Extensive intragenomic variations of the 18S rDNA V4 region in the toxigenic diatom species Pseudo-nitzschia multistriata revealed through high-throughput sequencing.

Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.

The high molecular diversity in Noctiluca scintillans is dominated by intra-genomic variations revealed by single cell high-throughput sequencing of 18S rDNA V4.

The application of high-throughput sequencing (HTS) technologies has revolutionized research on phytoplankton biodiversity by generating an unprecedented amount of molecular data in marine ecosystem surveys. However, high-level of molecular diversity uncovered in HTS-based metabarcoding analyses may lead to overinterpretation of phytoplankton diversity due to excessive intra-genomic variations (IGVs). The aims in this study are to explore the nature of phytoplankton molecular diversity and to test the hypothesis. We carried out single-cell metabarcoding analysis of 18S rDNA V4 sequences obtained in single Noctiluca scintillans cells isolated from various sites in coastal waters of China. Results showed that each single N. scintillans cell harbored a high level of IGVs with about 100 amplicon sequence variants (ASVs). The large numbers of non-dominant ASVs identified in N. scintillans cells, which might correspond to the larger numbers of ASVs annotated as N. scintillans and showed similar temporal dynamics in metabarcoding analyses, could inflate the inter-species diversity or intra-species genetic diversity. In addition, there were large numbers of additional ASVs that were not annotated as N. scintillans. These non-N. scintillans ASVs might represent diverse preys for N. scintillans, consistent with previous reports that N. scintillans may act as chance predator of a broad-spectrum preys. This single-cell study has unambiguously demonstrated that the existence of high levels of IGVs in N. scintillans and most likely many other phytoplankton species, demonstrating that the majority of the molecular diversity revealed in metabarcoding analysis, which were generally interpreted as the sum of inter-species diversity and intra-species diversity, actually included high levels of IGVs and should be interpreted with caution.

Rich diversity and active spatial-temporal dynamics of Thalassiosira species revealed by time-series metabarcoding analysis.

Thalassiosira is a species-rich genus in Bacillariophyta that not only contributes positively as primary producer, but also poses negative impacts on ecosystems by causing harmful algal blooms. Although taxonomical studies have identified a large number of Thalassiosira species, however, the composition of Thalassiosira species and their geographical distribution in marine ecosystems were not well understood due primarily to the lack of resolution of morphology-based approaches used previously in ecological expeditions. In this study, we systematically analyzed the composition and spatial-temporal dynamic distributions of Thalassiosira in the model marine ecosystem Jiaozhou Bay by applying metabarcoding analysis. Through analyzing samples collected monthly from 12 sampling sites, 14 Thalassiosira species were identified, including five species that were not previously reported in Jiaozhou Bay, demonstrating the resolution and effectiveness of metabarcoding analysis in ecological research. Many Thalassiosira species showed prominent temporal preferences in Jiaozhou Bay, with some displaying spring-winter preference represented by Thalassiosira tenera, while others displaying summer-autumn preference represented by Thalassiosira lundiana and Thalassiosira minuscula, indicating that the temperature is an important driving factor in the temporal dynamics. The application of metabarcoding analysis, equipped with appropriate molecular markers with high resolution and high specificity and databases of reference molecular marker sequences for potential all Thalassiosira species, will revolutionize ecological research of Thalassiosira species in Jiaozhou Bay and other marine ecosystems.

Chloroplast Genomes for Five Skeletonema Species: Comparative and Phylogenetic Analysis.

Skeletonema species are cosmopolitan coastal diatoms that exhibit important roles in ecological system. The chloroplast genomes (cpDNAs) have been proven to be important in the study of molecular evolution and genetic diversity. However, cpDNA of only a single Skeletonema species (S. pseudocostatum) has been constructed, hindering in-depth investigation on Skeletonema species. In this study, complete cpDNAs of five Skeletonema species were constructed with cpDNAs of four species S. marinoi, S. tropicum, S. costatum, and S. grevillea constructed for the first time. These cpDNAs had similar sizes and same numbers of genes. These cpDNAs were highly syntenic with no substantial expansions, contractions, or inversions. Interestingly, two copies of petF, which encodes ferredoxin with critical role in iron dependency, were found in all five Skeletonema species, with one copy in the cpDNA and another copy in the nuclear genome of each species. Selection analysis revealed that all PCGs of cpDNAs were undergoing purifying selection. Despite the high conservation of these cpDNAs, nine genomic regions with high sequence divergence were identified, which illustrated substantial variations that could be used as markers for phylogenetic inference and for tracking Skeletonema species in the field. Additionally, the numbers of simple sequence repeats varied among different cpDNAs, which were useful for detecting genetic polymorphisms. The divergence times estimated using PCGs of cpDNAs revealed that most of these species were established within ∼33 Mya, consistent with that estimated using mtDNAs. Overall, the current study deepened our understanding about the molecular evolution of Skeletonema cpDNAs.

Complete mitochondrial genome of Thalassiosira profunda (Mediophyceae, Bacillariophyta).

Thalassiosira is a species-rich genus with about 170 described species, many of which are harmful algal species with significant negative ecological impact. However, genome data of these species remain limited. In this study, the complete mitochondrial genome of Thalassiosira profunda (Hendey) Hasle 1973 was determined for the first time. The circular genome was 40,470 bp in length with GC content of 30.98%. It encodes 63 genes including 36 protein-coding genes (PCGs), 25 tRNA genes, and two rRNA genes. Phylogenetic analysis using concatenated PCGs suggested that T. profunda had a closer evolutionary relationship with Skeletonema marinoi of a different family (Skeletonemataceae) than Thalassiosira pseudonana, suggesting complex evolutionary relationship among species in these two families. Colinearity analysis also revealed fewer genome rearrangements between T. profunda and S. marinoi than that between T. profunda and T. pseudonana. This study suggests that mitochondrial genomes of many more species in the Thalassiosiraceae and Skeletonemataceae families are needed to disentangle the complex evolutionary relationships in the order of Thalassiosirales.

Complete mitochondrial genome of the harmful algal bloom species Thalassiosira nordenskioeldii (Mediophyceae, Bacillariophyta) from the east China sea.

Thalassiosira nordenskioeldii is a common harmful algal bloom (HAB) species with worldwide distribution. Although barcode sequences of this ecologically important species have been published, no genome data have been published for T. nordenskioeldii. In this study, we constructed the complete mitochondrial genome (mtDNA) of a T. nordenskioeldii strain isolated from the East China Sea. The T. nordenskioeldii mtDNA is circular and has a length of 47,038 bp and a GC content of 30.84%. The mtDNA encodes 69 genes, including 40 protein-coding (PCGs), 27 tRNA and two rRNA genes. Phylogenetic analysis using concatenated amino acid sequences of 31 shared PCGs from 37 diatom mtDNAs revealed that the mtDNA of T. nordenskioeldii was fully resolved in a clade with that of Thalassiosira profunda. The mtDNA of T. nordenskioeldii showed high collinearity with those of T. profunda and Skeletonema marinoi with only minor rearrangements. The completion of Thalassiosira mtDNAs will facilitate evolutionary studies on species of the order Thalassiosirales and the class Mediophyceae.