Molecular phylogeny of Spirotrichonymphea (Parabasalia) with emphasis on Spironympha, Spirotrichonympha, and three new genera Pseudospironympha, Nanospironympha, and Brugerollina.

Spirotrichonymphea, one of the six classes of phylum Parabasalia, are characterized by bearing many flagella in spiral rows, and they occur exclusively in the guts of termites. Phylogenetic relationships among the 13 described genera are not well understood due to complex morphological evolution and a paucity of molecular data. One such understudied genus is Spironympha. It has been variously considered a valid genus, a subgenus of Spirotrichonympha, or an "immature" life cycle stage of Spirotrichonympha. To clarify this, we sequenced the small subunit rRNA gene sequences of Spironympha and Spirotrichonympha cells isolated from the hindguts of Reticulitermes species and Hodotermopsis sjostedti and confirmed the molecular identity of H. sjostedti symbionts using fluorescence in situ hybridization. Spironympha as currently circumscribed is polyphyletic, with both H. sjostedti symbiont species branching separately from the "true" Spironympha from Reticulitermes. Similarly, the Spirotrichonympha symbiont of H. sjostedti branches separately from the "true" Spirotrichonympha found in Reticulitermes. Our data support Spironympha from Reticulitermes as a valid genus most closely related to Spirotrichonympha, though its monophyly and interspecific relationships are not resolved in our molecular phylogenetic analysis. We propose three new genera to accommodate the H. sjostedti symbionts and two new species of Spirotrichonympha from Reticulitermes.

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada.

BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

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.

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 Complete Protist Symbiont Communities of Coptotermes formosanus and Coptotermes gestroi: Morphological and Molecular Characterization of Five New Species.

Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann) (Blattoidea: Rhinotermitidae) are invasive subterranean termite pest species with a major global economic impact. However, the descriptions of the mutualistic protist communities harbored in their respective hindguts remain fragmentary. The C. formosanus hindgut has long been considered to harbor three protist species, Pseudotrichonympha grassii (Trichonymphida), Holomastigotoides hartmanni, and Cononympha (Spirotrichonympha) leidyi (Spirotrichonymphida), but molecular data have suggested that the diversity may be higher. Meanwhile, the C. gestroi community remains undescribed except for Pseudotrichonympha leei. To complete the characterization of these communities, hindguts of workers from both termite species were investigated using single-cell PCR, microscopy, cell counts, and 18S rRNA amplicon sequencing. The two hosts were found to harbor intriguingly parallel protist communities, each consisting of one Pseudotrichonympha species, two Holomastigotoides species, and two Cononympha species. All protist species were unique to their respective hosts, which last shared a common ancestor ~18 MYA. The relative abundances of protist species in each hindgut differed remarkably between cell count data and 18S rRNA profiles, calling for caution in interpreting species abundances from amplicon data. This study will enable future research in C. formosanus and C. gestroi hybrids, which provide a unique opportunity to study protist community inheritance, compatibility, and potential contribution to hybrid vigor.

Ultrastructure and Molecular Phylogeny of Iotanema spirale gen. nov. et sp. nov., a New Lineage of Endobiotic Fornicata with Strikingly Simplified Ultrastructure.

Fornicata (Metamonada) is a group of Excavata living in low-oxygen environments and lacking conventional mitochondria. It includes free-living Carpediemonas-like organisms from marine habitats and predominantly parasitic/commensal retortamonads and diplomonads. Current modest knowledge of biodiversity of Fornicata limits our ability to draw a complete picture of the evolutionary history in this group. Here, we report the discovery of a novel fornicate, Iotanema spirale gen. nov. et sp. nov., obtained from fresh feces of the gecko Phelsuma madagascariensis. Our phylogenetic analyses of the small subunit ribosomal RNA gene demonstrate that I. spirale is closely related to the free-living, marine strain PCS and the Carpediemonas-like organism Hicanonectes teleskopos within Fornicata. Iotanema spirale exhibits several features uncommon to fornicates, such as a single flagellum, a highly reduced cytoskeletal system, and the lack of the excavate ventral groove, but shares these characters with the poorly known genus Caviomonas. Therefore, I. spirale is accommodated within the family Caviomonadidae, which represents the third known endobiotic lineage of Fornicata. This study improves our understanding of character evolution within Fornicata when placed within the molecular phylogenetic context.

Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes.

BACKGROUND: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. RESULTS: We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. CONCLUSIONS: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

Protist symbionts of termites: diversity, distribution, and coevolution.

The symbiosis between termites and their hindgut protists is mutually obligate and vertically inherited. It was established by the late Jurassic in the cockroach ancestors of termites as they transitioned to wood feeding. Since then, protist symbionts have been transmitted from host generation to host generation by proctodeal trophallaxis (anal feeding). The protists belong to multiple lineages within the eukaryotic superphylum Metamonada. Most of these lineages have evolved large cells with complex morphology, unlike the non-termite-associated Metamonada. The species richness and taxonomic composition of symbiotic protist communities varies widely across termite lineages, especially within the deep-branching clade Teletisoptera. In general, closely related termites tend to harbour closely related protists, and deep-branching termites tend to harbour deep-branching protists, reflecting their broad-scale co-diversification. A closer view, however, reveals a complex distribution of protist lineages across hosts. Some protist taxa are common, some are rare, some are widespread, and some are restricted to a single host family or genus. Some protist taxa can be found in only a few, distantly related, host species. Thus, the long history of co-diversification in this symbiosis has been complicated by lineage-specific loss of symbionts, transfer of symbionts from one host lineage to another, and by independent diversification of the symbionts relative to their hosts. This review aims to introduce the biology of this important symbiosis and serve as a gateway to the diversity and systematics literature for both termites and protists. A searchable database with all termite-protist occurrence records and taxonomic references is provided as a supplementary file to encourage and facilitate new research in this field.

Ancient and pervasive expansion of adaptin-related vesicle coat machinery across Parabasalia.

The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems.

Molecular Phylogenetic Position of Microjoenia (Parabasalia: Spirotrichonymphea) from Reticulitermes and Hodotermopsis Termite Hosts.

Microjoenia are obligate symbionts of termites. The genus was erected in 1892 for small cells with many flagella that insert near, but not directly from, the cell apex, and an axostyle that can protrude from the cell posterior. Although ultrastructural studies have been carried out on three Microjoenia species to date, no molecular data have been directly attributed to any species. Microjoenia are classified within the parabasalian class Spirotrichonymphea, which is characterized by flagellar bands that emerge near the cell apex and proceed posteriorly in a right-handed helix. In Microjoenia, however, the flagellar bands are very short and proceed longitudinally or with a weakly observable helix. In this study, we have amplified and sequenced the 18S ribosomal RNA gene from individually isolated Microjoenia cells from Reticulitermes and Hodotermopsis hosts as part of an ongoing effort to understand the phylogeny of Spirotrichonymphea and their coevolution with termites. In our 18S rRNA gene phylogeny, Microjoenia forms the sister lineage to Spirotrichonympha, though many other evolutionary relationships within Spirotrichonymphea remain unresolved.

Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes.

Discoveries of diverse microbial eukaryotes and their inclusion in comprehensive phylogenomic analyses have crucially re-shaped the eukaryotic tree of life in the 21st century.1 At the deepest level, eukaryotic diversity comprises 9-10 "supergroups." One of these supergroups, the Metamonada, is particularly important to our understanding of the evolutionary dynamics of eukaryotic cells, including the remodeling of mitochondrial function. All metamonads thrive in low-oxygen environments and lack classical aerobic mitochondria, instead possessing mitochondrion-related organelles (MROs) with metabolisms that are adapted to low-oxygen conditions. These MROs lack an organellar genome, do not participate in the Krebs cycle and oxidative phosphorylation,2 and often synthesize ATP by substrate-level phosphorylation coupled to hydrogen production.3,4 The events that occurred during the transition from an oxygen-respiring mitochondrion to a functionally streamlined MRO early in metamonad evolution remain largely unknown. Here, we report transcriptomes of two recently described, enigmatic, anaerobic protists from the genus Anaeramoeba.5 Using phylogenomic analysis, we show that these species represent a divergent, phylum-level lineage in the tree of metamonads, emerging as a sister group of the Parabasalia and reordering the deep branching order of the metamonad tree. Metabolic reconstructions of the Anaeramoeba MROs reveal many "classical" mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Our findings suggest that the cenancestor of Metamonada likely had MROs with more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.

Combined morphological and phylogenomic re-examination of malawimonads, a critical taxon for inferring the evolutionary history of eukaryotes.

Modern syntheses of eukaryote diversity assign almost all taxa to one of three groups: Amorphea, Diaphoretickes and Excavata (comprising Discoba and Metamonada). The most glaring exception is Malawimonadidae, a group of small heterotrophic flagellates that resemble Excavata by morphology, but branch with Amorphea in most phylogenomic analyses. However, just one malawimonad, Malawimonas jakobiformis, has been studied with both morphological and molecular-phylogenetic approaches, raising the spectre of interpretation errors and phylogenetic artefacts from low taxon sampling. We report a morphological and phylogenomic study of a new deep-branching malawimonad, Gefionella okellyi n. gen. n. sp. Electron microscopy revealed all canonical features of 'typical excavates', including flagellar vanes (as an opposed pair, unlike M. jakobiformis but like many metamonads) and a composite fibre. Initial phylogenomic analyses grouped malawimonads with the Amorphea-related orphan lineage Collodictyon, separate from a Metamonada+Discoba clade. However, support for this topology weakened when more sophisticated evolutionary models were used, and/or fast-evolving sites and long-branching taxa (FS/LB) were excluded. Analyses of '-FS/LB' datasets instead suggested a relationship between malawimonads and metamonads. The 'malawimonad+metamonad signal' in morphological and molecular data argues against a strict Metamonada+Discoba clade (i.e. the predominant concept of Excavata). A Metamonad+Discoba clade should therefore not be assumed when inferring deep-level evolutionary history in eukaryotes.

Giardia/giardiasis - a perspective on diagnostic and analytical tools.

Giardiasis is a gastrointestinal disease of humans and other animals caused by species of parasitic protists of the genus Giardia. This disease is transmitted mainly via the faecal-oral route (e.g., in water or food) and is of socioeconomic importance worldwide. The accurate detection and genetic characterisation of the different species and population variants (usually referred to as assemblages and/or sub-assemblages) of Giardia are central to understanding their transmission patterns and host spectra. The present article provides a background on Giardia and giardiasis, and reviews some key techniques employed for the identification and genetic characterisation of Giardia in biological samples, the diagnosis of infection and the analysis of genetic variation within and among species of Giardia. Advances in molecular techniques provide a solid basis for investigating the systematics, population genetics, ecology and epidemiology of Giardia species and genotypes as well as the prevention and control of giardiasis.