Filters were then centrifuged again for 2 min at 800g. The filtrates click here were transferred to HPLC vials and stored at −20°C until measurement. Mass spectral experiments were performed on an ABI-SCIEX-4000 Q Trap (Applied Biosystems, Darmstadt, Germany), triple quadrupole mass spectrometer equipped with a TurboSpray® interface coupled

to an Agilent (Waldbronn, Germany) model 1100 LC. The LC equipment included a solvent reservoir, in-line degasser (G1379A), binary pump (G1311A), refrigerated autosampler (G1329A/G1330B), and temperature-controlled column oven (G1316A). After injection of 5 μL of sample, separation of lipophilic toxins was performed by reverse-phase chromatography on a C8 column (50 × 2 mm) packed with 3 μm Hypersil BDS 120 Å (Phenomenex, Aschaffenburg, Germany) and maintained at 25°C. The flow rate was 0.2 mL · min−1 and gradient elution was performed with two eluents, where eluent A was water and eluent B was methanol/water (95:5 v/v), both containing 2.0 mM ammonium formate and 50 mM formic acid. Initial conditions were elution with 5% B, followed by a linear gradient to 100% B within 10 min and isocratic elution until 10 min with 100% B. The program was then returned to initial conditions within 1 min followed by 9 min column equilibration (total run time: 30 min). Mass spectrometric parameters were as follows: curtain gas: 20 psi, CAD gas: medium, ion spray voltage: 5500 V, temperature: 650°C,

nebulizer gas: 40 psi, auxiliary gas: 70 selleck psi, interface heater: on, declustering potential: 121 V, entrance potential: 10 V, exit potential: 22 V, collision energy: 57 V. Selected reaction monitoring (SRM) experiments were carried out in positive ion mode by selecting the following transitions (precursor ion > fragment selleck chemicals llc ion): m/z 534 > >150, 536 > >150, 540 > 164, 552 > 150, 628 > 150, 640 > 164, 644 > 164, 650 > 164, 658 > 164, 674 > 164, 678 > 150, 678 > 164, 692 > 150, 692 > 164, 694 > 150, 694 > 164, 698 > 164, 706 > 164, 708 > 164, 710 > 150, 720 > 164, 722 > 164, 766 > 164 and 784 > 164. Dwell times of 40 ms were used for each transition. BI and ML methods returned phylogenetic

trees with identical topologies. In the BI tree shown in Figure 1, the A. ostenfeldii/A. peruvianum complex appears to be genetically highly structured with the sequences analyzed falling into six distinct phylogenetic groups. The clustering did not conform to the morphospecies distribution. Strains assigned morphologically to A. peruvianum and strains identified as A. ostenfeldii intermingled in the tree. Lower nodes were generally poorly resolved. Analysis of larger D1-D2 LSU data sets focusing on unique sequences and intra-strain variability largely confirmed the initial analysis. In the D1-D2 phylogeny (Fig. 2), all the groups indicated in Figure 1 were present as separate highly supported (>0.95 branches except for the group 2 which had a branch support of only 0.