Phaeocystis antarctica, a dominant phytoplankton species found in the Southern Ocean, is known for colony formation and large seasonal blooms. Blooms play a key role in Southern Ocean biogeochemistry, contributing to carbon export and the production of dimethylsulfoniopropionate (DMSP) and its climatically active breakdown product, dimethyl sulfide (DMS). Here we provide evidence of phagotrophy, i.e., mixotrophic behavior in the alga P. antarctica. Mixotrophy is widespread among marine protists and was recently identified in the temperate sister species P. globosa. We examined potential triggers for bacterivory using a 4 × 4 factorial design manipulating irradiance (diurnal changes versus static) and nutrient availability and evaluated bacterivory using fluorescent microspheres as tracers. Our results show that bacterivory in P. antarctica was driven more strongly by variability in irradiance than by nutrient availability, with greater ingestion occurring in treatments with diurnal cycles. Because macronutrient limitations are generally negligible in the Southern Ocean, bacterial ingestion of P. antarctica may represent an adaptive response to seasonal changes in light availability.

Rising temperature, variation of light levels, and changes in nutrient availability are among the top environmental factors known to have profound effects on growth, grazing, and photosynthesis in mixotrophic species. Polar species, in particular, are facing rapid changes in their environment. In the present study, phagotrophic and phototrophic responses of an Arctic chrysophyte, Dinobryon faculiferum, were investigated at different temperatures (2, 4, and 6 degrees C), light levels (0-200 mu mol photons m-2 s-1), and nutrient concentrations (12.5 and 50% f/2 + Si nutrient media in 32 PSU artificial seawater). Bacterivory by D. faculiferum, estimated by ingestion of fluorescent proxies, increased with rising temperature, while photosynthetic rates, determined by 14C uptake, were low under all experimental regimes; phagotrophy appeared to contribute more to this species' carbon budget than phototrophy. Furthermore, D. faculiferum survived long periods of darkness, implying it may not be an obligate phototroph, as found for some other species in the genus. In experiments limiting nitrogen and/or phosphorus, there was no effect of limiting phosphorus on either phototrophy or phagotrophy, but both were reduced in treatments with limited nitrogen, presumably impeding metabolic processes when strongly limited. Our results demonstrate the influences of abiotic environmental factors on phagotrophic and phototrophic responses, providing further understanding of how mixotrophic protists in polar systems may respond to a changing climate.

Mixotrophic chrysophytes are known for significant contributions to primary productivity and grazing within microbial loops in aquatic systems. Understanding how projected changes in a warming environment might alter physiological responses in mixotrophs is crucial. In this study, we investigated mixotrophic responses of the freshwater chrysophyte, Chrysolepidomonas dendrolepidota, under an array of temperatures (14-20 degrees C) and inorganic nutrient conditions (1%-50% DY-IV nutrient media). Photosynthetic rates of C. dendrolepidota were reduced in lower inorganic nutrient concentrations and increased with rising temperature. Bacterivory rates were not significantly different across inorganic nutrient regimes but decreased with rising temperature, and in some measure with decreasing bacterial abundance. The highest temperature tested promoted rapid initial growth in higher inorganic nutrient condition, with slower more continuous growth observed at lower temperatures. Our results indicate phototrophy and phagotrophy by C. dendrolepidota are strongly affected by temperature, shifting toward more phototrophic nutritional mode as temperature increased. While some mixotrophs have been shown to be more heterotrophic with increasing temperature, photosynthesis contributes more to C. dendrolepidota's carbon budget with increasing temperature. These findings demonstrate environmental factors can induce different physiological responses along gradient of photo-osmo-phago-mixotrophic abilities, which should be taken into consideration in future works involving models of climate-change impacts on phagotrophic phytoplankton.

During a cruise from October to November 2019, along the West Antarctic Peninsula, between 64.32 and 68.37°S, we assessed the diversity and composition of the active microbial eukaryotic community within three size fractions: micro- (> 20 μm), nano- (20–5 μm), and pico-size fractions (5–0.2 μm). The communities and the environmental parameters displayed latitudinal gradients, and we observed a strong similarity in the microbial eukaryotic communities as well as the environmental parameters between the sub-surface and the deep chlorophyll maximum (DCM) depths. Chlorophyll concentrations were low, and the mixed layer was shallow for most of the 17 stations sampled. The richness of the microplankton was higher in Marguerite Bay (our southernmost stations), compared to more northern stations, while the diversity for the nano- and pico-plankton was relatively stable across latitude. The microplankton communities were dominated by autotrophs, mostly diatoms, while mixotrophs (phototrophs-consuming bacteria and kleptoplastidic ciliates, mostly alveolates, and cryptophytes) were the most abundant and active members of the nano- and picoplankton communities. While phototrophy was the dominant trophic mode, heterotrophy (mixotrophy, phagotrophy, and parasitism) tended to increase southward. The samples from Marguerite Bay showed a distinct community with a high diversity of nanoplankton predators, including spirotrich ciliates, and dinoflagellates, while cryptophytes were observed elsewhere. Some lineages were significantly related—either positively or negatively—to ice coverage (e.g., positive for Pelagophyceae, negative for Spirotrichea) and temperature (e.g., positive for Cryptophyceae, negative for Spirotrichea). This suggests that climate changes will have a strong impact on the microbial eukaryotic community.

Foraminifera include diverse shell-building lineages found in a wide array of aquatic habitats from the deep-sea to intertidal zones to brackish and freshwater ecosystems. Recent estimates of morphological and molecular foraminifera diversity have increased the knowledge of foraminiferal diversity, which is critical as these lineages are used as bioindicators of past and present environmental perturbation. However, a comparative analysis of foraminiferal biodiversity between their major habitats (freshwater, brackish, intertidal, and marine) is underexplored, particularly using molecular tools. Here, we present a metabarcoding survey of foraminiferal diversity across different ecosystems using newly designed foraminifera-specific primers that target the hypervariable regions of the foraminifera SSU-rRNA gene (~250-300 bp long). We tested these primer sets on four foraminifera species and then across several environments: the intertidal zone, coastal ecosystems, and freshwater vernal pools. We retrieved 655 operational taxonomic units (OTUs); the majority of which are undetermined taxa that have no closely matching sequences in the reference database. Furthermore, we identified 163 OTUs with distinct habitat preferences. Most of the observed OTUs belonged to lineages of single-chambered foraminifera, including poorly explored freshwater foraminifera which encompass a clade of Reticulomyxa-like forms. Our pilot study provides the community with an additional set of newly designed and taxon-specific primers to elucidate foraminiferal diversity across different habitats.

Testate (shell-building) amoebae, such as the Arcellinida (Amoebozoa), are useful bioindicators for climate change. Though past work has relied on morphological analyses to characterize Arcellinida diversity, genetic analyses revealed the presence of multiple cryptic species underlying morphospecies. Here, we design and deploy Arcellinida-specific primers for the SSU-rDNA gene to assess the community composition on the molecular level in a pilot study of two samplings from a New England fen: (1) 36-cm horizontal transects and vertical cores; and (2) 26-m horizontal transects fractioned into four size classes (2-10, 10-35, 35-100, and 100-300 μm). Analyses of these data show the following: (1) a considerable genetic diversity within Arcellinida, much of which comes from morphospecies lacking sequences on GenBank; (2) communities characterized by DNA (i.e. active + quiescent) are distinct from those characterized by RNA (i.e. active, indicator of biomass); (3) active communities on the surface tend to be more similar to one another than to core communities, despite considerable heterogeneity; and (4) analyses of communities fractioned by size find some lineages (OTUs) that are abundant in disjunct size categories, suggesting the possibility of life-history stages. Together, these data demonstrate the potential of these primers to elucidate the diversity of Arcellinida communities in diverse habitats.

The availability of high-throughput sequencing (HTS) has transformed our understanding of the diversity of microbial eukaryotes (i.e., protists) across diverse habitats. Yet relating this biodiversity to function remains a challenge, particularly in the context of microbial food webs. Here we perform a set of microcosm experiments to evaluate the impact of changing predator and prey concentrations on a marine protist community, focusing on SAR (Stramenopila, Alveolata, and Rhizaria) lineages. We combine an estimate of taxonomic diversity through analysis of SSU-rDNA amplicons with metatranscriptomics, a proxy for function. We assess changes in a community sampled from New England waters with varying concentrations of predators (copepods) and prey (phytoplankton <15 μm in size). The greatest impact observed is on the diversity and function of the small plankton (2–10 μm, nanoplankton) community in the presence of high prey abundance (i.e., bloom conditions). Many SAR taxa in the nanosized fraction decrease with increasing phytoplankton abundance, while ciliates (from both the nano- and microsized fractions) increase. A large number of transcripts and function estimates in the nanoplankton decreased during our simulated phytoplankton bloom. We also find evidence of an interaction between increasing phytoplankton and copepod abundances on the microsized planktonic community, consistent with the hypothesis that phytoplankton and copepods exert bottom-up control and top-down control on the microsized protists, respectively. Together our analyses suggest that community function [i.e., diversity of gene families (GFs)] remains relatively stable, while the functions at the species level (i.e., transcript diversity within GFs) show a substantial reduction of function under bloom conditions. Our study demonstrated that interactions within plankton food webs are complex, and that the relationships between diversity and function for marine microeukaryotes remain poorly understood.

While the identification of microbial eukaryotes using metabarcoding tools is now widespread, additional data are needed to confirm molecular observations, to mark the difference between species and population variants, and to better understand the biogeography of microbial eukaryotes. In this issue of Molecular Ecology, Postel et al not only use three molecular approaches to identify subgroups of Fragilariopsis kerguelensis but also use morphology and physiology to better understand the relationship between the three genotypes. They revealed that (a) the three genotypes of the diatom F. kerguelensis show negligible gene flux; (b) two of the genotypes are geographically isolated with different physiology but still able to crossbreed; and (c) the remaining genotype is omnipresent but reproductively isolated.

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.

Estimating multiple sequence alignments (MSAs) and inferring phylogenies are essential for many aspects of comparative biology. Yet, many bioinformatics tools for such analyses have focused on specific clades, with greatest attention paid to plants, animals, and fungi. The rapid increase in high-throughput sequencing (HTS) data from diverse lineages now provides opportunities to estimate evolutionary relationships and gene family evolution across the eukaryotic tree of life. At the same time, these types of data are known to be error-prone (e.g., substitutions, contamination). To address these opportunities and challenges, we have refined a phylogenomic pipeline, now named PhyloToL, to allow easy incorporation of data from HTS studies, to automate production of both MSAs and gene trees, and to identify and remove contaminants. PhyloToL is designed for phylogenomic analyses of diverse lineages across the tree of life (i.e., at scales of >100 My). We demonstrate the power of PhyloToL by assessing stop codon usage in Ciliophora, identifying contamination in a taxon- and gene-rich database and exploring the evolutionary history of chromosomes in the kinetoplastid parasite Trypanosoma brucei, the causative agent of African sleeping sickness. Benchmarking PhyloToL's homology assessment against that of OrthoMCL and a published paper on superfamilies of bacterial and eukaryotic organellar outer membrane pore-forming proteins demonstrates the power of our approach for determining gene family membership and inferring gene trees. PhyloToL is highly flexible and allows users to easily explore HTS data, test hypotheses about phylogeny and gene family evolution and combine outputs with third-party tools (e.g., PhyloChromoMap, iGTP).

Vernal pools are dynamic freshwater ecosystems that dry during the summer. These unique habitats are vital to a number of well-studied animal species but there is little documentation of the diversity of the SAR-Stramenopila, Alveolata and Rhizaria-clade in vernal pools. Here, we characterize the protist community over a portion of the hydroperiod as the vernal pool transitions from its winter stage through its drying out in late summer. Our study focuses on the SAR clade, which encompasses a broad range of morphological diversity and a variety of trophic modes within the microbial food web. Using high-throughput sequencing, we investigate the total community (DNA) and the active (RNA) members on a temporal scale. These molecular data reveal seasonality within microbial communities, suggesting a larger community of autotrophs in the winter followed by an increase in heterotrophs in the summer. Our analysis also suggests the presence of a microbial seed bank, a collection of encysted protists, in the sediments below the pool. We hypothesize the seed bank allows for community turnover: taxa encyst in the sediment in poor environmental conditions and exit their cysts when favorable conditions occur. We also observe seasonal preference and partitioning of the environment within clades of close relatives, including taxa closely related to the ciliate Halteria and the oomycete Haptoglossa. These data provide insights into the seasonal patterns of a frequently overlooked group of organisms in this unusual environment.

Despite their diversity and ecological importance, many areas of the SAR—Stramenopila, Alveolata, and Rhizaria—clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well‐sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the “dark area” of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and “non‐vertebrate” hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.

The rocky intertidal zone represents a dynamic habitat marked by considerable species richness, which has been well-documented for invertebrates and macroalgae. This high biodiversity exists in the context of extreme fluctuations in abiotic factors such as temperature, salinity and pH that occur during each tidal cycle. Despite these attributes, few studies have focused on microbial diversity in tide pools, including analyses of the ciliate communities that are the focus of this study. We investigated the spatial and temporal distributions of ciliate species across the intertidal environment at sites in Maine and Connecticut, USA. Our study used a DNA fingerprinting technique, denaturing gradient gel electrophoresis (DGGE), which allows for genetic analyses of abundant community members. We investigated how ciliate diversity changed across several spatiotemporal scales: (1) between the open ocean and tide pools, (2) among different tide pools at varying distances from the low tide mark and (3) at differing times within a tidal cycle. In addition, we examined the differences between active and non-active members in these extreme environments by investigating diversity of both ribosomal DNA and RNA. In both Maine and Connecticut, we found abundant ciliate taxa that are either rare or absent in the open ocean, and that appear to quickly dominate tide pools once they are isolated from the open ocean. We also found that ciliate distributions within the tide pool community are complex and variable across spatial and temporal scales.

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).

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.

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.