at the end of January near st. R could have participated in reducing the bloom there (Calbet et al., 2003). During the cruise, P. schwartzii at the st. R area could have been grazing on A. minutum. Rotifers and tintinnida were abundant and co-occurred within the dinoflagellate patch (Fig. 7). As Synchaeta spp. are active grazers on dinoflagellates (Egloff, 1988) and A. minutum cells have often been observed inside tintinnida during bloom events (personal observation), microzooplankton played, probably, an important role in the bloom termination. Averaged ingestion rates 60 cells ind.1 day1 for rotifers and 45 cells ind.1 day1 for tintinnida (which are in the range reported by Calbet et al., 2003 and references therein) means a daily population decrease of 15%. If no growth in the A. minutum population is assumed, which may be quite reliable after 2 months of sustained biomass (>105 cells l1), the bloom would be finished in 5 days. That is in agreement with the sharp decrease of the dinoflagellate bloom between 18 and 24 February 2002 (Fig. 6). However, if the dinoflagellate population is actively growing micrograzers can only modulate population dynamics. 5. Conclusion In summary, the data obtained from the two Mediterranean regions indicates that A. minutum blooms occur in confined or semi-confined water areas. This may be attributed to the low flushing rates (tide amplitude is less than 20 cm in the Mediterranean). Long water residence time seems to be a main factor in the bloom maintenance phase, which could differentiate the Arenys bloom from the Syracuse one. The formation of density fronts due to local freshwater outflows could be one of the factors triggering the bloom, especially in Arenys de Mar harbour considering that it initiates near the freshwater entrance. This physical structure could be critical for the bloom initiation, avoiding cell dispersion and assuring high nutrient levels. Once a critical mass is achieved, the bloom expands over the whole harbour. Although Anderson (1998) suggested that Alexandrium species does not appear to have particularly high growth rates even under optimal physicochemical conditions, high growth rates were detected for A. minutum by in situ measures in Arenys (Garcés et al., 1998) where a dense seedbed has been found (Garcés et al., 2004). According to Probert (1999), the inoculum of a bloom only needs the excystment of a low proportion of the cysts in sediment beds. This explains the high-biomass blooms detected in Arenys de Mar. Thus, local M. Vila et al. / Harmful Algae 4 (2005) 673–695 conditions seem to be a key factor that makes a given locality particularly susceptible to A. minutum blooms. Finally, allelopathy is being considered as a relevant aspect of the Alexandrium ecology, playing a non-negligible role in species interaction, succession and, perhaps, bloom formation (Fistarol et al., 2004). Thus, inferring patterns of species interactions, as well as relationships between organism and (environmental) nutrient concentrations is a complex task. This is especially true in areas influenced by continental inputs (as Arenys and Syracuse) – involving a high small-scale, spatio-temporal variability of environmental parameters. Acknowledgements The authors thank J. Riba, S. Anglés and P. Tezanos (ICM, Catalonia) and A. Rabito (ARPA, Sicily) for their cooperation and efforts in facilitating the field programme. We thank the support of the Club Nautic Arenys de Mar for facilities during the cruise and sampling and especially Sr. D. Lucena for his enthusiastic collaboration. We thank I. Bravo, M. Fernandez and S. Fraga for supplying culture strains, Dr. F. Andreoni for technical support in sequencing analyses, R. Ventosa for nutrient analysis and Dr. A. Calbet for the rotifer identification. Financial support was provided by the EU project STRATEGY (EVK3CT-2001-00046), by the Agència Catalana de l’Aigua (Department de Medi Ambient, Generalitat de Catalunya) and CSIC through the contract ‘‘Plà de vigilància de fitoplàncton nociu i tòxic a la Costa Catalana’’ and by the Italian Ministery MiPAF, Department of Fisheries and Aquaculture (Projects 5C8 and 6C18). References Anderson, D.M., 1989. Toxic algal blooms and red tides: a global perspective. In: Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.), Red Tides: Biology, Environmental Science and Toxicology. Elsevier, New York, pp. 11–16. Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transitions. In: Anderson, D.M., Cambella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. NATO ASI Series, G41, pp. 29–48. 693 Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25 (4b), 704–726. Arzul, G., Seguel, M., Guzman, L., Erard-Le Denn, E., 1999. Comparison of allelopathic properties in three toxic Alexandrium species. J. Exp. Mar. Biol. Ecol. 232, 285–295. Arzul, G., Seguel, M., Clement, A., 2001. Effect of marine animal excretions on differential growth of phytoplankton species. ICES J. Mar. Sci. 58, 386–390. Bagøien, E., Miranda, A., Reguera, B., Franco, J.M., 1996. Effects of two paralytic shellfish toxin producing dinoflagellates on the pelagic harpacticoid copepod Euterpina acutifrons. Mar. Biol. 126, 361–369. Balech, E., 1989. Redescription of Alexandrium minutum Halim (Dinophyceae) type species of the genus Alexandrium. Phycologia 28 (2), 206–211. Balech, E., 1995. The Genus Alexandrium Halim (Dinoflagellata). Sherkin Island Marine Station, Sherkin Island Co., Cork, Ireland. Belin, C., 1993. 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