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Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Universal taxonomic frameworks have been critical tools to structure the fields of botany, zoology, mycology, and bacteriology as well as their large research communities. Animals, plants, and fungi have relatively solid, stable morpho-taxonomies built over the last three centuries, while bacteria have been classified for the last three decades under a coherent molecular taxonomic framework. By contrast, no such common language exists for microbial eukaryotes, even though environmental '-omics' surveys suggest that protists make up most of the organismal and genetic complexity of our planet's ecosystems! With the current deluge of eukaryotic meta-omics data, we urgently need to build up a universal eukaryotic taxonomy bridging the protist -omics age to the fragile, centuries-old body of classical knowledge that has effectively linked protist taxa to morphological, physiological, and ecological information. UniEuk is an open, inclusive, community-based and expert-driven international initiative to build a flexible, adaptive universal taxonomic framework for eukaryotes. It unites three complementary modules, EukRef, EukBank, and EukMap, which use phylogenetic markers, environmental metabarcoding surveys, and expert knowledge to inform the taxonomic framework. The UniEuk taxonomy is directly implemented in the European Nucleotide Archive at EMBL-EBI, ensuring its broad use and long-term preservation as a reference taxonomy for eukaryotes., (© 2017 The Author(s) Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

Marine protists are integral to marine food webs and exhibit complex relationships with other microbial taxa. Phagotrophic protists contribute significantly to carbon turnover in the sunlit ocean and evidence suggests grazing in the dark ocean can be significant as well. New in situ sampling technologies hold great promise for more accurately accessing these impacts. The molecular signatures of parasitic protists comprise significant fractions of many high-throughput sequencing datasets, suggesting a major role in controlling populations of their host(s). The prokaryotic symbionts of free-living protists can be numerous, and, particularly in low-oxygen to anoxic marine habitats, their collective metabolisms may contribute significantly to biogeochemical cycling. This short review addresses principally planktonic communities in the mesopelagic and bathypelagic dark ocean., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

High salt concentrations, absence of light, anoxia, and high hydrostatic pressure make deep hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea one of the most polyextreme habitats on Earth. Taking advantage of the unique chemical characteristics of these basins, we tested the effect of environmental selection and geographic distance on the structure of protistan communities. Terminal restriction fragment length polymorphism (T-RFLP) analyses were performed on water samples from the brines and seawater/brine interfaces of five basins: Discovery, Urania, Thetis, Tyro, and Medee. Using statistical analyses, we calculated the partitioning of diversity among the ten individual terminal restriction fragment (T-RF) profiles, based on peak abundance and peak incidence. While a significant distance effect on spatial protistan patterns was not detected, hydrochemical gradients emerged as strong dispersal barriers that likely lead to environmental selection in the DHAB protistan plankton communities. We identified sodium, magnesium, sulfate, and oxygen playing in concerto as dominant environmental drivers for the structuring of protistan plankton communities in the Eastern Mediterranean DHABs., (© 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Changes in ocean temperature and circulation patterns compounded by human activities are leading to oxygen minimum zone (OMZ) expansion with concomitant alteration in nutrient and climate active trace gas cycling. Here, we report the response of microbial eukaryote populations to seasonal changes in water column oxygen-deficiency using Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island British Columbia, as a model ecosystem. We combine small subunit ribosomal RNA gene sequencing approaches with multivariate statistical methods to reveal shifts in operational taxonomic units during successive stages of seasonal stratification and renewal. A meta-analysis is used to identify common and unique patterns of community composition between Saanich Inlet and the anoxic/sulfidic Cariaco Basin (Venezuela) and Framvaren Fjord (Norway) to show shared and unique responses of microbial eukaryotes to oxygen and sulfide in these three environments. Our analyses also reveal temporal fluctuations in rare populations of microbial eukaryotes, particularly anaerobic ciliates, that may be of significant importance to the biogeochemical cycling of methane in OMZs. Eukaryotic 18S rRNA gene sequences recovered from the Saanich Inlet water column on were deposited in Genbank under accession numbers HQ864863–HQ871151.

This is the second paper in a series of three that investigates eukaryotic microbial diversity and taxon distribution in the Cariaco Basin, Venezuela, the ocean's largest anoxic marine basin. Here, we use phylogenetic information, multivariate community analyses and statistical richness predictions to test whether protists exhibit habitat specialization within defined geochemical layers of the water column. We also analyze spatio-temporal distributions of protists across two seasons and two geographic sites within the basin. Non-metric multidimensional scaling indicates that these two basin sites are inhabited by distinct protistan assemblages, an observation that is supported by the minimal overlap in observed and predicted richness of sampled sites. A comparison of parametric richness estimations indicates that protistan communities in closely spaced-but geochemically different-habitats are very dissimilar, and may share as few as 5% of total operational taxonomic units (OTUs). This is supported by a canonical correspondence analysis, indicating that the empirically observed OTUs are organized along opposing gradients in oxidants and reductants. Our phylogenetic analyses identify many new clades at species to class levels, some of which appear restricted to specific layers of the water column and have a significantly nonrandom distribution. These findings suggest many pelagic protists are restricted to specific habitats, and likely diversify, at least in part due to separation by geochemical barriers.

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80,000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36,000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100,000-500,000 species), suggesting a degree of protistan endemism.

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.