Morphology, Phylogeny and Azaspiracid Profile of Azadinium Poporum (Dinophyceae) from the China Sea

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/236668617

Morphology, phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Article in Harmful Algae · January 2013 DOI: 10.1016/j.hal.2012.11.009

CITATIONS READS 32 298

5 authors, including:

Haifeng Gu Zhaohe Luo Third Institute of Oceanography China Third Institute of Oceanography China

48 PUBLICATIONS 420 CITATIONS 17 PUBLICATIONS 86 CITATIONS

SEE PROFILE SEE PROFILE

Urban Tillmann Alfred Wegener Institute Helmholtz Centre fo…

130 PUBLICATIONS 2,598 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Placing Marine Mixotrophs in Context View project

All content following this page was uploaded by Haifeng Gu on 05 February 2017.

The user has requested enhancement of the downloaded file.

Harmful Algae 21–22 (2013) 64–75

Contents lists available at SciVerse ScienceDirect

Harmful Algae

jo urnal homepage: www.elsevier.com/locate/hal

Morphology, phylogeny and azaspiracid proﬁle of Azadinium poporum

(Dinophyceae) from the China Sea

a, a b c b,

Haifeng Gu *, Zhaohe Luo , Bernd Krock , Mattias Witt , Urban Tillmann *

Third Institute of Oceanography, SOA, Xiamen 361005, China

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany

A R T I C L E I N F O A B S T R A C T

Article history: Azadinium poporum is a small dinoﬂagellate from the family Amphidomataceae which is known for the

Received 28 July 2012

production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean

Received in revised form 16 October 2012

waters. Here we present the ﬁrst report of its occurrence along the coast of China. Morphology of Chinese

Accepted 28 November 2012

A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch

sheath were revealed with light microscopy and conﬁrmed by transmission electron microscopy. Among

Keywords:

25 strains from the China Sea we identiﬁed two distinct ribotypes (referred to as ribotypes B and C). ITS

Azadinium

sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each

Azadinium poporum

other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains

Azaspiracids

China (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise

Genetic differentiation distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-

compensatory based changes were found within helix III of ITS indicating that they are conspeciﬁc.

Azaspiracid proﬁles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no

AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA

previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third

strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain

considerable amounts of toxic AZA-2 as the sole AZA, a ﬁnding that might elegantly explain the detection

of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with

subsequent shellﬁsh intoxication episodes for the Asian Paciﬁc.

1. Introduction been described as new (Tillmann et al., 2012), and this species is

the closest relative of Azadinium based on both molecular and

The recently erected dinoﬂagellate genus Azadinium Elbra¨chter morphological data. Amphidoma and Azadinium are now grouped

& Tillmann mainly attracts attention for its production of in the family Amphidomataceae, which forms an independent

azaspiracids, a recently discovered group of lipophilic phycotoxins lineage among other monophyletic major groups of the dino-

causing human intoxication via mussel consumption. With an phytes. Species of the genus Azadinium have so far been reported

epithecal afﬁnity to the Peridiniales and a hypothecal afﬁnity to the from the North Sea (Tillmann et al., 2009, 2010, 2011), the French

Gonyaulacales (Tillmann et al., 2009), the systematic position of and Irish coast of the eastern Atlantic (Salas et al., 2011; Ne´zan

the genus within the dinoﬂagellates is not yet clariﬁed. Three et al., 2012), the Argentinean coast (Akselman and Negri, 2012) and

species have so far been described, i.e. Azadinium spinosum the Korean coast (Potvin et al., 2011). Nevertheless, the presence of

Elbra¨chter & Tillmann, Azadinium obesum Tillmann & Elbra¨chter AZAs appeared to be distributed much more widely, reported in

and Azadinium poporum Tillmann & Elbra¨chter (Tillmann et al., Northern Africa, northern Europe, Chile, USA and China (James

2009, 2010, 2011). Recently, Amphidoma caudata Halldal has been et al., 2002; Magdalena et al., 2003; Taleb et al., 2006; Klontz et al.,

transferred to Azadinium based on both morphology and molecular 2009; Lopez-Rivera et al., 2010; Yao et al., 2010). The discrepancy

phylogeny (Ne´zan et al., 2012). Moreover, Amphidoma languida has between the distribution of Azadinium and AZAs suggest that

A. spinosum might have a wider distribution, or strains of other

Azadinium species could produce AZAs. Initially, A. spinosum was

the only species for which AZAs were reported. For A. spinosum

* Corresponding authors.

strain 3D9, the toxin proﬁle consisted of AZA-1, AZA-2 and an

E-mail addresses: [email protected] (H. Gu), [email protected]

(U. Tillmann). isomer of AZA-2 (Krock et al., 2009), which was later identiﬁed as

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 65

AZA-1 methyl ester and found to be an extraction artifact (Jauffrais coordinates and sample dates of the sites, see Table 1). The

et al., 2012). AZA-1 and -2 production was subsequently conﬁrmed sediment samples were stored in the dark at 4 8C until further

for A. spinosum strains from Denmark (Tillmann et al., 2011) and treatment. Approximately 2 g of wet sediment were mixed with

Ireland (Salas et al., 2011), indicating that production and proﬁle of 20 mL of ﬁltered seawater and sonicated for 2 min (100 W) to

known AZAs is a stable characteristic of the species A. spinosum. dislodge detrital particles. The watery slurry was incubated

Other related species/strains of Amphidomataceae have been directly in series of small containers in f/2-Si medium (Guillard

2 1

reported not to contain any of the known azaspiracids. However, and Ryther, 1962) at 20 8C, 90 mE m s under a 12:12 h

AZA production within Amphidomataceae probably is much more light:dark cycle (hereafter called ‘‘standard culture conditions’’).

complex and diverse; recent evidences indicate the presence of Azadinium cells are characterized by swimming at low speed,

new AZAs with a modiﬁed substitution pattern in A. poporum and interrupted by short, high-speed ‘jumps’ in various directions

A. languida (Krock et al., 2012). North Sea strains and the Korean (Tillmann et al., 2009). Cells exhibiting such a characteristic

isolate of A. poporum were found to produce AZAs with a swimming behavior were isolated by means of drawn-out Pasteur

characteristic m/z 348-fragment, however, in two different pipettes and established into clonal cultures. Only one strain was

variants with different masses in the North Sea strains and the established from one container to guarantee they represent true

Korean strain, respectively (Krock et al., 2012). clonal strains. Strains were maintained under standard culture

There is increasing evidence that AZAs are present in the Asian conditions.

Paciﬁc. In 2009, AZA-2 was isolated from a marine sponge collected

off Amami-Oshima Island (southern Japan) (Ueoka et al., 2009). 2.2. Light microscopy (LM)

Moreover, AZA1 was detected in shellﬁsh collected in several areas

of China (Yao et al., 2010), which encouraged us to search for Live cells were examined under a Zeiss Axio Imager microscope

Azadinium species along the coast of China. By incubating sediment (Carl Zeiss, Go¨ ttingen, Germany) equipped with both differential

samples we succeeded in obtaining 25 strains of A. poporum; their interference illumination and epiﬂuorescence. Light micrographs

morphology were examined in detail, and their partial large were obtained using a Zeiss Axiocam HRc digital camera.

subunit (LSU) and internal transcribed spacer regions (ITS1, ITS2 Approximately 1 mL of live, healthy culture in mid exponential

and 5.8S rDNA) sequences were compared with those of strains growth phase was transferred to a 1.5 mL microcentrifuge tube,

from Europe and Korea. Six of these strains were grown in larger and DAPI (4 ,6-diamidino-2-phenylindole dihydrochloride) stain

quantities and analyzed for the presence of AZAs. (Sigma–Aldrich, St. Louis, USA) was added at a ﬁnal concentration

of 10 mg mL . The cells were then incubated in the dark at room

2. Materials and methods temperature for 30 min. The cells were viewed and photographed

through a Zeiss Filterset (emission: BP 365-445; beamsplitter: FT

2.1. Sample collection and treatment 395). Cells in mid exponential growth phase were ﬁxed with 5%

Lugol’s solution and cell size was measured at 400 magniﬁca-

Sediment samples were collected in the Bohai Sea, East China tion. Fifty cells were measured for each strain of G60, G64, G66,

Sea and South China Sea using a grab sampler (Fig. 1, geographical and G68.

Fig. 1. Locations of sampling stations.

66 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75

Table 1

Strains of Azadinium poporum examined in the present study, including collection data and locations.

Species Strains LSU ribotype ITS ribotype Depth (m) Collection date Latitude (8N) Longitude (8E) Location

0 00 0 00

A. poporum G14 B B 14 2007.8.14 38854 60.00 117853 0.00 Bohai Sea, China

0 00 0 00

A. poporum G25 B B 14 2007.8.14 38854 60.00 117853 0.00 Bohai Sea, China

0 00 0 00

A. poporum G37 C C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G42 C C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G57 B B 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G58 C C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G59 C C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G60 C B 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G61 C C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G62 – C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G64 – C 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum G66 B B 41 2011.4.19 3080 0.00 122844 48.12 East China Sea

0 00 0 00

A. poporum AZDH04 – B 74 2011.5.4 28818 18.00 122819 30.00 East China Sea

0 00 0 00

A. poporum AZDH05 – B 74 2011.5.4 28818 18.00 122819 30.00 East China Sea

0 00 0 00

A. poporum AZDH11 – B 74 2011.5.4 28818 18.00 122819 30.00 East China Sea

0 00 0 00

A. poporum AZDH16 – B 74 2011.5.4 28818 18.00 122819 30.00 East China Sea

0 00 0 00

A. poporum AZDH23 – B 74 2011.5.4 28818 18.00 122819 30.00 East China Sea

0 00 0 00

A. poporum AZFC07 – B 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC12 – B 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC13 – C 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC14 – C 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC15 B B 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC16 C C 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum AZFC18 – B 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

0 00 0 00

A. poporum G68 – B 15 2011.4.28 21829 58.20 108813 53.20 South China Sea

2.3. Scanning electron microscopy Emitech, West Sussex, UK), sputter-coated with gold, and

examined using LEO 1530 Gemini SEM (Zeiss/LEO, Oberkochen,

For scanning electron microscopy (SEM), mid-exponential Germany).

batch cultures were collected by centrifugation at 5000 rpm. The

supernatant was removed and the cell pellet re-suspended in 60% 2.4. Transmission electron microscopy (TEM)

ethanol for 1 h at 8 8C to strip off the outer cell membrane. The

cells were pelleted by centrifugation and re-suspended in ﬁltered Cells were ﬁxed in 2.5% glutaraldehyde in phosphate buffer

seawater for 30 min at 8 8C. Cell pellets were re-suspended and (0.1 M PBS, pH 7.4) for 1 h, concentrated by centrifugation and

ﬁxed with 2.5% glutaraldehyde prepared with f/2-Si medium for washed three times with the above buffer for 10 min each. They

3 h at 8 8C. Cell pellets were washed twice with f/2-Si medium and were post-ﬁxed in 1% OsO4 made up with distilled water

ﬁxed with 2% OsO4 made up with ﬁltered seawater overnight at overnight at 4 8C and washed three times with 0.1 M PBS (pH

8 8C. The supernatant was removed and the cell pellet was applied 7.4) for 10 min each. Cells were dehydrated through a graded

to a coverslip coated with poly-L-lysine (molecular weight ethanol series (10, 30, 50, 70, 90 and 100%, 10 min at each step)

70,000–150,000). The cells were allowed to adhere to the followed by 100% acetone. The pellet was embedded in Spurr’s

coverslip for 30 min and then washed for 10 min in a 1:1 solution resin (Spurr, 1969) and sectioned with a Reichert Ultracut E

of distilled water and ﬁltered seawater, followed by a second wash microtome (Leica, Vienna, Austria), mounted on Formvar coated

in distilled water for 10 min. The samples were then dehydrated in grids, stained with uranyl acetate and lead citrate, and observed

a series of ethanol (10, 30, 50, 70, 90 and 3 in 100%, 10 min at each under a JEOL JEM-100 transmission electron microscope (JEOL,

step), critical point dried (K850 Critical Point Dryer, Quorum/ Tokyo, Japan).

Fig. 2. LM of live cells of Azadinium poporum. (A) Ventral view of strain G42, showing a large nucleus (N) and several pyrenoids (arrows). (B) Ventral view of strain G42,

showing a large nucleus (DAPI staining). (C) Ventral view of strain G42, showing a large pyrenoid (arrow). (D) Ventral view of strain G42, showing a large chloroplast (DAPI staining).

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 67

2.5. PCR ampliﬁcations and sequencing 2.7.1. Single reaction monitoring (SRM) measurements

Water was deionized and puriﬁed (Milli-Q, Millipore, Eschborn,

Total algal DNA was extracted from 10 mL of exponentially Germany) to 18 MV cm or better quality. Formic acid (90%, p.a.),

growing Azadinium cultures using a plant DNA extraction kit acetic acid (p.a.) and ammonium formate (p.a.) were purchased

(Sangon, Shanghai, China) according to the manufacturer’s from Merck (Darmstadt, Germany). The solvents, methanol and

protocol. Partial LSU rDNA, including the D1 and D2 domains acetonitrile, were high performance liquid chromatography (HPLC)

was ampliﬁed by polymerase chain reaction (PCR) using primers grade (Merck, Darmstadt, Germany).

D1R and D2C (Scholin et al., 1994). The total ITS1–5.8S–ITS2 was Mass spectral experiments were performed to survey for a wide

ampliﬁed using ITSA and ITSB primers (Adachi et al., 1996). array of AZAs. The analytical system consisted of an ABI-SCIEX-

The PCR protocol was as follows: initial denaturation for 4000 Q Trap, triple quadrupole mass spectrometer equipped with

3.5 min at 94 8C, followed by 35 cycles of 50 s denaturation at a TurboSpray interface coupled to an Agilent model 1100 LC.

94 8C, 50 s annealing at 45 8C, and 80 s extension at 72 8C, plus a The LC equipment included a solvent reservoir, in-line degasser

ﬁnal extension of 10 min at 72 8C. PCR products were sequenced (G1379A), binary pump (G1311A), refrigerated autosampler

directly in both directions using the ABI Big-Dye dye-terminator (G1329A/G1330B), and temperature-controlled column oven

technique (Applied Biosystems, Foster City, CA, USA), according to (G1316A).

the manufacturers’ recommendations. Sequences were deposited Separation of AZAs (5 mL sample injection volume) was

in the GenBank with accession numbers from KC286550 to performed by reverse-phase chromatography on a C8 phase. The

KC286582. analytical column (50 2 mm) was packed with 3 mm Hypersil

BDS 120 A˚ (Phenomenex, Aschaffenburg, Germany) and main-

2.6. Sequence alignment and phylogenetic analysis tained at 20 8C. The ﬂow rate was 0.2 mL min and gradient

elution was performed with two eluants, where eluant A was water

Multiple sequences were aligned using ‘MUSCLE’ (Edgar, 2004) and B was acetonitrile/water (95:5, v/v), both containing 2.0 mM

(http://www.ebi.ac.uk/Tools/msa/muscle/) with the default set- ammonium formate and 50 mM formic acid. Initial conditions

tings. The ITS sequences of Chinese A. poporum were added to were 8 min column equilibration with 30% B, followed by a linear

alignments comprising of closely related dinoﬂagellates from gradient to 100% B in 8 min and isocratic elution until 18 min with

GenBank. Completed alignments were saved as NEXUS ﬁles and 100% B then returning to initial conditions until 21 min (total run

imported into PAUP*4b10 software (Swofford, 2002) so that time: 29 min).

divergence rates could be estimated using simple uncorrected AZA proﬁles were determined in one period (0–18) min with

pair-wise (p) distance matrices. For maximum-likelihood (ML) curtain gas: 10 psi, CAD: medium, ion spray voltage: 5500 V,

analysis, we used the program JModeltest (Posada, 2008) to select temperature: ambient, nebulizer gas: 10 psi, auxiliary gas: off,

the most appropriate model of molecular evolution with Akaike interface heater: on, declustering potential: 100 V, entrance

information criterion (AIC). This test chose the general time- potential: 10 V, exit potential: 30 V. SRM experiments were

reversible (GTR) model of substitution (Rodriguez et al., 1990) carried out in positive ion mode by selecting the following

following a gamma distribution shape parameter (0.5400) transitions (precursor ion > fragment ion): (1) AZA-1 and AZA-6:

(GTR + G). ML trees were constructed in MEGA 5.05 (Tamura m/z 842 > 824 collision energy (CE): 40 V and m/z 842 > 672 (AZA-

et al., 2011). The robustness of tree topology was conducted using 1 only) CE: 70 V, (2) AZA-2: m/z 856 > 838 CE: 40 V and m/z

bootstrap with 1000 replications. 856 > 672 CE: 70 V, (3) AZA-3: m/z 828 > 810 CE: 40 V and m/z

A Bayesian reconstruction of the data matrix was performed 828 > 658 CE: 70 V, (4) AZA-4 and AZA-5: m/z 844 > 826 CE: 40 V,

with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) using the (5) AZA-7, AZA-8, AZA-9 and AZA-10: m/z 858 > 840 CE: 40 V, (6)

best-ﬁtting substitution model (GTR + G). Four Markov chain AZA-11 and AZA-12: m/z 872 > 854 CE: 40 V and (7) new

Monte Carlo (MCMC) chains ran for one million generations, compounds 816 > 798 CE: 40 V, 816 > 348 CE: 70 V, 830 > 812

sampling every 1000 generations. A majority rule consensus tree CE: 40 V, 830 > 348 CE: 70 V, 844 > 826 CE: 40 V 846 > 828 CE:

was created in order to examine the posterior probabilities of each 40 V, 846 > 348 CE: 70 V, 858 > 348 CE: 70 V, 860 > 842 CE: 40,

clade. 872 > 854 CE: 40 V, 920 > 902 CE: 40 V and 928 > 910 CE: 40 V.

Using Hidden Markov Models for the ﬂanking 5.8 and 28S rDNA AZAs were calibrated against an external standard solution of AZA-

regions, ITS-2 boundaries can be precisely detected (Keller et al., 1 (certiﬁed reference material (CRM) programme of the IMB-NRC,

2009). The secondary structures of ITS-2 rDNA sequences of 3 Halifax, Canada) and expressed as AZA-1 equivalents.

strains of A. poporum were predicted using the Mfold program

(Zuker, 2003) (http://mfold.rit.albany.edu/?q=mfold/RNA-Fold- 2.7.2. Precursor ion experiments

ing-Form). RNA structure was drawn with VARNA 3.8 (Darty Precursors of the fragments m/z 348 and m/z 362 were scanned

et al., 2009) and edited. in the positive ion mode from m/z 400 to 950 under the following

conditions: curtain gas: 10 psi, CAD: medium, ion spray voltage:

2.7. Chemical analysis of azaspiracids 5500 V, temperature: ambient, nebulizer gas: 10 psi, auxiliary

gas: off, interface heater: on, declustering potential: 100 V,

For AZA analysis, cultures of A. poporum were grown in 200 mL entrance potential: 10 V, collision energy: 70 V, exit potential:

Erlenmeyer ﬂasks under standard culture conditions. Around 10 12 V.

cells were collected by centrifugation at the exponential phase. Cell

pellets were extracted with 400 mL acetone by reciprocal shaking 2.7.3. Product ion spectra

at 6.5 m s with 0.9 g lysing matrix D (Thermo Savant, Illkirch, Product ion spectra were recorded in the Enhanced Product Ion

France) in a Bio101 FastPrep instrument (Thermo Savant, Illkirch, (EPI) mode in the mass range from m/z 150 to 930. Positive

France) for 45 s. Extracts were then centrifuged (Eppendorf 5415 R, ionization and unit resolution mode were used. The following

Hamburg, Germany) at 16,100 g at 4 8C for 15 min. Each parameters were applied: curtain gas: 10 psi, CAD: medium, ion

supernatant was transferred to a 0.45-mm pore-size spin-ﬁlter spray voltage: 5500 V, temperature: ambient, nebulizer gas:

(Millipore Ultrafree, Eschborn, Germany) and centrifuged for 30 s 10 psi, auxiliary gas: off, interface heater: on, declustering

at 800 g, and the resulting ﬁltrate being transferred into an LC potential: 100 V, collision energy spread: 0, 10 V, collision energy:

autosampler vial for LC–MS/MS analysis. 70 V.

68 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75

Fig. 3. LM and SEM of vegetative cells and cysts of Azadinium poporum. (A) Ventral view of strain G25 (SEM). (B) Dorsal view of strain G25 (SEM). (C) Ventral view of strain G64,

0 0

showing the ﬁrst apical plate (1 ) and pore plate (SEM). (D) Apical view of strain G64, showing a wide and symmetrical 3 (SEM). (E) Dorsal view of strain G64, showing a

narrow and symmetric 3 (SEM). (F) Location and detail of ventral pore (vp) in strain G25 (SEM). (G) Antapical view of strain G37, showing hypothecal plate pattern and

cingular plates (SEM). (H) Sulcal plates of strain G25, showing an anterior sulcal plate (Sa), a median sulcal plate (Sm), a right sulcal plate (Sd), a left sulcal plate (Ss), and a

posterior sulcal plate (Sp) (SEM). (I) A cyst generated in culture of strain G25, showing a bright accumulation body (arrow) (LM).

2.7.4. FTICR-MS measurements ing magnet (Bruker Biospin, Wissembourg, France) and Inﬁnity ICR

Mass spectra were acquired with a Solarix 12 T with Dual analyzer cell. The samples were ionized using a dual ion source in

source Fourier transform ion cyclotron resonance mass spectrom- electrospray positive ion mode (Bruker Daltonik GmbH, Bremen,

eter (FTICR-MS; Bruker Daltonik GmbH, Bremen, Germany) Germany). Sample solutions were continuously infused using a

equipped with a 12 T refrigerated actively shielded superconduct- syringe at a ﬂow rate of 2 mL min . The detection mass range was

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 69

Fig. 4. TEM micrographs of Azadinium poporum strain G42. (A) General view showing dinokaryon (N), chloroplasts (C), and pyrenoids (P). (B) Detail of the dinokaryon showing

a nucleolus (n), several chromosome (cr) and a pyrenoid (P) surrounded by a starch sheath (S). (C) Detail of a pyrenoid (P) and chloroplast (C), showing thylakoids in stacks of

twos or threes and a starch sheath (S).

set to m/z 150–3000. Ion accumulation time was set to 0.1 s. Data variable among cells of a single strain; plate 3 of some cells is

sets were acquired with 4 MW data points resulting in a resolving narrow and symmetric only in lateral view (Fig. 3E). Three

power of 450,000 at m/z 400. Spectra were zero-ﬁlled to process intercalary plates (1a, 2a and 3a) are present on the dorsal part of

size of 8 M data points before sine apodization. the epitheca. Plates 1a and 3a are large, whereas the four-sided 2a

Mass spectra were calibrated with arginine cluster using a is distinctly smaller (Fig. 3C–E).

linear calibration. A 10 mg mL solution of arginine in 50% The two antapical plates differ markedly in size, with the

0000

methanol was used to generate the clusters. For MS/MS experi- smaller plate 1 slightly displaced to the left (Fig. 3G). The sulcus is

ments accumulation time was up to several seconds, the isolation composed of an anterior sulcal plate (Sa), a median sulcal plate

window was 0.5 Da and collision energy set to 30 eV. (Sm), a right sulcal plate (Sd), a left sulcal plate (Ss), and a posterior

sulcal plate (Sp) (Fig. 3H).

3. Results In one of the strains (G25) the presence of a few distinct cysts

was observed. These cysts are ellipsoid, around 15 mm long and

3.1. Morphology 10 mm wide. They are full of pale granules, with a yellow

accumulation body inside (Fig. 3I).

Twenty-ﬁve strains of A. poporum were established by

incubating sediments along the coast of China (Table 1). The cells 3.2. Ultrastructure

are 9.9–17.1 mm long (mean = 13.6 1.6 mm, n = 200, strains G60,

G64, G66 and G68) and 7.6–12.9 mm wide (mean = 9.6 1.1 mm, A longitudinal section through the cell shows a large

n = 200) with a median length: width ratio of around 1.4. The large dinokaryon and irregularly formed chloroplast(s) scattered in

nucleus is spherical to slightly elongated and located in the central the periphery with several stalked pyrenoids (Fig. 4A). The

part of the cell (Fig. 2A and B). A few pyrenoids are visible in the light dinokaryon consists of multiple condensed chromosomes and a

microscope (Fig. 2A and C). A presumably single chloroplast is spherical nucleolus (Fig. 4B). The thylakoids are grouped in twos or

situated in the periphery of the cell (Fig. 2D). threes to form lamellae, and the mitochondria consist of tubular

Strains of G25, G37, G42, G60, G64, and G66 were subjected to cristae. Stalked pyrenoids are surrounded by a starch sheath, and

detailed examination under SEM, and all of them showed similar their matrix is penetrated by thylakoids slightly (Fig. 4C).

morphology. The epitheca is conical and the hypotheca is

0 00 000

hemispherical with a plate pattern of Po, X, 4 , 6 , 6 C, 5S, 6 , 3.3. Molecular analysis and phylogeny

0000

2 (Fig. 3A–E). The cingulum is deep and wide, descending of less

than one cingulum width (Fig. 3A). The cingulum composes of six Two distinct types of LSU/ITS sequences were recorded among

plates of almost equal size, with the ﬁrst one being relatively the 25 Chinese strains of A. poporum. Fifteen strains from the Bohai

narrow. The apical pore is round, located in the center of a pore Sea, East China Sea and South China Sea share nearly identical

plate and is covered by a cover plate (Fig. 3D). There is an obvious LSU/ITS sequences and are referred to as ribotype B, and 10 strains

ventral pore located at the junction of the apical pore and the ﬁrst from the East China Sea and South China Sea share nearly identical

0 0 0

two apical plates (1 and 2 ) (Fig. 3F). The ﬁrst apical plate (1 ) is LSU/ITS sequences and referred to as ribotype C (Table 1).

slightly asymmetrical (Fig. 3A). The dorsal apical plate 3 normally LSU and ITS sequences of representative strains (G25 and G42)

is wide and symmetrical (Fig. 3C). However, the shape of 3 can be were compared with related Azadinium strains (Table 2). For LSU

Table 2

Partial LSU and ITS sequences comparison of Azadinium poporum strains from China with those of related species from elsewhere. The numeral refers to different nucleotide

positions and the percentage in bracket refers to the similarity out of partial LSU sequences (736 bp) and ITS region sequences.

LSU/ITS A. poporum G42 Korean A. poporum A. poporum UTHC8 A. obesum 2E10 A. spinosum UTHE2

A. poporum G25 16(97.8%)/11(98.3%) 0(100%)/3(99.5%) 26(96.5%)/16(97.5%) 44(93.9%)/46(92.8%) 39(94.8%)/72(88.8%)

A. poporum G42 16(97.8%)/14(97.5%) 24(96.8%)/15(97.7%) 46(93.9%)/45(93.0%) 38(94.8%)/72(88.8%)

70 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75

Fig. 5. Phylogeny of Azadinium poporum inferred from ITS and 5.8S rDNA sequences using maximum likelihood (ML) and Bayesian inference (BI). Branch lengths are drawn to

scale, with the scale bar indicating the number of nt substitutions per site. Numbers on branches are statistical support values to clusters on the right of them (left: ML

bootstrap support values; right: Bayesian posterior probabilities).

sequences comparison, strain G25 (ribotype B) and G42 (ribotype helix pairing) in helices I, II and IV. Helices III does not show any

C) differ from each other at 16 positions out of 736 bp (97.8% difference among the three ribotypes. The helices IV of ribotype A

similarity). Strain G25 share identical LSU sequence with the is 3 pairs shorter than that of ribotypes B and C (Fig. 6).

Korean strain of A. cf. poporum, but differs from A. poporum strain

UTHC8 of Europe (referred to as ribotype A) at 26 positions (96.5% 3.4. AZA proﬁles

similarity).

For ITS sequences comparison, strain G25 (ribotype B) and G42 Out of the six analyzed strains (G25, G42, G60, G64, G66 and

(ribotype C) differ from each other at 11 positions (98.3% G68) ﬁve strains showed different AZA proﬁles (Table 4). Whereas

similarity). Strain G25 differs from Korean strain of A. cf. poporum strain G60 was found not to produce any AZAs in measurable

at 3 positions (99.5% similarity), from A. poporum strain UTHC8 of quantities (limit of detection: ca. 0.005 fg cell ), strains G42, G64

Europe (referred to as ribotype A) at 16 positions (97.5% similarity). and G68 were found to contain AZA-2 (retention time: 12.39 min,

The ML and BI analysis generated similar phylogenetic trees Fig. 7) as the sole azaspiracid. Cell quotas of AZA-2 ranged from 1.8

(Fig. 5). Azadinium poporum comprise 3 well supported clades, to 23.0 fg cell . Strain G66 produced an AZA of the m/z 362-

referred to as ribotypes A, B, and C. Ribotype B includes strains fragment-type with a molecular mass of 871 Da and a retention

along the coast of China as well as a Korean strain. Ribotype C

includes strains from the East China Sea and South China Sea, and

Table 3

ribotype A consists of strains from Europe. They are a nearest sister

Estimated genetic distance (p) between ITS regions of Azadinium species.

clade to A. spinosum and A. obesum with strong support.

The ITS region p value range from 0.017 to 0.022 within A. Species (strains) 1 2 3 4 5

poporum, while those between species range from 0.043 to 0.078

1 A. poporum (G25) –

(Table 3). The ITS-2 secondary structure of 3 strains, representing 2 A. poporum (G42) 0.01711 –

ribotypes A, B and C was predicted. All of them show four main 3 A. poporum (UTHC5) 0.02172 0.02182 –

4 A. obesum (2E10) 0.05 0.05013 0.04349 –

helices (I–IV) and display several hemi-compensatory base

5 A. spinosum (UTHE2) 0.07811 0.07812 0.07808 0.05131 –

changes (hemi-CBCs, compensatory change on only one side of a

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 71

Fig. 6. ITS-2 rDNA secondary structure model of Azadinium poporum strain G25 (ribotype B). The additional loop and stem structures are shown for the Chinese strain G37

(ribotype C) and European strain UTHC5 (ribotype A).

time of 11.38 min (provisionally named compound A) (Fig. 8). The

cell quota of this AZA in strain G66 was estimated as 0.9–

1.9 fg cell expressed as AZA-1 equivalents.

In contrast, strain G25 produced an AZA recently found in a

Korean isolate of A. cf poporum with a characteristic m/z 348

fragment and a molecular mass of 857 Da (retention time:

11.98 min) at a cell quota of 1.4 fg cell (expressed as AZA-1

equivalent). Interestingly strain G25 additionally contained two

early eluting AZA-related compounds of the m/z 348-fragment-

type with molecular masses of 919 and 927 Da with retention

times of 9.97 and 10.73 min, respectively, with cell quotas of

1 1

0.02 fg cell and 0.14 fg cell , which were not detected in the

Korean isolate (Fig. 9).

4. Discussion

4.1. Morphology

There are a number of morphological key characters separating

species in the genus of Azadinium, i.e. the presence of an antapical

spine, the arrangement of the ﬁrst precingular plate 1 (whether in

contact to the ﬁrst intercalary plate or not), and the location of a

ventral pore (Tillmann et al., 2011). It is particularly the distinct

position of the ‘‘ventral’’ pore located at the junction of the pore

plate and the ﬁrst two apical plates that identiﬁed all Chinese

strains as A. poporum. Without any exception, all our strains are

also in accordance with other morphological descriptions of the

Table 4

Azaspiracid proﬁles produced in Chinese strains of Azadinium poporum (X:

presence,8: not detected).

Strains AZA-2 cpd A cpd 1 cpd B cpd C

(871 Da) (857 Da) (919 Da) (927 Da)

G25 8 8 X X X

G42 X 8 8 8 8

G64 X 8 8 8 8

G66 8 X 8 8 8

G68 X 8 8 8 8

Fig. 7. (A) CID mass spectrum of a standard solution of AZA-2, (B) of AZA-2 detected

cpd, compound. in A. poporum strain G64.

72 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75

type material of A. poporum (Tillmann et al., 2011). Cells of the reported in A. poporum are also found in many other dinoﬂagellates

Chinese isolates are close in size to that of the Korean (Potvin et al., (Dodge and Crawford, 1971; Schnepf and Elbra¨chter, 1999).

2011) and European (Tillmann et al., 2011) isolate. Moreover, Ultrastructural information on pyrenoids of other Azadinium

median length and width ratio of the strains from different areas species is not yet available.

are similar. In our material, the position of the ventral pore was In one strain, thick walled immotile stages identiﬁed as cysts

invariable, whereas in the Korean A. poporum strain, this pore, as a were observed. Given that the successful isolation strategy was

rare exception, was observed to be located on the 1 plate (Potvin based on incubating sediment samples, the presence of cysts of

et al., 2011). Comparing the Korean and European strains of A. Azadinium was quite likely, but nevertheless cysts have not been

poporum it has been discussed that the 3 apical plate of Korean A. depicted before. However, we do not have any data indicating the

poporum strain is usually symmetric on the AP (antero-posterior) nature of these cysts, whether they might be vegetative pellicle

and lateral axes, whereas the 3 apical plate of the European strains cysts or resting cysts with long dormancy period. Clearly, more

is symmetric only on the AP axis (Potvin et al., 2011; Tillmann et al., data and observations are needed to clarify the whole life cycle of

2011). Our ﬁnding that the shape of plate 3 is variable within a Azadinium.

single strain indicates that this feature is not suitable as a

morphological criterion. Asian and North Sea isolates thus seem to 4.2. Phylogeny and genetic differentiation

lack any distinct morphological difference and that support the

notion that A. poporum from Europe and Asia are conspeciﬁc. Invariably, isolates successfully brought to culture turned out to

Light- and transmission electron microscopy revealed the be A. poporum. The failure to detect other Azadinium species, i.e. A.

presence of a number of pyrenoids per cell in A. poporum (see. obesum and A. spinosum, does not necessarily mean their absence

Figs. 2 and 4A) and thus conﬁrm the original description in which along the Chinese coast. They might not have cyst stages, might not

up to four pyrenoids per cell have been described (Tillmann et al., have survived our isolation procedure or just escaped our

2011). Pyrenoids are electron-dense bodies located in the detection. Clearly, the application of molecular tools designed to

chloroplast stroma of most eukaryotic algae, which has been detect and quantify species of Azadinium (To¨be et al., 2012) need to

suggested to be one of the factors underlying the high afﬁnity for be applied. A. poporum has so far been reported from a few

CO2 in algal photosynthesis (Kuchitsu et al., 1988). The presence/ locations in the North Sea and the Korean coast (Potvin et al., 2011;

absence, location, number and types of pyrenoids have been Tillmann et al., 2011). Here we extend its distribution to the Bohai

regarded as useful taxonomic characters between genera or Sea, East China Sea and South China Sea and uncover two

species (Schnepf and Elbra¨chter, 1999; Tillmann et al., 2011) genetically distinct populations, both also distinct from the

and has in particular been discussed as a potential feature visible in European isolates. Genetic distances between European and

light microscopy to differentiate species of Azadinium (Tillmann Asian strains are slightly larger compared to genetic distances

et al., 2011). Stalked pyrenoids with a starch sheath as here among the Asian clades (Table 2), which probably reﬂect the large

Fig. 8. (A) CID mass spectrum range from m/z 150 to 730 of compound A. (B) AZA-1; characteristic group fragments of both compounds are identical. (C) Molecular ion cluster

of compound A. (D) of compound 1 (Krock et al., 2012); both compounds show identical fragmentation patterns: six water losses from the [M+H] ion (solid arrows and m/z

values), a loss of CO2 followed by ﬁve water losses (dashed arrows and m/z values) and a loss of C2H6O3 followed by two waterlosses (pointed arrows and m/z values).

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 73

intermediate ITS sequences of A. poporum were not encountered.

In any case, in order to be able to experimentally test the

hypothesis that they are reproductively compatible, knowledge

about the life cycle of Azadinium is a prerequisite.

4.3. Toxin proﬁles

Our detailed search for the presence of AZAs in six of the

Chinese A. poporum strains revealed a large variability in AZA

proﬁle among strains. No AZA with any of the characteristic

fragments m/z 348 and m/z 362 above the detection limit of

0.005 fg cell could be found in one of the strains. In the other

strains, three novel AZAs were detected. Full structural elucidation

of these compounds will require NMR spectroscopy. Nevertheless,

interpretation of fragmentation patterns, comparison to those of

known AZAs and high resolution mass measurements using

Fourier transform ion cyclotron resonance-mass spectrometry

(FTICR-MS) allows for the prediction of structural elements of

these unknown compounds.

For the ﬁrst new compound with a mass of 871 Da produced by

strain G66 (provisionally named compound A), the collision

induced dissociation (CID) spectrum (Fig. 8) showed identical

fragment mass peaks as AZA-1 up to m/z 672, which is produced by

the A-ring cleavage of AZA-1 (Rehmann et al., 2008). This means

that AZA-1 and compound A share the same A to I-ring system.

However, in contrast to AZA-1 the molecular ion cluster pattern of

compound A is identical with those of two new AZAs (provisionally

named compounds 1 and 2) (Fig. 8) recently described in Korean

and Danish isolates of A. poporum (Krock et al., 2012), which leads

to the conclusion that compound A as well as compounds 1 and 2

share the same carboxylic side chain. This structural proposal is

consistent with the sum formulas of its molecular and fragment

ions measured by high resolution mass spectrometry (HRMS).

Tentatively, mass spectral information suggests that compound A

is 3-hydroxy-8-methyl-AZA-1.

Strain G25 was found to contain a m/z 348-type AZA previously

found in a Korean strain of A. poporum (Krock et al., 2012), In

addition, strain G25 contained two minor compounds with typical

Fig. 9. (A) CID mass spectrum of compound B (m/z 920). (B)CID mass spectrum of CID spectra of AZAs: a compound (provisionally named compound

compound C (m/z 928) detected in A. poporum strain G25. C) with a molecular mass of 927 Da and a sum formula of

C51H77NO14 as determined by HRMS. Its CID spectrum shows the

same fragment ions of compound 1, which is also present in this

geographical distance between both areas. A. poporum ribotype B is strain. This is strong evidence that compound 1 and C share the

widespread along the coast of China and also occur in Korea (Potvin same A-I-ring structure. The elemental difference to compound 1 is

et al., 2011). In contrast, A. poporum ribotype C was exclusively C4H6O and the fact that no other abundant fragments other than

recorded in the East China Sea and South China Sea. Strains of the those of compound 1 are formed, leads to the conclusion that

ribotype C might be speciﬁc for warm-temperate regions or simply compound B may be a diol ester of compound 1. Another

remained undetected in Northern China, since only a few strains compound (provisionally named compound B) with a molecular

were obtained from there. The absence of the European A. poporum mass of 919 Da also shows the characteristic fragment ion pattern

ribotype A in China suggests that it may have a limited distribution. of the m/z 348-type AZAs, but with atypical fragment ion

In any case, the knowledge on A. poporum from Europe is currently intensities. Another obvious difference to all other AZAs is the

restricted to three strains from the same location and clearly more complete lack of water losses from the pseudo molecular ion. These

strains from Europe and other areas of the word are needed to fully differences make it difﬁcult to make any reasonable predictions

assess the genetic variability of A. poporum. about its structure. Furthermore, the concentration of this

The sympatric occurrence of two distinct ribotypes of A. compound in our sample was too low (cell quota ca. 0.02 fg cell )

poporum in China may be explained by the fact that they have 1) to obtain exact masses of most of the fragment ions, which

diverged recently. Relatively low pairwise distance based on ITS further hampers structural analysis of this compound.

sequences implies that they are conspeciﬁc (Wayne Litaker et al., It is interesting to note the relative dominance of AZA-2 in three

2007). Total loss of gamete compatibility may generally be out of six strains of Chinese A. poporum. For all available A.

observed before the critical region of helix III contains a CBC spinosum strains, AZA-2 is present only in conjunction to AZA-1, as

(Coleman, 2009). The observed lack of CBC in helix III among it is the case for North Sea plankton samples (James et al., 2003;

different types of A. poporum thus support that they are Krock et al., 2009). Nevertheless, there are reports of samples

conspeciﬁc. Strains with distinct ITS sequences can be sexually consisting of AZA-2 as the dominant compound, the most

compatible (Brosnahan et al., 2010; Casteleyn et al., 2008), and interesting one being the isolation of AZA-2 from the marine

natural hybrids has been reported in several diatom species sponge Echinoclathria sp. collected off Amami-Oshima island in

(Casteleyn et al., 2009; D’Alelio et al., 2009). Nevertheless, southern Japan (Ueoka et al., 2009). Although azaspiracid

74 H. Gu et al. / Harmful Algae 21–22 (2013) 64–75

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and

poisoning has not yet appeared in Japanese shellﬁsh, the necessity

high throughput. Nucleic Acids Research 32 (5), 1792–1797.

of the identiﬁcation of the AZA-2 origin in the sponge was

Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella

underlined (Ueoka et al., 2009). In view of our ﬁndings it is likely nana Hustedt and Detonula confervacea Cleve. Canadian Journal of Microbiology

8, 229–239.

that AZA-2 producing A. poporum is this source.

James, K.J., Furey, A., Lehane, M., Ramstad, H., Aune, T., Hovgaard, P., Morris, S.,

Interestingly, there are reports from Morocco (Taleb et al., 2006)

Higman, W., Satake, M., Yasumoto, T., 2002. First evidence of an extensive

and Portugal (Vale et al., 2008) with an AZA-proﬁle in shellﬁsh northern European distribution of azaspiracid poisoning (AZP) toxins in shell-

ﬁsh. Toxicon 40 (7), 909–915.

samples consisting of just AZA-2, quite different from any shellﬁsh

James, K.J., Moroney, C., Roden, C., Satake, M., Yasumoto, T., Lehane, M., Furey, A.,

sample from Ireland, Norway, Spain or France (Twiner et al., 2008).

2003. Ubiquitous benign alga emerges as the cause of shellﬁsh contamination

Again, it is conceivable that also AZA-2 producing A. poporum and responsible for the human toxic syndrome, azaspiracid poisoning. Toxicon 41

not A. spinosum may be the algal source of these shellﬁsh (2), 145–151.

Jauffrais, T., Herrenknecht, C., Se´chet, V., Sibat, M., Tillmann, U., Krock, B., Kilcoyne, J.,

contaminations in the North Atlantic.

Miles, C.O., McCarron, P., Amzil, Z., Hess, P., 2012. Quantitative analysis of

To conclude, there is increasing evidence that azaspiracid

azaspiracids in Azadinium spinosum cultures. Analytical and Bioanalytical

production among Amphidomataceae is quite diverse. In particu- Chemistry 403 (3), 833–846.

Keller, A., Schleicher, T., Schultz, J., Mu¨ ller, T., Dandekar, T., Wolf, M., 2009. 5.8S–28S

lar, A. poporum appears to be a rich source of different azaspiracid

rRNA interaction and HMM-based ITS2 annotation. Gene 430 (1–2), 50–57.

compounds. The two different types of AZAs characterized by

Klontz, K.C., Abraham, A., Plakas, S.M., Dickey, R.W., 2009. Mussel-associated

different fragments (Krock et al., 2012) (the m/z 362 Type’’ and the azaspiracid intoxication in the United States. Annals of Internal Medicine

150 (5), 361.

m/z 348 type) were previously found to be restricted to A.

Krock, B., Tillmann, U., John, U., Cembella, A.D., 2009. Characterization of azaspir-

spinosum (m/z 362 type) and A. poporum/A. languida (m/z 348

acids in plankton size-fractions and isolation of an azaspiracid-producing

type), but it is now clear that both types may occur among strains dinoﬂagellate from the North Sea. Harmful Algae 8 (2), 254–263.

Krock, B., Tillmann, U., Voß, D., Koch, B.P., Salas, R., Witt, M., Potvin, E´ ., Jeong, H.J.,

of A. poporum.

1 2012. New azaspiracids in Amphidomataceae (Dinophyceae). Toxicon, http://

The cell quota of the new 871 Da AZA of 2 fg cell in strain dx.doi.org/10.1016/j.toxicon.2012.05.007.

G66 was relatively low but cell quota of AZA-2 in Chinese strains of Kuchitsu, K., Tsuzuki, M., Miyachi, S., 1988. Changes of starch localization within the

chloroplast induced by changes in CO concentration during growth of Chla-

up to 23 fg cell were in the same range as total AZAs reported for 2

mydomonas reinhardtii: independent regulation of pyrenoid starch and stroma

European A. spinosum strains (Salas et al., 2011; Tillmann et al.,

starch. Plant and Cell Physiology 29 (8), 1269–1278.

2009). Whereas toxic effects and toxicity of the new compounds Lopez-Rivera, A., O’Callaghan, K., Moriarty, M., O’Driscoll, D., Hamilton, B., Lehane,

need to be investigated, toxicity of AZA-2 is known (Twiner et al., M., James, K., Furey, A., 2010. First evidence of azaspiracids (AZAs): a family of

lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and

2008). When compared to AZA-1, AZA-2 is even more potent with a

mussels (Mytilus chilensis) collected in two regions of Chile. Toxicon 55 (4),

toxic equivalent factor of 1.8 (mouse bioassay, Ofuji et al., 1999) or 692–701.

even 8.3 (cytotoxicity, Twiner et al., 2012). Thus, more data on the Magdalena, A.B., Lehane, M., Krys, S., Ferna´ndez, M.L., Furey, A., James, K.J., 2003. The

first identification of azaspiracids in shellfish from France and Spain. Toxicon 42

ecophysiology and spatio-temporal distribution of A. poporum are

(1), 105–108.

needed in order to fully evaluate the risk potential of A. poporum

Ne´zan, E., Tillmann, U., Bilien, G., Boulben, S., Che`ze, K., Zentz, F., Salas, R., Chome´rat,

blooms and subsequent shellﬁsh contamination episodes in the N., 2012. Taxonomic revision of the dinoﬂagellate Amphidoma caudata: transfer

to the genus Azadinium (Dinophyceae) and proposal of two varieties, based on

Asian Paciﬁc and North Atlantic.

morphological and molecular phylogenetic analyses. Journal of Phycology,

http://dx.doi.org/10.1111/j.1529-8817.2012.01159.x.

Acknowledgments Ofuji, K., Satake, M., McMahon, T., Silke, J., James, K.J., Naoki, H., Oshima, Y.,

Yasumoto, T., 1999. Two analogs of azaspiracid isolated from mussels, Mytilus

edulis, involved in human intoxication in Ireland. Natural Toxins 7 (3), 99–102.

This project was supported by the National Scientiﬁc-Basic Posada, D., 2008. jModelTest: phylogenetic model averaging. Molecular Biology and

Evolution 25 (7), 1253–1256.

Special Fund of China (Grant No. 2009FY210400) and the National

Potvin, E´ ., Jeong, H.J., Kang, N.S., Tillmann, U., Krock, B., 2011. First report of

Natural Science Foundation of China (Grant No. 0900081). We

the photosynthetic dinoﬂagellate genus Azadinium in the Paciﬁc Ocean:

thank Woldgang Drebing for technical assistance with LC–MS/MS morphology and molecular characterization of Azadinium cf. poporum. Jour-

nal of Eukaryotic Microbiology, http://dx.doi.org/10.1111/j.1550-7408.2011.

analysis and Mike Twiner for many helpful comments and

corrections.[TS] 00600.x.

Rehmann, N., Hess, P., Quilliam, M.A., 2008. Discovery of new analogs of the marine

biotoxin azaspirazid in blue mussels Mytilus edulis by ultra-performance liquid

References chromatography/tandem mass spectrometry. Rapid Communication Mass

Spectrometry 22, 549–558.

Rodriguez, F., Oliver, J., Marin, A., Medina, J.R., 1990. The general stochastic model of

Adachi, M., Sako, Y., Ishida, Y., 1996. Analysis of Alexandrium (Dinophyceae) species

nucleotide substitution*. Journal of Theoretical Biology 142 (4), 485–501.

using sequences of the 5.8S ribosomal DNA and internal transcribed spacer

Ronquist, F., Huelsenbeck, J., 2003. MrBayes 3: Bayesian phylogenetic inference

regions. Journal of Phycology 32 (3), 424–432.

under mixed models. Bioinformatics 19, 1572–1574.

Akselman, R., Negri, R.M., 2012. Blooms of Azadinium cf. spinosum Elbra¨chter et

Salas, R., Tillmann, U., John, U., Kilcoyne, J., Burson, A., Cantwell, C., Hess, P., Jauffrais,

Tillmann (Dinophyceae) in northern shelf waters of Argentina, Southwestern

T., Silke, J., 2011. The role of Azadinium spinosum (Dinophyceae) in the produc-

Atlantic. Harmful Algae, http://dx.doi.org/10.1016/j.hal.2012.05.004.

tion of azaspiracid shellﬁsh poisoning in mussels. Harmful Algae 10, 774–783.

Brosnahan, M.L., Kulis, D.M., Solow, A.R., Erdner, D.L., Percy, L., Lewis, J., Anderson,

Schnepf, E., Elbra¨chter, M., 1999. Dinophyte chloroplasts and phylogeny—a review.

D.M., 2010. Outbreeding lethality between toxic Group I and nontoxic Group III

Grana 38 (2–3), 81–97.

Alexandrium tamarense spp. isolates: predominance of heterotypic encystment

Scholin, C.A., Herzog, M., Sogin, M., Anderson, D.M., 1994. Identiﬁcation of group-

and implications for mating interactions and biogeography. Deep Sea Research

and strain-speciﬁc genetic markers for globally distributed Alexandrium (Dino-

Part II: Topical Studies in Oceanography 57 (3–4), 175–189.

phyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. Journal of

Casteleyn, G., Adams, N.G., Vanormelingen, P., Debeer, A.E., Sabbe, K., Vyverman, W.,

Phycology 30 (6), 999–1011.

2009. Natural hybrids in the marine diatom Pseudo-nitzschia pungens (Bacillar-

Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron

iophyceae): genetic and morphological evidence. Protist 160 (2), 343–354.

microscopy. Journal of Ultrastructure Research 26, 31–43.

Casteleyn, G., Chepurnov, V.A., Leliaert, F., Mann, D.G., Bates, S.S., Lundholm, N.,

Swofford, D., 2002. Sinauer Associates; Sunderland, MA: 2002. PAUP*. Phylogenetic

Rhodes, L., Sabbe, K., Vyverman, W., 2008. Pseudo-nitzschia pungens (Bacillar-

analysis using parsimony (* and other methods), version 4, b10.

iophyceae): a cosmopolitan diatom species? Harmful Algae 7 (2), 241–257.

Taleb, H., Vale, P., Amanhir, R., Benhadouch, A., Sagou, R., Chaﬁk, A., 2006. First

Coleman, A.W., 2009. Is there a molecular key to the level of. Molecular Phyloge-

detection of azaspiracids in mussels in north west Africa. Journal of Shellﬁsh

netics and Evolution 50 (1), 197–203.

Research 25 (3), 1067–1070.

D’Alelio, D., Amato, A., Kooistra, W.H.C.F., Procaccini, G., Casotti, R., Montresor, M.,

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:

2009. Internal transcribed spacer polymorphism in Pseudo-nitzschia multistriata

molecular evolutionary genetics analysis using maximum likelihood, evolu-

(Bacillariophyceae) in the Gulf of Naples: recent divergence or intraspeciﬁc

tionary distance, and maximum parsimony methods. Molecular Biology and

hybridization? Protist 160 (1), 9–20.

Evolution 28 (10), 2731–2739.

Darty, K., Denise, A., Ponty, Y., 2009. VARNA: interactive drawing and editing of the

Tillmann, U., Elbra¨chter, M., John, U., Krock, B., 2011. A new non-toxic species in the

RNA secondary structure. Bioinformatics 25 (15), 1974.

dinoﬂagellate genus Azadinium: A. poporum sp. nov. European Journal of Phy-

Dodge, J., Crawford, R., 1971. A ﬁne structural survey of dinoﬂagellate pyrenoids and

cology 46 (1), 74–87.

food reserves. Botanical Journal of the Linnean Society 64 (2), 105–115.

H. Gu et al. / Harmful Algae 21–22 (2013) 64–75 75

Tillmann, U., Elbra¨chter, M., John, U., Krock, B., Cembella, A., 2010. Azadinium obesum Twiner, M.J., Rehmann, N., Hess, P., Doucette, G.J., 2008. Azaspiracid shellﬁsh

(Dinophyceae), a new nontoxic species in the genus that can produce azaspir- poisoning: a review on the chemistry, ecology, and toxicology with an emphasis

acid toxins. Phycologia 49 (2), 169–182. on human health impacts. Marine Drugs 6 (2), 39–72.

Tillmann, U., Elbra¨chter, M., Krock, B., John, U., Cembella, A., 2009. Azadinium Ueoka, R., Ito, A., Izumikawa, M., Maeda, S., Takagi, M., Shin-ya, K., Yoshida, M., van

spinosum gen. et sp. nov. (Dinophyceae) identiﬁed as a primary producer of Soest, R.W.M., Matsunaga, S., 2009. Isolation of azaspiracid-2 from a marine

azaspiracid toxins. European Journal of Phycology 44 (1), 63–79. sponge Echinoclathria sp. as a potent cytotoxin. Toxicon 53 (6), 680–684.

Tillmann, U., Salas, R., Gottschling, M., Krock, B., O’Driscoll, D., Elbra¨chter, M., 2012. Vale, P., Bire, R., Hess, P., 2008. Conﬁrmation by LC–MS/MS of azaspiracids in

Amphidoma languida sp. nov. (Dinophyceae) reveals a close relationship be- shellﬁsh from the Portuguese north-west coast. Toxicon 51, 1449–1456.

tween Amphidoma and Azadinium Protist 163 (5) 701–719. Wayne Litaker, R., Vandersea, M.W., Kibler, S.R., Reece, K.S., Stokes, N.A., Lutzoni, F.M.,

To¨ be, K., Joshi, A.R., Messtorff, P., Tillmann, U., Cembella, A., John, U., 2012. Yonish, B.A., West, M.A., Black, M.N.D., Tester, P.A., 2007. Recognizing dinoﬂa-

Molecular discrimination of taxa within the dinoﬂagellate genus Azadinium, gellate species using ITS rDNA sequences. Journal of Phycology 43 (2), 344–355.

the source of azaspiracid toxins. Journal of Plankton Research, http://dx.doi.org/ Yao, J., Tan, Z., Zhou, D., Guo, M., Xing, L., Yang, S., 2010. Determination of

10.1093/plankt/fbs077. azaspiracid-1 in shellﬁshes by liquid chromatography with tandem mass

Twiner, M., El-Ladki, R., Kilcoyne, J., Doucette, G.J., 2012. Comparative effects of the spectrometry. Chinese Journal of Chromatography 28 (4), 363–367.

marine algal toxins azaspiracid-1,-2, and -3 on Jurkat T lymphocyte cells. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization

Chemical Research in Toxicology 25 (3), 747–754. prediction. Nucleic Acids Research 31 (13), 3406–3415.

View publication stats