Abstract: Pseudo-nitzschia is a group of wide-distributed planktonic diatoms. Some species produce the neurotoxin domoic acid (DA). Herein, two new Pseudo-nitzschia species were described from Chinese coastal waters and South China Sea after combining morphological and molecular data, together with biogeographical traits. Pseudo-nitzschia punctionis sp. nov. was similar to P. bipertita morphologically, but differed in poroid structure which was undivided in P. punctionis but divided in two sectors in P. bipertita. This difference corresponded to the presence of two HCBCs in ITS2 secondary structure. Pseudo-nitzschia polymorpha sp. nov. was sister to P. limii phylogenetically, but distinct by the various shapes of perforations on the valve and copula, which was supported by four HCBCs. In a metabarcoding analysis, multiple new ribotypes were identified within the two new species, and intra-specific genetic divergences were analyzed. Metabarcoding data revealed that P. punctionis had a broader temperature range (12.9-30.5℃) than P. polymorpha (22.3-30.5℃). Within the two new species, different traits were found among the ASVs regarding to the temperature and biogeography, representing different microevolutionary directions under environmental selection. The two new species had different biogeographical traits when compared to their closely-related species. Domoic acid was detected in strains of P. punctionis at a concentration of 13.5-17.7 fg cell^-1, but the toxin was not found in strains of P. polymorpha. A combination of characters based on laboratory strains and field metabarcoding data provided more data for delimiting Pseudo-nitzschia species and gave new insights on their diversity and biogeography.

Key words: China, Pseudo-nitzschia, morphology, phylogeny, metabarcoding, domoic acid