Charles Bachy

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study

Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial ‘rare biosphere’. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular methods relates to that revealed by classical morphology approaches is practically nonexistent. To address this issue, we used different approaches to assess the diversity of tintinnid ciliates, a species-rich group in which species can be easily distinguished morphologically. We studied two Mediterranean marine samples with different patterns of tintinnid diversity. We estimated tintinnid diversity in these samples employing morphological observations and both classical cloning and sequencing and pyrosequencing of two different markers, the 18S rDNA and the internal transcribed spacer (ITS) regions, applying a variety of computational approaches currently used to analyze pyrosequence reads. We found that both molecular approaches were efficient in detecting the tintinnid species observed by microscopy and revealed similar phylogenetic structures of the tintinnid community at the species level. However, depending on the method used to analyze the pyrosequencing results, we observed discrepancies with the morphology-based assessments up to several orders of magnitude. In several cases, the inferred number of operational taxonomic units (OTUs) largely exceeded the total number of tintinnid cells in the samples. Such inflation of the OTU numbers corresponded to ‘rare biosphere’ taxa, composed largely of artifacts. Our results suggest that a careful and rigorous analysis of pyrosequencing data sets, including data denoising and sequence clustering with well-adjusted parameters, is necessary to accurately describe microbial biodiversity using this molecular approach.

Eukaryotic algal phytochromes span the visible spectrum

Significance Photosynthetic organisms exploit photosensory proteins to respond to changing light conditions. In land plants, phytochromes use the ratio of red to far-red light to detect shading by neighboring plants, leading to changes in growth and development. Light conditions can be more variable for algae because of the wavelength-dependent attenuation of light by water and because of ocean mixing. We studied phytochromes from taxonomically diverse eukaryotic algae from groups considered important for coastal ecosystems and the global carbon cycle. These proteins detect light throughout the visible spectrum (blue, green, orange, red, and far-red). Extensive spectral tuning has evolved within these algae, presumably reflecting aquatic light environments. These studies should ultimately facilitate engineering of crop plant species for diverse light environments. Plant phytochromes are photoswitchable red/far-red photoreceptors that allow competition with neighboring plants for photosynthetically active red light. In aquatic environments, red and far-red light are rapidly attenuated with depth; therefore, photosynthetic species must use shorter wavelengths of light. Nevertheless, phytochrome-related proteins are found in recently sequenced genomes of many eukaryotic algae from aquatic environments. We examined the photosensory properties of seven phytochromes from diverse algae: four prasinophyte (green algal) species, the heterokont (brown algal) Ectocarpus siliculosus, and two glaucophyte species. We demonstrate that algal phytochromes are not limited to red and far-red responses. Instead, different algal phytochromes can sense orange, green, and even blue light. Characterization of these previously undescribed photosensors using CD spectroscopy supports a structurally heterogeneous chromophore in the far-red–absorbing photostate. Our study thus demonstrates that extensive spectral tuning of phytochromes has evolved in phylogenetically distinct lineages of aquatic photosynthetic eukaryotes.

Diversity and Vertical Distribution of Microbial Eukaryotes in the Snow, Sea Ice and Seawater Near the North Pole at the End of the Polar Night

Our knowledge about the microorganisms living in the high Arctic Ocean is still rudimentary compared to other oceans mostly because of logistical challenges imposed by its inhospitable climate and the presence of a multi-year ice cap. We have used 18S rRNA gene libraries to study the diversity of microbial eukaryotes in the upper part of the water column (0–170 m depth), the sea ice (0–1.5 m depth) and the overlying snow from samples collected in the vicinity of the North Pole (N88°35′, E015°59) at the very end of the long polar night. We detected very diverse eukaryotes belonging to Alveolata, Fungi, Amoebozoa, Viridiplantae, Metazoa, Rhizaria, Heterokonta, and Telonemia. Different alveolates (dinoflagellates and Marine Alveolate Groups I and II species) were the most abundant and diverse in gene libraries from water and sea ice, representing 80% of the total number of clones and operational taxonomic units. Only contaminants and/or species from continental ecosystems were detected in snow, suggesting wind- and animal- or human-mediated cosmopolitan dispersal of some taxa. By contrast, sea ice and seawater samples harbored a larger and more similar inter-sample protist diversity as compared with snow. The North Pole was found to harbor distinctive eukaryotic communities along the vertical gradient with an unparalleled diversity of core dinoflagellates, largely dominant in libraries from the water column, as compared to other oceanic locations. In contrast, phototrophic organisms typical of Arctic sea ice and plankton, such as diatoms and prasinophytes, were very rare in our samples. This was most likely due to a decrease of their populations after several months of polar night darkness and to the presence of rich populations of diverse grazers. Whereas strict phototrophs were scarce, we identified a variety of likely mixotrophic taxa, which supports the idea that mixotrophy may be important for the survival of diverse protists through the long polar night.

Marine algae and land plants share conserved phytochrome signaling systems

Significance Phytochromes are photosensory signaling proteins widely distributed in unicellular organisms and multicellular land plants. Best known for their global regulatory roles in photomorphogenesis, plant phytochromes are often assumed to have arisen via gene transfer from the cyanobacterial endosymbiont that gave rise to photosynthetic chloroplast organelles. Our analyses support the scenario that phytochromes were acquired prior to diversification of the Archaeplastida, possibly before the endosymbiosis event. We show that plant phytochromes are structurally and functionally related to those discovered in prasinophytes, an ecologically important group of marine green algae. Based on our studies, we propose that these phytochromes share light-mediated signaling mechanisms with those of plants. Phytochromes presumably perform critical acclimative roles for unicellular marine algae living in fluctuating light environments. Phytochrome photosensors control a vast gene network in streptophyte plants, acting as master regulators of diverse growth and developmental processes throughout the life cycle. In contrast with their absence in known chlorophyte algal genomes and most sequenced prasinophyte algal genomes, a phytochrome is found in Micromonas pusilla, a widely distributed marine picoprasinophyte (<2 µm cell diameter). Together with phytochromes identified from other prasinophyte lineages, we establish that prasinophyte and streptophyte phytochromes share core light-input and signaling-output domain architectures except for the loss of C-terminal response regulator receiver domains in the streptophyte phytochrome lineage. Phylogenetic reconstructions robustly support the presence of phytochrome in the common progenitor of green algae and land plants. These analyses reveal a monophyletic clade containing streptophyte, prasinophyte, cryptophyte, and glaucophyte phytochromes implying an origin in the eukaryotic ancestor of the Archaeplastida. Transcriptomic measurements reveal diurnal regulation of phytochrome and bilin chromophore biosynthetic genes in Micromonas. Expression of these genes precedes both light-mediated phytochrome redistribution from the cytoplasm to the nucleus and increased expression of photosynthesis-associated genes. Prasinophyte phytochromes perceive wavelengths of light transmitted farther through seawater than the red/far-red light sensed by land plant phytochromes. Prasinophyte phytochromes also retain light-regulated histidine kinase activity lost in the streptophyte phytochrome lineage. Our studies demonstrate that light-mediated nuclear translocation of phytochrome predates the emergence of land plants and likely represents a widespread signaling mechanism in unicellular algae.

Molecular Phylogeny of Tintinnid Ciliates (Tintinnida, Ciliophora)

We investigated the phylogeny of tintinnids (Ciliophora, Tintinnida) with 62 new SSU-rDNA sequences from single cells of 32 marine and freshwater species in 20 genera, including the first SSU-rDNA sequences for Amphorides, Climacocylis, Codonaria, Cyttarocylis, Parundella, Petalotricha, Undella and Xystonella, and 23 ITS sequences of 17 species in 15 genera. SSU-rDNA phylogenies suggested a basal position for Eutintinnus, distant to other Tintinnidae. We propose Eutintinnidae fam. nov. for this divergent genus, keeping the family Tintinnidae for Amphorellopsis, Amphorides and Steenstrupiella. Tintinnopsis species branched in at least two separate groups and, unexpectedly, Climacocylis branched among Tintinnopsis sensu stricto species. Tintinnopsis does not belong to the family Codonellidae, which is restricted to Codonella, Codonaria, and also Dictyocysta (formerly in the family Dictyocystidae). The oceanic genus Undella branched close to an undescribed freshwater species. Metacylis, Rhabdonella and Cyttarocylis formed a well supported clade with several Tintinnopsis species at a basal position. Petalotricha ampulla and Cyttarocylis cassis SSU-rDNA and ITS sequences were identical or almost identical. Therefore, we propose Cyttarocylis ampulla comb. nov. for them. Intensive use of single-cell isolation and sequencing revealed unexpected complexity in the evolutionary history of these relatively well-studied ciliates. Notably, the diversity of freshwater forms suggests multiple marine-freshwater invasions.

Probing the evolution, ecology and physiology of marine protists using transcriptomics

Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

Intron Invasions Trace Algal Speciation and Reveal Nearly Identical Arctic and Antarctic Micromonas Populations

Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, new families of spliceosomal introns that are repeated hundreds of times in individual genomes have recently been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence–absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting that their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture (Micromonas Clade E2) isolated from a polar region where abundance of this alga is increasing due to climate induced changes. The same two families were detected in metagenomic data from Antarctica—a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our research illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation, and global distribution of this sentinel marine alga.

Updating Biodiversity Studies in Loricate Protists: The Case of the Tintinnids (Alveolata, Ciliophora, Spirotrichea)

Species determination is crucial in biodiversity research. In tintinnids, identification is based almost exclusively on the lorica, despite its frequent intraspecific variability and interspecific similarity. We suggest updated procedures for identification and, depending on the aim of the study, further steps to obtain morphological, molecular, and ecological data. Our goal is to help improving the collection of information (e.g. species re‐/descriptions and DNA barcodes) that is essential for generating a natural tintinnid classification and a reliable reference for environmental surveys. These suggestions are broadly useful for protistologists because they exemplify data integration, quality/effort compromise, and the need for scientific collaborations.

Microbial Ecology: Finding Structure in the Rare Biosphere

Protists (unicellular eukaryotes) play important roles in marine ecosystems but are tremendously diverse and many remain uncharacterized. Deep-sequencing of a universal marker gene has helped resolve community composition patterns among rare and abundant protistan sequence groups in coastal European waters.

Newly discovered deep-branching marine plastid lineages are numerically rare but globally distributed

Ocean surface warming is resulting in an expansion of stratified, low-nutrient environments, a process referred to as ocean desertification [1]. A challenge for assessing the impact of these changes is the lack of robust baseline information on the biological communities that carry out marine photosynthesis. Phytoplankton perform half of global biological CO2 uptake, fuel marine food chains, and include diverse eukaryotic algae that have photosynthetic organelles (plastids) acquired through multiple evolutionary events [1-3]. While amassing data from ocean ecosystems for the Baselines Initiative (6,177 near full-length 16S rRNA gene sequences and 9.4 million high-quality 16S V1-V2 amplicons) we identified two deep-branching plastid lineages based on 16S rRNA gene data. The two lineages have global distributions, but do not correspond to known phytoplankton. How the newly discovered phytoplankton lineages contribute to food chains and vertical carbon export to the deep sea remains unknown, but their prevalence in expanding, low nutrient surface waters suggests they will have a role in future oceans.