Dinoflagellate cysts from surface sediments of Syracuse Bay (Western Ionian Sea, Mediterranean)

ARTICLE IN PRESS Deep-Sea Research II 57 (2010) 243–247

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Dinoflagellate cysts from surface sediments of Syracuse Bay (Western Ionian Sea, Mediterranean) Fernando Rubino a,n, Manuela Belmonte a, Carmela Caroppo a, Mariagrazia Giacobbe b a b

Istituto per l’Ambiente Marino Costiero, CNR, Talassografico ‘‘A. Cerruti’’, Via Roma 3, 74100 Taranto, Italy Istituto per l’Ambiente Marino Costiero, CNR, Spianata S. Raineri 86, 98122 Messina, Italy

a r t i c l e in fo

abstract

Available online 18 September 2009

The occurrence and abundance of dinoflagellate cysts were investigated for the first time at an Ionian locality along the south-eastern coast of Sicily, subject to spring–summer harmful algal events. Thirtyfour cyst morphotypes were recognized belonging to 24 taxa identified at least at the genus level. Cyst abundance in surface sediments ranged from 43 to 828 cysts g  1 dry weight, with the highest numbers recorded at the most restricted station. Germination experiments allowed confirmation of species identification determined by cyst analysis and provided clonal cultures of Alexandrium minutum and Gymnodinium nolleri, two of the bloom-forming species in the area. This represents the first record of G. nolleri for the Mediterranean Sea. & 2009 Elsevier Ltd. All rights reserved.

Keywords: Plankton Resting stages Dinoflagellates Toxic species Mediterranean Sea Syracuse Bay

1. Introduction Many marine plankters, including species causing red tides, shellfish poisoning and other harmful events, have a resting stage (cyst) as part of their life cycle (Blackburn and Parker, 2005). In confined basins, usually characterised by low hydrodynamic conditions and high productivity, cysts produced in the water column sink to the sediments where they may remain for many years, constituting a reservoir of potential biodiversity (Dale, 1983; Belmonte et al., 1995). The presence of benthic resting stages in the life cycle of planktonic organisms has pointed the need to re-articulate the classical ecological models of plankton dynamics in the neritic habitat (e.g., Boero, 1994), and to reconsider pelagic–benthic fluxes not only in terms of biogeochemical cycles (Marcus and Boero, 1998). Ideally, we should study the pelagic/benthic system as a whole, in spite of the classical methodological compartmentation (Boero et al., 1996). Cysts found in the sediment will indicate to some extent those planktonic species that might develop in the water column (Moscatello et al., 2004). Therefore, knowledge of cyst deposits can provide us with valuable information as to future events in a given area and, thus, are of strategic importance in anticipating toxic or non-toxic plankton blooms (Stock et al., 2007). The objective of the present work was to investigate, for the first time, the occurrence of dinoflagellate cysts in the sediments of Syracuse Bay, a Mediterranean locality where toxic phytoplankton blooms recurrently occur (Vila et al., 2005; Giacobbe et al., 2006). Blooms

n

Corresponding author. Tel.: +39 099 4542 225; fax: +39 099 4542 215. E-mail address: [email protected] (F. Rubino).

0967-0645/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr2.2009.09.011

of Alexandrium minutum, Lingulodinium polyedrum and other dinoflagellates in this eutrophic area of the Mediterranean may reach proportions of 106 cell l  1 during the spring–summer season, coinciding in some cases with PSP toxicity of farmed shellfish that may exceed the safety threshold for human consumption. Syracuse Bay represents an optimal site for such a study as it is a semi-enclosed, shallow, highly productive area, hosting shellfish farms and receiving freshwater inputs. The species composition and abundance of dinoflagellate cysts at this Mediterranean locality are reported, providing new insights into our understanding of harmful bloom dynamics at this site, as well as novel records of species for the area.

2. Methods 2.1. Study site Syracuse Bay (371030 N, 151170 E) (Fig. 1) is located on the SE coast of Sicily (Ionian Sea), inside a wide gulf bounded to the north by Ortigia Island and to the south by the Maddalena Peninsula. The bay measures some 3.5 km along the NS axis, and some 2.0 km along the EW axis (total area700 ha). Water depths are 0.5–8 m over the sampling area and 25–30 m at the entrance to the bay. The harbour (Porto Grande) is located close to the inner city and is subject to freshwater inputs (riverine and springwater). Moreover, for about 20 years, the area has received urban discharge from Syracuse, amounting to 9  106 m  3 y  1 of purified sewage through the Grimaldi-Pantanelli canal. The bay has a limited water exchange, a high nutrient supply from organic

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Fig. 1. Sampling stations in Syracuse Bay, Ionian coast of Sicily (March 2007).

Table 1 Geographic coordinates of the sampling stations in Syracuse Bay and main chemical–physical data registered at water surface during the sample collections (March 2007). Station 1 2 3 4

Name Harbour Aretusa springwater Anapo-Ciane river Shellfish farm

Latitude N 0

371103 51’’ 371030 21’’ 371030 24’’ 371020 09’’

Longitude E 0

15117 01’’ 151170 40’’ 151160 22’’ 151170 20’’

Depth (m)

T (1C)

S (ppt)

Diss. O2(%)

PO4(lM)

NH4(lM)

NO3(lM)

NO2(lM)

3.5 4.0 1.0 6.0

19.50 18.30 19.70 18.40

33.80 21.60 18.60 31.60

85.00 79.20 84.80 127.90

0.50 0.52 0.80 0.20

1.25 2.01 1.38 1.27

14.84 1.21 13.54 1.53

0.36 0.23 0.06 0.05

and inorganic sources (see Table 1) and is characterised by evident eutrophy with regular water discoloration events especially during the spring–summer period. 2.2. Hydrography and nutrients Temperature, salinity and dissolved oxygen were measured at the water surface at each station using a WTW Multiline F/Set-3 multiparametric probe (Table 1). Surface water samples for nutrient analyses were frozen immediately after collection. Concentrations of nitrate, nitrite and phosphate were determined in the laboratory following the Strickland and Parsons (1972) method, and ammonia according to the Aminot and Chaussepied (1983) method. Nutrient samples were analysed using a spectrophotometer (Cary 50, Varian). 2.3. Sediment sample collection and analysis Surface sediment samples (top 3 cm) were collected by SCUBA diving at four stations (Fig. 1; Table 1) in March 2007. Once collected, samples were stored in the dark at 4 1C until processing. After homogenization of the entire sediment sample, subsamples of 3–5 ml were screened through a 20-mm mesh (Endecott’s LTD steel sieves, ISO3310-1, London, England), using natural filtered (0.45 mm) seawater. The retained fraction was gently ultrasonicated for 1 min and sieved again through 125, 75 and 20-mm mesh sizes, producing a fine-grained fraction containing protistan cysts (20–75 mm) and a 475 mm fraction with larger dinoflagellate resting stages (e.g., Polykrikos spp. and Lingulodinium spp.). Material retained on the 125-mm mesh was discarded. No chemicals were used to disaggregate sediments in order to

avoid the dissolution of calcareous and siliceous cyst walls. Qualitative and quantitative analyses were carried out on an inverted microscope (Zeiss Axiovert S100 equipped with a Nikon Coolpix 990 digital camera) at 200 and 320 magnifications. Both full (i.e. presumably viable) and empty (germinated) cysts were recorded. At least 200 viable cysts were counted for the finest fraction, although for station 3 quantitative analysis was performed on 1/10 of the whole sample volume due to the very low cyst abundance (coarse sediment). In all samples the 475 mm fraction was entirely examined. Cysts were identified using published descriptions and germination experiments. Identification was performed at the species level. When this was not possible, higher taxa were considered. As a rule, biological names were used. Only for Bicarinellum tricarinelloides has the palaeontological name been used because the motile stage has not yet been described. Quantitative data are reported only for living cysts, as cysts g  1 of dry weight sediment. 2.4. Germination experiments and culturing Single viable cysts were isolated for germination with a micropipette and placed into Nunclon microwells (Nalge Nunc International, Roskilde, Denmark) containing E1 ml of natural sterilised seawater. Cysts were incubated at 21 1C, 14:10 h LD cycle and 100 mE m  2 s  1 irradiance and were examined daily until germination or were discarded after 30 days of unsuccessful incubation. Clonal cultures of A. minutum and Gymnodinium nolleri were established from single excysted cells. Each cell was placed into a new microwell filled with diluted (1/10 in volume) Guillard’s f/2 culture medium (Guillard, 1975). Once a minimum number of 20

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swimming cells were observed, they were transferred into 270 ml culture flasks. Culture incubation conditions were the same as for incubated cysts. Two cultures for each species are maintained in the collection of the IAMC-CNR Talassografico of Taranto (A. minutum CNR-TA-AM-E9A12, A. minutum CNR-TA-AM-B8A12; G. nolleri CNR-TA-GN-B7A14, G. nolleri CNR-TA-GN-C3A14).

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Three species dominated at each station (Fig. 12): A. minutum, G. nolleri (Figs. 5a, b), L. polyedrum (two cyst types, Table 2, see Ellegaard, 2000), Pentapharsodinium tyrrhenicum and Scrippsiella trochoidea accounting for more than 80% of the total cyst abundance. Resting stages of 3 Alexandrium species were collected, i.e., A. minutum, A. cf. tamarense and one unidentified species (Fig. 2a, b), with A. minutum cysts occurring at all the stations sampled. Cysts of this latter species represented 3–36% of the total stock of dinoflagellate cysts found at each sampling point, with the highest incidence at the springwater station (Station 2, Sal.=21.6, Table 1); 4–18% of the cyst stock was contributed by G. nolleri, 18–45% by L. polyedrum and 6–37% by S. trochoidea. Among organic-walled cysts there were some interesting observations, such as those of two Gymnodiniales species. The first (Fig. 3a) is a cyst reported mainly from Korea and Japan but recently collected from Mediterranean sediments (Rubino et al., 2009). This cyst geminated producing a motile stage identified as Cochlodinium polykrikoides (Fig. 3b). The second (Fig. 6a, b) has a

3. Results Thirty-four dinoflagellate cyst morphotypes were recognized (see list in Table 2) and thirty-one of them were identified at least at the genus level. Two morphotypes remained undetermined and are reported here as Dinophyta sp.1 and Dinophyta sp.2 (Figs. 2–11). Total cyst abundance ranged from 43 to 828 cysts g  1 (Table 2). The highest abundance was registered at the most restricted station (St.1) and the lowest cyst abundance was observed at Station 3, a very shallow site located in front of a small river mouth.

Table 2 Cyst densities (cysts g  1 dw sediment) reported for each taxon at the 4 sampling stations.

Dinophyta Alexandrium minutum Halim Alexandrium cf. tamarense (Lebour) Balech Alexandrium sp. Bicarinellulm tricarinelloides Versteegh Cochlodinium polykrikoides Margalef Diplopsalis lenticula Bergh Diplopsalis sp. Gonyaulax sp. Gymnodinium impudicum (Fraga & Bravo) Hansen & Moestrup Type a Gymnodinium impudicum (Fraga & Bravo) Hansen & Moestrup Type b Gymnodinium nolleri Ellegaard & Moestrup Gymnodiniales sp. Lingulodinium polyedrum (Stein) Dodge Lingulodinium polyedrum (stein) Dodge Early type Pentapharsodinium tyrrhenicum (Balech) Montresor et al. Type a Pentapharsodinium tyrrhenicum (Balech) Montresor et al. Type b Polykrikos kofoidii Chatton Polykrikos schwartzii Butschli Protoperidinium conicum (Gran) Balech Protoperidinium leonis (Pavillard) Balech Protoperidinium oblongum (Aurivillius) Parke & Dodge Type a Protoperidinium oblongum (Aurivillius) Parke & Dodge Type b Protoperidinium thorianum (Paulsen) Balech Protoperidinium sp. Scrippsiella lachrymosa Lewis Scrippsiella trochoidea (Stein) Loeblich Large type Scrippsiella trochoidea (Stein) Loeblich Medium type Scrippsiella trochoidea (Stein) Loeblich Small type Scrippsiella trochoidea (Stein) Loeblich Smooth type Scrippsiella sp. 1 Scrippsiella sp. 2 Scrippsiella sp. 3 Dinophyta sp. 1 Dinophyta sp. 2 No. of morphotypes Cysts g  1dry weight sediment

St.1 Harbour

St.2 Aretusa springwater

St.3 Anapo-Ciane river

St.4 Shellfish farm

Germination

48

207 3 3

11

12

  

2 2 3 2 5

3 3 6 12

5

   

3

 6 133

5 20

319

102

21 51 2 72 5

   

12

 54

69

24

 3 6 2



5 3

 3

 5



24 21 3

3

5

9

5

15

92

77

65

9

13

43

12 30

5 8 15 15 5 3

10 12 14

   

12

24 828

21 573

32

2 43

19 365

  

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Figs. 2–11. Cysts of planktonic dinoflagellates isolated from surface sediments of Syracuse Bay in March 2007. Scale bars 20 mm. 2.Alexandrium sp. (a) Living ellipsoidal cyst with small inner granules concentrated in the central part and a red accumulation body. (b) Germinated motile planomeiocyte. 3.Cochlodinium polykrikoides. (a) Living cyst with granular content and a central red accumulation body. (b) Germinated motile cell. 4.Gymnodinium impudicum. Cyst type ‘‘a’’, non-sticky, with a large red accumulation body and a rounded outline. 5.Gymnodinium nolleri. (a) Living cyst with granular content and a well-visible red accumulation body. (b) Two-cell motile chain. 6. Gymnodiniales sp. (a) Living cyst with an ellipsoidal inner body and the red accumulation body. (b) Empty cyst with apical archeopyle and a sort of paracingulum sharpened by two parallel ridges running along the girth of the cyst. 7.Scrippsiella sp.1. Living cyst with granular content. 8.Scrippsiella sp.2. Living cyst with short spines and granular content. 9.Scrippsiella sp.3. Living cyst with calcareous wall and short spines. 10. Dinophyta sp.1. Living cyst with granular content and a small red accumulation body. 11. Dinophyta sp.2. Living cyst with large accumulation bodies and short spines.

complete documentation, so it is reported here as Gymnodiniales sp. Two cyst types of Gymnodinium impudicum were identified. One is the well-known sticky cyst originally described by Sonneman and Hill (1997), while the other is spheroidal, non-sticky, with a large red accumulation body (Fig. 4). The wall is smooth, covered by some mucus material and the archeopyle is an irregular slit in the centre of the body. Cyst diameter was 31 mm. This cyst has not previously been described in the literature. Finally, three different cyst types (Figs. 7–9) are reported here as Scrippsiella species on the basis of the observation of excysted cells.

4. Discussion

Fig. 12. Cyst bank produced by dinoflagellates at the sampling stations in Syracuse Bay. The dominant species are indicated in the legend. Blank spaces in each bar indicate all the other taxa.

distinctive morphology, with an ellipsoidal body inside the roughly cylindrical wall that showed apical and antapical protrusions and an evident paracingulum made by two parallel ridges running along the entire girth of the cyst. A red body was visible in the central part or towards the apex. The archeopyle is tremic, sensu Matsuoka (1985). The cyst was 37.5 mm long and 28 mm wide (mean, n =3). One specimen germinated producing a swimming cell tentatively identified as Cochlodinium sp. Unfortunately after few days of active swimming the cell died, preventing

In this study, we have provided the first data about abundance and composition of dinoflagellate cyst assemblages, including toxic species, in Syracuse Bay, a critical area of the Mediterranean Sea subject to recurrent harmful algal blooms. Although this is a preliminary survey, the data highlight the presence, in surface sediments, of a diverse and abundant dinoflagellate cyst deposit. The data also highlight a close correspondence between water column and sediments, as the main bloom-forming species (A. minutum, G. nolleri, L. polyedrum and S. trochoidea) were dominant in the sediments. As vegetative cells of these species were rarely observed in the water column in periods other than the spring–summer, this indicates that the seed population for Syracuse blooms may start from an inoculum of excysted cells, the cue for cyst germination possibly being warmer temperatures, together with increased light availability in this season.

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This initial investigation of dinoflagellate cysts in Syracuse sediments allowed us to detect the presence of species that have never been observed in the water column, e.g. P. tyrrhenicum, Protoperidinium thorianum and Scrippsiella lachrymosa. The finding of Gymnodinium impudicum is also considered to be the first record for the area. Two cyst morphotypes produced by this species were found. One of these, here reported as G. impudicum type a (Fig. 4), has never been reported in the literature. This cyst is commonly found in surface sediments of another semi-enclosed Mediterranean area, the Mar Piccolo of Taranto in high abundance (Rubino, pers. observ.). Further analyses are in progress to ascertain its role in the life cycle of the species. A second interesting morphotype was identified as Gymnodiniales sp. (Figs. 6a, b). This cyst appears identical to those reported as ‘‘morphotype A’’ by Morquecho and Lechuga-Deve´ze (2004) from Bahia Concepcion and as Gymnodinioid sp.1 from Lisbon Bay (Ribeiro and Amorim, 2008). Our findings confirmed this last observation, i.e. the existence of a new cyst morphotype among those produced by species of the order Gymnodiniales. Finally, the detection of G. nolleri cysts provided confirmation of the presence in Syracuse Bay of this species that is responsible for periodic, high-mass blooms. In Europe this dinoflagellate has been reported, so far, only from Atlantic coasts (e.g. Ellegaard and Oshima, 1998; Figueroa et al., 2006; Bravo and Ramilo, 1999); therefore our data represent the first record of G. nolleri for the Mediterranean Sea. In conclusion, even though a small number of samples was analysed from only a few stations, cyst deposits produced by planktonic dinoflagellates were discovered in the sediments of Syracuse Bay. A tight relationship between pelagic and benthic domains is indicated from this study, suggesting that cyst germination may be an important factor in the onset of harmful blooms in this Mediterranean site. Thus a priority of future (local) surveys should be fine cyst mapping, accompanied by studies of the timing of cyst germination and its triggering factors, including the role of resuspension events.

Acknowledgments We thank Dr. R.I. Figueroa and S. Fraga (IEO Vigo, Spain) for their assistance in identifying Gymnodinium nolleri; A. Rabito and S. Di Grande (DAP, ARPA Syracuse, Italy) for their help with sampling; S. Borz`ı, A. Marini and F. Azzaro (IAMC-CNR of Messina, Italy) for technical assistance and nutrient data, respectively. This study was funded by the European Commission, SEED Project no.GOCE-CT-2005-003875. References Aminot, A., Chaussepied, M., 1983. Manuel des analyses chimiques en milieu marin. Centre National pour l’Exploitation des Oceans, Brest (France), 365.

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