Linda K. Medlin

The origin and evolution of the diatoms: their adaptation to a planktonic existence

Publisher Summary This chapter reviews the evolution of the diatoms and of various groups within the diatoms and evaluates the significance of particular shared derived traits. The chapter also covers the question of why the diatoms dominate the modem marine phytoplankton, why they as members of the chromist lineages win over red and green unicellular phytoplankton, why heterokontophytes win over dinoflagellates and haptophytes, and why diatoms win over other heterokontophytes. Such insights would be valuable to climatologists trying to discover if there are any historical analogues for the current Earth system, with atmospheric CO 2 now at levels unknown since the mid-Tertiary and an ocean pH that within a few centuries may be lower than at any time since the end of the Carboniferous. The study of molecular evolution among closely related organisms or groups of organisms helps us to provide the foundations for the application of the use of genes and their products for understanding ecosystem resilience, to understand the key mechanisms that have given rise to the similarities and differences within and between species genomes, to understand the consequences of variation in genome sizes, gene families, and the exploitation of sequence polymorphisms in organisms, to appreciate how combinations of a diversity of kinds of mutations, genetic drift and selection have given rise to todays organisms, to see how the comparative biological approaches within and between species give additional insight into genome structure, evolution, and function.

Phylogenetic relationships of the ‘golden algae’ (haptophytes, heterokont chromophytes) and their plastids

The phylogenetic relationships of the “golden algae”, like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the “golden algae”. Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/ chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the “golden algae”, viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).

Molecular Detection, Quantification, and Diversity Evaluation of Microalgae

This study reviews the available molecular methods and new high-throughput technologies for their practical use in the molecular detection, quantification, and diversity assessment of microalgae. Molecular methods applied to other groups of organisms can be adopted for microalgal studies because they generally detect universal biomolecules, such as nucleic acids or proteins. These methods are primarily related to species detection and discrimination among various microalgae. Among current molecular methods, some molecular tools are highly valuable for small-scale detection [e.g., single-cell polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and biosensors], whereas others are more useful for large-scale, high-throughput detection [e.g., terminal restriction length polymorphism, isothermal nucleic acid sequence-based amplification, loop-mediated isothermal amplification, microarray, and next generation sequencing (NGS) techniques]. Each molecular technique has its own strengths in detecting microalgae, but they may sometimes have limitations in terms of detection of other organisms. Among current technologies, qPCR may be considered the best method for molecular quantification of microalgae. Metagenomic microalgal diversity can easily be achieved by 454 pyrosequencing rather than by the clone library method. Current NGS, third and fourth generation technologies pave the way for the high-throughput detection and quantification of microalgal diversity, and have significant potential for future use in field monitoring.

Picomonas judraskeda Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes, Formerly Known as ‘Picobiliphytes’

In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe Picomonas judraskeda gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for Picomonas judraskeda, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’.

Thalassiosira species (Bacillariophyceae) from a Scottish sea-loch

During the spring diatom blooms of 1992 and 1993 in Loch Creran, Scotland, regular water column and sediment samples were collected, examined, and species composition of the phytoplankton determined. Thalassiosira species were found to be a major bloom constituent. Species were identified from specimens cultured from sediments and isolated from vertical net hauls, using light and scanning electron microscopy. Of the 17 species positively determined, only Thalassiosira angulata, T. anguste-lineata, T. eccentrica, T. gravida, T. minima, T. nordenskioeldii, T. rotula and T. tenera have been previously reported in Scottish waters. Other species, for example Thalassiosira pseudonana and T. punctigera, have been noted elsewhere in the British Isles but not along the Scottish west coast. Several species, namely Thalassiosira concaviuscula, T. constricta, T. delicatula, T. kuschirensis, T. oceanica, T. pacifica and T. tealata, are new records for the British Isles. One unidentified taxon is illustrated. Descriptions, illustrations and a key are provided for the 18 taxa.

Introduction to project MIDTAL: its methods and samples from Arcachon Bay, France

Microalgae worldwide regularly cause harmful effects, considered from the human perspective, in that they cause health problems and economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. These episodes encompass a broad range of phenomena collectively referred to as “harmful algal blooms” (HABs). For adequate management of these phenomena, monitoring of microalgae is required. However, effective monitoring is time-consuming because cell morphology as determined by light microscopy may be insufficient to give definitive species and toxin attribution. In the European Union FP7 project MIDTAL (Microarrays for the Detection of Toxic Algae), we achieved rapid species identification using rRNA genes as the target. These regions can be targeted for probe design to recognise species or even strains. We also included antibody reactions to specific toxins produced by these microalgae because, even when cell numbers are low, toxins can be present and can accumulate in the shellfish. Microarrays are the state-of-the-art technology in molecular biology for the processing of bulk samples for detection of target RNA/DNA sequences. After 36xa0months, we have completed RNA-cell number–signal intensity calibration curves for 18 HAB species and the analysis of monthly field samples from five locations from year 1. Results from one location, Arcachon Bay (France), are reported here and compared favourably with cell counts in most cases. In general, the microarray was more sensitive than the cell counts, and this is likely a reflection in the difference in water volume analysed with the volume filtered for the microarray an order of magnitude greater.

New Insights into Plagiogrammaceae (Bacillariophyta) Based on Multigene Phylogenies and Morphological Characteristics with the Description of a New Genus and Three New Species

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

Are Prorocentrum hoffmannianum and Prorocentrum belizeanum (DINOPHYCEAE, PROROCENTRALES), the same species? An integration of morphological and molecular data.

The taxonomic assignment of Prorocentrum species is based on morphological characteristics; however, morphological variability has been found for several taxa isolated from different geographical regions. In this study, we evaluated species boundaries of Prorocentrum hoffmannianum and Prorocentrum belizeanum based on morphological and molecular data. A detailed morphological analysis was done, concentrating on the periflagellar architecture. Molecular analyses were performed on partial Small Sub‐Unit (SSU) rDNA, partial Large Sub‐Unit (LSU) rDNA, complete Internal Transcribed Spacer Regions (ITS1‐5.8S‐ITS2), and partial cytochrome b (cob) sequences. We concatenated the SSU‐ITS‐LSU fragments and constructed a phylogenetic tree using Bayesian Inference (BI) and maximum likelihood (ML) methods. Morphological analyses indicated that the main characters, such as cell size and number of depressions per valve, normally used to distinguish P. hoffmannianum from P. belizeanum, overlapped. No clear differences were found in the periflagellar area architecture. Prorocentrum hoffmannianum and P. belizeanum were a highly supported monophyletic clade separated into three subclades, which broadly corresponded to the sample collection regions. Subtle morphological overlaps found in cell shape, size, and ornamentation lead us to conclude that P. hoffmanianum and P. belizeanum might be considered conspecific. The molecular data analyses did not separate P. hoffmannianum and P. belizeanum into two morphospecies, and thus, we considered them to be the P. hoffmannianum species complex because their clades are separated by their geographic origin. These geographic and genetically distinct clades could be referred to as ribotypes: (A) Belize, (B) Florida‐Cuba, (C1) India, and (C2) Australia.

A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2-9.6% and 1.3-12.0% respectively. The method was applied to the analysis of aquatic samples (n=206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n=22), cylindrospermopsin (n=25), microcystin-RR (n=17), microcystin-LR (n=12), microcystin-LY (n=1), microcystin-LF (n=1) and nodularin (n=5). For microcystins, the levels detected ranged from 0.001 to 1.51μg/L, with two samples showing combined levels above the guideline set by the WHO of 1μg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.

Testing a Microarray to Detect and Monitor Toxic Microalgae in Arcachon Bay in France

Harmful algal blooms (HABs) occur worldwide, causing health problems and economic damages to fisheries and tourism. Monitoring agencies are therefore essential, yet monitoring is based only on time-consuming light microscopy, a level at which a correct identification can be limited by insufficient morphological characters. The project MIDTAL (Microarray Detection of Toxic Algae)—an FP7-funded EU project—used rRNA genes (SSU and LSU) as a target on microarrays to identify toxic species. Furthermore, toxins were detected with a newly developed multiplex optical Surface Plasmon Resonance biosensor (Multi SPR) and compared with an enzyme-linked immunosorbent assay (ELISA). In this study, we demonstrate the latest generation of MIDTAL microarrays (version 3) and show the correlation between cell counts, detected toxin and microarray signals from field samples taken in Arcachon Bay in France in 2011. The MIDTAL microarray always detected more potentially toxic species than those detected by microscopic counts. The toxin detection was even more sensitive than both methods. Because of the universal nature of both toxin and species microarrays, they can be used to detect invasive species. Nevertheless, the MIDTAL microarray is not completely universal: first, because not all toxic species are on the chip, and second, because invasive species, such as Ostreopsis, already influence European coasts.