Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

Contractile vacuoles: a rapidly expanding (and occasionally diminishing?) understanding.

Osmoregulation is the homeostatic mechanism essential for the survival of organisms in hypoosmotic and hyperosmotic conditions. In freshwater or soil dwelling protists this is frequently achieved through the action of an osmoregulatory organelle, the contractile vacuole. This endomembrane organelle responds to the osmotic challenges and compensates by collecting and expelling the excess water to maintain the cellular osmolarity. As compared with other endomembrane organelles, this organelle is underappreciated and under-studied. Here we review the reported presence or absence of contractile vacuoles across eukaryotic diversity, as well as the observed variability in the structure, function, and molecular machinery of this organelle. Our findings highlight the challenges and opportunities for constructing cellular and evolutionary models for this intriguing organelle.

Meteora sporadica, a protist with incredible cell architecture, is related to Hemimastigophora.

"Kingdom-level" branches are being added to the tree of eukaryotes at a rate approaching one per year, with no signs of slowing down.1,2,3,4 Some are completely new discoveries, whereas others are morphologically unusual protists that were previously described but lacked molecular data. For example, Hemimastigophora are predatory protists with two rows of flagella that were known since the 19th century but proved to represent a new deep-branching eukaryote lineage when phylogenomic analyses were conducted.2Meteora sporadica5 is a protist with a unique morphology; cells glide over substrates along a long axis of anterior and posterior projections while a pair of lateral "arms" swing back and forth, a motility system without any obvious parallels. Originally, Meteora was described by light microscopy only, from a short-term enrichment of deep-sea sediment. A small subunit ribosomal RNA (SSU rRNA) sequence was reported recently, but the phylogenetic placement of Meteora remained unresolved.6 Here, we investigated two cultivated Meteora sporadica isolates in detail. Transmission electron microscopy showed that both the anterior-posterior projections and the arms are supported by microtubules originating from a cluster of subnuclear microtubule organizing centers (MTOCs). Neither have a flagellar axoneme-like structure. Sequencing the mitochondrial genome showed this to be among the most gene-rich known, outside jakobids. Remarkably, phylogenomic analyses of 254 nuclear protein-coding genes robustly support a close relationship with Hemimastigophora. Our study suggests that Meteora and Hemimastigophora together represent a morphologically diverse "supergroup" and thus are important for resolving the tree of eukaryote life and early eukaryote evolution.

Kaonashia insperata gen. et sp. nov., a eukaryotrophic flagellate, represents a novel major lineage of heterotrophic stramenopiles.

Eukaryotrophic protists are ecologically significant and possess characteristics key to understanding the evolution of eukaryotes; however, they remain poorly studied, due partly to the complexities of maintaining predator-prey cultures. Kaonashia insperata, gen. nov., et sp. nov., is a free-swimming biflagellated eukaryotroph with a conspicuous ventral groove, a trait observed in distantly related lineages across eukaryote diversity. Di-eukaryotic (predator-prey) cultures of K. insperata with three marine algae (Isochrysis galbana, Guillardia theta, and Phaeodactylum tricornutum) were established by single-cell isolation. Growth trials showed that the studied K. insperata clone grew particularly well on G. theta, reaching a peak abundance of 1.0 × 105 ± 4.0 × 104 cells ml-1 . Small-subunit ribosomal DNA phylogenies infer that K. insperata is a stramenopile with moderate support; however, it does not fall within any well-defined phylogenetic group, including environmental sequence clades (e.g. MASTs), and its specific placement remains unresolved. Electron microscopy shows traits consistent with stramenopile affinity, including mastigonemes on the anterior flagellum and tubular mitochondrial cristae. Kaonashia insperata may represent a novel major lineage within stramenopiles, and be important for understanding the evolutionary history of the group. While heterotrophic stramenopile flagellates are considered to be predominantly bacterivorous, eukaryotrophy may be relatively widespread amongst this assemblage.

The function of the feeding groove of 'typical excavate' flagellates.

Phagotrophic flagellates are the main consumers of bacteria and picophytoplankton. Despite their ecological significance in the 'microbial loop', many of their predation mechanisms remain unclear. 'Typical excavates' bear a ventral groove, where prey is captured for ingestion. The consequences of feeding through a 'semi-rigid' furrow on the prey size range have not been explored. An unidentified moving element called 'the wave' that sweeps along the bottom of the groove toward the site of phagocytosis has been observed in a few species; its function is unclear. We investigated the presence, behavior, and function of the wave in four species from the three excavate clades (Discoba, Metamonada, and Malawimonadida) and found it present in all studied cases, suggesting the potential homology of this feature across all three groups. The wave displayed a species-specific behavior and was crucial for phagocytosis. The morphology of the feeding groove had an upper-prey size limit for successful prey captures, but smaller particles were not constrained. Additionally, the ingestion efficiencies were species dependent. By jointly studying these feeding traits, we speculate on adaptations to differences in food availability to better understand their ecological functions.

Characterisation and Cultivation of New Lineages of Colponemids, a Critical Assemblage for Inferring Alveolate Evolution.

There are several alveolate groups outside the well-studied trio - ciliates, dinoflagellates, and apicomplexans - that are crucial for understanding the evolution of this major taxon. One such assemblage is the "colponemids", which are eukaryotrophic biflagellates, usually with a ventral groove associated with the posterior flagellum. Previous phylogenetic studies show colponemids forming up to three distinct deep branches within alveolates (e.g. sister groups to Myzozoa or all other alveolates). We have developed dieukaryotic (predator-prey) cultures of four colponemid isolates. One represents the first stable culture of the halophile Palustrimonas (feeding on Pharyngomonas), while SSU rDNA phylogenies show the other isolates as two distinct new lineages. Neocolponema saponarium gen. et sp. nov. is a swimming alkaliphile with a large groove, which feeds on a kinetoplastid. Loeffela hirca gen. et sp. nov. is halophilic, has a subtle groove, usually moves along surfaces, and feeds on Pharyngomonas and Percolomonas. Prey capture in both new genera is raptorial, involves a specialized structure/region to the right of the proximal posterior flagellum, and presumed extrusomes. The relationships amongst Myzozoa, ciliates, and the (now) five described colponemid clades are unresolved, signaling that colponemid diversity represents both a challenge and important resource for tracing deep alveolate evolution.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation.

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialized parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae and fungi) like some parasites but then devour their cell contents by phagocytosis.1 Besides prey recognition and attachment, this complex behavior involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure.2 Yet the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g., locomotion and cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g., lytic polysaccharide monooxygenases) belong to families that are largely uncharacterized, emphasizing the potential of non-fungal microeukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor agilis, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behavior and new directions for cell biology research on non-model eukaryotes.

Diplonemids - A Review on "New" Flagellates on the Oceanic Block.

Diplonemids are a group of flagellate protists, that belong to the phylum Euglenozoa alongside euglenids, symbiontids and kinetoplastids. They primarily inhabit marine environments, though are also found in freshwater lakes. Diplonemids have been considered as rare and unimportant eukaryotes for over a century, with only a handful of species described until recently. However, thanks to their unprecedented diversity and abundance in the world oceans, diplonemids now attract increased attention. Recent improvements in isolation and cultivation have enabled characterization of several new genera, warranting a re-examination of all available knowledge gathered so far. Here we summarize available data on diplonemids, focusing on the recent advances in the fields of diversity, ecology, genomics, metabolism, and endosymbionts. We illustrate the life stages of cultivated genera, and summarise all reported interspecies associations, which in turn suggest lifestyles of predation and parasitism. This review also includes the latest classification of diplonemids, with a taxonomic revision of the genus Diplonema. Ongoing efforts to sequence various diplonemids suggest the presence of large and complex genomes, which correlate with the metabolic versatility observed in the model species Paradiplonema papillatum. Finally, we highlight its successful transformation into one of few genetically tractable marine protists.

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist.

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida, Rhizaria).

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D'Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage ("clade C") of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for "clade C."

Description of Imasa heleensis, gen. nov., sp. nov. (Imasidae, fam. nov.), a Deep-Branching Marine Malawimonad and Possible Key Taxon in Understanding Early Eukaryotic Evolution.

Malawimonadida is a deep-level (arguably "kingdom-scale") lineage of eukaryotes whose phylogenetic affinities are uncertain but of great evolutionary interest, as the group is suspected to branch close to the root of the tree of eukaryotes. Part of the difficulty in placing Malawimonadida phylogenetically is its tiny circumscription: at present, it comprises only two described and one cultured but undescribed species, all of them are freshwater suspension-feeding nanoflagellates. In this study, we cultivated and characterised Imasa heleensis gen. nov., sp. nov. (Imasidae fam. nov.), the first marine malawimonad to be described. Light and electron microscopy observations show that Imasa is largely similar to other malawimonads, but more frequently adheres to the substrate, often by means of a pliable posterior extension. Phylogenetic analyses based on two ribosomal RNA genes and four translated protein-coding genes using three different taxon sets place Imasa as sister to the three freshwater malawimonad strains with strong support. Imasa's mitochondrial genome is circular-mapping and shows a similar gene complement to other known malawimonads. We conclude that Imasa represents an important expansion of the range of taxa available for future evolutionary study.

Evolutionary History of Mitochondrial Genomes in Discoba, Including the Extreme Halophile Pleurostomum flabellatum (Heterolobosea).

Data from Discoba (Heterolobosea, Euglenozoa, Tsukubamonadida, and Jakobida) are essential to understand the evolution of mitochondrial genomes (mitogenomes), because this clade includes the most primitive-looking mitogenomes known, as well some extremely divergent genome information systems. Heterolobosea encompasses more than 150 described species, many of them from extreme habitats, but only six heterolobosean mitogenomes have been fully sequenced to date. Here we complete the mitogenome of the heterolobosean Pleurostomum flabellatum, which is extremely halophilic and reportedly also lacks classical mitochondrial cristae, hinting at reduction or loss of respiratory function. The mitogenome of P. flabellatum maps as a 57,829-bp-long circular molecule, including 40 coding sequences (19 tRNA, two rRNA, and 19 orfs). The gene content and gene arrangement are similar to Naegleria gruberi and Naegleria fowleri, the closest relatives with sequenced mitogenomes. The P. flabellatum mitogenome contains genes that encode components of the electron transport chain similar to those of Naegleria mitogenomes. Homology searches against a draft nuclear genome showed that P. flabellatum has two homologs of the highly conserved Mic60 subunit of the MICOS complex, and likely lost Mic19 and Mic10. However, electron microscopy showed no cristae structures. We infer that P. flabellatum, which originates from high salinity (313‰) water where the dissolved oxygen concentration is low, possesses a mitochondrion capable of aerobic respiration, but with reduced development of cristae structure reflecting limited use of this aerobic capacity (e.g., microaerophily).

EukRef-excavates: seven curated SSU ribosomal RNA gene databases.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

The Molecular Diversity of Phagotrophic Euglenids Examined Using Single-cell Methods.

Euglenids are a diverse group of euglenozoan flagellates that includes phototrophs, osmotrophs, and phagotrophs. Despite making up most of the phylogenetic diversity of euglenids, phagotrophs remain understudied, and recent work has focused on 'deep-branching' groups. Spirocuta is the large clade encompassing all flexible euglenids including the phototroph and primary osmotroph clades, plus various phagotrophs. Understanding the phylogenetic diversity of phagotrophic spirocutes is crucial for tracing euglenid evolution, including how phototrophs arose. We used single-cell approaches to greatly increase sampling of SSU rDNA for phagotrophic euglenids, particularly spirocutes, including the first sequences from Urceolus, Jenningsia, Chasmostoma, and Sphenomonas, and expanded coverage for Dinema and Heteronema sensu lato, amongst others. Urceolus monophyly is unconfirmed. Organisms referred to Jenningsia form two distinct clades. Heteronema vittatum and similar cells branch separately from Heteronema (c.f.) globuliferum and Teloprocta/Heteronema scaphurum, while Dinema appears as 2-3 clades. Sphenomonas is monophyletic and the deepest branch within Petalomonadida. The census of genera markedly underestimates the phylogenetic diversity of phagotrophs, but taxonomic restraint is necessary when sequences are not available from type species or reasonable surrogates. SSU rDNA phylogenies do not resolve most deep relationships within Spirocuta, but identify units of diversity to sample in future multigene analyses.

Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

The New Tree of Eukaryotes.

For 15 years, the eukaryote Tree of Life (eToL) has been divided into five to eight major groupings, known as 'supergroups'. However, the tree has been profoundly rearranged during this time. The new eToL results from the widespread application of phylogenomics and numerous discoveries of major lineages of eukaryotes, mostly free-living heterotrophic protists. The evidence that supports the tree has transitioned from a synthesis of molecular phylogenetics and biological characters to purely molecular phylogenetics. Most current supergroups lack defining morphological or cell-biological characteristics, making the supergroup label even more arbitrary than before. Going forward, the combination of traditional culturing with maturing culture-free approaches and phylogenomics should accelerate the process of completing and resolving the eToL at its deepest levels.

A single-cell genome reveals diplonemid-like ancestry of kinetoplastid mitochondrial gene structure.

Euglenozoa comprises euglenids, kinetoplastids, and diplonemids, with each group exhibiting different and highly unusual mitochondrial genome organizations. Although they are sister groups, kinetoplastids and diplonemids have very distinct mitochondrial genome architectures, requiring widespread insertion/deletion RNA editing and extensive trans-splicing, respectively, in order to generate functional transcripts. The evolutionary history by which these differing processes arose remains unclear. Using single-cell genomics, followed by small sub unit ribosomal DNA and multigene phylogenies, we identified an isolated marine cell that branches on phylogenetic trees as a sister to known kinetoplastids. Analysis of single-cell amplified genomic material identified multiple mitochondrial genome contigs. These revealed a gene architecture resembling that of diplonemid mitochondria, with small fragments of genes encoded out of order and or on different contigs, indicating that these genes require extensive trans-splicing. Conversely, no requirement for kinetoplastid-like insertion/deletion RNA-editing was detected. Additionally, while we identified some proteins so far only found in kinetoplastids, we could not unequivocally identify mitochondrial RNA editing proteins. These data invite the hypothesis that extensive genome fragmentation and trans-splicing were the ancestral states for the kinetoplastid-diplonemid clade but were lost during the kinetoplastid radiation. This study demonstrates that single-cell approaches can successfully retrieve lineages that represent important new branches on the tree of life, and thus can illuminate major evolutionary and functional transitions in eukaryotes. This article is part of a discussion meeting issue 'Single cell ecology'.

Combined cultivation and single-cell approaches to the phylogenomics of nucleariid amoebae, close relatives of fungi.

Nucleariid amoebae (Opisthokonta) have been known since the nineteenth century but their diversity and evolutionary history remain poorly understood. To overcome this limitation, we have obtained genomic and transcriptomic data from three Nuclearia, two Pompholyxophrys and one Lithocolla species using traditional culturing and single-cell genome (SCG) and single-cell transcriptome amplification methods. The phylogeny of the complete 18S rRNA sequences of Pompholyxophrys and Lithocolla confirmed their suggested evolutionary relatedness to nucleariid amoebae, although with moderate support for internal splits. SCG amplification techniques also led to the identification of probable bacterial endosymbionts belonging to Chlamydiales and Rickettsiales in Pompholyxophrys. To improve the phylogenetic framework of nucleariids, we carried out phylogenomic analyses based on two datasets of, respectively, 264 conserved proteins and 74 single-copy protein domains. We obtained full support for the monophyly of the nucleariid amoebae, which comprise two major clades: (i) Parvularia-Fonticula and (ii) Nuclearia with the scaled genera Pompholyxophrys and Lithocolla. Based on these findings, the evolution of some traits of the earliest-diverging lineage of Holomycota can be inferred. Our results suggest that the last common ancestor of nucleariids was a freshwater, bacterivorous, non-flagellated filose and mucilaginous amoeba. From the ancestor, two groups evolved to reach smaller (Parvularia-Fonticula) and larger (Nuclearia and related scaled genera) cell sizes, leading to different ecological specialization. The Lithocolla + Pompholyxophrys clade developed exogenous or endogenous cell coverings from a Nuclearia-like ancestor. This article is part of a discussion meeting issue 'Single cell ecology'.