Content uploaded by Esther Garcés
Author content
All content in this area was uploaded by Esther Garcés
Content may be subject to copyright.
AQUATIC MICROBIAL ECOLOGY
Aquat Microb Ecol
Vol. 26: 51–60, 2001 Published October 26
INTRODUCTION
Epiphytic (in close association with macroalgae) and
benthic (in coral rubble, sand and detritus) dinofla-
gellates are relevant because the species Gambierdis-
cus toxicus causes ciguatera (Yasumoto et al. 1977,
Adachi & Fukuyo 1979). Ciguatera, or ciguatera fish
poisoning, is a human disease caused by the ingestion
of contaminated marine finfish from tropical and sub-
tropical regions, which results in gastrointestinal and
neurological disorders and sometimes death. Polyether
toxins (ciguatoxins and maitotoxins, among others)
that are produced by marine epiphytic dinoflagellates
(Steidinger 1983) may cause these symptoms. Macro-
algae (and epiphytic assemblages of harmful dinofla-
gellates) are eaten by herbivorous fish, which then
become toxic. Thus, the toxins are biologically concen-
trated within the food chain (Steidinger & Baden 1984).
A dinoflagellate assemblage in the genera Gam-
bierdiscus, Ostreopsis, Coolia, Prorocentrum and
Amphidinium (Ballantine et al. 1985, Carlson & Tindall
1985, Bomber & Aikman 1989, Bourdeau et al. 1995,
Faust 1995) has also been reported in ciguatera-
endemic areas. In particular, Prorocentrum lima, P.
concavum, Ostreopsis siamensis and O. ovata have
been implicated in ciguatera fish poisoning based on
distribution, toxicity to mice and the presence of a fat-
soluble toxic fraction (Yasumoto et al.1980, Nakajima
et al.1981). These organisms form epiphytic communi-
ties associated with coral reefs, or rather with macro-
algae attached to coral surfaces. These assemblages
may vary in species composition and cell concentration
© Inter-Research 2001
*E-mail: magda@icm.csic.es
Potentially toxic epiphytic dinoflagellate
assemblages on macroalgae in the NW
Mediterranean
Magda Vila*, Esther Garcés, Mercedes Masó
Institut de Ciències del Mar, Passeig Marítim de la Barceloneta, 37– 49, 08003 Barcelona, Catalonia, Spain
ABSTRACT: A potentially toxic epiphytic dinoflagellate assemblage on macroalgae was studied for
1 yr in a shallow protected rocky habitat in Palamós (Costa Brava, NW Mediterranean). The assem-
blage was monitored on 4 macroalgae: Corallina elongata (Rhodophyceae), Dictyota dichotoma, Dilo-
phus fasciola and Halopteris scoparia (Phaeophyceae). The dominant dinoflagellates were Ostreop-
sis sp., and the accompanying species were Coolia monotis and Prorocentrum lima. The diatom
Coscinodiscus sp. was an abundant component of the assemblage. Ostreopsis followed the same sea-
sonal pattern on the 4 macroalgae selected. Substrate was not significantly different for the dinofla-
gellate assemblages. Ostreopsis was present both in the water column and in the sand concomitant
with maximal cell densities on macroalgae. Small-scale sampling revealed that all the epiphytic
organisms prefer slightly shaken habitats. While Ostreopsis sp. prefers shaken to slightly shaken
waters, Coolia monotis prefers slightly shaken to calm ones. The dinoflagellate assemblage follows a
clear seasonal pattern, achieving maximum cell concentration during spring and summer without
significant relative changes in the species composition. The epiphytic assemblage was widespread
along the Catalan coast and Majorca, although dinoflagellates were found to be more abundant in
the Costa Brava. In Corsica, diatoms dominated the assemblage, whereas Ostreopsis sp. was a minor
component.
KEY WORDS: Ostreopsis · Coolia monotis · Prorocentrum lima · Benthic dinoflagellates · Ciguatera
fish poisoning
Resale or republication not permitted without written consent of the publisher
Aquat Microb Ecol 26: 51– 60, 2001
between sites (Tindall & Morton 1998). The mixed
association of toxic dinoflagellates may contribute to
the polymorphism of the clinical features of ciguatera
(Yasumoto et al. 1987).
Ostreopsidaceae species are widespread in most epi-
phytic and benthic dinoflagellate communities from
ciguatera-endemic regions of the world (35° N to
35° S). Thus, the geographic distribution of Ostreopsis
siamensis, O. lenticularis and O. ovata is similar to that
of Gambierdiscus toxicus (Tindall & Morton 1998),
with 2 notable exceptions: O. siamensis and O. ovata
have been reported in the Mediterranean Sea (Taylor
1979, Tognetto et al. 1995). Nevertheless, data are
limited on the incidence of Ostreopsis in the waters of
the Mediterranean Sea and on the magnitude of po-
tentially toxic epiphytic dinoflagellate assemblage
attached to macroalgae.
In this study, we quantified epiphytic dinoflagellate
assemblages on the Catalan coast, NW Mediterranean.
The potentially toxic epiphytic dinoflagellate assem-
blage associated with macroalgae was examined dur-
ing an annual cycle in a rocky habitat. In addition,
small-scale spatial variability and middle-scale spatial
distribution were analysed to shed some light on the
epiphytic dinoflagellate assemblages in the NW
Mediterranean.
The dominant dinoflagellate Ostreopsis sp. could not
be assigned to any described species. Thus, a brief de-
scription of the species with scanning electron mi-
crophotographs is included for further considerations.
MATERIAL AND METHODS
Sampling sites. Epiphytic dinoflagellates on selected
macroalgae (Rhodophyceae and Phaeophyceae) were
quantified for 1 yr (July 1997 to July 1998). The sampled
macroalgae grow in multispecies assemblages attached
to stones in the infralittoral. Macroalgal specimens (in
triplicate) were collected weekly at 20 to 40 cm depths
during summer (July and August 1997) and monthly
during the rest of the year. The sampling site was a
shallow protected rocky habitat in Palamós (Catalan
sea, NW Mediterranean). During the summers of 1997
and 1998, a more extensive study was carried out. Four-
teen stations were sampled, mainly along the Costa
Brava (northern Catalan coast) and in 2 other Mediter-
ranean areas (Majorca and Corsica) (Fig. 1).
Sampling methods. A subsample (15 to 20 g fresh
weight [FW]) was carefully cut and placed with tweez-
ers in a small glass bottle containing 10 ml of formalde-
hyde-filtered seawater. Since the macroalgae commu-
nity is dynamic and the species composition varies
throughout the year, at every sampling date the avail-
able macroalgae were taken to cover the annual cycle.
The main macroalgae analysed were Corallina elon-
gata (Rhodophyceae), Dictyota dichotoma, Dilophus
fasciola and Halopteris scoparia (Phaeophyceae). C.
elongata is present throughout the year; thus, we have
studied the seasonal patterns in this macroalga. In
addition, other macroalgae were collected sporadi-
cally, mainly during the cold season, when the 4 target
species were very scarce. The additional macroalgae
sampled were Jania corniculata, Pterocladia capil-
lacea, Laurencia gr. obtusa, Rissoella verruculosa,
Ceramium ciliatum, Peyssonnelia squamaria (Rhodo-
phyceae), Dictyopteris membranacea, Padina pavon-
ica (Phaeophyceae) and Ulva sp. (Chlorophyceae).
Surface water (0.5 m) and sediment samples were
collected in 150 and 50 ml bottles and preserved with
formaldehyde (1% final concentration) for dinoflagel-
late examination. Nutrient samples were taken and
frozen immediately and analysed for nitrate, nitrite,
ammonia, phosphate and silicate as described by
Grasshoff et al. (1983). Temperature and salinity were
measured.
52
N
12º
4º
0º
6º
48º
44º
40º
36º
6º
4º
14
18
15
1617
8º
Fig. 1. Study area and stations sampled. 1: Cala Garbet; 2:
Portlligat; 3: Canyelles Grosses; 4: Cala Portitxol; 5: Cala
Pedrosa; 6: La Foradada; 7: Palamós (sampling site); 8: Gran
de Palamós; 9: Cala Canyet; 10: Santa Cristina d’Aro; 11: El
Xuclador; 12: Sant Pol de Mar; 13: Vallcarca; 14: Riu Sènia;
15: Deià; 16: Cala Fornells; 17: Portals Vells; 18: Campomoro;
1–14 in Catalonia; 15–17 in Majorca; 18 in Corsica
2º3º
42º
41º
N
1
8
2
3
4
5
6
7
10
11
12
13
9
Sampling site
Vila et al.: Epiphytic dinoflagellates in the Mediterranean Sea
Once in the laboratory, macroalgae bottles were
shaken vigorously for 1 min to dislodge the epiphytic
organisms. Macroalgae were removed and the sample
was settled for 6 h in 10 ml counting chambers. An
appropriate area of the chamber was then scanned
(Throndsen 1995) for epiphytic organism counting at
63 to 200×magnification using a Leica-Leitz DM-IL
inverted microscope (Leica Mikroskopie und Systeme
GmbH, Wetzlar). Samples were examined and counted
for epiphytic microalgal species. When high densities
of organisms were found in the sample, only a sub-
sample was examined. Macroalgae were processed for
fresh weight (FW) and dry weight (DW) measure-
ments. DW was measured after the macroalgae were
dried in an oven at 60°C. FW and DW were highly
correlated (regression analysis, r2> 0.98). Thus, we
worked with FW, as is usual in other studies. One-way
ANOVA was performed to test differences between
the 4 macroalgae for each epiphyte dinoflagellate
(4 ×4) (STATISTICA for Windows, Statsoft, Tulsa, OK).
The analysis was done during the warmer months to
avoid seasonal interactions. Water samples for phyto-
plankton quantification were settled for 24 h in 50 ml
counting chambers and they were then examined as
above. Sediment samples (around 30 g) were sonicated
in filtered seawater for 10 to 15 s and sieved. The 20 to
135 µm fraction was examined in an inverted micro-
scope, as described above.
In April 1998, small-scale samples were taken at
Stn 7 to study the spatial variability of epiphytic organ-
isms on Corallina elongata in relation to hydrody-
namism. Three hydrodynamic regimens were defined:
shaken, slightly shaken and calm. Shaken regimens
were observed in sites where macroalgae were
directly hit by waves (high hydrodynamism); calm reg-
imens corresponded to sites where macroalgae were
protected from the waves by rock barriers (low hydro-
dynamism); and the slightly shaken regimens were
intermediate. Macroalgal samples were collected from
the 3 habitats and processed as described above.
Three sites were sampled at each habitat and 3 repli-
cates were analysed. An ANOVA nested design was
used (3 ×3 ×3) (STATISTICA for Windows).
During summer 1997 and 1998, 14 stations were
sampled, mainly along the Costa Brava (northern
region of the Catalan coast, Fig. 1) to limit the geo-
graphical distribution. Samples were also taken from
Corsica (summer 1998) and Majorca (Balearic Islands,
summer 2000) and qualitatively examined for the
epiphytic assemblage to determine the extent of the
phenomenon in the NW Mediterranean.
Identification of dinoflagellates. Samples were
fixed with 4% glutaraldehyde for scanning electron
microscopy. One millilitre of fixed sample was filtered
through a 13 mm diameter and 0.8 µm pore size Nucle-
pore PC polycarbonate membrane filter (Costar,
Europe Ltd, Badhoevedorp). Samples were washed in
distilled water and dehydrated in an ethanol series (30,
50, 70, 80, 90, 100%) at 4°C, critical point dried with
CO2and examined under a Hitachi S-570 scanning
electron microscope (Nissei Sangyo Co. Ltd, Tokyo;
modified from Faust et al. 1996).
RESULTS
Taxonomy of the epiphytic assemblages
Natural populations of benthic dinoflagellates and
diatoms formed a mucilaginous matrix on the macroal-
gal thallus and aggregated therein (Fig. 2A,B). Cells
remained motile within the matrix and loosely linked
to macroalgae, as revealed by light microscopy. When
the epiphyte assemblage was dense, the brownish
mucilaginous matrix covering the surface of the algae
was visible to the naked eye.
The dinoflagellate epiphyte assemblage on macroal-
gae comprised Coolia monotis, Prorocentrum lima and
especially Ostreopsis sp.The highest Ostreopsis sp.
concentration was 596 ×103cells g–1 FW macroalga on
Halopteris scoparia in July 1997, or 6270 ×103cells g–1
DW macroalgae. P. mexicanum and P. emerginatum
were occasionally recorded as minor components of
the community. The benthic diatom Coscinodiscus sp.
was an abundant constituent, sometimes accompanied
by other diatoms such as Striatella sp. and Cylin-
drotheca closterium. Polychaete and crustacean larvae
were often observed.
The morphological features of the dominant species,
Ostreopsis sp., do not match those described else-
where (Fukuyo 1981, Norris et al. 1985, Quod 1994,
Faust & Morton 1995, Faust et al. 1996, Faust 1999).
Ostreopsis cells were usually quite large, pointed
towards the sulcus in apical view and compressed
anteroposteriorly (about 22 µm). The dorsoventral
diameter (length) was 63 to 90 µm (average 75 µm) and
the transdiameter (width) 34 to 56 µm (average 45 µm)
(Fig. 2C,D,E). The pore plate (Po) was about 10 µm
long. On the epitheca, the 1’ plate was large and in
contact with plates Po/2’, 3’, 1’’, 2’’, 6’’ and 7’’. The
external part of the thecal plates was covered with 1
size of pores (0.1 to 0.2 µm diameter).
There may be confusion in the literature about the
morphology of Ostreopsis siamensis and O. lenticu-
laris. O. siamensis, which was first described by
Schmidt (1902), was redescribed by Fukuyo (1981),
when he also described O. lenticularis and O. ovata. O.
siamensis and O. lenticularis were similar in size but
differed in the presence of dissimilar thecal pore sizes.
O. siamensis was found to have 1 size of thecal pore
53
Aquat Microb Ecol 26: 51– 60, 2001
whereas O. lenticularis had 2 sizes (Fukuyo 1981). O.
ovata was smaller and had 1 size of thecal pores. In
contast, Faust et al. (1996) found that O. siamensis was
bigger than O. lenticularis and had 2 sizes of thecal
pores, while O. lenticularis had 1 pore size. Our spe-
cies description agrees with the O. siamensis (in cell
size and number of pore sizes) described by Fukuyo
(1981), but not with that described by Faust et al.
(1996). It also agrees with O. ovata in the number of
pore sizes, although this organism is smaller than our
species. Thus, given the confusion, it was not assigned
any specific name. Taxonomical studies and genetic
assays are in progress (A. Penna in press).
Preferred habitat and seasonal variability
The temporal variability in the physico-chemical
characteristics of the study site (Stn 7) is shown in
Fig. 3A. Water temperature showed marked seasonal-
ity (range 11.5 to 26.3°C). The study site received, dur-
ing periods of rain, freshwater from a small river.
Accordingly, salinity oscillated between 37.2 and
38.1 psu. Nutrient concentrations ranged from 0.11 to
0.86 µM for phospate, 0.76 to 7.74 µM for DIN and
0.17 to 4.51 µM for silicate. A clear temporal variation
was not observed. However, discrete high concentra-
tions of DIN and silicate were observed during winter
and spring.
The concentration of Ostreopsis sp. in 3 habitats
(attached on macroalgae, in the water column and in
sand) at Stn 7 is shown in Fig. 3B. Ostreopsis sp. was
also the dominant species in the water column and
sand. The 3 habitats showed a clear seasonal pattern,
with high biomass from late winter until late summer.
Ostreopsis sp. in the water column achieved high cell
concentrations (>104cells l–1) concomitant with maxi-
mal cell densities on macroalgae (104to 105cells g–1
FW). Cell densities in the water column and on
macroalgae were positively and significantly corre-
54
Fig. 2. Mucilaginous
matrix of epiphytic
dinoflagellates on a
macroalga observed
under (A) light micro-
scope and (B) scanning
electron microscope
(SEM). Ostreopsis sp.
cells viewed with SEM
in (C) epithecal view,
(D) hypothecal view
and (E) left lateral view
Vila et al.: Epiphytic dinoflagellates in the Mediterranean Sea
lated (n = 18, Pearson’s r = 0.82, p < 0.001), espe-
cially for Corallina elongata (n = 11, Pearson’s r =
0.79, p < 0.05) and Halopteris scoparia (n = 8,
Pearson’s r = 0.91, p < 0.05). Cell densities in sand
followed the same seasonal pattern (although this
was not statistically significant, since we worked
near the detection limit) as in the water column
and on macroalgae.There were few Coolia
monotis and Prorocentrum lima in the water col-
umn (maximum cell concentrations 4600 and 330
cells l–1, respectively) and they were mostly
absent from sand, except on some sampling days
(cell concentrations <2 cells g–1).
Ostreopsis followed the same seasonal pattern
on the 4 substrates selected (Corallina elongata,
Dictyota dichotoma, Dilophus fasciola and Halop-
teris scoparia) (Fig. 4A). Maximum concentrations
were found from March to September. Substrate
(macroalgae) was not significant (ANOVA, p >
0.05) for Ostreopsis sp., Prorocentrum lima and
Coolia monotis, but was significant for Cocinodis-
cus sp. (ANOVA, p < 0.05). The analysis was per-
formed during the warmest months to avoid sea-
sonal interactions. Table 1 presents the epiphytic
dinoflagellates on additional macroalgal species
examined all year round. The epiphytic assem-
blage of dinoflagellates was the same, with Ostre-
opsis sp. the most abundant species Coscinodis-
cus also reached high cell numbers (Table 1).
The distribution of potentially toxic epiphytic
dinoflagellate and Coscinodiscus assemblages on
Corallina elongata (Ostreopsis sp., Coolia mono-
tis, Prorocentrum lima and Coscinodiscus sp.) and
monthly relative species abundance are shown in
55
Macroalgae Season n Average (cells g–1 FW) Relative abundance (%)
Os Co Pl Cs Os Co Pl Cs
Pterocladia capillacea Summer 2 2591 90 647 3574 38 1 9 52
Jania corniculata 1 1181 47 331 189 68 3 19 11
Laurencia gr. obtusa Autumn 2 867 0 397 785 42 0 19 38
Laurencia gr. obtusa 3 1410 4 35 16 96 0 2 1
Pterocladia capillacea Winter 4 156 0 1 0 99 0 1 0
Ulva sp. 1 109 0 5 34 74 0 4 23
Risoella verruculosa 10000
Jania corniculata 4 242 5 2 124 65 1 0 33
Pterocladia capillacea 3 133 1 1 39 76 1 1 23
Ceramium ciliatum Spring 3 74147 4681 55 11190 82 5 0 12
Dictyopteris membranacea 3 47976 9557 238 16529 65 13 0 22
Laurencia gr. obtusa 3 134512 11801 130 6740 88 8 0 4
Peyssonnelia squamaria 3 4209 1030 46 2354 55 13 1 31
Padina pavonica 2 56404 33825 481 25475 49 29 0 22
Table 1. Epiphytic assemblage composition in macroalgae (other than 4 target species) from the sampling site (Palamós) year
round. Average epiphytic cell concentration on macroalgae (cells g–1 fresh weight [FW]) and relative abundance (%). Os: Ostre-
opsis sp.; Co: Coolia monotis; Pl: Prorocentrum lima; Cs: Coscinodiscus sp.
B
Fig. 3. (A) Temporal physico-chemical properties of the study area.
Nutrient samples from July to September 1997 were taken approx-
imately 200 m away from the sampling site. Sal: salinity; Temp:
temperature. (B) Seasonal abundance of Ostreopsis sp. in the
macroalgae, in the water column and on the sediment at the sam-
pling site. Cells on macroalgae are the averaged values for the 4
macroalgae sampled. Bars indicate SE. Missing bars correspond to
macroalgae that were not sampled. FW: fresh weight
A
Aquat Microb Ecol 26: 51– 60, 2001
Fig. 4B and C, respectively. The 4 epiphytic species fol-
lowed the same seasonal pattern (n = 34, Pearson’s r =
0.87, p < 0.05). Although Ostreopsis sp. was the most
dominant species, the relative abundances varied
(Fig. 4C). For instance, in April C. monotis achieved
18% relative abundance in C. elongata, whereas it
reached 64% in the other substrate, Dictyota
dichotoma (absolute abundance of 143 ×103cells g–1
FW). P. lima occasionally achieved high relative abun-
dance (28 to 49%), which does not reflect high cell
densities (<200 cells P. lima g–1 FW). On the contrary, it
is caused by low densities on the whole epiphytic com-
munity (80 to 700 cells g–1 FW).
The spatial variability of the epiphytic assemblage
on Corallina elongata is shown in Fig. 5 (April 1998).
The photophilic communities of C. elongata are typi-
cal of shaken or turbulent environments. In the area
sampled, macroalgae were scarce in the calm area
(where fine sandy deposition was observed) and
abundant in slightly shaken or shaken areas. Cell
densities of all epiphytic organisms were the highest
in slightly shaken sites. The dominant species were
Ostreopsis sp. and Coolia monotis, whereas cell densi-
ties on Coscinodiscus sp. (<104cells g–1 FW) and Pro-
rocentrum lima were very low (<102cells g–1 FW).
Ostreopsis sp. was more abundant than C. monotis in
shaken sites (75 vs 19%) and less abundant in calm
sites (8 vs 81%). In slightly shaken sites, the preferred
habitat for Ostreopsis sp. and C. monotis, they were
co-dominant (41 vs 51%) (Fig. 5). Differences among
the 3 regimens (shaken, slightly shaken and calm)
were significant (ANOVA, p < 0.05), but those within
groups (3 sites, 3 replicates) were not significant
(ANOVA, p > 0.05).
Cell abundances are shown from the broader sam-
pling area along Costa Brava in Table 2. The epi-
56
Fig 5. Epiphytic averaged densities (Ostreopsis sp., Coolia
monotis, Prorocentrum lima and Coscinodiscus) on Corallina
elongata in 3 hydrodynamic regimens (shaken, slightly
shaken and calm). Samples were taken in the sampling
station in April 1998 (when cell densities where high, see
arrow in Fig 4B)
Fig. 4. (A) Seasonal abundance of epiphyte Ostreopsis sp. on
the 4 macroalgae: Corallina elongata, Dictyota dichotoma,
Dilophus fasciola and Halopteris scoparia. (B) Seasonal
abundance and (C) percentage of the major epiphytic organ-
isms (Ostreopsis sp., C. monotis, P. lima and Coscinodiscus
sp.) on C. elongata from July 1997 to July 1998. Asterisks
indicate the months in which the macroalgae were not col-
lected. Arrow indicates the day on which sampling was inten-
sive to record the spatial variability associated with 3 hydro-
dynamic regimens
C
A
B
Vila et al.: Epiphytic dinoflagellates in the Mediterranean Sea
phytic assemblage (4 microalgal species enumerated)
is shown on various macroalgae. Dinoflagellates were
most abundant at Stns 6 to 11 (which are the nearest
to the sampled site; Fig. 1). A similar assemblage (on
the fourth main species) was found in Majorca, in
contrast with Corsica, where diatoms dominated the
community and Ostreopsis sp. was a minor compo-
nent.
DISCUSSION
The epiphytic dinoflagellate assemblage in the
Mediterranean: comparison with other places
This is not the first report of the genus Ostreopsis in
the Mediterranean Sea. Taylor (1979) described an
association of Ostreopsis siamensis, Coolia monotis,
57
Macroalgae Stn Abundance (cells g–1 FW) Relative abundance (%)
Os Co Pl Cs Os Co Pl Cs
Catalonia, summer 1997 and 1998
Phaeophyceae
Dictyota dichotoma 1 312 37 73 0 74 9 17 0
Dictyota dichotoma 2 228 2151 554 196 7 69 18 6
Halopteris scoparia 2 0 63 98 0 0 39 61 0
Dilophus fasciola 3 414 61 97 170 56 8 13 23
Halopteris scoparia 3 10 10 105 0 8 8 85 0
Halopteris scoparia 4 0 0 0 1 0 0 0 100
Dictyota dichotoma 5 0 104 367 389 0 12 43 45
Dictyota dichotoma 6 17939 76 284 132 97 0 2 1
Halopteris scoparia 670200250750
Dictyota dichotoma 8 159 26 53 160 40 7 13 40
Halopteris scoparia 8 282 0 311 256 33 0 37 30
Dilophus fasciola 9 0 342 683 506 0 22 45 33
Dilophus fasciola 10 10917 363 60 30 96 3 1 0
Dictyota dichotoma 11 21 10 7573 2195 0 0 77 22
Halopteris scoparia 11 129 39 741 107 13 4 73 11
Dictyota dichotoma 12 0 40 71 0 0 36 64 0
Dictyota dichotoma 13 6 18 0 0 26 74 0 0
Dictyota dichotoma 14 0 0 0 0 0 0 0 0
Rhodophyta
Corallina elongata 1 0 0 7 0 0 0 100 0
Corallina elongata 2 4 59 28 4 4 63 29 4
Corallina elongata 3 25 18 55 0 25 19 56 0
Corallina elongata 4 0 36 7 58 0 36 7 57
Corallina elongata 5 0 0 2 1 11 0 56 33
Corallina elongata 6 77136 1322 1542 331 96 2 2 0
Jania +Corallina 9 24 416 559 309 2 32 43 24
Jania corniculata 10 6543 239 84 6 95 3 1 0
Corallina elongata 11 1090 88 651 158 55 4 33 8
Corallina elongata 12 0 3 0 0 0 100 0 0
Corallina elongata 14 103 0 6 0 94 0 6 0
Majorca, summer 2000
Phaeophyceae
Halopteris scoparia 15 – – + ++
Dictyopteris membranacea 15 ++++ ++ + +
Dilophus fasciola 15 +++ ++ ++ –
Padina pavonica 15 ++++ ++ ++ ++
Halopteris scoparia 16 +++ – + ++++
Halopteris scoparia 17 – – + –
Dictyota +Dilophus 17 ++ + + +
Corsica, summer 1998
Phaeophyceae + Rhodophyta Other diatoms*
Mixed sample 18 ++ – – – ++++
(Corallina, Jania, Dictyota, Halopteris...)
Table 2. Epiphytic assemblage composition in macroalgae from other stations in the NW Mediterranean. Average epiphytic cell
concentration on macroalgae (cells g–1 FW) and relative abundance (%). See Table 1 for species abbreviations. –: absent; +: pre-
sent; ++: low abundance; +++: high abundance; ++++: very high abundance. *Diatoms other than Coscinodiscus
Aquat Microb Ecol 26: 51– 60, 2001
Oxyrrhis marina and Amphidinium sp. in the NW
Mediterranean (Vilefranche-sur-Mer). The occurrence
of O. ovata in the water column was also documented
in the Thyrrhenian Sea (Tognetto et al. 1995). More-
over, low cell concentrations of Ostreopsis cf. siamen-
sis (<200 cells l–1) have been recorded in Andalusia
(Mamán et al. 2000) and Catalonia (Vila et al. 2001).
An association dominated by C. monotis and low con-
centrations of Ostreopsis sp. were reported in other
Mediterranean localities (Ganzirri Lagoon, Sicily)
(Gangemi 2001). However, long-term epiphytic associ-
ations had not been quantified in the Mediterranean
Sea. In this study, the epiphytic microscopic assem-
blage mentioned in the previous section was also
detected in samples from Majorca and Corsica. Thus,
this association is probably common in the NW
Mediterranean Sea.
A similar association, consisting of Ostreopsis sia-
mensis, O. lenticularis, O. ovata, Prorocentrum lima, P.
compressum and Coolia monotis, has been recorded in
northern New Zealand. The dominant species, O. sia-
mensis, accounted for 64 to 85% of the total epiphytic
flora during summer (Chang et al. 2000). Although
Gambierdiscus toxicus and O. lenticularis are co-dom-
inant in many tropical regions (Bagins et al. 1985, Bal-
lantine et al. 1985, Carlson & Tindall 1985, Gillespie et
al. 1985, Bomber & Aikman 1989), G. toxicus has been
recorded only once (and in extremely low concentra-
tions) in northern New Zealand (Chang et al. 2000)
and never on the Catalan coast. There may be a latitu-
dinal gradient that implies different species composi-
tion within the benthic association. What is certain is
that these epiphytic assemblages are not restricted to
tropical and subtropical waters but are present in tem-
perate water as well.
The epiphytic and benthic dinoflagellates from New
Zealand seem to be associated with the lipid-soluble
toxins detected in shellfish from the studied area.
However, the link between toxins and the presence of
Ostreopsis siamensis is not yet clear (Chang et al.
2000). The epiphytic community toxicity in Catalonia
had been tested by injecting the extract (intraperi-
toneally) into mice (modified from the Association of
Official Analytic Chemists 1980, Yasumoto et al. 1980).
The organic fraction was not toxic, whereas the water-
soluble fraction killed the mice in 20 min. The symp-
toms observed in mice were not PSP symptoms.
Instead, they were reminiscent of neurotoxic symp-
toms (E. Cacho pers. comm.). Signs of paralytic shell-
fish poisoning (PSP) were also not detected by HPLC
analysis (J. M. Franco pers. comm.). Our preliminary
results suggest that the toxin is present in the water-
soluble fraction, in disagreement with the study car-
ried out in New Zealand and in agreement with Tindall
et al. (1990), who identified a water-soluble toxin very
similar to maitotoxin (ostreotoxin) in O. lenticularis.
However, the specific toxicity of the Mediterranean
epiphytic community requires further research.
Preferred habitat and seasonal variability
Dinoflagellates in this study were epiphytic on
macroalgae, and low densities were detected in the
water column and on the sediments. Numerous species
of macroalgae host significant numbers of epiphytic
dinoflagellates. They include members of Rodophyta,
Phaeophyta, Chlorophyta and Cyanophyta (Tindall &
Morton 1998). The macroalgae tested in this study,
which correspond mainly to Rodophyta and Phaeo-
phyta, supported high densities of epiphytic dinofla-
gellates. The highest density detected for Ostreopsis
sp. in this study was 5.9 ×105cells g–1 FW in Halopteris
scoparia during July 1997. To our knowledge, this is
one of the highest densities of epiphytic species ever
reported. For example, the highest density of Ostreop-
sis lenticularis was estimated to be 2.35 ×105cells g–1
FW on the macroalga Dictyota at Laurel Reef, Puerto
Rico (Ballantine et al. 1985) and that of Gambierdiscus
toxicus was estimated to be 5.0 ×105cells g–1 FW on
Jania in a Gambier Island reef (Yasumoto et al. 1980).
At Virgin Islands, Coolia monotis density was 1.2 ×106
cells g–1 FW macroalgae and that of Prorocentrum
mexicanum was 1.5 ×106cells g–1 FW macroalgae
(Carlson & Tindall 1985). However, the maximum den-
sities of epiphytic species commonly range from 102to
104cells g–1 FW macroalgae (Tindall & Morton 1998).
Significant differences in epiphytic densities between
macroalgae were not detected in this study, in agree-
ment with Taylor (1985), Lobel et al. (1988) and
Bomber et al. (1989), who stated that epiphytic dinofla-
gellates prefer 3-dimensional, flexible, high-surface
area algae, like the macroalgae sampled in this study,
rather than a particular macroalgal species or phylum.
Cell densities around 10 ×103to 20 ×103cell l–1 were
sometimes recorded during warm months in the water
column at the sampling site (Stn 7). However, bloom
concentrations of Ostreopsis were never detected in
the water column and Ostreopsis was very scarce on
the sediment. The positive and significant correlation
of Ostreopsis sp. concentrations in the water column
and sediment with those on macroalgae probably indi-
cates that the former were resuspended or released
from the surface of macroalgae. The presence of Ostre-
opsis sp. in the water column on the Catalan coast has
been well documented since the beginning of routine
monitoring in 1995 (Vila et al. 2001). Ostreopsis sp. in
the water column has occasionally been detected, but
in cell densities lower than 100 cells l–1. High densities
of Ostreopsis sp. were observed only in Garraf harbour
58
Vila et al.: Epiphytic dinoflagellates in the Mediterranean Sea
(5 ×103to 78 ×103cells l–1) during autumn 1997 (near
Stn 13), concomitant with wrested macroalgae that
were floating during the sampling days, and in Blanes
harbour (98 ×103cells l–1) on October 27, 1997 (near
Stn 10), after a heavy storm. Thus, the preferred habi-
tat of Ostreopsis sp. in the Catalan sea is epiphytic on
macroalgae. In coral reef areas, these organisms were
mostly associated with macroalgae located between
0.5 and 3 m (Ballantine et al. 1985, Carlson & Tindall
1985, Bomber & Aikman 1989), but macroalgae
attached to mangrove roots and dead coral pavement
did not support high numbers of dinoflagellates (Carl-
son & Tindall 1985). In contrast, dead corals colonised
by algal turf to various extents had higher epiphytic
dinoflagellate densities than macroalgal substrates in
Mayotte Island (SW Indian Ocean) (Grzebyk et al.
1994, Quod 1994).
The dinoflagellate assemblage on macroalgae fol-
lows a clear seasonal pattern in response to several
factors, probably the same factors that trigger spring
growth of macroalgae in the area (Ballesteros 1992):
increase in temperature and irradiance at the begin-
ning of spring, calmer sea (the spatial variability due to
hydrodynamic regimens is significant) and availability
of substrate. No significant correlations were observed
between epiphytic organisms and water temperature
or nutrients. Gillespie et al. (1985) showed that period-
icity in the densities of Gambierdiscus toxicus was not
directly linked to temperature. They found the maxi-
mum density in water at temperatures near 20°C and
before the maximum temperature was reached. Here,
no clear seasonal pattern in the relative abundance of
epiphytic organisms was observed (e.g., substitution of
dinoflagellates vs diatoms). The dominant dinoflagel-
late was mostly Ostreopsis sp., although Coolia mono-
tis and Prorocentrum lima occasionally achieved high
absolute and relative numbers. The mechanisms that
trigger species abundance are unclear, but changes in
the hydrodynamic regime many be involved. Spatial
variability (Fig. 5) indicates that although C. monotis
and Ostreopsis sp. are better adapted to slightly
shaken environments, C. monotis outnumbers Ostre-
opsis sp. in calm waters; however, the former is
excluded by Ostreopsis sp. in shaken waters.
In autumn, the input of external energy from storms
and rains exerts a negative effect on the macroalgae,
which simplify the macroalgal community structure
and reduce the biomass (Ballesteros 1992). These con-
ditions may also affect all epiphytic organisms. Differ-
ences in cell concentration during warm and cold
months may also result from the carrying capacity of
macroalgae (Lobel et al. 1988), which varies according
to hydrodynamic characteristics (Tindall & Morton
1998). Each species of macroalga has a characteristic
surface area or space, which, once occupied, can sup-
port no additional cells. In high turbulence conditions,
this space is limited to the surface layer, whereas in
stagnant conditions dinoflagellates multiply to fill all
the spaces within the macroalgal canopy. In highly tur-
bulent conditions, when no additional cells can be sup-
ported by macroalgae, dinoflagellates continuously
migrate to adjacent areas. Bomber et al. (1989) sug-
gested that the presence of Prorocentrum spp., Coolia
monotis and Ostreopsis siamensis in the water column
is due to vertical migration, which facilitates cell redis-
tribution and concentration. Thus, the epiflora does not
strictly depend on a given macroalga because under
certain circumstances (e.g., macroalga death) they can
migrate and colonise other algae.
The high cell concentrations recorded during sum-
mer for the 4 target macroalgae are attributed to the
less shaken and more stable water environments,
similar to the Type II system described by Tindall &
Morton (1998). On the other hand, the low cell concen-
trations recorded during winter months, which are
probably associated with more shaken fluxes, can be
compared to the Type I system.
In conclusion, the epiphytic associations of Ostreop-
sis sp., Coolia monotis, Prorocentrum lima and Coscin-
odiscus sp. are characteristic along the Catalan coast,
NW Mediterranean. Further research in other areas of
the Mediterranean Sea is required to define the spatial
distribution and to determine whether such an associa-
tion exists.
Acknowledgements. We thank M. Delgado, E. Ballesteros
and collaborators for sampling assistance, E. Ballesteros for
the taxonomical identification of macroalgae, S. Fraga for
comments and suggestions on scanning electron microscopy
protocols and Ostreopsis taxonomy, and E. Cacho and J. M.
Franco for toxicity analysis. J. M. Fortuño, R. Fernández and
R. Ventosa provided technical assistance. The authors thank
J. Camp for critical reading of the manuscript and his support
of our work. Financial support was provided by the ACA
(Departament de Medi Ambient, Generalitat de Catalunya)
and CSIC.
LITERATURE CITED
Adachi R, Fukuyo Y (1979) The thecal structure of a marine
toxic dinoflagellate Gambierdiscus toxicus gen. et sp. nov.
collected in a ciguatera-endemic area. Bull Jpn Soc Sci
Fish 45:67–71
Association of Official Analytic Chemists (1980) Standard
mouse bioassay for paralytic shellfish toxins. In: Horwitz
W (ed) Official methods of analysis, 13th edn. Association
of Official Analytic Chemists, Washington, p 298–299
Bagins R, Bennet J, Prieur C, Legrand AM (1985) The dynam-
ics of three toxic benthic dinoflagellates and the toxicity of
ciguateric surgeonfish in French Polynesia. In: Anderson
DM, White AW, Baden DG (eds) Toxic dinoflagellates.
Elsevier Science Publisher, New York, p 177–182
Ballantine DL, Bardales AT, Tosteson TR, Durst HD (1985)
Seasonal abundance of Gambierdiscus toxicus and Ostre-
59
Aquat Microb Ecol 26: 51– 60, 2001
opsis sp. in coastal waters of southwest Puerto Rico. Proc
5th Int Coral Reef Congress, Tahiti 4:417–422
Ballesteros E (1992) Els vegetals i la zonació litoral: espècies,
comunitats i factors que influeixen en la seva distribució.
Institut d’Estudis Catalans, Barcelona
Bomber JW, Aikman KE (1989) The ciguatera dinoflagellates.
Biol Oceanogr 6:291–311
Bomber JW, Rubio MG, Norris DR (1989) Epiphytism of
dinoflagellates associated with ciguatera: substrate speci-
ficity and nutrition. Phycologia 28:360– 368
Bourdeau P, Durand-Clement M, Ammar M, Fessard V (1995)
Ecological and toxicological characteristics of benthic
dinoflagellates in a ciguateric area (Saint Barthelemy:
French West Indies). In: Lassus P, Arzul G, Erard E, Gen-
tien P, Marcaillou C (eds) Harmful marine algal blooms.
Lavoisier, London, p 133–137
Carlson RD, Tindall DR (1985) Distribution and periodicity of
toxic dinoflagellates in the Virgin Islands. In: Anderson
DM, White AW, Baden DG (eds) Toxic dinoflagellates.
Elsevier, New York, p 171–176
Chang FH, Shimizu Y, Hay B, Stewart R, Mackay G, Tasker R
(2000) Three recently recorded Ostreopsis spp. (Dino-
phyceae) in New Zealand: temporal and regional distribu-
tion in the upper North Island from 1995 to 1997. NZ J Mar
Freshw Res 34(1):29–39
Faust MA (1995) Benthic, toxic dinoflagellates: an overview.
In: Lassus P, Arzul G, Erard E, Gentien P, Marcaillou C
(eds) Harmful marine algal blooms. Lavoisier, London,
p 847–854
Faust MA (1999) Three new Ostreopsis species (Dino-
phyceae): O.marinus sp. nov., O. belizeanus sp. nov., and
O. carribeanus sp. nov. Phycologia 38(2):92– 99
Faust MA, Morton SL (1995) Morphology and ecology of the
marine dinoflagellate Ostreopsis labens sp. nov. (Dino-
phyceae). J Phycol 31:456– 463
Faust MA, Morton SL, Quod JP (1996) Further SEM study of
marine dinoflagellates: the genus Ostreopsis (Dino-
phyceae). J Phycol 32:1053–1065
Fukuyo Y (1981) Taxonomical study on benthic dinoflagel-
lates collected in Coral Reefs. Bull Jpn Soc Sci Fish 47(8):
967–978
Gangemi E (2001) Dinoflagellati nocivi nelle fitocenosi di un
ecosistema litorale della Sicilia Orientale (Pantano
Grande — Lagune di Capo Peloro, Me). PhD thesis, Uni-
versità di Messina, Sicily
Gillespie NC, Holmes MJ, Burke JB, Doley J (1985) Distribu-
tion and periodicity of Gambierdiscus toxicus in Queens-
land, Australia. In: Anderson DM, White AW, Baden DG
(eds) Toxic dinoflagellates. Elsevier, New York, p 183–188
Grasshoff K, Ehrhardt M, Kremling K (1983) Methods of sea
water analysis. Ed Verlag Chemie, Weinheim
Grzebyk D, Berland B, Thomassin BA, Bossi C, Arnoux A
(1994) Ecology of ciguateric dinoflagellates in the coral
reef complex of Maynotte Island (S.W. Indian Ocean).
J Exp Mar Biol Ecol 178:51–66
Lobel PS, Anderson DM, Durand-Clement M (1988) Assess-
ment of ciguatera dinoflagellate populations: sample vari-
ability and algal substrate selection. Biol Bull 175:91–101
Mamán L, Fernández L, Ocaña A, Marco JJ, Morales J,
Caballos M, Márquez I, Aguilar M (2000) Seguimiento de
fitoplancton tóxico en la costa de Andalucía durante los
años 1997 y 1998. In: Márquez I (ed) VI Reunión Ibérica de
Fitoplancton Tóxico y Biotoxinas Sevilla, Junta de
Andalucía, p 41–49
Nakajima I, Oshima Y, Yasumoto T (1981) Toxicity of benthic
dinoflagellates in Okinawa. Bull Jpn Soc Sci Fish 47:
1029–1033
Norris DR, Bomber JW, Balech E (1985) Benthic dinoflagel-
lates associated with ciguatera from Florida Keys. I. Ostre-
opsis heptagona sp. nov. In: Anderson DM, White AW,
Baden DG (eds) Toxic dinoflagellates. Elsevier, New York,
p 39– 44
Penna A, Gangemi E, Vila M, Giacobbe MG, Garcés E, Flo-
rean S, Androni F, Magnani M (in press) A preliminary
survey of Ostreopsidaceae (Dinophyta) in the Mediteran-
nean: molecular and morphological analyses of different
isolates. Phycologia 40(Suppl):51
Quod JP (1994) Ostreopsis mascarensis sp. nov. (Dino-
phyceae), dinoflagellé toxique associé à la ciguatera dans
l’Océan Indien. Cryptogamie. Algol 15(4):243–251
Schmidt J (1902) Flora of Koh Chang. Contritution to the
knowledge of the vegetation in the Gulf of Siam. Part IV.
Peridiniales. J Bot 23:212–218
Steidinger KA (1983) A re-evaluation of toxic dinoflagellate
biology and ecology. In: Round FE, Chapman D (ed)
Progress in phycological research. New York, Elsevier,
p 147–188
Steidinger KA, Baden DG (1984) Toxic marine dinoflagel-
lates. In: Spectot DL (ed) Dinoflagellates. Academic Press,
New York, p 201–261
Taylor FJR (1979) A description of the benthic dinoflagellate
associated with maitotoxin and ciguatoxin, including
observations on Hawaiian material. In: Taylor DL, Seliger
HH (eds) Toxic dinoflagellate blooms. Elsevier North
Holland, New York, p 71–76
Taylor JFR (1985) The distribution of the dinoflagellate Gam-
bierdiscus toxicus in eastern Caribbean. In: Salvat B (ed)
Proc 5th Int Coral Reef Congress, Moorea, p 423 –428
Throndsen J (1995) Estimating cell numbers. In: Hallegraeff
GM, Anderson DM, Cembella AD (eds) Manual on harm-
ful marine microalgae. IOC Manuals and Guides No. 33.
UNESCO, Paris, p 63– 80
Tindall DR, Miller DM, Tindall PM (1990) Toxicity of Ostreop-
sis lenticularis from the British and United States Virgin
Islands. In: Graneli E, Sundström B, Edler L, Anderson DM
(eds) Toxic marine phytoplankton. Elsevier, New York,
p 424– 429
Tindall DR, Morton SL (1998) Community dynamics and
physiology of epiphytic/benthic dinoflagellates associated
with Ciguatera. In: Anderson DA, Cembella AD, Hallegra-
eff GM (eds) Physiological ecology of harmful algal
blooms. Springer-Verlag, Berlin, p 293–313
Tognetto L, Bellato S, Moro I, Andreoli C (1995) Occurrence
of Ostreopsis ovata (Dinophyceae) in the Tyrrhenian Sea
during summer 1994. Bot Mar 38:291–295
Vila M, Camp J, Garcés E, Masó M, Delgado M (2001) High
resolution spatio-temporal detection of HABs in confined
waters of the NW Mediterranean. J Plankton Res 23(5):
497–514
Yasumoto T, Nakajima I, Bagins R, Adachi R (1977) Finding of
a dinoflagellate as a likely culprit of ciguatera. Bull Jpn
Soc Sci Fish 43:1021–26
Yasumoto T, Oshima Y, Murakami Y, Nakajima I, Bagnis R,
Fukuyo Y (1980) Toxicity of benthic dinoflagellates found
in coral reef. Bull Jpn Soc Sci Fish 46(1):327– 331
Yasumoto T, Seino N, Murakami Y, Muata M (1987) Toxins
produced by benthic dinoflagellates. Biol Bull 172:
128–131
60
Editorial responsibility: David Caron,
Los Angeles, California, USA
Submitted: April 18, 2001; Accepted: July 16, 2001
Proofs received from author(s): October 17, 2001
