(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.分子检测方法为浮游生物样本中有毒微藻的常规检测与定量提供了极具潜力的技术手段。为此，本研究针对海洋甲藻属亚历山大藻（Alexandrium）的2个物种——微小亚历山大藻（A. minutum）、塔姆图特亚历山大藻（A. tamutum），以及塔玛亚历山大藻物种复合体（A. tamarense species complex）的3个类群/核糖型：Group I/北美型（NA）、Group II/地中海型（ME）、Group III/西欧型（WE），开发了靶向小亚基（SSU）或大亚基（LSU）核糖体亚基（rRNA）的新型TaqMan小沟结合探针（TaqMan minor groove binding probe）与引物。 针对实时荧光定量聚合酶链式反应（qPCR）设计的引物与探针具备物种特异性，且在利用培养的亚历山大藻菌株纯DNA开展qPCR交叉验证实验时，展现出极高的扩增效率。本研究从苏格兰东海岸采集的自然浮游生物群落样本中，评估了该qPCR检测体系作为分子工具，用于检测亚历山大藻物种及类群并估算其相对细胞丰度的适用性。将检测结果与亚历山大藻属（Alexandrium spp.）的倒置显微镜细胞计数（乌特莫尔法，Utermöhl technique）结果，以及对应样本的麻痹性贝类毒素（PSP）浓度进行了比对分析。 qPCR检测结果显示，塔玛亚历山大藻Group I（北美型）与塔姆图特亚历山大藻为丰度最高的亚历山大藻类群，且二者均与浮游生物样本的PSP毒素含量呈显著正相关。塔玛亚历山大藻Group III（西欧型）细胞几乎在所有采样站点均有检出，但丰度较低。本研究未在任何样本中检出微小亚历山大藻与塔玛亚历山大藻Group II（地中海型）细胞，这表明至少在本次采样时段，该特定北海海域不存在这两个类群。塔玛亚历山大藻Group I与Group III的同域分布现象，进一步支持了如下假说：塔玛亚历山大藻物种复合体的各个类群/核糖型实为隐存种，而非隶属于同一物种的不同变种。