Top-Down and Bottom-Up Controls on Microeukaryotic Diversity (i.e., Amplicon Analyses of SAR Lineages) and Function (i.e., Metatranscriptome Analyses) Assessed in Microcosm Experiments

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 (phytoplanktonμm 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

Seed Bank and Seasonal Patterns of the Eukaryotic SAR (Stramenopila, Alveolata and Rhizaria) Clade in a New England Vernal Pool

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

Diversity of microbial eukaryotes along the West Antarctic Peninsula in austral spring

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Grattepanche, J.-D., Jeffrey, W., Gast, R., & Sanders, R. Diversity of microbial eukaryotes along the West Antarctic Peninsula in austral spring. Frontiers in Microbiology, 13, (2022): 844856, https://doi.org/10.3389/fmicb.2022.844856.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.This work was supported by the National Science Foundation (Grant Nos. ANT 1744767 to RS, ANT 1744663 to RG, and ANT 1744638 to WJ). This research was based, in part, upon sequencing conducted using the Rhode Island Genomics and Sequencing Center, which was supported in part by the National Science Foundation (MRI Grant No. DBI-0215393 and EPSCoR Grant Nos. 0554548 and EPS-1004057), the US Department of Agriculture (Grant Nos. 2002-34438-12688 and 2003-34438-13111), and the University of Rhode Island. This research includes calculations carried out on Temple University HPC resources supported in part by the National Science Foundation through major research instrumentation (Grant No. 1625061) and by the US Army Research Laboratory under (Contract No. W911NF-16-2-0189)

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)

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

PhyloToL: A Taxon/Gene-Rich Phylogenomic Pipeline to Explore Genome Evolution of Diverse Eukaryotes

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 \u3e100 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\u27s 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)

Microbial Diversity in the Eukaryotic SAR Clade: Illuminating the Darkness Between Morphology and Molecular Data

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 above

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

Succession of primary producers and micrograzers in a coastal ecosystem dominated by Phaeocystis globosa blooms

The community structures and succession of phytoplankton, protozooplankton and copepods were studied from February 2007 to July 2009 in a coastal area of the eastern English Channel subject to Phaeocystis globosa blooms. While diatom blooms preceded P. globosa blooms each year, the community structure and stock of heterotrophic protists appeared to be related to the dominant P. globosa life cycle stages. In 2007, the dominance of large colonies (> 100 mu m, up to 316 mu g C L(-1)), which resulted in a high biomass of healthy free cells (up to 132 mu g C L(-1)), accompanied high spirotrich ciliate stocks (up to 58 mu g C L(-1)) and high abundances of the copepods Acartia clausi and Temora longicornis (up to 11 ind. L(-1)). In 2008, the bloom which lasted a shorter period of time was dominated by large colonies (up to 328 mu g C L(-1)) and fewer free cells (up to 98 mu g C L(-1)). This corresponded with a lower abundance of grazers, with stocks of heterotrophic protists and copepods 1.6 times and 2.2 times lower, respectively. In 2009, the P. globosa bloom was again dominated by large colonies and < 100 mu m diatoms. This corresponded to a dominance of heterotrophic dinoflagellates among the protists (62% of the total heterotrophic protist biomass) and Acartia clausi (55% of the copepod abundance). Overall, heterotrophic dinoflagellates appeared to be likely the most important group of phytoplankton grazers

Rapid Turnover of Ciliate Community Members in New England Tide Pools

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