Helgol Mar Res (2007) 61:157–165 DOI 10.1007/s10152-007-0063-x ORIGINAL ARTICLE First records of the benthic, bloom-forming, non-toxic dinoXagellate Thecadinium yashimaense (Dinophyceae) in Europe: with special emphasis on the invasion in the North Sea Mona Hoppenrath · Malte Elbrächter · Hannelore Halliger · Reinoud P. T. Koeman · Alexander Krakhmalnyy · Barbara Surek · Katrin Erler · Bernd Luckas Received: 1 June 2006 / Revised: 12 December 2006 / Accepted: 8 January 2007 / Published online: 22 February 2007 © Springer-Verlag and AWI 2007 Abstract Thecadinium yashimaense was recorded for Introduction the Wrst time in France, Great Britain, The Nether- lands, and Germany. The invasion and establishment The number of introduced and established marine of the species in the German Bight was documented species of the North Sea coasts is about 80 and com- reliably and is presented here. The geographic expan- prises invertebrates, macroalgae, and protists (Reise sion of the species from the North PaciWc to the North et al. 1999). It is a very diYcult task to distinguish Atlantic Ocean is discussed. This bloom-forming, between real exotics and misinterpreted native species marine, sand-dwelling dinoXagellate was shown to be (pseudo-exotics), which were discovered late but may non-toxic. Also Thecadinium kofoidii, the type species have been around long before without being noticed of the genus, was analyzed for potential toxin produc- (Reise et al. 1999). This is especially true for small tion and turned out to be non-toxic as well. organisms like phytoplankton and protist taxa. Meth- odological problems and extrinsic factors (Lee and Keywords Benthos · DinoXagellate · Invasion · Patterson 1998) make it diYcult to prove the exotics- Plankton · Thecadinium · Toxins status of protists (Elbrächter 1999). There are only a few examples of well documented invasions of planktonic Communicated by H.-D. Franke. M. Hoppenrath R. P. T. Koeman Biologische Anstalt Helgoland, Koeman en Bijkerk BV, Kerklaan 30, Alfred Wegener Institute for Polar and Marine Research, Haren, The Netherlands Kurpromenade, 27498 Helgoland, Germany A. Krakhmalnyy M. Hoppenrath (&) N.G. Kholodny Institute of Botany, Department of Botany, University of British Columbia, National Academy of Sciences of the Ukraine, 6270 University Boulevard, 2 Tereschenkovskaya St., 01001 Kiev, Ukraine Vancouver, BC, V6T 1Z4 Canada e-mail: [email protected] B. Surek Culture Collection of Algae at the University M. Elbrächter of Cologne (CCAC), Universität zu Köln, Deutsches Zentrum für Marine Biodiversität, Gyrhofstraße 15, 50931 Köln, Germany Wadden Sea Station Sylt, Forschungsinstitut Senckenberg, K. Erler · B. Luckas Hafenstr. 43, 25992 List/Sylt, Germany Institut für Ernährungswissenschaften, Friedrich-Schiller-Universität, H. Halliger Dornburger Str. 25, 07743 Jena, Germany Wadden Sea Station Sylt, Alfred Wegener Institute for Polar and Marine Research, Hafenstr. 43, 25992 List/Sylt, Germany 123 158 Helgol Mar Res (2007) 61:157–165 diatoms into the North Sea, e.g., Odontella sinensis known for nearly 40 years, Wrst recognized at the North (Greville) Grunow (as Biddulphia sinensis Greville, in American PaciWc coast, but misidentiWed at that time Ostenfeld 1908), Thalassiosira punctigera (Castracane) (Baillie 1971; Hoppenrath et al. 2004). Six marine, Hasle (Dürselen and Rick 1999), and Coscinodiscus sand-dwelling Thecadinium species were observed in wailesii Gran et Angst (Boalch and Harbour 1977; the German Wadden Sea (Hoppenrath 2000b; Dürselen and Rick 1999; Rick and Dürselen 1995; Hoppenrath et al. 2004). Here we present the Wrst Robinson et al. 1980). Flagellates have rarely been records of T. yashimaense in the North Sea and the documented as invasive species (Elbrächter 1999), and reliably documented invasion. Shortly before submit- the only known examples are Karenia mikimotoi ting the manuscript we learnt about an isolate of (Miyake et Kominami ex Oda) Hansen et Moestrup T. yashimaense from France, and we have integrated (as Gyrodinium aureolum Hulburt, in Braarud and this information too. Heimdal 1970; Hickel et al. 1971) and Alexandrium leei Balech (Koeman 1997). The possible vectors for the dispersal of non-indige- Methods nous species are aquaculture, migratory birds, and ocean going vessels. The transport of protists via bal- Samples and cultures last water is documented (Galil and Hülsmann 1997; HallegraeV and Bolch 1991, 1992; HallegraeV et al. Benthic sand samples were collected with a spoon 1990). Surviving the harsh conditions inside ballast during low tide, and dinoXagellates were separated water tanks is a prerequisite for a successful invasion, from the sand by extraction with melting seawater-ice especially for phototrophic species. Most of them will (Uhlig 1964) through a Wne Wlter (Hoppenrath et al. die because of darkness, but a few cells and cysts can 2004). They were accumulated in a Petri dish beneath stay alive (Dickmann and Zhang 1999; HallegraeV and the Wlter and were then identiWed with a Leitz Flu- Bolch 1992; Yoshida et al. 1996). The ability to form overt FS invert-microscope at 40 to 250£ magniWca- resting cysts is only known for a minority of dinoXagel- tion. Sampling sites were in bare, eulittoral regions, late species (HallegraeV and Bolch 1992). The aware- south of List Harbor (55°00.85ЈN; 08°06.30ЈE; tidal ness of a possible survival of resting cells of Xat of 100 m) and at the “Oddewatt” northeast of List phototrophic species is relatively new (e.g., Dickmann (55°01.80ЈN; 08°26.00ЈE; tidal Xat of 500 m). For and Zhang 1999; Zhang and Dickmann 1999). Resting more details on the sampling sites, see Hoppenrath cells can survive for lengthy periods in ballast water (2000c). tanks (C.J.S. Bolch, personal communication). There is Net samples from surface water near List (55°01.30ЈN; also the possibility that species may naturally expand 08°27.10ЈE) and of the Helgoland Reede Station their range via ocean currents. (54°11.30ЈN, 7°54.00ЈE) were collected weekly (Drebes Galil and Hülsmann (1997) showed that the protists and Elbrächter 1976; Hoppenrath 2004). Nets of 20 and found in ballast water tanks belong mainly to the benthic 80 m mesh size were used. The samples were brought to communities. Deciding whether a newly recorded ben- the laboratory and living organisms were identiWed in thic protist species is introduced to the North Sea habitat small Petri dishes with an inverted light microscope or has been overlooked in the past, is nearly impossible equipped with seawater-immersion objectives. in most cases because knowledge about the species Cells were isolated by micropipetting and cultivated assemblage is missing for nearly all taxa (for example, see in Petri dishes supplied with f/2 medium (Guillard and Houpt and Hoppenrath 2006). There are no monitoring Ryther 1962) at 16°C under a 14:10 LD cycle at about programs for benthic microalgae/protists, as there are for 10 mol/s m2 light intensity. the phytoplankton community. Moreover, intensive tax- Thecadinium yashimaense isolated from the PaciWc onomic investigations and published accounts are missing coast of British Columbia, Canada, was available as in many cases. Sand-dwelling dinoXagellates, euglenoids, culture in the Provasoli-Guillard National Center for and ciliates of the North Frisian Wadden Sea are an Culture of Marine Phytoplankton (CCMP 1890, see exception (Hoppenrath 2000a; Hartwig 1973), and dino- Hoppenrath et al. 2004) and was also used for the toxin Xagellates and euglenoids are also documented for the analysis. Two new isolates from Sylt and Helgoland, Danish Wadden Sea (Larsen 1985, 1987). Germany, were deposited in the CCMP (culture Thecadinium yashimaense Yoshimatsu, Toriumi et numbers CCMP 2726 and CCMP 2667). Dodge was described from Japan (Yoshimatsu et al. The observations of T. yashimaense in The Nether- 2004). There was some taxonomic confusion about this lands were made in two water-bodies in the south- species (Hoppenrath et al. 2005). The taxon has been western part of the country: Lake Grevelingen, a 123 Helgol Mar Res (2007) 61:157–165 159 semi-stagnant saltwater lake, and the Oosterschelde (OA), dinophysistoxins (DTXs), pectenotoxins estuary, which is a marine tidal basin. Locations were (PTXs), yessotoxins (YTXs), and azaspiracides (AZ Lake Grevelingen (DREISR: 51°42.88ЈN, 3°59.96ЈE) As). The ASP toxin domoic acid (DA) is a water solu- and Oosterschelde (HAMMOT: 51°39.61ЈN, 3°51.16ЈE; ble amino acid. LODSGT: 51°29.66ЈN, 4°07.92ЈE; WISSKKE: 51°36.10ЈN, 3°43.24ЈE; ZIJPE: 51°38.68ЈN, 4°05.82ЈE). Sample preparation On these locations samples were collected at least monthly. Recently the species was also observed in the Independent of the determination method, the toxins north-eastern part at Rottumerplaat (ROTTMPT3: have to be quantitatively extracted from the biomass. 53°33.58ЈN, 6°33.51ЈE), west of the island of Borkum. Filters with algal material were homogenized with 1 ml For storage reason the samples of 1l were Wxed with methanol/water (50:50) using an ultrasonic probe. The acidic Lugol’s solution. Algae were enumerated by suspension was centrifuged and Wltrated (Hummert analyzing 1–40 ml of the Wxed samples with the aid et al. 2002). In addition, for determination of PSP tox- of an inverted microscope after sedimentation of the ins the extraction was carried out with 0.03 N acetic cells in a counting chamber (Utermöhl 1958). The- acid (Luckas 2000). cate dinoXagellates were counted after rinsing with distilled water and staining with CalcoXuar-white Determination of PSP toxins with an epiXuorescence microscope. When neces- sary a small needle was used to manipulate individ- The common methods applied for PSP determination ual cells. are based on ion-pair chromatography of underivatized The Thecadinium/Amphidiniopsis culture (strain PSP toxins followed by post-column oxidation and M1408) from the Culture Collection of Algae at the Xuorescence detection (Luckas et al. 2003). Recently, University of Cologne (CCAC; Surek and Melkonian such an HPLC/FD method was modiWed to lower the 2004) turned out to be T.