Microbial Eukaryotes Research Articles

Giant Virus Infection Signatures Are Modulated by Euphotic Zone Depth Strata and Iron Regimes of the Subantarctic Southern Ocean.

Viruses can alter the abundance, evolution, and metabolism of microorganisms in the ocean, playing a key role in water column biogeochemistry and global carbon cycles. Large efforts to measure the contribution of eukaryotic microorganisms (e.g., protists) to the marine food web have been made, yet the in situ activities of the ecologically relevant viruses that infect these organisms are not well characterized. Viruses within the phylum Nucleocytoviricota ("giant viruses") are known to infect a diverse range of ecologically relevant marine protists, yet how these viruses are influenced by environmental conditions remains under-characterized. By employing metatranscriptomic analyses of in situ microbial communities along a temporal and depth-resolved gradient, we describe the diversity of giant viruses at the Southern Ocean Time Series (SOTS), a site within the subpolar Southern Ocean. Using a phylogeny-guided taxonomic assessment of detected giant virus genomes and metagenome-assembled genomes, we observed depth-dependent structuring of divergent giant virus families mirroring dynamic physicochemical gradients in the stratified euphotic zone. Analyses of transcribed metabolic genes from giant viruses suggest viral metabolic reprogramming of hosts from the surface to a 200-m depth. Lastly, using on-deck incubations reflecting a gradient of iron availability, we show that modulating iron regimes influences the activity of giant viruses in the field. Specifically, we show enhanced infection signatures of giant viruses under both iron-replete and iron-limited conditions. Collectively, these results expand our understanding of how the water column's vertical biogeography and chemical surroundings affect an important group of viruses within the Southern Ocean. IMPORTANCE The biology and ecology of marine microbial eukaryotes is known to be constrained by oceanic conditions. In contrast, how viruses that infect this important group of organisms respond to environmental change is less well known, despite viruses being recognized as key microbial community members. Here, we address this gap in our understanding by characterizing the diversity and activity of "giant" viruses within an important region in the sub-Antarctic Southern Ocean. Giant viruses are double-stranded DNA (dsDNA) viruses of the phylum Nucleocytoviricota and are known to infect a wide range of eukaryotic hosts. By employing a metatranscriptomics approach using both in situ samples and microcosm manipulations, we illuminated both the vertical biogeography and how changing iron availability affects this primarily uncultivated group of protist-infecting viruses. These results serve as a foundation for our understanding of how the open ocean water column structures the viral community, which can be used to guide models of the viral impact on marine and global biogeochemical cycling.

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary events leading to the diversification of this genus over the last 20 million years. Here, we show the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

Cryo-electron microscopy of extracellular vesicles associated with the marine toxic dinoflagellate Alexandrium minutum

Extracellular Vesicles (EVs) are likely an important strategy of transport and communication in marine microbial community. Their isolation and characterization from axenic culture of microbial eukaryotes represents a technological challenge not fully solved. Here, for the first time, we isolated EVs from a near-axenic culture of the toxic dinoflagellate Alexandrium minutum. Pictures of the isolated vesicles were done with Cryo TEM (Cryogenic Transmission Electron Microscopy). Based on their morphotype the EVs were clustered in five major groups (rounded, rounded electron-dense, lumen electron-dense, double and irregular) and each EV was measured resulting in an average size of 0.36 µm of diameter. Taking in account that in prokaryotes it has been demonstrated that EVs play an important role in the mechanism of toxicity, this descriptive work aims to be the first step to study the possible role of EVs in the toxicity of dinoflagellates.

Organic rice cultivation enhances the diversity of above-ground arthropods but not below-ground soil eukaryotes

Organic farming generally has greater biodiversity compared to conventional farming. However, few studies have examined both above- and below-ground biodiversity in organic farming of the rice field, and little is known about whether and how biodiversity is affected by the duration of organic farming. In this study, we conducted a two-year investigation on one conventional farm, as well as two organic farms with different management durations (1 and 5 years before sampling). Compared to conventional farming, species richness increased by 40% for arthropods in organic farming, with 75% for pests and 35% for natural enemies, but there were no differences for soil microbial eukaryotes. No significant difference in species diversity of arthropods and soil microbial eukaryotes was found between two organic farms, which may indicate that five years is not yet long enough to achieve significant changes in arthropod and eukaryotic communities. We used a compatibility index (Cc-o) to evaluate the trade-off between biodiversity, rice yield, and economic benefits of conversion from conventional to organic farming. Results showed that the yield-based value of Cc-o was close to 0 for above-ground arthropods and negative for soil eukaryotes. However, Cc-o values based on economic benefits were positive for all taxa. Our study indicates that both one year and five years durations of organic farming could improve the diversity of above-ground arthropods, but not that of below-ground soil eukaryotes in the rice field. There was an equivalent gain in biodiversity and loss in yield for above-ground arthropods under organic farming, but win-win scenarios may occur when the economic benefits are considered.

Origins of genome-editing excisases as illuminated by the somatic genome of the ciliate Blepharisma

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly ofBlepharismastoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons. All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.

Impact of Nesting Mode, Diet, and Taxonomy in Structuring the Associated Microbial Communities of Amazonian Ants

Studies of ant biodiversity are important to understand their group better, as well as to extend our knowledge on the evolution of their associated organisms. Host-associated microbial communities, and particularly bacterial communities, are shaped by different host factors such as habitat, diet, and phylogeny. Here, we studied the structures of bacterial and microbial eukaryote communities associated with Amazonian ants collected from two habitats: the rainforest and the city. We collected 38 ant species covering a large taxonomic range, and we used 16S rRNA and 18S rRNA amplicon sequencing to study the impact of the host’s ecological and phylogenetic factors on their microbial communities. Our results show that (1) habitat does not structure ant microbial communities, (2) ant diet and nesting mode impact bacterial communities, while only nesting mode structures microbial eukaryote communities, and (3) microbial diversity is not correlated with host phylogeny, although several ant genera have conserved bacterial communities. As we continue to uncover the diversity and function of insect-associated microbes, this work explores how host ecology and evolutionary history shape ant microbial communities.

Formation of multicellular colonies by choanoflagellates increases susceptibility to capture by amoeboid predators.

Many heterotrophic microbial eukaryotes are size-selective feeders. Some microorganisms increase their size by forming multicellular colonies. We used choanoflagellates, Salpingoeca helianthica, which can be unicellular or form multicellular colonies, to study the effects of multicellularity on vulnerability to predation by the raptorial protozoan predator, Amoeba proteus, which captures prey with pseudopodia. Videomicrography used to measure the behavior of interacting S. helianthica and A. proteus revealed that large choanoflagellate colonies were more susceptible to capture than were small colonies or single cells. Swimming colonies produced larger flow fields than did swimming unicellular choanoflagellates, and the distance of S. helianthica from A. proteus when pseudopod formation started was greater for colonies than for single cells. Prey size did not affect the number of pseudopodia formed and the time between their formation, pulsatile kinematics and speed of extension by pseudopodia, or percent of prey lost by the predator. S. helianthica did not change swimming speed or execute escape maneuvers in response to being pursued by pseudopodia, so size-selective feeding by A. proteus was due to predator behavior rather than prey escape. Our results do not support the theory that the selective advantage of becoming multicellular by choanoflagellate-like ancestors of animals was reduced susceptibility to protozoan predation.

Spatial and temporal dynamics of coastal benthic microbial communities along a salinity gradient

The Baltic Sea is a unique brackish water ecosystem that has been studied for decades; however, knowledge about the diversity of the benthic communities of bacteria and microbial eukaryotes within this system is sparse. Using an amplicon sequencing approach, we evaluated alpha and beta diversity of shallow-water coastal microbial sediment communities and their relationship to several environmental factors, on both a large spatial scale in the Baltic Sea and the adjacent North Sea, as well as on a temporal scale at selected sites along the salinity gradient in the Baltic Sea. We found salinity to be among the drivers of alpha diversity for bacteria but not for eukaryotes; however, community network structure appeared to change between sites of different salinity. Furthermore, beta diversity analyses revealed that communities in the poly- to mesohaline sections of the study area were affected to a higher degree by temperature, nutrient and sediment characteristics. This study shows that the structure of coastal microbial communities is to some degree related to salinity, but other factors also play a role.

Exploring the diversity of microeukaryotic communities in New England tide pools

Though historically understudied, due in large part to most species being uncultivable, microbial eukaryotes (i.e. protists) are abundant and widespread across diverse habitats. Recent advances in molecular techniques, including metabarcoding, allow for the characterization of poorly known protist lineages. This study surveys the diversity of SAR (Stramenopila, Alveolata, and Rhizaria), a major eukaryotic clade that is estimated to represent about half of all eukaryotic diversity. SAR lineages use varied metabolic strategies like mixotrophy in dinoflagellates (Alveolata), parasitism in apicomplexans (Alveolata) and labyrinthulids (Stramenopila), and life cycle stages that include encystment and attachment (e.g. in ciliates, Alveolata) to survive in highly dynamic habitats. Using metabarcoding primers designed specifically to target a portion of the 18S small subunit ribosomal RNA (SSU-rRNA) gene of SAR lineages, we compare protist community composition from tide pools in Acadia National Park, Maine, USA. We characterize over 500 lineages, here operational taxonomic units (OTUs), many of which are found abundant in the tide pool environment. We also find that communities vary by month sampled and exhibit patterns by size (i.e. macro-, micro-, and nano-sized). Taken together, these data allow us to further catalog protist diversity in extreme environments (e.g. those subject to extreme fluctuations in temperature and salinity during tidal cycles). Such data are critical in the explorations of biodiversity patterns among microorganisms on our rapidly changing planet.

Free living ciliated protists from the chemoautotrophic cave ecosystem of Frasassi (Italy)

This study provides the first report on a community of free-living ciliated protists from the chemoautotrophic cave ecosystem of Frasassi, Italy. This subterranean groundwater ecosystem represents a hotspot of biodiversity that still needs to be fully explored with particular reference to microbial eukaryotes such as protist ciliates. A total of 33 taxa of ciliates were identified along with one species each of flagellate, heliozoans and naked amoebae, from four main sampling sites, namely, Grotta Solfurea (GSO), Lago Verde (LVE), Ramo Solfureo (RSO), and Pozzo dei Cristalli (PDC). The last consists of small microhabitats/ponds presenting different chemical–physical and biological parameters, such as sulfur and nutrient contents and the presence of bacterial biofilms. Furthermore, an analysis of the cryptic ciliate species biosphere as a ‘seedbank’ of diversity against cave ecosystem disturbance was also performed. This study also highlights some peculiar adaptations of cave-dwelling ciliates not described in their noncave-dwelling conspecifics, such as the extreme photosensitivity of Urocentrum turbo, the cannibalism of Coleps hirtus, the variable number of thorns in Aspidisca species as a defensive response to predation, and the frequent reorganization of ciliary structures in Euplotes aediculatus. The 18S rDNA sequences were generated for five species and were compared with those of the noncave-dwelling conspecifics. Finally, our results shed light on the still largely unknown ciliate diversity in the chemosynthesis-based sulfidic groundwater ecosystem of Frasassi.

Quantifying relative contributions of biotic interactions to bacterial diversity and community assembly by using community characteristics of microbial eukaryotes

Biotic interactions are known as a major control on microbial diversity. However, biotic interactions have rarely been quantified in an adequate manner, often leaving much residual variation unexplained in microbial biogeographic studies. Herein, we propose a holistic approach to disentangle the relative importance of inter-domain interactions in shaping microbial diversity by incorporating community-level characteristics. Taking coastal bacterioplankton on a regional scale as an example, we designated a range of community characteristics of pico- and nanoeukaryotes derived from metabarcoding and flow cytometric data as inter-domain interacting proxies, which were then considered in statistical modeling. We found that the bacterial diversity indices and community structure were much more accurately explained by a number of eukaryotic characteristics than by the measured environmental variables and/or spatial variables alone, as were the richness, relative abundances, and assemblage structures of major bacterial taxa. In co-occurrence networks, the nodes of characteristics that had more edges (links) were frequently the best explanatory variables for bacterial diversity indices. Over 70% of total variation in bacterial community structure could be explained by three categories of biotic interactions: parasitism (27%), fungi-bacterial competition (32%), and trophic structure and bacterivory (13%). This study showcases a methodological framework to infer different types of inter-domain interactions at play, and stresses the importance of non-grazing interacting processes in shaping bacterial diversity and community assembly.

Ciliate Morpho-Taxonomy and Practical Considerations before Deploying Metabarcoding to Ciliate Community Diversity Surveys in Urban Receiving Waters.

Disentangling biodiversity and community assembly effects on ecosystem function has always been an important topic in ecological research. The development and application of a DNA metabarcoding method has fundamentally changed the way we describe prokaryotic communities and estimate biodiversity. Compared to prokaryotes (bacteria and archaea), the eukaryotic microbes (unicellular eukaryotes) also fulfill extremely important ecological functions in different ecosystems regarding their intermediate trophic positions. For instance, ciliated microbes (accounting for a substantial portion of the diversity of unicellular eukaryotes) perform pivotal roles in microbial loops and are essential components in different ecosystems, especially in water purification processes. Therefore, the community composition of ciliated species has been widely utilized as a proxy for water quality and biological assessment in urban river ecosystems and WWTPs (wastewater treatment plants). Unfortunately, investigating the dynamic changes and compositions in ciliate communities relies heavily on existing morpho-taxonomical descriptions, which is limited by traditional microscopic approaches. To deal with this dilemma, we discuss the DNA-based taxonomy of ciliates, the relative merits and challenges of deploying its application using DNA metabarcoding for surveys of ciliate community diversity in urban waterbodies, and provide suggestions for minimizing relevant sources of biases in its implementation. We expect that DNA metabarcoding could untangle relationships between community assembly and environmental changes affecting ciliate communities. These analyses and discussions could offer a replicable method in support of the application of evaluating communities of ciliated protozoa as indicators of urban freshwater ecosystems.

Revisiting fecal metatranscriptomics analyses of macaques with idiopathic chronic diarrhoea with a focus on trichomonad parasites.

Trichomonads, anaerobic microbial eukaryotes members of the phylum Parabasalia, are common obligate extracellular symbionts that can lead to pathological or asymptomatic colonization of various mucosal surfaces in a wide range of animal hosts. Results from previous in vitro studies have suggested a number of intriguing mucosal colonization strategies by Trichomonads, notably highlighting the importance of interactions with bacteria. However, in vivo validation is currently lacking. A previous metatranscriptomics study into the cause of idiopathic chronic diarrhoea in macaques reported the presence of an unidentified protozoan parasite related to Trichomonas vaginalis. In this work, we performed a reanalysis of the published data in order to identify the parasite species present in the macaque gut. We also leveraged the information-rich metatranscriptomics data to investigate the parasite behaviour in vivo. Our results indicated the presence of at least 3 genera of Trichomonad parasite; Tetratrichomonas, Pentatrichomonas and Trichomitus, 2 of which had not been previously reported in the macaque gut. In addition, we identified common in vivo expression profiles shared amongst the Trichomonads. In agreement with previous findings for other Trichomonads, our results highlighted a relationship between Trichomonads and mucosal bacterial diversity which could be influential in health and disease.

Distribution of Soil Microbes in Urban Parks: An Effect of Under-Tree Crown and Hillside Position on Testate Amoeba Assemblages in Subtropics (Shenzhen, China)

Testate amoebae (TA) are important components of soil ecosystems, where they play an essential role in belowground food webs. In this study, we examined species composition, diversity, and structure of TA assemblages in soils beneath tree crowns (microscale) at three slope positions (mesoscale) in subtropical urban parks in Shenzhen, China. Forty-two species of TA belonging to 18 genera were identified in 81 samples. TA assemblages were the most diverse and abundant in the center of the tree crown comparing with other under-tree habitats. Foot of the hills harbored higher TA abundance and diversity comparing with upper locations along the hillslopes. The distribution of TA was mostly driven by under-crown and hillside positions but not by elementary environmental conditions such as pH, moisture content and thickness of leaf litter layer. None of later factors were sufficient in shaping TA assemblage composition. The findings of our study suggest that in regional studies of soil microbial eukaryotes both micro- (i.e., under-crown) and mesoscale (i.e., the slope position) heterogeneity should be considered.

First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora).

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex.

Trophic State Drives the Diversity of Protists in a Tropical River (New River, Belize).

Land use disrupts the ecosystem functioning of freshwater systems and significantly affects trophic state. Consequently, biodiversity is severely affected by changes to the ecosystem. Microbial eukaryotes (i.e., protists) play an essential role in ecosystem functioning, contributing to biogeochemical processes, nutrient cycling, and food webs. Protist composition is a useful biological quality parameter for monitoring aquatic ecosystems and determining aquatic system health. In this study, we investigated the effects of land usage and trophic state on the communities of microbial eukaryotes in the New River (Belize, C.A.). Land use and trophic state both significantly affected protist community compositions, with impacted and mesotrophic sampled sites having higher biodiversity when compared to other sites. Autotrophic organisms dominated indirectly impacted and eutrophic sites, while impacted and mesotrophic sites had proportional ratios of autotrophic and heterotrophic organisms. Our study highlights the significant effects of trophic gradients on protistan community composition, even at the local scales.

Microbial predators form a new supergroup of eukaryotes.

Molecular phylogenetics of microbial eukaryotes has reshaped the tree of life by establishing broad taxonomic divisions, termed supergroups, that supersede the traditional kingdoms of animals, fungi and plants, and encompass a much greater breadth of eukaryotic diversity1. The vast majority of newly discovered species fall into a small number of known supergroups. Recently, however, a handful of species with no clear relationship to other supergroups have been described2-4, raising questions about the nature and degree of undiscovered diversity, and exposing the limitations of strictly molecular-based exploration. Here we report ten previously undescribed strains of microbial predators isolated through culture that collectively form a diverse new supergroup of eukaryotes, termed Provora. The Provora supergroup is genetically, morphologically and behaviourally distinct from other eukaryotes, and comprises two divergent clades of predators-Nebulidia and Nibbleridia-that are superficially similar to each other, but differ fundamentally in ultrastructure, behaviour and gene content. These predators are globally distributed in marine and freshwater environments, but are numerically rare and have consequently been overlooked by molecular-diversity surveys. In the age of high-throughput analyses, investigation of eukaryotic diversity through culture remains indispensable for the discovery of rare but ecologically and evolutionarily important eukaryotes.

The description of a new brackish water ciliate species from China, Trachelostyla aestuarina n. sp., with a species key and biogeographic investigation for Trachelostyla (Ciliophora, Sporadotrichida)

The ciliated protists are a diverse group of microbial eukaryotes that play an important role in the functioning of microbial food webs. The genus Trachelostyla Borror, 1972, comprised a few species with a non-twisted distinctly cephalized shape. The overall taxonomy has been challenging for this group, and the understanding of their diversity and distribution is therefore relatively limited. To further investigate this group, we report here on two species from the East China Sea, Trachelostyla aestuarina n. sp. and Trachelostyla caudata Borror, 1972, including their in vivo morphology, infraciliature, and phylogenetic relationships based on 18S rRNA gene sequences. The physiological reorganization stages of the latter were also investigated. T. aestuarina is characterized by a combination of features including a bipartite cell shape, a size of 150–220 × 15–35 μm, scattered cortical granules, and multiple macronuclear nodules (37–55). T. caudata has only 10 cirri in the frontal region, fewer than its congeners; thus, we improved the definition of the genus. The global distribution of Trachelostyla was summarized based on the available literature and a key to the valid species provided. This work improves the overall knowledge of hypotrich biodiversity and provides underpinning for future researchers in global regions for environmental monitoring and resource investigations.

Comparative genomics reveals insight into the evolutionary origin of massively scrambled genomes.

Ciliates are microbial eukaryotes that undergo extensive programmed genome rearrangement, a natural genome editing process that converts long germline chromosomes into smaller gene-rich somatic chromosomes. Three well-studied ciliates include Oxytricha trifallax, Tetrahymena thermophila, and Paramecium tetraurelia, but only the Oxytricha lineage has a massively scrambled genome, whose assembly during development requires hundreds of thousands of precisely programmed DNA joining events, representing the most complex genome dynamics of any known organism. Here we study the emergence of such complex genomes by examining the origin and evolution of discontinuous and scrambled genes in the Oxytricha lineage. This study compares six genomes from three species, the germline and somatic genomes for Euplotes woodruffi, Tetmemena sp., and the model ciliate O. trifallax. We sequenced, assembled, and annotated the germline and somatic genomes of E. woodruffi, which provides an outgroup, and the germline genome of Tetmemena sp. We find that the germline genome of Tetmemena is as massively scrambled and interrupted as Oxytricha's: 13.6% of its gene loci require programmed translocations and/or inversions, with some genes requiring hundreds of precise gene editing events during development. This study revealed that the earlier diverged spirotrich, E. woodruffi, also has a scrambled genome, but only roughly half as many loci (7.3%) are scrambled. Furthermore, its scrambled genes are less complex, together supporting the position of Euplotes as a possible evolutionary intermediate in this lineage, in the process of accumulating complex evolutionary genome rearrangements, all of which require extensive repair to assemble functional coding regions. Comparative analysis also reveals that scrambled loci are often associated with local duplications, supporting a gradual model for the origin of complex, scrambled genomes via many small events of DNA duplication and decay.

Globally-distributed microbial eukaryotes exhibit endemism at deep-sea hydrothermal vents.

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.