Peer-reviewed.Azaspiracids (AZAs) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin. It took about twelve years from the first human poisoning event until a culprit for AZA production was unambiguously identified and described as a novel species, Azadinium spinosum, within a newly created genus. Since then, knowledge on the genus has increased considerably, and an update on the current circumscription of the genus is presented here including various aspects of morphology, phylogeny, biogeography, and toxin production. There are currently five described species: A. spinosum, A. obesum, A. poporum, A. caudatum, and A. polongum. As indicated by molecular sequence variation detected in field samples, there are probably more species to recognize. Moreover, Amphidoma languida has been described recently, and this species is the closest relative of Azadinium based on both molecular and morphological data. Amphidoma and Azadinium are now grouped in the family Amphidomataceae, which forms an independent lineage among other monophyletic major groups of dinophytes. Initially, azaspiracids have been detected in A. spinosum only, but AZA production within the Amphidomataceae appears complex and diverse: A new type of azaspiracid, with a number of structural variants, has been detected in A. poporum and Amphidoma languida, and AZA-2 has now been detected in Chinese strains of A. poporum

Elbrächter, M.

Gottschling, M.

Gu, H.

Jeong, H.J.

Krock, B.

Nézan, E.

Potvin, E.

Salas, R.

Soehner, S.

Tillmann, U.

Marine Institute Open Access Repository (OAR)

 Proceedings 15th ICHA, accepted manuscript   The dinophycean genus Azadinium and related species – morphological and molecular characterization, biogeography, and toxins  Urban Tillmann1*, Malte Elbrächter2, Marc Gottschling3, Haifeng Gu4, Hae Jin Jeong5, Bernd Krock1, Elisabeth Nézan6, Éric Potvin5, Rafael Salas7 and Sylvia Soehner3  1* Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany, E-Mail: urban.tillmann@awi.de, 2 Alfred Wegener Institute, Hafenstr. 43, D-25992 List/Sylt, Germany 3 Department Biologie, Systematische Botanik und Mykologie, GeoBio-Center, Ludwig-Maximilians-Universität München, Menzinger Str. 67, D-80638 München, Germany, 4 Third Institute of Oceanography, SOA, Xiamen 361005, China, 5 School of Earth and Environmental Sciences, Seoul National University, Seoul 151-747, Republic of Korea, 6 IFREMER, Station de Biologie Marine, Place de la Croix, BP 40537, 29185 CONCARNEAU Cedex, France 7 Marine Institute, Oranmore, Galway, Ireland  Abstract  Azaspiracids (AZAs) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin. It took about twelve years from the first human poisoning event until a culprit for AZA production was unambiguously identified and described as a novel species, Azadinium spinosum, within a newly created genus. Since then, knowledge on the genus has increased considerably, and an update on the current circumscription of the genus is presented here including various aspects of morphology, phylogeny, biogeography, and toxin production. There are currently five described species: A. spinosum, A. obesum, A. poporum, A. caudatum, and A. polongum. As indicated by molecular sequence variation detected in field samples, there are probably more species to recognize. Moreover, Amphidoma languida has been described recently, and this species is the closest relative of Azadinium based on both molecular and morphological data. Amphidoma and Azadinium are now grouped in the family Amphidomataceae, which forms an independent lineage among other monophyletic major groups of dinophytes. Initially, azaspiracids have been detected in A. spinosum only, but AZA production within the Amphidomataceae appears complex and diverse: A new type of azaspiracid, with a number of structural variants, has been detected in A. poporum and Amphidoma languida, and AZA-2 has now been detected in Chinese strains of A. poporum.  Keywords: Azadinium, azaspiracids, Amphidomataceae, shellfish toxin   Introduction Among the known marine toxins responsible for shellfish contamination, azaspiracids (AZAs) are the most recently discovered group of lipophilic compounds of microalgal origin. Compared to the knowledge on toxin structure, detection methods, and toxicology, convincing clarification of the aetiology of azaspiracid-poisoning was seriously lacking for quite a long time. AZA toxins are known for their seasonal and episodic accumulation in suspension-feeding shellfish (Salas et al. 2011) – a situation similar with several other marine biotoxins from microalgal origin – and thus, a planktonic source has been suspected from the outset. In addition, due to their polyether structural features, a dinophyte origin of AZAs has ab initio been suspected. Subsequently, it did not came as a surprise that it was a dinophycean species, Protoperidinium crassipes, which was first claimed to be the source of AZAs (James et al. 2003). However, production of AZAs by P. crassipes could not be verified since then (Gribble 2006). Moreover, in contrast to other identified producers of phycotoxins, which are all primarily phototrophic, P. crassipes is a large (> 50 µm) heterotrophic species, known to prey upon other dinophytes (Gribble and Anderson 2006).  During a research cruise with RV Poseidon in the North Sea in 2007, this issue became quite evident when toxin analysis of fractionated plankton samples clearly showed that (1) high amounts of AZAs were found at stations where P. crassipes was absent, (2) AZAs could be found in isolated cells of the predatory ciliate Favella ehrenbergii, and (3) in fractionated plankton samples, the Proceedings 15th ICHA, accepted manuscript     largest AZA amounts were found in the small size (<20 µm) class (Krock et al. 2009). All these indications led to the isolation of a small dinophyte, which was shown to produce AZA-1 and -2 in axenic culture (Krock et al. 2009) and which was identified as the a species, Azadinium spinosum, in a newly erected genus (Tillmann et al. 2009). Since then, knowledge on the genus has increased considerably. Here, we present an update on the current circumscription of the genus including various aspects on morphological and molecular characterisation, biogeography, and toxin production.  The species Considering the short period since the first identification of Azadinium, the known diversity of the genus has increased rapidly and now comprises five species. Azadinium spinosum, the type species of the genus, as well as Azadinium obesum were firstly isolated from the same water sample taken from the North Sea off Scotland (Tillmann et al. 2009, 2010). Later, Azadinium poporum was described from three clones isolated from the southern North Sea off the Danish coast (Tillmann et al. 2011). Azadinium caudatum, which was initially described in 1953 by Halldal as Amphidoma caudata, was recently transferred to the genus Azadinium (Nézan et al. 2012). Both DNA sequence and morphometric data clearly showed that the species occurred with two distinct varieties, var. caudatum and var. margalefii. They are easy to distinguish by the different shape of the antapical projection. Azadinium polongum – isolated from the Shetland Islands – is the most recently described species of Azadinium (Tillmann et al. 2012b).  With the description of Amphidoma languida, a genus closely related to Azadinium could be identified (Tillmann et al. 2012a). Amphidoma languida has been isolated concurrently with the Irish strain of A. spinosum from Bantry Bay, Ireland (Tillmann et al. 2012a, Salas et al. 2011).    Proceedings 15th ICHA, accepted manuscript   Morphological and molecular characterization With the exception of A. caudatum, all species of Azadinium and Amphidoma languida are of small size and of similar shape (Tab. 1). They are photosynthetically active with rather typical peridinin pigment profiles (e.g., Tillmann et al. 2009). They presumably possess a single chloroplast which is parietally arranged. As a result of a distinct starch cup, stalked pyrenoid(s) are visible in light microscopy for a number of species. All species of Azadinium consistently show the Kofoidean plate pattern Po, cp, X, 4´, 3a, 6´´, 6C, 5S, 6´´´, 2´´´´. Amphidoma languida exhibits the same hypothecal plate pattern but differs in epithecal plates by having six apical plates and no intercalary series. A very characteristic feature of all species is the prominent apical pore complex (APC) composed of a pore (Po) plate with a central round pore covered by a cover plate (cp) and an X-plate with a characteristic three-dimensional struc-ture. The arrangement of the five sulcal plates is very characteristic for all Amphidomataceae, with a large plate Sa invading the epitheca and a peculiar and conservative Ss plate running from the plates C1 to C6. The second antapical plate may bear a small spine (A. spinosum, A. polon-gum), a distinct horn with a spine (A. caudatum), or a prominent antapical pore (A. languida). All species of Azadinium and Amphidoma languida have a conspicuous ventral pore. However, the position of that pore differs between the species (Tab. 1). Other species determining characters, as the presence or absence of stalked pyrenoid(s), or the shape and arrangement of certain epithecal plates, are listed in Table 1. Morphology, and in particular the plate tabulation with five different rows of plates, undoubtedly classify the genus Azadinium as a member of the dinophycean subclass Peridiniphycidae. This subclass is currently subdivided into two orders, the Peridiniales and Gonyaulacales, with a number of differences discussed in detail by Fensome et al. (1993). Azadinium clearly exhibits morphological characteristics of both of these orders. The hypothecal plate arrangement, and the presence of six precingular, six postcingular, and six cingular plates, suggest a relationship to the Gonyaulacales. Other general features including the mode of cell division, the plate suture and growth band structure, and the presence of a ventral pore in Azadinium seem likewise to reveal a relationship to the Gonyaulacales. However, the epithecal plate arrangement with four apical and three symmetric intercalary plates implies an affinity to the Peridiniales. Moreover, the shape and composition of the APC is typical of the Peridiniales. With the description of Amphidoma languida, the taxonomic affiliation of Azadinium at the family level was recently clarified. Amphidoma was found to be closely related to Azadinium with such possible morphological synapomorphies as the cingular and hypothecal plate arrangement, the number and arrangement of sulcal plates, and the characteristic APC. Amphidoma and Azadinium were thus placed in the family Amphidomataceae by Tillmann et al. (2012a). Molecular phylogenies of the Amphi-domataceae based on ribosomal RNA sequence data supported the morphological considerations, but were not able to fully resolve the phylogenetic position of the group within the Dinophyceae (Tillmann et al. 2012a, b). Both morphology and molecular phylogeny thus did not allow for a clear order affiliation and leaves Azadium and the family Amphidomataceae with an unclear order affiliation.  Fig. 1: Global records ofAzadinium/Amphidoma languida.(1): Tillmann et al. 2009 , 2010. (2): Tillmann et al. 2011. (3): Salas et al. 2011, Tillmann et al. 2012a. (4): Tillmann et al. 2012b. (5): Tillmann, unpubl. (6) Nezan et al. 2012. (7): Margalef et al. 1954. (8) Rampi 1969. (9): Adriana Zingone, pers. com. (10): Tillmann & Busch, unpubl. (11) Hernandez-Becerril et al. 2012. (12): Akselman & Negri2012. (13): Checklist Black Sea phytoplankton. (14): Consuelo CarbonelMoore, pers. com. (15): Potvin et al. 2012. (16): Gu et al. 2013 1234551391014 14161516161211876  Global distribution Although initially described from the North Sea, the genus Azadinium probably is distributed worldwide (Fig. 1). In the North Sea, all five described species have been observed (Tillmann et al. 2009, 2010, 2011, 2012b). Based on full characterization of local strains in terms of morphology and sequence information (records 1-5, 6, 9, 15, 16) or based on a few records of single specimens detected by scanning plankton samples by light (record 7-8 referring to the large A. caudatum, record 10) or electron microscopy (records 11-14), an increasing number of global records could be added to a distribution map (Fig. Proceedings 15th ICHA, accepted manuscript   1). Nevertheless, knowledge on the biogeography of the genus currently is rather limited and patchy. As a consequence of an increasing awareness of the genus, however, the availability of FISH and QPCR as species-specific detection methods (Töbe et al. 2013, Potvin et al. 2013), and due to the increasing use of “next generation” high throughput sequencing with environmental samples, it is expected that our knowledge on the biogeography of the Amphidomataceae will increase rapidly.  Tab. 2: Origin and AZA profile of strains of Amphidomataceae according to Tillmann et al. 2012b, Krock et al. 2012 and Gu et al. 2013. Species Strain Origin AZA A. spinosum 3D9 Scotland 2007 AZA-1 AZA-2 AZA-716UTHE2 Denmark 2008 SM2 Ireland 2009 Shet F6 Shetland 2011 A. obesum 2E10 Scotland 2007 - A. polongum Shet B2 Shetland 2011 - A. caudatum AC1/2 Scotland 2011 - A. poporum UTHD4 UTHC5 UTHC8 Denmark 2008 AZA-846HJ2010 Korea 2010 AZA-858G25 Bohai Sea 2007 AZA-858-920, -928G42 East China Sea 2011 AZA-2 G64 East China Sea 2011 AZA-2 G60 East China Sea 2011 - G66 East China Sea 2011 AZA-872G68 South China Sea2011 AZA-2 Amphidoma languida SM1 Ireland 2009 AZA-816,-830  Toxins Multiple strains of the type species A. spinosum from different locations have consistently been found to produce AZA-1, AZA-2, and AZA-716 (Tillmann et al. 2012b). In contrast, A. obesum, A. poporum, and Amphidoma languida have initially been described as non-toxigenic, as none of the known AZAs could be found (Tillmann et al. 2010, 2011, 2012a). However, we recently detected four new AZAs in a number of different species. Compared to the previously known AZAs, these new analogs are characterized by a missing methyl group at C39 thus forming a characteristic m/z 348 fragment (Krock et al. 2012). Thus, it is evident that the species diversity within this group is also reflected by a high chemical diversity (Tab. 2). We know now that AZA production can also be found in the related genus Amphidoma (Krock et al. 2012). Six different AZA compounds have been found in strains of A. poporum (Tab. 2), with a large variability of AZA-profile among different strains (Krock et al. 2012, Gu et al. 2013). AZAs were not detected in all cultivated Amphidomataceae (e.g., A. obesum, A. caudatum var. margalefii, and A. polongum), but we cannot exclude the presence of yet unknown and thus undetectable AZA-related compounds.  References Akselman R, Negri A (2012). Harmful Algae 19: 30-38. Dodge JD, Saunders RD (1985). Arch. Protistenkd. 129: 89-99. 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Potvin E, Jeong HJ, Kang NST, Tillmann U, Krock B (2012). J. Eukaryot. Microbiol. 59: 145-156. Potvin E, Hwang YJ, Yoo YD, Kim JS, Jeong HJ (2013). Aquat. Microb. Ecol. 68: 143-158. Rampi L (1969). Natura (Milano) 60: 49-56. Salas R, Tillmann U, John U, Kilcoyne J, Burson A, Cantwell C, Hess P, Jauffrais T, Silke J (2011). Harmful Algae 10: 774-783. Tillmann U, Elbrächter M, John U, Krock B (2011). Eur. J. Phycol. 46: 74-87. Tillmann U, Elbrächter M, John U, Krock B, Cembella A (2010). Phycologia 49: 169-182. Tillmann U, Elbrächter M, Krock B, John U, Cembella A (2009). Eur. J. Phycol. 44: 63-79. Tillmann U, Salas R, Gottschling M, Krock B, O´Drisol D, Elbrächter M (2012a). Protist 163: 701-719. Tillmann U, Soehner S, Nézan E, Krock B (2012b). Harmful Algae 20: 142-155.  Töbe K, Joshi AR, Messtorff P, Tillmann U, Cembella A, John U (2013). J. Plankton Res. 35: 225-230. 

The dinophycean genus Azadinium and related species – morphological and molecular characterization, biogeography, and toxins

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The dinophycean genus Azadinium and related species – morphological and molecular characterization, biogeography, and toxins

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