Mixoplankton and mixotrophy: future research priorities.

Phago-mixotrophy, the combination of photoautotrophy and phagotrophy in mixoplankton, organisms that can combine both trophic strategies, have gained increasing attention over the past decade. It is now recognized that a substantial number of protistan plankton species engage in phago-mixotrophy to obtain nutrients for growth and reproduction under a range of environmental conditions. Unfortunately, our current understanding of mixoplankton in aquatic systems significantly lags behind our understanding of zooplankton and phytoplankton, limiting our ability to fully comprehend the role of mixoplankton (and phago-mixotrophy) in the plankton food web and biogeochemical cycling. Here, we put forward five research directions that we believe will lead to major advancement in the field: (i) evolution: understanding mixotrophy in the context of the evolutionary transition from phagotrophy to photoautotrophy; (ii) traits and trade-offs: identifying the key traits and trade-offs constraining mixotrophic metabolisms; (iii) biogeography: large-scale patterns of mixoplankton distribution; (iv) biogeochemistry and trophic transfer: understanding mixoplankton as conduits of nutrients and energy; and (v) in situ methods: improving the identification of in situ mixoplankton and their phago-mixotrophic activity.

The Mixoplankton Database (MDB): Diversity of photo-phago-trophic plankton in form, function, and distribution across the global ocean.

Protist plankton are major members of open-water marine food webs. Traditionally divided between phototrophic phytoplankton and phagotrophic zooplankton, recent research shows many actually combine phototrophy and phagotrophy in the one cell; these protists are the "mixoplankton." Under the mixoplankton paradigm, "phytoplankton" are incapable of phagotrophy (diatoms being exemplars), while "zooplankton" are incapable of phototrophy. This revision restructures marine food webs, from regional to global levels. Here, we present the first comprehensive database of marine mixoplankton, bringing together extant knowledge of the identity, allometry, physiology, and trophic interactivity of these organisms. This mixoplankton database (MDB) will aid researchers that confront difficulties in characterizing life traits of protist plankton, and it will benefit modelers needing to better appreciate ecology of these organisms with their complex functional and allometric predator-prey interactions. The MDB also identifies knowledge gaps, including the need to better understand, for different mixoplankton functional types, sources of nutrition (use of nitrate, prey types, and nutritional states), and to obtain vital rates (e.g. growth, photosynthesis, ingestion, factors affecting photo' vs. phago' -trophy). It is now possible to revisit and re-classify protistan "phytoplankton" and "zooplankton" in extant databases of plankton life forms so as to clarify their roles in marine ecosystems.

Genome architecture used to supplement species delineation in two cryptic marine ciliates.

The purpose of this study is to determine which taxonomic methods can elucidate clear and quantifiable differences between two cryptic ciliate species, and to test the utility of genome architecture as a new diagnostic character in the discrimination of otherwise indistinguishable taxa. Two cryptic tintinnid ciliates, Schmidingerella arcuata and Schmidingerella meunieri, are compared via traditional taxonomic characters including lorica morphometrics, ribosomal RNA (rRNA) gene barcodes and ecophysiological traits. In addition, single-cell 'omics analyses (single-cell transcriptomics and genomics) are used to elucidate and compare patterns of micronuclear genome architecture between the congeners. The results include a highly similar lorica that is larger in S. meunieri, a 0%-0.5% difference in rRNA gene barcodes, two different and nine indistinguishable growth responses among 11 prey treatments, and distinct patterns of micronuclear genomic architecture for genes detected in both ciliates. Together, these results indicate that while minor differences exist between S. arcuata and S. meunieri in common indices of taxonomic identification (i.e., lorica morphology, DNA barcode sequences and ecophysiology), differences exist in their genomic architecture, which suggests potential genetic incompatibility. Different patterns of micronuclear architecture in genes shared by both isolates also enable the design of species-specific primers, which are used in this study as unique "architectural barcodes" to demonstrate the co-occurrence of both ciliates in samples collected from a NW Atlantic estuary. These results support the utility of genomic architecture as a tool in species delineation, especially in ciliates that are cryptic or otherwise difficult to differentiate using traditional methods of identification.

Genetic differentiation and phylogeography of rotifer Polyarthra dolichoptera and P. vulgaris populations between Southeastern China and eastern North America: High intercontinental differences.

Genetic differentiations and phylogeographical patterns of small organisms may be shaped by spatial isolation, environmental gradients, and gene flow. However, knowledge about genetic differentiation of rotifers at the intercontinental scale is still limited. Polyarthra dolichoptera and P. vulgaris are cosmopolitan rotifers that are tolerant to environmental changes, offering an excellent model to address the research gap. Here, we investigated the populations in Southeastern China and eastern North America and evaluated the phylogeographical patterns from their geographical range sizes, geographic-genetic distance relationships and their responses to spatial-environmental factors. Using the mitochondrial cytochrome c oxidase subunit I gene as the DNA marker, we analyzed a total of 170 individuals. Our results showed that some putative cryptic species, also known as entities were widely distributed, but most of them were limited to single areas. The divergence of P. dolichoptera and P. vulgaris indicated that gene flow between continents was limited while that within each continent was stronger. Oceanographic barriers do affect the phylogeographic pattern of rotifers in continental waters and serve to maintain genetic diversity in nature. The genetic distance of P. dolichoptera and P. vulgaris populations showed significant positive correlation with geographic distance. This might be due to the combined effects of habitat heterogeneity, long-distance colonization, and oceanographic barriers. Furthermore, at the intercontinental scale, spatial distance had a stronger influence than environmental variables on the genetic differentiations of both populations. Wind- and animal-mediated transport and even historical events of continental plate tectonics are potential factors for phylogeography of cosmopolitan rotifers.

Distribution Patterns of Ciliate Diversity in the South China Sea.

Ciliates are abundant microplankton that are widely distributed in the ocean. In this paper, the distribution patterns of ciliate diversity in the South China Sea (SCS) were analyzed by compiling community data from previous publications. Based on morphological identification, a total of 592 ciliate species have been recorded in the SCS. The ciliate communities in intertidal, neritic and oceanic water areas were compared in terms of taxonomy, motility and feeding habit composition, respectively. Significant community variation was revealed among the three areas, but the difference between the intertidal area and the other two areas was more significant than that between neritic and oceanic areas. The distributions of ciliates within each of the three areas were also analyzed. In the intertidal water, the community was not significantly different among sites but did differ among habitat types. In neritic and oceanic areas, the spatial variation of communities among different sites was clearly observed. Comparison of communities by taxonomic and ecological traits (motility and feeding habit) indicated that these traits similarly revealed the geographical pattern of ciliates on a large scale in the SCS, but to distinguish the community variation on a local scale, taxonomic traits has higher resolution than ecological traits. In addition, we assessed the relative influences of environmental and spatial factors on assembly of ciliate communities in the SCS and found that environmental selection is the major process structuring the taxonomic composition in intertidal water, while spatial processes played significant roles in influencing the taxonomic composition in neritic and oceanic water. Among ecological traits, environmental selection had the most important impact on distributions.

Combined Genome and Transcriptome Analyses of the Ciliate Schmidingerella arcuata (Spirotrichea) Reveal Patterns of DNA Elimination, Scrambling, and Inversion.

Schmidingerella arcuata is an ecologically important tintinnid ciliate that has long served as a model species in plankton trophic ecology. We present a partial micronuclear genome and macronuclear transcriptome resource for S. arcuata, acquired using single-cell techniques, and we report on pilot analyses including functional annotation and genome architecture. Our analysis shows major fragmentation, elimination, and scrambling in the micronuclear genome of S. arcuata. This work introduces a new nonmodel genome resource for the study of ciliate ecology and genomic biology and provides a detailed functional counterpart to ecological research on S. arcuata.

Perspectives from Ten Years of Protist Studies by High-Throughput Metabarcoding.

During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding.

Incubation and grazing effects on spirotrich ciliate diversity inferred from molecular analyses of microcosm experiments.

We used an experimental approach of analyzing marine microcosms to evaluate the impact of both predation (top-down) and food resources (bottom-up) on spirotrich ciliate communities. To assess the diversity, we used two molecular methods-denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS). We carried out two types of experiments to measure top-down (adult copepods as predators) and bottom-up effects (phytoplankton as food resources) on the spirotrich ciliates. We observed both strong incubation effects (untreated controls departed from initial assessment of diversity) and high variability across replicates within treatments, particularly for the bottom-up experiments. This suggests a rapid community turn-over during incubation and differential susceptibility to the effects of experimental manipulation. Despite the variability, our analyses reveal some broad patterns such as (1) increasing adult copepod predator abundance had a greater impact on spirotrich ciliates than on other microbial eukaryotes; (2) there was no evidence for strong food selection by the dominant spirotrich ciliates.

The diversity and biogeography of abundant and rare intertidal marine microeukaryotes explained by environment and dispersal limitation.

Benthic microeukaryotes are key ecosystem drivers in marine sandy beaches, an important and dynamic environment; however, little is known about their diversity and biogeography on a large spatial scale. Here, we investigated the community composition and geographical distributions of benthic microeukaryotes using high-throughput sequencing of the 18S rRNA gene and quantified the contributions of environmental factors and spatial separation on the distribution patterns of both rare and abundant taxa. We collected 36 intertidal samples at 12 sandy beaches from four regions that spanned distances from 0.001 to 12,000 km. We found 12,890 operational taxonomic units (OTUs; 97% sequence identity level) including members of all eukaryotic super-groups and several phyla of uncertain position. Arthropoda and Diatomeae dominated the sequence reads in abundance, but Ciliophora and Discoba were the most diverse groups across all samples. About one-third of the OTUs could not be definitively classified at a similarity level of 80%, supporting the view that a large number of rare and minute marine species may have escaped previous characterization. We found generally similar geographical patterns for abundant and rare microeukaryotic sub-communities, and both showed a significant distance-decay similarity trend. Variation partitioning showed that both rare and abundant sub-communities exhibited a slightly stronger response to environmental factors than spatial (distance) factors. However, the abundant sub-community was strongly correlated with variations in spatial, environmental and sediment grain size factors (66% of variance explained), but the rare assemblage was not (16%). This suggests that different or more complex mechanisms generate and maintain diversity in the rare biosphere in this habitat.

Dartintinnus alderae n. g., n. sp., a Brackish Water Tintinnid (Ciliophora, Spirotrichea) with Dual-ended Lorica Collapsibility.

A tintinnid ciliate isolated from waters of the Thames River (Connecticut, USA) is described through combined in vivo observation, protargol impregnation, and phylogenetic analysis. The novel genus Dartintinnus and its type species, D. alderae are distinct from established tintinnid taxa by a lorica that collapses on both anterior and posterior ends. Dartintinnus is placed in the family Eutintinnidae based on a hyaline, elongated lorica opened at both ends, a ciliary pattern including a ventral kinety, at least one dorsal kinety, and right, left and lateral fields, and a sister relationship with Eutintinnus in gene trees. Main differences between D. alderae and Eutintinnus species include a 5.5-6.5% divergence in the small subunit rRNA gene, the geometry of the lorica (resembling an isosceles tetrahedron when collapsed vs. a cylinder, respectively), the number of macronuclear nodules (two vs. four), and the number of dorsal kineties (one vs. usually two). Considering the features of the new genus, we improve the diagnosis of the family Eutintinnidae, including the presence of a lateral ciliary field that had been overlooked in some Eutintinnus species. This work exemplifies the potential for novel diversity, even in these relatively well-studied protists, and the importance of an integrated approach for the description of tintinnid taxa.

Distribution of Abundant and Active Planktonic Ciliates in Coastal and Slope Waters Off New England.

Despite their important role of linking microbial and classic marine food webs, data on biogeographical patterns of microbial eukaryotic grazers are limited, and even fewer studies have used molecular tools to assess active (i.e., those expressing genes) community members. Marine ciliate diversity is believed to be greatest at the chlorophyll maximum, where there is an abundance of autotrophic prey, and is often assumed to decline with depth. Here, we assess the abundant (DNA) and active (RNA) marine ciliate communities throughout the water column at two stations off the New England coast (Northwest Atlantic)-a coastal station 43 km from shore (40 m depth) and a slope station 135 km off shore (1,000 m). We analyze ciliate communities using a DNA fingerprinting technique, Denaturing Gradient Gel Electrophoresis (DGGE), which captures patterns of abundant community members. We compare estimates of ciliate communities from SSU-rDNA (abundant) and SSU-rRNA (active) and find complex patterns throughout the water column, including many active lineages below the photic zone. Our analyses reveal (1) a number of widely-distributed taxa that are both abundant and active; (2) considerable heterogeneity in patterns of presence/absence of taxa in offshore samples taken 50 m apart throughout the water column; and (3) three distinct ciliate assemblages based on position from shore and depth. Analysis of active (RNA) taxa uncovers biodiversity hidden to traditional DNA-based approaches (e.g., clone library, rDNA amplicon studies).

Integrating dimensions of biodiversity in choreotrichs and oligotrichs of marine plankton.

Choreotrichs and oligotrichs are the main ciliate groups in marine plankton, where they play major roles as trophic intermediaries. We have studied these groups with a variety of approaches to combine the three dimensions of biodiversity-taxonomy, genetics and function. Here we revisit our findings with an integrative perspective, and highlight future directions. In our studies, the correspondence between classical taxonomy (mostly based on morphology) and the increasingly available genetic data (DNA sequences) is examined at the individual, population, species, and assemblage levels. We use a combination of single-cell and environmental sequencing to quantify diversity, track distribution patterns, and explain biogeography processes. Comparativelly, we know little about how the morphological and genetic estimates of diversity relate to function, but we expect to better link these aspects by incorporating modern -omics approaches. For example, we have pioneered functional transcriptomic analyses in these groups by contrasting a heterotrophic choreotrich and a mixotrophic oligotrich. These data provide a tremendous resource to start building reference databases needed to measure differential expression of key functional genes, either experimentally or directly in the environment.

Phylogeny, classification and diversity of Choreotrichia and Oligotrichia (Ciliophora, Spirotrichea).

Ciliated protists in the subclasses Choreotrichia and Oligotrichia are major components of marine plankton. Despite their ecological relevance, there are uncertainties in their systematics and diversity. We retrieved and curated all the GenBank ribosomal DNA (rDNA) sequences from these groups, which were analyzed in two ways. The first approach was based on morphologically-identified sequences (including those of two families and six genera newly studied here by single-cell sequencing), and aimed at improving phylogenetic inferences using concatenated sequences of three rDNA loci. Based on phylogenetic and morphological support, we update the taxonomic classification of these subclasses into 23 families, including the re-established Favellidae. We also propose an informal naming system for incertae sedis taxa, namely Tintinnopsis and five related genera that are spread among eleven lineages. The second approach included unidentified environmental sequences, and was used to explore potentially novel diversity in these subclasses. Our results support high proportions of both synonyms in tintinnids and uncharacterized taxa in choreotrichids and oligotrichs. One previously unidentified, environmental clade is here linked to our new Leegaardiellidae sequences. Our curation of almost 4000 rDNA sequences exemplifies known issues of public repositories, and suggests caution in both the use and contribution to these unique resources for evolutionary and diversity studies.

Patchiness of Ciliate Communities Sampled at Varying Spatial Scales along the New England Shelf.

Although protists (microbial eukaryotes) provide an important link between bacteria and Metazoa in food webs, we do not yet have a clear understanding of the spatial scales on which protist diversity varies. Here, we use a combination of DNA fingerprinting (denaturant gradient gel electrophoresis or DGGE) and high-throughput sequencing (HTS) to assess the ciliate community in the class Spirotrichea at varying scales of 1-3 km sampled in three locations separated by at least 25 km-offshore, midshelf and inshore-along the New England shelf. Analyses of both abundant community (DGGE) and the total community (HTS) members reveal that: 1) ciliate communities are patchily distributed inshore (i.e. the middle station of a transect is distinct from its two neighboring stations), whereas communities are more homogeneous among samples within the midshelf and offshore stations; 2) a ciliate closely related to Pelagostrobilidium paraepacrum 'blooms' inshore and; 3) environmental factors may differentially impact the distributions of individual ciliates (i.e. OTUs) rather than the community as a whole as OTUs tend to show distinct biogeographies (e.g. some OTUs are restricted to the offshore locations, some to the surface, etc.). Together, these data show the complexity underlying the spatial distributions of marine protists, and suggest that biogeography may be a property of ciliate species rather than communities.

Unexpected biodiversity of ciliates in marine samples from below the photic zone.

Marine microbial eukaryotes play critical roles in planktonic food webs and have been described as most diverse in the photic zone where productivity is high. We used high-throughput sequencing (HTS) to analyse the spatial distribution of planktonic ciliate diversity from shallow waters (<30 m depth) to beyond the continental shelf (>800 m depth) along a 163 km transect off the coast of New England, USA. We focus on ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), as these taxa are major components of marine food webs. We did not observe the decrease of diversity below the photic zone expected based on productivity and previous analyses. Instead, we saw an increase of diversity with depth. We also observed that the ciliate communities assessed by HTS cluster by depth layer and degree of water column stratification, suggesting that community assembly is driven by environmental factors. Across our samples, abundant OTUs tend to match previously characterized morphospecies while rare OTUs are more often undescribed, consistent with the idea that species in the rare biosphere remain to be characterized by microscopy. Finally, samples taken below the photic zone also reveal the prevalence of two uncharacterized (i.e. lacking sequenced morphospecies) clades - clusters X1 and X2 - that are enriched within the nano-sized fraction (2-10 µm) and are defined by deletions within the region of the SSU-rDNA analysed here. Together, these data reinforce that we still have much to learn about microbial diversity in marine ecosystems, especially in deep-waters that may be a reservoir for rare species and uncharacterized taxa.

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

Our knowledge on microbial biogeography depends on the way we define and study diversity. In contrast to most microbes, some protist lineages have conspicuous structures that allow comparisons of diversity concepts and measures-those based on molecules and those based on morphology. We analyzed a group of shell-bearing planktonic ciliates, the tintinnids, in a coast-to-ocean gradient using high-throughput sequencing and microscopy. First, we compared molecular operational taxonomic units (OTUs) and morphospecies in terms of assemblage composition, distribution and relationships with the environment. OTUs revealed potentially novel and rare taxa, while morphospecies showed clearer correlations with environmental factors, and both approaches coincided in supporting a coastal versus oceanic pattern. Second, we explored which processes influence assembly across the environmental gradient examined. Assemblage fluctuations were associated with significant distance-decay and changes in morphospecies size and prey proxies, thus suggesting niche partitioning as a key structuring mechanism. Our conclusion is that molecules and morphologies generally agreed, but they provided complementary data, the first revealing hidden diversity, and the latter making better connections between distribution patterns and ecological processes. This highlights the importance of linking genotypes and phenotypes (using multidisciplinary analyses and/or reliable databases of barcoded species), to understand the diversity, biogeography and ecological roles of microbes.

Space station image captures a red tide ciliate bloom at high spectral and spatial resolution.

Mesodinium rubrum is a globally distributed nontoxic ciliate that is known to produce intense red-colored blooms using enslaved chloroplasts from its algal prey. Although frequent enough to have been observed by Darwin, blooms of M. rubrum are notoriously difficult to quantify because M. rubrum can aggregate into massive clouds of rusty-red water in a very short time due to its high growth rates and rapid swimming behavior and can disaggregate just as quickly by vertical or horizontal dispersion. A September 2012 hyperspectral image from the Hyperspectral Imager for the Coastal Ocean sensor aboard the International Space Station captured a dense red tide of M. rubrum (10(6) cells per liter) in surface waters of western Long Island Sound. Genetic data confirmed the identity of the chloroplast as a cryptophyte that was actively photosynthesizing. Microscopy indicated extremely high abundance of its yellow fluorescing signature pigment phycoerythrin. Spectral absorption and fluorescence features were related to ancillary photosynthetic pigments unique to this organism that cannot be observed with traditional satellites. Cell abundance was estimated at a resolution of 100 m using an algorithm based on the distinctive yellow fluorescence of phycoerythrin. Future development of hyperspectral satellites will allow for better enumeration of bloom-forming coastal plankton, the associated physical mechanisms, and contributions to marine productivity.

Discrimination of closely related species in tintinnid ciliates: new insights on crypticity and polymorphism in the genus Helicostomella.

This study focuses on the utility of molecular markers for the discrimination of closely related species in tintinnid ciliates. We analyzed the ecologically important genus Helicostomella by sequencing part of the large-subunit rDNA (LSU rDNA) and the 5.8S rDNA combined with the internally transcribed spacer regions 1 and 2 (5.8S rDNA-ITS) from forty-five individuals collected in NW and SW Atlantic waters and after culturing. Although all described Helicostomella species represent a continuum of morphologies, forms with shorter or longer loricae would correspond to different species according to previous molecular data. Here we observed that long forms show both crypticity (i.e. two almost identical long forms with different DNA sequences) and polymorphism (i.e. some long forms develop significantly shorter loricae after culturing). Reviewing all available tintinnid sequences, we found that 1) three Helicostomella clusters are consistent with different species from a molecular perspective, although these clusters are neither clearly differentiated by their loricae nor unambiguously linked to described species, 2) Helicostomella is closely related (probably to the family or genus level) to four "Tintinnopsis-like" morphospecies, and 3) if considered separately, neither LSU rDNA nor 5.8S rDNA-ITS completely discriminate closely related species, thus supporting the use of multi-gene barcodes for tintinnids.

De novo transcriptomes of a mixotrophic and a heterotrophic ciliate from marine plankton.

Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich Strombidinopsis sp., and their respective algal food using Illumina RNAseq. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We detected approximately 10,000 and 7,600 amino acid sequences for S. rassoulzadegani and Strombidinopsis sp., respectively. About half of these transcripts had significant BLASTP hits (E-value <10-6) against previously-characterized sequences, mostly from the model ciliate Oxytricha trifallax. Transcriptomes from both the mixotroph and the heterotroph species provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance and functioning in the transcriptome of this ciliate. While chloroplasts are known sources of reactive oxygen species (ROS), we found the same complement of antioxidant pathways in both ciliates, except for one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy.

Diversity of diversity: conceptual and methodological differences in biodiversity estimates of eukaryotic microbes as compared to bacteria.

Recent advances such as high-throughput sequencing (HTS) have changed conceptions about the magnitude of diversity on Earth. This is especially true for microbial lineages, which have seen the discovery of great numbers of rare forms in places such as the human gut as well as diverse environments (e.g., freshwater, marine, and soil). Given the differences in perceptions of diversity for bacterial and eukaryotic microbes, including divergent species concepts, HTS tools used to eliminate errors and population-level variation in bacteria may not be appropriate for microbial eukaryotes and may eliminate valid species from the data. We discuss here how the nature of biodiversity varies among microbial groups and the extent to which HTS tools designed for bacteria are useful for eukaryotes.