| Summary: | Domoic acid (DA) is produced by almost half of the species belonging to the diatom genus <i>Pseudo-nitzschia</i> and causes amnesic shellfish poisoning (ASP). It is, therefore, important to investigate the diversity and toxin production of <i>Pseudo-nitzschia</i> species for ASP risk assessments. Between 2018 and 2020, seawater samples were collected from various sites around Aotearoa New Zealand, and 130 clonal isolates of <i>Pseudo-nitzschia</i> were established. Molecular phylogenetic analysis of partial large subunit ribosomal DNA and/or internal transcribed spacer regions revealed that the isolates were divided into 14 species (<i>Pseudo-nitzschia americana</i>, <i>Pseudo-nitzschia arenysensis</i>, <i>Pseudo-nitzschia australis</i>, <i>Pseudo-nitzschia calliantha</i>, <i>Pseudo-nitzschia cuspidata</i>, <i>Pseudo-nitzschia delicatissima</i>, <i>Pseudo-nitzschia fraudulenta</i>, <i>Pseudo-nitzschia galaxiae</i>, <i>Pseudo-nitzschia hasleana</i>, <i>Pseudo-nitzschia multiseries</i>, <i>Pseudo-nitzschia multistriata</i>, <i>Pseudo-nitzschia plurisecta</i>, <i>Pseudo-nitzschia pungens</i>, and <i>Pseudo-nitzschia</i> cf. <i>subpacifica</i>). The <i>P. delicatissima</i> and <i>P. hasleana</i> strains were further divided into two clades/subclades (I and II). Liquid chromatography-tandem mass spectrometry was used to assess the production of DA and DA isomers by 73 representative strains. The analyses revealed that two (<i>P. australis</i> and <i>P. multiseries</i>) of the 14 species produced DA as a primary analogue, along with several DA isomers. This study is the first geographical distribution record of <i>P. arenysensis</i>, <i>P.</i><i>cuspidata</i>, <i>P. galaxiae</i>, and <i>P. hasleana</i> in New Zealand coastal waters.
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