Koliella bifissiva sp. nov (Chlorellaceae, Chlorophyta) and Analysis of Its Organelle Genomes.

Chlorellacean members are common in aquatic or subaerial habitats, and many of them have significant economic value. Taxonomic reports and organelle genome data for the Nannochloris clade, an important subgroup within this family, are limited, hindering the understanding and exploitation of this clade. In this study, a fusiform-celled strain, FACHB-3607, was isolated from a pond in China. Through examination of morphological characteristics and phylogenetic analyses of rbcL, 18S rDNA, and ITS, it was identified as a new species within the Nannochloris clade, named Koliella bifissiva sp. nov. In addition, this study provided a first insight into the organellar genomes of the genus Koliella. The K. bifissiva chloroplast had a 99.8 kb genome, and the mitochondrion had a 40.8 kb genome, which are moderate sizes within the Nannochloris clade. Phylogenomic analysis showed that K. bifissiva is most closely related to Nannochloris sp. "desiccata", followed by Marvania. In contrast, Picochlorum was the most distantly related species. The organelle genomes of the Nannochloris clade display dynamic evolution, reflected in variations in genome size, gene content and order, and selection pressure. This research enhances our knowledge of species diversity and evolutionary history in the Nannochloris clade.

Chromosome-scale genome assembly reveals insights into the evolution and ecology of the harmful algal bloom species Phaeocystis globosa Scherffel.

The phytoplankton Phaeocystis globosa plays an important role in sulfur cycling and climate control, and can develop harmful algal blooms (HABs). Here we report a chromosome-scale reference genome assembly of P. globosa, which enable in-depth analysis of molecular underpinnings of important ecological characteristics. Comparative genomic analyses detected two-rounds of genome duplications that may have fueled evolutionary innovations. The genome duplication may have resulted in the formation of dual HiDP and LoDP dimethylsulphoniopropionate (DMSP) biosynthesis pathways in P. globosa. Selective gene family expansions may have strengthened biological pathways critical for colonial formation that is often associated with the development of algal blooms. The copy numbers of rhodopsin genes are variable in different strains, suggesting that rhodopsin genes may play a role in strain-specific adaptation to ecological factors. The successful reconstruction of the P. globosa genome sets up an excellent platform that facilitates in-depth research on bloom development and DMSP metabolism.

Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

Genetic Diversity and Geographical Distribution of the Red Tide Species Coscinodiscus granii Revealed Using a High-Resolution Molecular Marker.

Diatoms are responsible for approximately 40% of the global primary photosynthetic production and account for up to 20% of global carbon fixation. Coscinodiscus granii is a red tide forming species of the phylum Bacillariophyta that has been detected in a wide range of coastal regions, suggesting the possibility of the existence of high genetic diversity with differential adaptation. Common molecular markers including 18S rDNA, 16S rDNA, ITS, cox1, and rbcL do not provide sufficient resolution for distinguishing intra-species genetic diversity, hindering in-depth research on intra-species genetic diversity and their spatial and temporal dynamics. In this project, we aimed to develop molecular markers with high resolution and specificity for C. granii, attempting to identify different taxa of this species, which will set up a stage for subsequent functional assays. Comparative genomics analysis of the mtDNAs of C. granii strains identified a genomic region with high genomic variations, which was used to guide the development of a molecular marker with high resolution and high specificity. This new molecular marker, which was named cgmt1 (C. granii mitochondrial 1), was 376 bp in size and differentiated C. granii samples collected in coastal regions of China into three different clades. Preliminary analysis of field samples collected in various coastal regions in China revealed that C. granii clades were almost exclusively found in the Bohai Sea and the north Yellow Sea. This newly developed molecular marker cgmt1 could be used for tracking intra-species genetic diversity and biogeographic distribution of C. granii in different ecosystems.

Genetic Analysis of a Large-Scale Phaeocystis globosa Bloom Offshore Qingdao, China.

A sudden large-scale bloom event of the haptophyte Phaeocystis globosa that lasted over one month in the winter of 2021 was observed offshore Qingdao, China. This P. globosa bloom event was unusual as it was the first P. globosa bloom recorded in Qingdao offshore. Furthermore, the temperature at which this event occurred was much lower than that of previous P. globosa blooms in China. We hypothesize that the P. globosa strains that drove the development of this bloom offshore Qingdao were genetically unique and have a competitive advantage in the environmental conditions. To test this hypothesis, we analyzed P. globosa genetic diversity and the temporal dynamics of the bloom, using the high-resolution molecular markers pgcp1 and cox1 that we developed recently. The analysis revealed that the genetic compositions of P. globosa offshore Qingdao were rather limited, containing two dominant genotypes and other rare genotypes with low abundance, representing a small portion of the genetic diversities identified in coastal waters in China, and were rather different from the P. globosa genotypes outside of the Jiaozhou Bay before the P. globosa bloom in the winter of 2021. This suggested only certain strains contribute to the development of blooms under certain environmental conditions. The genetic composition may indicate the unusual timing and scale of this P. globosa event.

Metabarcoding analysis of microbiome dynamics during a Phaeocystis globosa bloom in the Beibu Gulf, China.

Phaeocystis globosa is an ecologically important haptophyte that can form harmful algal blooms (HABs). In this study, we used 16S rDNA V3-V4 amplicon sequencing data to explore the ecological mechanisms underlying a P. globosa bloom in the Beibu Gulf, China. Using field samples collected from three time points of a bloom, we observed a distinct succession in the bacteria, archaea and phytoplankton community composition throughout the bloom. We also observed temporal variation in response to the bloom at the nucleotide level, which supports a previously underappreciated amount of intragroup variation in the niches taken up by microbes during HABs. We developed a preliminary model for the development and progression of the P. globosa bloom using the spatial-temporal dynamics of P. globosa and the bacteria, archaea, phytoplankton and environmental variables. We also identified microbes with putative interactions with P. globosa during the bloom by identifying microbes correlated with P. globosa in interaction networks, identifying particle-associated microbes and exploring the P. globosa colony microbiome using sequences from whole P. globosa colonies collected during the bloom. This study revealed novel insight into the development of P. globosa HABs and many testable hypotheses that will guide future research on the mechanisms of P. globosa HABs.

Complete mitochondrial genome of Coscinodiscus granii (Coscinodiscophyceae, Bacillariophyta).

Coscinodiscus is a genus common in marine phytoplankton, with some species thought to have a significant negative ecological impact. However, the availability of their genome sequences is rather limited. Here, we assembled and annotated the first complete mitochondrial genome (mtDNA) of the species Coscinodiscus granii L.F.Gough 1905, as part of our efforts to gain a better understanding of the genetic characteristics of Coscinodiscus taxa at a genomic level. The circular mtDNA was 34,970 bp in length and encoded 60 genes, including 32 protein-coding genes (PCGs), 24 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and two conserved open reading frames (orfs). The overall GC content of C. granii mtDNA was 24.30%, which was slightly lower than that of C. wailesii (25.00%), the first species in the genus Coscinodiscus whose mtDNA has been reported, and higher than that of Melosira undulata (21.60%), the first species in the class Coscinodiscophyceae whose mtDNA has been reported. As expected for congeneric species, phylogenetic analysis using concatenated amino acid sequences of 27 shared PCGs suggested that C. granii has a closer evolutionary relationship with C. wailesii. Coscinodiscus was found to be monophyletic in the phylogeny. The complete mtDNAs of more Coscinodiscus species will facilitate the exploration of the evolutionary relationships of species in the Class Coscinodiscophyceae.

Research on the biology and ecology of the harmful algal bloom species Phaeocystis globosa in China: Progresses in the last 20 years.

Chinese researchers have made substantial progresses in research on the harmful algal bloom (HAB) species Phaeocystis globosa since the first P. globosa bloom outbreak in the Chinese coastal waters in 1997. However, as many results, especially the earlier ones, were published in non-English literature, much of the research on P. globosa biology, ecology, and biogeochemistry made by Chinese researchers have been unknown to colleagues from other countries. We review current knowledge on taxonomy, morphology, genetics, physiology, survival strategies and mitigation of P. globosa gained by Chinese researchers from the past two decades. P. globosa is the only Phaeocystis species that causes blooms in the Chinese waters, although other Phaeocystis species including P. jahnii and P. cordata have been detected in Chinese coastal regions. P. globosa has a complex life history with at least two morphotypes including a haploid flagellate and a diploid colonial cell. Colonial P. globosa blooms typically occur in winter after a diatom bloom in coastal waters of the South China Sea. P. globosa in Chinese coastal waters usually has extremely large colonial sizes, up to 3 cm in diameter, an order of magnitude greater than that observed in European coastal waters. The development of giant colonies is associated with enhanced sinking rate, limited nutrient diffusion, as well as decreased stability of colonies. The Chinese P. globosa strains also showed strong genetic diversity and physiological plasticity, being able to grow and develop into colonies at higher temperature and irradiance relative to that in European waters. High genetic diversity of P. globosa was revealed by developing high-resolution and high-specificity molecular markers including Phaeocystis globosa chloroplast 1 (pgcp1). Due to the severe impact of P. globosa on ecology and economy, much effort has been made to mitigate P. globosa blooms including the application of modified clays. Overall, P. globosa in the Chinese waters demonstrate unique genetic, phenotypical and physiological features that differ from P. globosa in other ocean regions. Further studies are needed to explore how environmental factors trigger the occurrence of P. globosa blooms and ascertain the impact of P. globosa blooms on the environment.

High genetic diversity of the harmful algal bloom species Phaeocystis globosa revealed using the molecular marker COX1.

Accumulating evidence indicates that the haptophyte species Phaeocystis globosa, which plays an important role in climate control and may cause harmful algal blooms (HABs), displays a rich genetic diversity that may be responsible for differences in colonial sizes, different toxicity during blooms, and differential optimum growth temperature. In this project, we demonstrated that COX1 can be used as an effective molecular marker for its low intra-genome variations and high resolution in differentiating different P. globosa strains. Analyzing 57 P. globosa strains and seven field samples revealed high P. globosa genetic diversity with at least seven distinct clades. This study not only demonstrated for the first time that the common molecular marker COX1 can be used for differentiating P. globosa strains with high-resolution, and for tracking dynamics of different P. globosa strains during bloom development, but also revealed that P. globosa had high genetic diversity in the field.

Large Differences in the Haptophyte Phaeocystis globosa Mitochondrial Genomes Driven by Repeat Amplifications.

The haptophyte Phaeocystis globosa is a well-known species for its pivotal role in global carbon and sulfur cycles and for its capability of forming harmful algal blooms (HABs) with serious ecological consequences. Its mitochondrial genome (mtDNA) sequence has been reported in 2014 but it remains incomplete due to its long repeat sequences. In this study, we constructed the first full-length mtDNA of P. globosa, which was a circular genome with a size of 43,585 bp by applying the PacBio single molecular sequencing method. The mtDNA of this P. globosa strain (CNS00066), which was isolated from the Beibu Gulf, China, encoded 19 protein-coding genes (PCGs), 25 tRNA genes, and two rRNA genes. It contained two large repeat regions of 6.7 kb and ∼14.0 kb in length, respectively. The combined length of these two repeat regions, which were missing from the previous mtDNA assembly, accounted for almost half of the entire mtDNA and represented the longest repeat region among all sequenced haptophyte mtDNAs. In this study, we tested the hypothesis that repeat unit amplification is a driving force for different mtDNA sizes. Comparative analysis of mtDNAs of five additional P. globosa strains (four strains obtained in this study, and one strain previously published) revealed that all six mtDNAs shared identical numbers of genes but with dramatically different repeat regions. A homologous repeat unit was identified but with hugely different numbers of copies in all P. globosa strains. Thus, repeat amplification may represent an important driving force of mtDNA evolution in P. globosa.

The complete mitochondrial genome and phylogenetic analysis of Coscinodiscus wailesii (Coscinodiscophyceae, Bacillariophyta).

Coscinodiscus is a species-rich genus with about 400 species described, many of which are harmful algal bloom species with significant negative ecological impact. Despite of their importance in primary production and as harmful algal bloom species, genome data for species in this genus is limited. No mitochondrial genome (mtDNA) of any species in this genus has been reported. Here, the complete mtDNA sequence of the Coscinodiscus wailesii Gran & Angst 1931 was constructed and analyzed. The circular mtDNA was 36,071 bp in length, encoding 64 genes, including 34 protein coding genes (PCGs), 24 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and 4 conserved open reading frames (orfs). The overall AT content of C. wailesii mtDNA was 75.00%, which was slightly lower than that of Melosira undulate (78.40%). Maximum likelihood (ML) phylogenetic analysis using 29 shared protein-coding genes revealed that C. wailesii clustered well with M. undulata, which was the only species of class Coscinodiscophyceae whose mtDNA has been fully constructed. The complete mtDNAs of more Coscinodiscus species will be valuable for studying the evolutionary relationships among species in the genus Coscinodiscus and in the Class of Coscinodiscophyceae.

Development of a high-resolution molecular marker for tracking Phaeocystis globosa genetic diversity through comparative analysis of chloroplast genomes.

The phytoplankton Phaeocystis globosa thrives in a wide range of marine regions and plays an important role in climate control. It can also form harmful algal blooms (HABs) that threaten environments and impact important coastal infrastructures. Mechanisms underlying the formation of P. globosa blooms still remain poorly understood. Accumulating evidence suggests that P. globosa has high genetic diversity and different P. globosa strains may have differential contributions to the development of P. globosa blooms. However, due to the lack of molecular markers with adequate resolution for distinguishing P. globosa genetic diversity, such differential contributions by different P. globosa strains could not be accurately ascertained. As such, high-resolution molecular markers need to be developed and applied to distinguish P. globosa genetic diversity. In this study, we undertook to define high-resolution molecular marker by assembling and comparing the whole chloroplast genomes of P. globosa strains isolated from different regions of the world. Through comparative analysis of P. globosa cpDNAs and detection of single nucleotide variations (SNVs), a molecular marker pgcp1 with improved resolution was developed. The pgcp1 demonstrated the highest resolution compared with other regions including 18S rDNA V4 region, 28S rDNA D1-D2 region and rbcL region, through genetic distance and phylogenetic analysis of 13 P. globosa strains. Molecular analysis of environmental samples and strains collected in multiple expeditions from a wide range of ocean regions including multiple regions in China, Vietnam, Thailand and Western Pacific using pgcp1 as the molecular marker displayed high genetic diversity of P. globosa with at least four major P. globosa clades. In conclusion, we have developed pgcp1 as a high-resolution molecular marker for the harmful algal bloom species P. globosa, which can be used to track intra-species genetic diversity and dynamics of P. globosa during bloom development.

Morphology and molecular phylogeny of coccoid green algae Coelastrella sensu lato (Scenedesmaceae, Sphaeropeales), including the description of three new species and two new varieties.

The genus Coelastrella was established by Chodat (Bull. Soc. Bot. Geneve, 13 [1922] 66), and was characterized as being unicellular or in few-celled aggregations with many longitudinal ribs on the cell wall. Many species of this genus showed strong ability to accumulate carotenoids and oils, so they have recently attracted much attention from researchers due to its potential applicability in the energy and food industries. In this study, a total of 23 strains of Coelastrella were sampled from China, and three new species and two new varieties were described: C. thermophila sp. nov., C. yingshanensis sp. nov., C. tenuitheca sp. nov., C. thermophila var. globulina var. nov., C. rubescens var. oocystiformis var. nov. Besides 18S rDNA and ITS2 sequences, we have newly sequenced the tufA gene marker for this taxon. Phylogenetic analysis combined with morphological studies revealed four morphotypes within the Coelastrella sensu lato clade, which contained the morphotype of original Coelastrella, original Scotiellopsis, Asterarcys, and morphotype of C. vacuolata and C. tenuitheca sp. nov. The relationships between morphological differences and phylogenic diversity based on different markers were discussed. Our results support that 18S rDNA was too conserved to be used a species-specific or even a genus-specific marker in this clade. The topology of tufA gene-based phylogenetic tree had a better match with the morphological findings.

Evolutionary Analysis of Unicellular Species in Chlamydomonadales Through Chloroplast Genome Comparison With the Colonial Volvocine Algae.

This study is the first determination of six chloroplast genomes of colonial volvocine algae, Colemanosphaera charkowiensis, Volvulina compacta, Pandorina colemaniae, Pandorina morum, Colemanosphaera angeleri, and Yamagishiella unicocca. Based on 55 chloroplast protein-coding genes, we compared the nonsynonymous (dN) and synonymous (dS) substitution rates between colonial volvocine algae and the other unicellular Chlamydomonadales species. When refer to the dN, we found 27 genes were significantly different, among them, 19 genes were significant higher in unicellular species (FDR-adjusted P < 0.05). When refer to the dS, we found 10 genes were significantly different, among them, 6 genes were significant higher in unicellular species (FDR-adjusted P < 0.05). Then we identified 14 putative fast-evolving genes and 11 putative positively selected genes of unicellular species, we analyzed the function of positively selected sites of the overlap genes of putative fast-evolving and positively selected genes, and found some sites were close to the important functional region of the proteins. Photosynthesis is the process to transform and store solar energy by chloroplast, it plays a vital role in the survival of algae, this study is the first to use the chloroplast genomes to analysis the evolutionary relationship between colonial and unicellular species in Chlamydomonadales. We found more genes have higher substitution rates in unicellular species and proposed that the fast-evolving and positively selected two genes, psbA and psbC, may help to improve the photosynthetic efficiency of unicellular species in Chlamydomonadales.

Phylogenic Diversity and Taxonomic Problems of the Dictyosphaerium Morphotype within the Parachlorella Clade (Chlorellaceae, Trebouxiophyceae).

The Parachlorella clade was put forward as a group within the family Chlorellaceae in 2004. Recent molecular analyses have revealed that Dictyosphaerium morphotype algae form several independent lineages within the Parachlorella clade, and new genera and species have been established. In this study, we focus on the diversity of Dictyosphaerium morphotype algae within the Parachlorella clade, based on 42 strains from China. We used combined analyses of morphology and molecular data based on SSU and internal transcribed spacer region (ITS) rDNA sequences to characterize these algae. In addition, the secondary structure of ITS2 was compared to delineate new lineages. Our results revealed high phylogenic diversity of Dictyosphaerium morphotype algae, and we describe five distinct lineages. We examined the morphological features of these five lineages, and morphological differences are difficult to find compared with other Dictyosphaerium morphotype algae. The five distinct lineages were not described as new genera currently. We lastly discuss the taxonomic problems regarding the Dictyosphaerium morphotype within the Parachlorella clade, and possible solutions are considered.

Euchlorocystis gen. nov. and Densicystis gen. nov., Two New Genera of Oocystaceae Algae from High-altitude Semi-saline Habitat (Trebouxiophyceae, Chlorophyta).

The Oocystaceae family is generally considered to contain common freshwater eukaryotic microalgae, and few are reported living in semi-saline habitats. Our latest ecological survey in Qinghai Lake and Angzicuo Lake, both large, closed, high-altitude, semi-saline lakes located on the Qinghai-Tibet plateau in China, revealed Oocystaceae species as a dominant group among plankton. Since limited knowledge exists about semi-saline species in the Oocystaceae family, a taxonomical study was carried out using morphological and phylogenetic methods. Using this approach, four new strains of Oocystaceae were identified and successfully cultured in the lab. Molecular results correlated with morphological characters and resolved these species into at least three genera. A new genus, Euchlorocystis, with type species Euchlorocystis subsalina, is described here as having the distinctive morphology of multiple pyrenoids per chloroplast among Oocystaceae, and an independent phylogenetic position at the base of the Oocystaceae. Similarly, the genus Densicystis, with type species Densicystis glomerata, is newly proposed here as having a unique colony morphology of dozens or hundreds of little cells tightly embedded in ellipsoid to round mucilage masses. Oocystis marina, originally described from the Baltic Sea, was also identified in Qinghai Lake and Angzicuo Lake and phylogenetically positioned in the semi-saline clade of the Oocystaceae. The result that a marine species was detected in the closed inland lakes implies a further need to reevaluate the origins of these species.

Three novel species of coccoid green algae within the Watanabea clade (Trebouxiophyceae, Chlorophyta).

Coccoid green algae are extremely diverse despite their simple coccoid phenotype, a phenotype that may be the result of convergent evolution. In this study, we used a polyphasic approach combining molecular phylogenetic analyses, morphology and ultrastructure to investigate isolated coccoid strains from China, and our results reveal three new lineages of Trebouxiophyceae: the novel genus and species Mysteriochloris nanningensis gen. et sp. nov., and the two novel species Phyllosiphon coccidium sp. nov. and Desertella yichangensis sp. nov. (Trebouxiophyceae, Chlorophyta). We provide a detailed characterization of the novel microalgae which they are autosporic coccoid unicells and have parietal chloroplasts. In phylogenies based on 18S rDNA sequences and the chloroplast ribulose-bisphosphate carboxylase gene (rbcL), these three algae are nested within the Watanabea clade and are different from any known algae. M. nanningensis FACHB-1787 is not really close to any known algae within the Watanabea clade. Phyllosiphoncoccidium FACHB-2212 is within the Phyllosiphon lineages. D. yichangensis FACHB-1793 is closely related to Desertella californica and described as a representative of a novel species of the genus Desertella.