Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria.

BACKGROUND: Ascetosporea (Endomyxa, Rhizaria) is a group of unicellular parasites infecting aquatic invertebrates. They are increasingly being recognized as widespread and important in marine environments, causing large annual losses in invertebrate aquaculture. Despite their importance, little molecular data of Ascetosporea exist, with only two genome assemblies published to date. Accordingly, the evolutionary origin of these parasites is unclear, including their phylogenetic position and the genomic adaptations that accompanied the transition from a free-living lifestyle to parasitism. Here, we sequenced and assembled three new ascetosporean genomes, as well as the genome of a closely related amphizoic species, to investigate the phylogeny, origin, and genomic adaptations to parasitism in Ascetosporea. RESULTS: Using a phylogenomic approach, we confirm the monophyly of Ascetosporea and show that Paramyxida group with Mikrocytida, with Haplosporida being sister to both groups. We report that the genomes of these parasites are relatively small (12-36 Mb) and gene-sparse (~ 2300-5200 genes), while containing surprisingly high amounts of non-coding sequence (~ 70-90% of the genomes). Performing gene-tree aware ancestral reconstruction of gene families, we demonstrate extensive gene losses at the origin of parasitism in Ascetosporea, primarily of metabolic functions, and little gene gain except on terminal branches. Finally, we highlight some functional gene classes that have undergone expansions during evolution of the group. CONCLUSIONS: We present important new genomic information from a lineage of enigmatic but important parasites of invertebrates and illuminate some of the genomic innovations accompanying the evolutionary transition to parasitism in this lineage. Our results and data provide a genetic basis for the development of control measures against these parasites.

Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden.

Freshwater bivalves play key ecological roles in lakes and rivers, largely contributing to healthy ecosystems. The freshwater pearl mussel, Margaritifera margaritifera, is found in Europe and on the East coast of North America. Once common in oxygenated streams, M. margaritifera is rapidly declining and consequently assessed as a threatened species worldwide. Deterioration of water quality has been considered the main factor for the mass mortality events affecting this species. Yet, the role of parasitic infections has not been investigated. Here, we report the discovery of three novel protist lineages found in Swedish populations of M. margaritifera belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa). These lineages are closely related-but clearly separated-from the tadpole parasite Nematopsis temporariae. In one lineage, which is specifically associated with mortality events of M. margaritifera, we found cysts containing single vermiform zoites in the gills and other organs of diseased individuals using microscopy and in situ hybridization. This represents the first report of a parasitic infection in M. margaritifera that may be linked to the decline of this mussel species. We propose a tentative life cycle with the distribution of different developmental stages and potential exit from the host into the environment.

A novel kleptoplastidic symbiosis revealed in the marine centrohelid Meringosphaera with evidence of genetic integration.

Plastid symbioses between heterotrophic hosts and algae are widespread and abundant in surface oceans. They are critically important both for extant ecological systems and for understanding the evolution of plastids. Kleptoplastidy, where the plastids of prey are temporarily retained and continuously re-acquired, provides opportunities to study the transitional states of plastid establishment. Here, we investigated the poorly studied marine centrohelid Meringosphaera and its previously unidentified symbionts using culture-independent methods from environmental samples. Investigations of the 18S rDNA from single-cell assembled genomes (SAGs) revealed uncharacterized genetic diversity within Meringosphaera that likely represents multiple species. We found that Meringosphaera harbors plastids of Dictyochophyceae origin (stramenopiles), for which we recovered six full plastid genomes and found evidence of two distinct subgroups that are congruent with host identity. Environmental monitoring by qPCR and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) revealed seasonal dynamics of both host and plastid. In particular, we did not detect the plastids for 6 months of the year, which, combined with the lack of plastids in some SAGs, suggests that the plastids are temporary and the relationship is kleptoplastidic. Importantly, we found evidence of genetic integration of the kleptoplasts as we identified host-encoded plastid-associated genes, with evolutionary origins likely from the plastid source as well as from other alga sources. This is only the second case where host-encoded kleptoplast-targeted genes have been predicted in an ancestrally plastid-lacking group. Our results provide evidence for gene transfers and protein re-targeting as relatively early events in the evolution of plastid symbioses.

Contrasting outcomes of genome reduction in mikrocytids and microsporidians.

BACKGROUND: Intracellular symbionts often undergo genome reduction, losing both coding and non-coding DNA in a process that ultimately produces small, gene-dense genomes with few genes. Among eukaryotes, an extreme example is found in microsporidians, which are anaerobic, obligate intracellular parasites related to fungi that have the smallest nuclear genomes known (except for the relic nucleomorphs of some secondary plastids). Mikrocytids are superficially similar to microsporidians: they are also small, reduced, obligate parasites; however, as they belong to a very different branch of the tree of eukaryotes, the rhizarians, such similarities must have evolved in parallel. Since little genomic data are available from mikrocytids, we assembled a draft genome of the type species, Mikrocytos mackini, and compared the genomic architecture and content of microsporidians and mikrocytids to identify common characteristics of reduction and possible convergent evolution. RESULTS: At the coarsest level, the genome of M. mackini does not exhibit signs of extreme genome reduction; at 49.7 Mbp with 14,372 genes, the assembly is much larger and gene-rich than those of microsporidians. However, much of the genomic sequence and most (8075) of the protein-coding genes code for transposons, and may not contribute much of functional relevance to the parasite. Indeed, the energy and carbon metabolism of M. mackini share several similarities with those of microsporidians. Overall, the predicted proteome involved in cellular functions is quite reduced and gene sequences are extremely divergent. Microsporidians and mikrocytids also share highly reduced spliceosomes that have retained a strikingly similar subset of proteins despite having reduced independently. In contrast, the spliceosomal introns in mikrocytids are very different from those of microsporidians in that they are numerous, conserved in sequence, and constrained to an exceptionally narrow size range (all 16 or 17 nucleotides long) at the shortest extreme of known intron lengths. CONCLUSIONS: Nuclear genome reduction has taken place many times and has proceeded along different routes in different lineages. Mikrocytids show a mix of similarities and differences with other extreme cases, including uncoupling the actual size of a genome with its functional reduction.

A Mitosome With Distinct Metabolism in the Uncultured Protist Parasite Paramikrocytos canceri (Rhizaria, Ascetosporea).

Ascetosporea are endoparasites of marine invertebrates that include economically important pathogens of aquaculture species. Owing to their often-minuscule cell sizes, strict intracellular lifestyle, lack of cultured representatives and minimal availability of molecular data, these unicellular parasites remain poorly studied. Here, we sequenced and assembled the genome and transcriptome of Paramikrocytos canceri, an endoparasite isolated from the European edible crab Cancer pagurus. Using bioinformatic predictions, we show that P. canceri likely possesses a mitochondrion-related organelle (MRO) with highly reduced metabolism, resembling the mitosomes of other parasites but with key differences. Like other mitosomes, this MRO is predicted to have reduced metabolic capacity and lack an organellar genome and function in iron-sulfur cluster (ISC) pathway-mediated Fe-S cluster biosynthesis. However, the MRO in P. canceri is uniquely predicted to produce ATP via a partial glycolytic pathway and synthesize phospholipids de novo through the CDP-DAG pathway. Heterologous gene expression confirmed that proteins from the ISC and CDP-DAG pathways retain mitochondrial targeting sequences that are recognized by yeast mitochondria. This represents a unique combination of metabolic pathways in an MRO, including the first reported case of a mitosome-like organelle able to synthesize phospholipids de novo. Some of these phospholipids, such as phosphatidylserine, are vital in other protist endoparasites that invade their host through apoptotic mimicry.

Unravelling the Phylogeny of a Common Intestinal Protist: Intrageneric Diversity of Endolimax.

Endolimax nana is a common endobiont of the human intestine, but members of the genus have also been reported in non-human hosts and in non-intestinal organs. Limited information is available regarding the genetic diversity of Endolimax, which is necessary to delineate species, host specificity and potential differences in clinical impact on the host. Here, we used cloning of PCR products followed by Sanger sequencing and next-generation PacBio Sequencing to obtain Endolimax-related nuclear ribosomal gene sequences and undertook a phylogenetic analysis to gain additional insight into the taxonomy of Endolimax and related organisms. The new sequences confirmed that E. nana forms a discrete clade within the Archamoebae and is related to Endolimax piscium and Iodamoeba. However, we identified substantial sequence divergence within E. nana and evidence for two distinct clades, which we propose to name E. nana ribosomal lineage 1 and E. nana ribosomal lineage 2. Both of the sequencing approaches applied in the study helped us to improve our understanding of genetic diversity across Endolimax, and it is likely that wider application of next-generation sequencing technologies will facilitate the generation of Endolimax-related DNA sequence data and help complete our understanding of its phylogenetic position and intrageneric diversity.

Global patterns and rates of habitat transitions across the eukaryotic tree of life.

The successful colonization of new habitats has played a fundamental role during the evolution of life. Salinity is one of the strongest barriers for organisms to cross, which has resulted in the evolution of distinct marine and non-marine (including both freshwater and soil) communities. Although microbes represent by far the vast majority of eukaryote diversity, the role of the salt barrier in shaping the diversity across the eukaryotic tree is poorly known. Traditional views suggest rare and ancient marine/non-marine transitions but this view is being challenged by the discovery of several recently transitioned lineages. Here, we investigate habitat evolution across the tree of eukaryotes using a unique set of taxon-rich phylogenies inferred from a combination of long-read and short-read environmental metabarcoding data spanning the ribosomal DNA operon. Our results show that, overall, marine and non-marine microbial communities are phylogenetically distinct but transitions have occurred in both directions in almost all major eukaryotic lineages, with hundreds of transition events detected. Some groups have experienced relatively high rates of transitions, most notably fungi for which crossing the salt barrier has probably been an important aspect of their successful diversification. At the deepest phylogenetic levels, ancestral habitat reconstruction analyses suggest that eukaryotes may have first evolved in non-marine habitats and that the two largest known eukaryotic assemblages (TSAR and Amorphea) arose in different habitats. Overall, our findings indicate that the salt barrier has played an important role during eukaryote evolution and provide a global perspective on habitat transitions in this domain of life.

Photophysiological response of Symbiodiniaceae single cells to temperature stress.

Photosynthetic dinoflagellates in the family Symbiodiniaceae engage in symbiosis with scleractinian corals. As coral 'bleaching' is partly governed by the thermal sensitivity of different Symbiodiniaceae lineages, numerous studies have investigated their temperature sensitivity. However, the systematic identification of single-cells with increased temperature resistance among these dinoflagellates has remained inaccessible, mostly due to a lack of technologies operating at the microscale. Here, we employed a unique combination of microfluidics, miniaturized temperature control, and chlorophyll fluorometry to characterize the single-cell heterogeneity among five representative species within the Symbiodiniaceae family under temperature stress. We monitored single-cell maximum quantum yields (Fv/Fm) of photosystem (PS) II under increasing temperature stress (22‒39 °C, + 1 °C every 15 min), and detected a significant Fv/Fm reduction at lineage-specific temperatures ranging from 28 °C to 34 °C alongside a 40- to 180- fold increase in intraspecific heterogeneity under elevated temperatures (>31 °C). We discovered that the initial Fv/Fm of a cell could predict the same cell's ability to perform PSII photochemistry under moderate temperature stress (<32 °C), suggesting its use as a proxy for measuring the thermal sensitivity among Symbiodiniaceae. In combination, our study highlights the heterogeneous thermal sensitivity among photosynthetic Symbiodiniaceae and adds critical resolution to our understanding of temperature-induced coral bleaching.

On the origin of TSAR: morphology, diversity and phylogeny of Telonemia.

Telonemia is a poorly known major phylum of flagellated eukaryotes with a unique combination of morphological traits. Phylogenomics recently revealed the phylogenetic position of telonemids as sister to SAR, one of the largest groups of eukaryotes, comprising Stramenopiles, Alveolata and Rhizaria. Due to this key evolutionary position, investigations of telonemids are of critical importance for elucidating the origin and diversification of an astounding diversity of eukaryotic forms and life strategies. To date, however, only two species have been morphologically characterized from Telonemia, which do not represent this genetically very diverse group. In this study, we established cultures for six new telonemid strains, including the description of five new species and a new genus. We used these cultures to update the phylogeny of Telonemia and provide a detailed morphological and ultrastructural investigation. Our data elucidate the origin of TSAR from flagellates with complex morphology and reconstruction of the ancestral structure of stramenopiles, alveolates and rhizarians, and their main synapomorphic characters. Since telonemids are a common component of aquatic environments, the features of their feeding, behaviour and ecological preferences observed in clonal cultures and the results of global metabarcoding analysis contribute to a deeper understanding of organization of microbial food webs.

Single cell genomics reveals plastid-lacking Picozoa are close relatives of red algae.

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids.

Diversity and ecology of protists revealed by metabarcoding.

Protists are the dominant eukaryotes in the biosphere where they play key functional roles. While protists have been studied for over a century, it is the high-throughput sequencing of molecular markers from environmental samples - the approach of metabarcoding - that has revealed just how diverse, and abundant, these small organisms are. Metabarcoding is now routine to survey environmental diversity, so data have rapidly accumulated from a multitude of environments and at different sampling scales. This mass of data has provided unprecedented opportunities to study the taxonomic and functional diversity of protists, and how this diversity is organised in space and time. Here, we use metabarcoding as a common thread to discuss the state of knowledge in protist diversity research, from technical considerations of the approach to important insights gained on diversity patterns and the processes that might have structured this diversity. In addition to these insights, we conclude that metabarcoding is on the verge of an exciting added dimension thanks to the maturation of high-throughput long-read sequencing, so that a robust eco-evolutionary framework of protist diversity is within reach.

PhyloFisher: A phylogenomic package for resolving eukaryotic relationships.

Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic "single-copy orthogroup" datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset.

The long-time orphan protist Meringosphaera mediterranea Lohmann, 1902 [1903] is a centrohelid heliozoan.

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophyte-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exist so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the west coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 µm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S V9 metabarcoding dataset, which revealed four OTUs with 94.8%-98.2% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily.

Phylogenomic Insights into the Origin of Primary Plastids.

The origin of plastids was a major evolutionary event that paved the way for an astonishing diversification of photosynthetic eukaryotes. Plastids originated by endosymbiosis between a heterotrophic eukaryotic host and cyanobacteria, presumably in a common ancestor of the primary photosynthetic eukaryotes (Archaeplastida). A single origin of primary plastids is well supported by plastid evidence but not by nuclear phylogenomic analyses, which have consistently failed to recover the monophyly of Archaeplastida hosts. Importantly, plastid monophyly and nonmonophyletic hosts could be explained under scenarios of independent or serial eukaryote-to-eukaryote endosymbioses. Here, we assessed the strength of the signal for the monophyly of Archaeplastida hosts in four available phylogenomic data sets. The effect of phylogenetic methodology, data quality, alignment trimming strategy, gene and taxon sampling, and the presence of outlier genes were investigated. Our analyses revealed a lack of support for host monophyly in the shorter individual data sets. However, when analyzed together under rigorous data curation and complex mixture models, the combined nuclear data sets supported the monophyly of primary photosynthetic eukaryotes (Archaeplastida) and recovered a putative association with plastid-lacking Picozoa. This study represents an important step toward better understanding deep eukaryotic evolution and the origin of plastids. [Archaeplastida; Bayesian; chloroplast; maximum likelihood; mixture model; ortholog; outlier loci; paralog; protist.].

A molecular timescale for eukaryote evolution with implications for the origin of red algal-derived plastids.

In modern oceans, eukaryotic phytoplankton is dominated by lineages with red algal-derived plastids such as diatoms, dinoflagellates, and coccolithophores. Despite the ecological importance of these groups and many others representing a huge diversity of forms and lifestyles, we still lack a comprehensive understanding of their evolution and how they obtained their plastids. New hypotheses have emerged to explain the acquisition of red algal-derived plastids by serial endosymbiosis, but the chronology of these putative independent plastid acquisitions remains untested. Here, we establish a timeframe for the origin of red algal-derived plastids under scenarios of serial endosymbiosis, using Bayesian molecular clock analyses applied on a phylogenomic dataset with broad sampling of eukaryote diversity. We find that the hypotheses of serial endosymbiosis are chronologically possible, as the stem lineages of all red plastid-containing groups overlap in time. This period in the Meso- and Neoproterozoic Eras set the stage for the later expansion to dominance of red algal-derived primary production in the contemporary oceans, which profoundly altered the global geochemical and ecological conditions of the Earth.

Automated Removal of Non-homologous Sequence Stretches with PREQUAL.

Large-scale multigene datasets used in phylogenomics and comparative genomics often contain sequence errors inherited from source genomes and transcriptomes. These errors typically manifest as stretches of non-homologous characters and derive from sequencing, assembly, and/or annotation errors. The lack of automatic tools to detect and remove sequence errors leads to the propagation of these errors in large-scale datasets. PREQUAL is a command line tool that identifies and masks regions with non-homologous adjacent characters in sets of unaligned homologous sequences. PREQUAL uses a full probabilistic approach based on pair hidden Markov models. On the front end, PREQUAL is user-friendly and simple to use while also allowing full customization to adjust filtering sensitivity. It is primarily aimed at amino acid sequences but can handle protein-coding nucleotide sequences. PREQUAL is computationally efficient and shows high sensitivity and accuracy. In this chapter, we briefly introduce the motivation for PREQUAL and its underlying methodology, followed by a description of basic and advanced usage, and conclude with some notes and recommendations. PREQUAL fills an important gap in the current bioinformatics tool kit for phylogenomics, contributing toward increased accuracy and reproducibility in future studies.

Perspectives from Ten Years of Protist Studies by High-Throughput Metabarcoding.

During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding.

Predatory colponemids are the sister group to all other alveolates.

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which support their monophyly and position as the sister lineage to all other known alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but they are diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates and the origin of myzocytosis and plastids.

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.