Update of the Planktonic Diatom Genus Pseudo-nitzschia in Aotearoa New Zealand Coastal Waters: Genetic Diversity and Toxin Production

Abstract

Domoic acid (DA) is produced by almost half of the species belonging to the diatom genus Pseudo-nitzschia and causes amnesic shellfish poisoning (ASP). It is, therefore, important to investigate the diversity and toxin production of Pseudo-nitzschia 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 Pseudo-nitzschia 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 (Pseudo-nitzschia americana, Pseudo-nitzschia arenysensis, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia cuspidata, Pseudo-nitzschia delicatissima, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia galaxiae, Pseudo-nitzschia hasleana, Pseudo-nitzschia multiseries, Pseudo-nitzschia multistriata, Pseudo-nitzschia plurisecta, Pseudo-nitzschia pungens, and Pseudo-nitzschia cf. subpacifica). The P. delicatissima and P. hasleana 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 (P. australis and P. multiseries) of the 14 species produced DA as a primary analogue, along with several DA isomers. This study is the first geographical distribution record of P. arenysensis, P.cuspidata, P. galaxiae, and P. hasleana in New Zealand coastal waters.

Keywords: Pacific Ocean; amnesic shellfish poisoning; domoic acid; geographical distribution; liquid chromatography-tandem mass spectrometry; molecular phylogenetic analysis; phytoplankton.

Figure 1

Molecular phylogenetic tree of 52 Pseudo-nitzschia species based on the ITS region sequences (253 sequences, 809 positions) using maximum likelihood (ML) analysis. See Figure 2 for details of 25 species. Strains from New Zealand are shown in colour fonts. Blue and green fonts indicate potential ‘low and no ASP risk’ species in New Zealand, respectively. A Black or coloured circle indicates a strain used in the LSU rDNA D1–D3 tree shown in Figure 3. Nodal support represents ML bootstrap value/Bayesian inference (BI) posterior probability. Nodal support under 50 in ML or 0.50 in BI is shown as a minus sign (−). A node that was not present in the BI tree is labelled as np. A scale bar indicates the number of nucleotide substitutions per site. *¹: A sequence obtained from holotype material is shown in bold font. *²: A sequence having only ITS 2 region. *³: A reduction ratio of reduced nodal length calculated from original nodal length. *⁴: Clade separation reported by the present study. *⁵: Subclade separation reported by Ajani et al. (2020) [17]. *⁶: Pseudo-nitzschia cuspidata strain MER was re-assigned as P. cf. cuspidata by Ajani et al. (2021) [18].

Figure 2

Molecular phylogenetic tree of 25 Pseudo-nitzschia species based on the ITS region sequences (144 sequences, 809 positions) using maximum likelihood (ML) analysis. Strains from New Zealand are shown in colour fonts. Red, blue, and green fonts indicate potential ‘high, low, and no ASP risk’ species in New Zealand, respectively. A Black or coloured circle indicates a strain used in the LSU rDNA D1–D3 tree shown in Figure 3. Nodal support represents ML bootstrap value/Bayesian inference (BI) posterior probability. Nodal support under 50 in ML or 0.50 in BI is shown as a minus sign (−). A node that was not present in the BI tree is labelled as np. A scale bar indicates the number of nucleotide substitutions per site. *¹: A sequence obtained from holotype material is shown in bold font. *²: A sequence obtained from epitype material is shown in bold font. *³: A reduction ratio of reduced nodal length calculated from original nodal length. *⁴: Clade separation reported by the present study. *⁵: Subclade separation reported by Stonik et al. (2018) [14]. *⁶: Subclade separation reported by Gai et al. (2018) [15]. *⁷: Clade separation reported by Kim et al. (2015) [16].

Figure 3

Molecular phylogenetic tree of 47 Pseudo-nitzschia species based on the LSU rDNA D1–D3 sequences (179 sequences, 660 positions) using maximum likelihood (ML) analysis. Strains from New Zealand are shown in colour fonts. Red, blue, and green fonts indicate potential ‘high, low, and no ASP risk’ species in New Zealand, respectively. A Black or coloured circle indicates a strain used in the ITS region trees shown in Figure 1 and Figure 2. Nodal support represents ML bootstrap value/Bayesian inference (BI) posterior probability. Nodal support under 50 in ML or 0.50 in BI is shown as a minus sign (−). A node that was not present in the BI tree is labelled as np. A scale bar indicates the number of nucleotide substitutions per site. *¹: A sequence obtained from holotype material is shown in bold font. *²: A reduction ratio of reduced nodal length calculated from original nodal length. *³: Clade separation reported by the present study. *⁴: Clade separation reported by Kim et al. (2015) [16]. *⁵: Clade separation reported by McDonald et al. (2007) [19]. *⁶: Subclade separation reported by Gai et al. (2018) [15]. *⁷: Subclade separation reported by Stonik et al. (2018) [14]. *⁸: Strain 0BD041219-B1, originally reported as P. delicatissima by Dermastia et al. (unpublished data), is assigned as P. cf. arenysensis as its sequence was almost identical to those of P. cf. arenysensis reported recently by Giulietti et al. (2021) [20]. *⁹: Subclade separation reported by Ajani et al. (2020) [17]. *¹⁰: Pseudo-nitzschia cf. heimii strain CAWB106 is assigned as P. subpacifica based on morphological characters following discussions in Rhodes et al. (2013) [10]. *¹¹: Pseudo-nitzschia pungens strains CV4, CV5, 3b, and 51b [21,22] were isolated from California, USA where only P. pungens clade II was reported to date. These strains were tentatively assigned as putative clade II of P. pungens in the present study. *¹²: Pseudo-nitzschia cuspidata strain MER was re-assigned as P. cf. cuspidata by Ajani et al. (2021) [18].

Figure 4

Sampling and distribution map of 14 Pseudo-nitzschia species in New Zealand coastal waters assessed between 2018 and 2020. The locations of 22 sampling sites (grey circles) and their site codes are shown. The numbers in each pie chart indicate the total number of clonal strains genetically identified by the molecular phylogenies. Red, blue, and green indicate potential ‘high, low, and no ASP risk’ species in New Zealand, respectively. Sampling details, including sampling site code, are shown in Table S1.

Figure 5

Structures of domoic acid (DA) and DA isomers. Adapted from Quilliam et al. (1995) [23]. *¹ DA isomers not analysed in the present study.

Figure 6

Domoic acid (DA) and DA isomers profiles of Pseudo-nitzschia australis and P. multiseries strains from New Zealand. (A) DA and DA isomers cell quota (pg cell⁻¹); (B) DA and DA isomers proportion (%). Asterisks: Strain was cultured using f/2−Si medium at *¹ three, *² four, *³ seven, *⁴ nine, or *⁵ ten months after the strain was established. *⁶: Three P. multiseries strains (G010Ps10, CAWB149, and G001Ps04) produced very low cell quotas of iso-DA E (0.002–0.005 pg cell⁻¹, 0.1–0.2%), resulting in difficulty in seeing these data in Figure 6 (A,B).

Figure 7

Total ion chromatogram of DA and DA isomers (combined 312 > 266 and 312 > 161) of DA and DA isomers standards and a Pseudo-nitzschia extract. (A) 1 µL injection of 1/50 dilution of NRCC DA-f DA and DA isomers standard; (B) 1 µL injection of iso-DA C standard (950 ng mL⁻¹); (C) 1 µL injection of an extract of P. multiseries strain G001Ps04.

Figure 8

Distribution map of 16 Pseudo-nitzschia species in New Zealand coastal waters between 1993 and 2020. Red, blue, and green indicate potential ‘high, low, and no ASP risk’ species in New Zealand, respectively. *¹: Species identification should be treated as tentative, as discussed in Section 3.1.