Advancing the Taxonomy of the Diatom Pseudo-nitzschia Through an Integrative Study Conducted in the Central and Southeastern Adriatic Sea

Abstract

The marine diatom genus Pseudo-nitzschia comprises cosmopolitan phytoplankton species commonly present in the Adriatic Sea. Species within the genus Pseudo-nitzschia have been of significant concern because they produce domoic acid (DA), which can cause amnesic shellfish poisoning (ASP). In this study, we identified Pseudo-nitzschia species along the Central and Southeastern Adriatic Sea, where monthly sampling carried out from February 2022 to February 2024 allowed for comprehensive species documentation. Pseudo-nitzschia species cell cultures isolated from the study areas were morphologically and molecularly analysed. Morphological analyses were performed using a scanning electron microscope (FE-SEM/STEM), while molecular analyses were conducted, targeting the ITS1-5.8S-ITS2, LSU, and rbcL regions, to confirm species identity. This integrative approach led to the identification of eight species: Pseudo-nitzschia allochrona, Pseudo-nitzschia calliantha, Pseudo-nitzschia delicatissima, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia mannii, Pseudo-nitzschia multistriata, Pseudo-nitzschia pseudodelicatissima, and Pseudo-nitzschia subfraudulenta. Our findings underscore the value of a combined approach for reliable species identification and contribute to the development of genetic sequence databases that support the advancement of next-generation methods such as metabarcoding. This research emphasises the importance of combined morphological and molecular methods for the differentiation of the cryptic and pseudo-cryptic Pseudo-nitzschia species.

Keywords: Bayesian inference; FE-SEM/STEM; ITS1-5.8S-ITS2; LSU; Pseudo-nitzschia spp.; diatoms; molecular analysis; morphology; phylogeny; rbcL.

Figure 1

Micrographs of Pseudo-nitzschia allochrona: (a) colony in girdle view, LM; (b) whole valve, SEM; (c) central part of the valve face with central nodule, SEM; (d,e) valve ends, SEM; (f) girdle band with rows of perforations, SEM. Scale bars: (a,b) 5 µm; (c–f) 500 nm.

Figure 2

Micrographs of Pseudo-nitzschia calliantha: (a) whole valve, SEM; (b) colony in girdle view, LM; (c) central part of the valve face with central nodule, SEM; (d) central part of the valve face with central nodule, STEM; (e) poroid structure with sector detail, SEM; (f) poroid structure with sector detail, STEM; (g,h) valve ends, SEM; (i) valve end, STEM; (j) girdle band with rows of perforations, SEM; (k) girdle band with rows of perforations, STEM. Scale bars: (a,b) 10 µm; (g–i) 1 µm; (c–f,j,k) 500 nm.

Figure 3

Micrographs of Pseudo-nitzschia delicatissima: (a–c) whole valve, SEM; (d) central part of the valve face with central nodule, SEM; (e) central part of the valve face with central nodule, STEM; (f,g) valve ends, SEM; (h) girdle band with rows of perforations, SEM; (i) girdle band with rows of perforations, STEM. Scale bars: (a) 10 µm; (b,c) 5 µm; (d–i) 1 µm.

Figure 4

Micrographs of Pseudo-nitzschia fraudulenta: (a) whole valve, SEM; (b) central part of the valve face with central nodule, SEM; (c) poroid structure with sector detail, SEM; (d) girdle band with rows of perforations, SEM; (e,f) valve ends, SEM. Scale bars: (a) 10 µm; (b) 2 µm; (c,e,f) 1 µm; (d) 500 nm.

Figure 5

Micrographs of Pseudo-nitzschia mannii: (a) whole valve, SEM; (b) colony in girdle view, LM; (c) central part of the valve face with central nodule, SEM; (d) central part of the valve face with central nodule, STEM; (e) poroid structure with sector detail, SEM; (f) poroid structure with sector detail, STEM; (g,h) valve ends, SEM; (i) girdle band with rows of perforations, SEM; (j) girdle band with rows of perforations STEM. Scale bars: (a,b) 10 µm; (c,d) 1 µm; (g–i) 1 µm; (e,f,j) 500 nm.

Figure 6

Micrographs of Pseudo-nitzschia multistriata: (a) whole valve, SEM; (b) colony in girdle view, LM; (c) central part of the valve face without central nodule, SEM; (d) central part of the valve face without central nodule, STEM; (e,f) valve ends, SEM; (g) girdle band with rows of perforations, SEM; (h) girdle band with rows of perforations, STEM; (i,j) valve ends, STEM. Scale bars: (a,b) 10 µm; (e,f,i,j) 1 µm; (c,d,g,h) 500 nm.

Figure 7

Micrographs of Pseudo-nitzschia pseudodelicatissima: (a) whole valve, SEM; (b) central part of the valve face with central nodule, SEM; (c) central part of the valve face with central nodule, STEM; (d,e) valve ends, SEM; (f) girdle band with rows of perforations, SEM; (g) girdle band with rows of perforations, STEM. Scale bars: (a) 10 µm; (d,e) 1 µm; (b,c,f,g) 500 nm.

Figure 8

Micrographs of Pseudo-nitzschia subfraudulenta: (a) whole valve, SEM; (b) colony in girdle view, LM; (c) central part of the valve face with central nodule, SEM; (d) central part of the valve face with central nodule, STEM; (e) detail of the valve striae with one row of pores, SEM; (f) poroid structure with sector detail, SEM; (g) poroid structure with sector detail, STEM; (h) girdle band with rows of perforations, SEM; (i,j) valve ends, SEM; (k) girdle band with rows of perforations, STEM. Scale bars: (a,b) 10 µm; (c–e,i,j) 1 µm; (f–h,k) 500 nm.

Figure 9

Phylogenetic tree reconstruction created using Bayesian tree based on ITS marker GTR+G+I; ngen = 5,000,000 Monte Carlo Markov Chain generations. Bayesian inference (BI) posterior probabilities (PP) > 0.90 are shown. Isolates sequenced in this study are presented in red. The scale bar represents 0.2 substitutions per site.

Figure 10

Phylogenetic tree reconstruction created using Bayesian tree based on LSU sequences GTR+G+I; ngen = 4,000,000 Monte Carlo Markov Chain generations. Bayesian inference (BI) posterior probabilities (PP) > 0.90 are shown. Isolates sequenced in this study are presented in red. The scale bar represents 0.01 substitutions per site.

Figure 11

Phylogenetic tree reconstruction using Bayesian tree based on rbcL sequences GTR+G+I; ngen = 4,000,000 Monte Carlo Markov Chain generations. Bayesian inference (BI) posterior probabilities (PP) > 0.90 are shown. Isolates sequenced in this study are presented in red. The scale bar represents 0.02 substitutions per site.

Figure 12

Combined phylogenetic tree reconstruction using Bayesian tree based on ITS, LSU, and rbcL marker GTR+G+I; ngen = 10,000,000 Monte Carlo Markov Chain generations. Bayesian inference (BI) posterior probabilities (PP) > 0.90 are shown. Isolates sequenced in this study are presented in red. The scale bar represents 1.0 substituions per site.

Figure 13

Morphometric data regarding observed Pseudo-nitzschia species: (a) length; (b) width; (c) number of fibulae in 10 µm; (d) number of interstriae in 10 µm; (e) number of poroids in 1 µm; (f) number of sectors within poroids; and (g) number of band interstriae in 10 µm.

Figure 14

The study stations: (a) V—Velebit Channel, (b) S—Šibenik Bay, (c) K—Kaštela Bay, (d) and M—Mali Ston Bay.