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).
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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
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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.
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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,
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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).
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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.
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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
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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).
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