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- Describe characteristics associated with Rhizaria
The Rhizaria supergroup includes many of the amoebas, most of which have threadlike or needle-like pseudopodia. Pseudopodia function to trap and engulf food particles and to direct movement in rhizarian protists. These pseudopods project outward from anywhere on the cell surface and can anchor to a substrate. The protist then transports its cytoplasm into the pseudopod, thereby moving the entire cell. This type of motion, called cytoplasmic streaming, is used by several diverse groups of protists as a means of locomotion or as a method to distribute nutrients and oxygen.
Foraminiferans, or forams, are unicellular heterotrophic protists, ranging from approximately 20 micrometers to several centimeters in length; they occasionally resemble tiny snails. As a group, the forams exhibit porous shells, called tests, that are built from various organic materials and typically hardened with calcium carbonate. The tests may house photosynthetic algae, which the forams can harvest for nutrition. Foram pseudopodia extend through the pores and allow the forams to move, feed, and gather additional building materials. Foraminiferans are also useful as indicators of pollution and changes in global weather patterns.
The life-cycle involves an alternation between haploid and diploid phases. The haploid phase initially has a single nucleus, and divides to produce gametes with two flagella. The diploid phase is multinucleate, and after meiosis fragments to produce new organisms. The benthic forms has multiple rounds of asexual reproduction between sexual generations.
A second subtype of Rhizaria, the radiolarians, exhibit intricate exteriors of glassy silica with radial or bilateral symmetry. Radiolarians display needle-like pseudopods that are supported by microtubules which radiate outward from the cell bodies of these protists and function to catch food particles. The shells of dead radiolarians sink to the ocean floor, where they may accumulate in 100 meter-thick depths. Preserved, sedimented radiolarians are very common in the fossil record.
- The needle-like pseudopodia are used to carry out a process called cytoplasmic streaming which is a means of locomotion or distributing nutrients and oxygen.
- Two major subclassifications of Rhizaria include Forams and Radiolarians.
- Forams are characterized as unicellular heterotrophic protists that have porous shells, referred to as tests, which can contain photosynthetic algae that the foram can use as a nutrient source.
- Radiolarians are characterized by a glassy silica exterior that displays either bilateral or radial symmetry.
- pseudopodia: temporary projections of eukaryotic cells
- test: the external calciferous shell of a foram
Chromista is a biological kingdom consisting of single-celled and multicellular eukaryotic species that share similar features in their photosynthetic organelles (plastids).  It includes all protists whose plastids contain chlorophyll c, such as some algae, diatoms, oomycetes, and protozoans. It is probably a polyphyletic group whose members independently arose as a separate evolutionary group from the common ancestor of all eukaryotes.  As it is assumed the last common ancestor already possessed chloroplasts of red algal origin, the non-photosynthetic forms evolved from ancestors able to perform photosynthesis. Their plastids are surrounded by four membranes, and are believed to have been acquired from some red algae.
Chromista as a biological kingdom was created by British biologist Thomas Cavalier-Smith in 1981 to differentiate some protists from typical protozoans and plants.  According to Cavalier-Smith, the kingdom originally included only algae, but his later analysis indicated that many protozoans also belong to the new group. As of 2018, the kingdom is as diverse as kingdoms Plantae and Animalia, consisting of eight phyla. Notable members include marine algae, potato blight, dinoflagellates, Paramecium, brain parasite (Toxoplasma) and malarial parasite (Plasmodium). 
Single Cell Transcriptomics, Mega-Phylogeny, and the Genetic Basis of Morphological Innovations in Rhizaria
The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport, and cytokinesis, and is largely responsible for the diversity of morphologies among eukaryotes. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton, we provide the first single cell transcriptomes from uncultured, free-living unicellular eukaryotes: the polycystine radiolarian Lithomelissa setosa (Nassellaria) and Sticholonche zanclea (Taxopodida). A phylogenomic approach using 255 genes finds Radiolaria and Foraminifera as separate monophyletic groups (together as Retaria), while Cercozoa is shown to be paraphyletic where Endomyxa is sister to Retaria. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these on the revised phylogeny of Rhizaria reveal lineage-specific gene duplications and neofunctionalization of α and β tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution in uncultured protist species.
Keywords: Radiolaria Rhizaria SAR cytoskeleton phylogeny protists single-cell transcriptomics.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
The phytomyxean P. brassicae and S. subterranea are parasites of the plant genera Brassica and Solanum, respectively, and the in vitro callus samples were prepared according to an unpublished protocol (Bulman et al. submitted). Consequently, an unknown amount of plant contamination was expected in the phytomyxean ESTs. An initial blast examination showed that many of the phytomyxid-callus contigs had high similarity to plant sequences and were thus possibly derived from the host cells. We took advantage of the large amount of data available for Brassica and Solanum to filter out these plant sequences and simplify data searching for constructing the single-gene alignments (see Methods).
A total of 167 gene alignments with at least one rhizarian species represented in each were constructed for phylogenetic analyses. Based on recently published results suggesting close evolutionary affinities between Rhizaria, stramenopiles and alveolates, forming the so-called SAR group [3, 5, 6], representatives for these 3 groups were included. The full dataset comprised 10 rhizarians, 9 stramenopiles, and 9 alveolates. In order to reduce the risks of artifacts, 11 green plant taxa were chosen to root our phylogenies because 1) of the availability of complete genomes for many lineages, thus considerably reducing the amount of missing data in the outgroup 2) they have evolved more slowly comparatively to most of the SAR species and 3) their relative evolutionary proximity to the SAR group in the tree of eukaryotes . However, an alternative outgroup, haptophytes, was also tested as it was proposed to be more closely related to the SAR group (data not shown) . We did not select it for the final analyses because only medium-sized EST datasets are available for a limited number of species, except for one complete genome (Emiliana huxleyi), and the intra-Rhizaria relationships remained identical to the trees rooted using the green plants (see below). Each single-gene dataset was thoroughly tested by bootstrapped maximum likelihood (ML) analyses for deep paralogy or suspicious relationships possibly indicative of lateral gene transfer (LGT) or contamination. The acanthareans are known to harbor zooxanthellae symbionts and polycystine radiolarians are hosts of prasinophytes, dinoflagellates and other alveolates . Accordingly, non-acantharean sequences were expected to be found. Out of the 167 selected genes, we could identify 1 sequence related to haptophytes in Astrolonche and Phyllostaurus, 2 sequences of dinoflagellate origin in Astrolonche, 5 and 2 sequences of general plant affinity in Astrolonche and Phyllostaurus, respectively, and, surprisingly, 25 sequences in Astrolonche and 21 sequences in Phyllostaurus clearly belonging to streptophytes (angiosperms). It is not clear to us why streptophyte sequences were present in our acantharean dataset, but one possible explanation could be that the samples were contaminated with a small amount of pollen. All these contaminant sequences were removed from our alignments. The curated protein alignments were concatenated into a supermatrix amounting to 36,735 unambiguously aligned amino acid positions (global percentage of missing data: 40% see Additional file 1 for details) that was subjected to phylogenetic analyses.
Phylogenetic analyses of the supermatrix
We analyzed our concatenated alignment using probabilistic methods of tree reconstruction with (i) empirical site-homogeneous models of sequence evolution in ML (LG model) and Bayesian (WAG model) frameworks and (ii) site-heterogeneous mixture model in a Bayesian framework (CAT model). Figures 1 and 2 depict the relationships inferred from these analyses. The "LG" and "WAG" trees received maximal bootstrap support proportions (BP) and posterior probabilities (PP), respectively, for nearly all nodes (Figure 1). As expected, the 3 major groups included in this study, i.e. rhizarians, stramenopiles and alveolates, were strongly recovered, and the relationships between and within them corresponded to previously published trees [4, 6, 7]. All 3 analyses robustly supported the monophyly of the four rhizarian phyla: Foraminifera, Acantharea, Phytomyxea and Cercozoa. Notably, Foraminifera were placed as a sister group to Acantharea with 100% BP ("LG" and "CAT") and 1.0 PP ("WAG" and "CAT"). The branching order within Rhizaria was identical and highly supported in the "LG" and WAG" trees, with the Foraminifera + Acantharea clade being sister to Cercozoa and a group composed of Phytomyxea and Gromia (Figure 1). On the other hand, the site-heterogeneous CAT model inferred a different topology that received low to high PP and BP, recovering the Foraminifera + Acantharea group in an internal position, sister to Gromia (0.93 PP 90% BP) and more closely related to Phytomyxea (0.51 PP 50% BP) to the exclusion of Cercozoa (Figure 2). Because the discrepancies between the "LG" and "CAT" topologies are an indication that some relationships may be artifactual, we estimated the fit of these 2 models based on a cross-validation test (see Methods). The "CAT" model was found to much better fit the data than the "LG" model with a score averaged over 10 replicates of 1547 ± 71 (all replicates favoured the "CAT" model), indicating that the topology in Figure 1 is likely the results of biases not correctly handled by the site-homogeneous models (LG and WAG).
Phylogeny of SAR as inferred by RAxML and the LG model of evolution. The tree was rooted using the green plants. Species with new genomic data generated in this study are in bold. An identical topology was also recovered using MrBayes and the WAG model of evolution. Black dots correspond to 100% ML bootstrap support (BP) and 1.0 Bayesian posterior probabilities (PP). Numbers at nodes represent BP (above) and PP (below) when not maximal. The area of the yellow circles are proportional to the number of genes included in the supermatrix for each taxon. The scale bar represents the estimated number of amino acid substitutions per site.
Phylogeny of SAR as inferred by PhyloBayes and the CAT model of evolution. Consensus tree between 2 independent Markov chains, rooted with green plants. Species with new genomic data generated in this study are in bold. Black dots correspond to 1.0 PP and 100% BP and values at nodes PP and BP when not maximal. The area of the yellow circles are proportional to the number of genes included in the supermatrix for each taxon. The scale bar represents the estimated number of amino acid substitutions per site.
To better evaluate these differences, a topology comparison analysis using the approximately unbiased (AU) test was performed . Both trees in figure 1 (P = 0.916) and in figure 2 (P = 0.084) were not rejected at the 5% significance level. This test was based on the comparison of trees obtained with 2 non-nested models, "LG" (Figure 1) and "CAT" (Figure 2), using the "LG" empirical matrix. Hence, if the topology in Figure 2 had been rejected, it would not have been very informative because the "CAT" model could still have inferred the true tree. In the present case, however, the LG-based AU test kept the "CAT" tree among the trees possibly correctly describing the relationships within Rhizaria, thus strengthening the branching pattern showed in Figure 2. In addition, a topology with Acantharea alone in a sister position to the rest of Rhizaria was also tested in order to estimate the likelihood of the basal branching of Radiolaria seen in some SSU trees (see  for a discussion). This topology was strongly rejected (P = 7e-09), further supporting the association of Foraminifera and Radiolaria.
Evaluating the branching order within Rhizaria
In our trees, both foraminiferans and acanthareans appeared as fast-evolving taxa. This raised a concern about their potentially erroneous grouping due to the long branch attraction (LBA) artifact  that would affect not only the position of these diverging lineages but also the relationships among all rhizarian groups. To evaluate for the possibility of LBA, we first conducted a fast-evolving taxa removal experience in which, in turn, the most diverging foraminiferan representative Reticulomyxa filosa (Figure 3), both foraminiferan species (Figure 4), and the acanthareans (Figure 5) were discarded. The removal of R. filosa had no impact on the sister relationship of foraminiferans and acanthareans: both groups remained monophyletic with maximum support. However, this slightly different taxon sampling largely affected the branching order among the rhizarian groups. The "LG" model robustly placed Gromia as the most closely related lineage to the Foraminifera + Acantharea group (93% BP), and Phytomyxea were recovered as sister to this assemblage with 87% BP (Figure 3A). The "CAT" model inferred the same topology (Figure 3B), which also corresponded to the full tree inferred with this model (Figure 2) but, interestingly, the support values increased from 0.92 to 1.0 PP and from 0.51 to 0.92 PP for the node joining Foraminifera + Acantharea + Gromia and the node uniting Phytomyxea to this group, respectively. Similarly, when Foraminifera were removed altogether, both models again recovered the "CAT" topology (Figure 2) with high BP and PP values, exactly as in absence of Reticulomyxa only (Figure 3). Finally, discarding Acantharea led in both "LG" and "CAT" analyses to the basal position of Foraminifera (98% BP 0.73 PP) and the sister position of Gromia to Phytomyxea (100% BP 0.9 PP), as observed in the complete "LG" tree (Figure 5 and Figure 1).
Phylogeny of SAR without Reticulomyxa. RAxML with "LG" model (A) and PhyloBayes with "CAT" model (B) phylogenies of the SAR group, rooted with the green plants. The foraminiferan species Reticulomyxa filosa was removed from the alignment for inferring these trees. Black dots correspond to 100% ML bootstrap support (BP) in (A) and 1.0 Bayesian posterior probabilities (PP) in (B). Numbers at nodes represent BP (A) or PP (B) when not maximal. The scale bar represents the estimated number of amino acid substitutions per site.
Phylogeny of SAR without Foraminifera. RAxML with "LG" model (A) and PhyloBayes with "CAT" model (B) phylogenies of the SAR group, rooted with the green plants. Both foraminiferan taxa Reticulomyxa filosa and Quinqueloculina sp. were removed from the alignment for inferring these trees. Black dots correspond to 100% ML bootstrap support (BP) in (A) and 1.0 Bayesian posterior probabilities (PP) in (B). Numbers at nodes represent BP (A) or PP (B) when not maximal. The scale bar represents the estimated number of amino acid substitutions per site.
Phylogeny of SAR without Acantharea. RAxML with "LG" model (A) and PhyloBayes with "CAT" model (B) phylogenies of the SAR group, rooted with the green plants. Both acantharean taxa Astrolonche sp. and Phyllostaurus sp. were removed from the alignment for inferring these trees. Black dots correspond to 100% ML bootstrap support (BP) in (A) and 1.0 Bayesian posterior probabilities (PP) in (B). Numbers at nodes represent BP (A) or PP (B) when not maximal. The scale bar represents the estimated number of amino acid substitutions per site.
To assess the robustness of the Foraminifera-Acantharea clade and to further investigate the two competing topologies for intra-Rhizaria relationships (Figure 1 and 2), we then conducted a site removal analysis in which the fastest-evolving sites were progressively removed from the original alignment. The rationale behind this analysis is that fast-evolving sites are more likely to be saturated and not correctly interpreted as convergence by tree reconstruction methods, thus strongly influencing the potential artifactual grouping of highly diverging lineages . Specifically, we tested 14 shorter alignments ranging from 35,230 aa to 14,281 aa and reconstructed phylogenetic trees with LG and CAT models at each step to determine the support value for several nodes of interest (Figure 6). First, the highly supported association between Foraminifera and Acantharea was not affected by the removal of fast-evolving sites, with almost no decrease in bootstrap values even for the smallest number of positions remaining in the alignment. This result provides additional evidence that the grouping of Foraminifera and Acantharea is not caused by artifacts of tree reconstruction. Second, we monitored the bootstrap supports for the sister position of Gromia with respect to Phytomyxea, the basal position of the Foraminifera-Acantharea clade (as observed in the "LG" tree, Figure 1), as well as the alternatives: the sister grouping of Gromia to the Foraminifera-Acantharea group, and the basal position of Cercozoa (as observed in the "CAT" tree, Figure 2). Interestingly, as the fast-evolving sites were removed, the bootstrap values for the phylogenetic relationships obtained in the LG-based analysis of the complete dataset decreased (Figure 6, blue line) and, at the same time, the branching order supported by the CAT-based reconstruction gained statistical significance (Figure 6, red line). When 13'379 fast-evolving positions were removed, the LG-based analysis converged with high support (94% BP 0.99 PP) towards the topology that was weakly suggested by the CAT-based analysis of the complete dataset for the association of Phytomyxea, Gromia, Foraminifera and Acantharea, before diverging likely due to lack of phylogenetic signal in the shortest alignments. The position of Gromia remained more ambiguous throughout the removal process and, although the support for the association with Phytomyxea rapidly decreased to below 50% BP, its sisterhood to the Foraminifera-Acantharea clade suggested by the "CAT" model did not gain significance.
Site removal analysis. Figures (A) and (B) illustrate the bipartitions that were sought in the pool of trees generated by bootstrapped ML reconstructions, corresponding to the "LG" (blue) and "CAT" (red) relationships, respectively. The monitored relationships are indicated as followed: star: Foraminifera-Acantharea grouping square: Gromia sister to Phytomyxea diamond: basal position of the Foraminifera-Acantharea clade within Rhizaria cross: Gromia sister to the Foraminifera-Acantharea clade circle: basal position of Cercozoa to the rest of rhizarian lineages. (C) Dependence of the bootstrap support values (BP) for the monitored relationships on the number of removed fast-evolving sites, marked for each of the 14 shorter alignments. The blue and red lines correspond to the BP of nodes found in the "LG" and "CAT" trees, respectively. The black line corresponds to the BP for the Foraminifera-Acantharea grouping. The vertical dashed line shows the step (13'379 positions removed) where the supports for the sister position of Retaria reached a minimum and the support for the sister position of Cercozoa a maximum. Numbers next to the marks are PP obtained with the "CAT" model (PhyloBayes), resulting from the pooling of all trees after burnin of 2 independent chains and corresponding to the bifurcations found. At removal steps 1, 2, 4 and 5 only one PP value is shown next to the cross mark, indicating that the CAT model could not infer the position of Phytomyxea within Rhizaria (multifurcation). The y-axis represents the BP and the x-axis the length of the alignments after the removal of sites.
Although our multigene analysis represents the broadest rhizarian sampling to date, three important rhizarian groups, Haplosporidia, Filoreta, and Polycystinea, are still missing. Therefore, we performed a separate phylogenetic analysis based on actin, the only protein-coding gene sequenced in all rhizarian groups. ML and Bayesian analyses of our alignment (317 amino acid positions), containing 73 rhizarians and 6 stramenopiles as outgroup, indicated that the acantharean Astrolonche possesses 2 actin paralogues branching as sister groups to 2 of the actin paralogues present in Foraminifera (Figure 7). The only actin sequence found in Phyllostaurus grouped with Astrolonche as sister to the foraminiferal paralogue 2. Sister to this clade were two previously obtained actin sequences of the polycystinean radiolarians Thalassicolla pellucida and Collozoum inerme, and their grouping with Acantharea and Foraminifera was strongly supported in Bayesian inferences (0.99 and 1.0 with PhyloBayes and MrBayes, respectively) but not supported in ML (31% BP). However, the relationships between these 3 groups remained unresolved, leaving open the question of a possible radiolarian monophyly. For both paralogues, Haplosporidia appeared as sister to the Foraminifera + Radiolaria clade, albeit without much support.
Actin phylogeny of Rhizaria. ML phylogeny of Rhizaria based on actin, rooted with stramenopiles as outgroup. Numbers at nodes represent the bootstrap values obtained with RAxML ("LG" model) and the posterior probabilities obtained with PhyloBayes ("LG" model) and MrBayes ("WAG" model). For clarity, only the values for the deep nodes and the nodes of interest for this study are shown The scale bar represents the estimated number of amino acid substitutions per site. The branches leading to Acantharea and Foraminifera in actin paralogs 1 and 2 are in bold.
In addition to the multigene and actin analyses, we screened our newly generated data for the presence of molecular signatures characteristic of Rhizaria. First, polyubiquitin sequences were searched for the 1 or 2 amino acid insertion previously described at the monomer-monomer junction in all Rhizaria except in Radiolaria [17, 22]. We found threonine (T) in 4 sequences of Astrolonche and one sequence of Phyllostaurus and alanine (A) in 6 sequences of Phyllostaurus (Figure 8A). The presence of 2 different amino acids in Phyllostaurus was surprising, but this is not exceptional as it has already been observed in Lotharella amoeboformis (AY099125) where both A and S insertions have been found . A serine (S) was also found in the Gromia sphaerica sequence, which was identical to the available polyubiquitin of Gromia oviformis (AY571670). In addition, a new polyubiquitin sequence amplified from the phagomyxid Maullinia ectocarpii was included.
Specific insertions in Rhizaria. Rhizarian specific insertions of (A) 1-2 residues between monomers in polyubiquitin and (B) 2 residues at position 103 in the 60S ribosomal protein L10a. Numbers above the alignment shows the sequence position in the Mus protein. Species names in bold indicate new sequences generated in this study.
Interestingly, we identified a new insertion of 2 and 4 amino acids in the 60S ribosomal protein L10a, a characteristic also apparently unique to Rhizaria. A phenylalanine (F), an asparagine (N), and a serine (S) followed by a lysine (K) were inserted at position 104 in G. sphaerica, R. filosa, B. natans, and Paracercomonas sp., respectively (Figure 8B). In G. sphaerica, the sequence contained 2 additional inserted amino acids, i.e. a valine (V) and a glycine (G). Unfortunately, this gene was not present in the acantharean dataset and several attempts to amplify it by PCR failed. Blast searches against GenBank-nr and dbEST revealed no other known rpl10a gene containing this insertion.
On the phenology of protists: Recurrent patterns reveal seasonal variation of protistan (Rhizaria: Cercozoa, Endomyxa) communities in tree canopies
Tree canopies are colonized by billions of highly specialized microorganisms that are well adapted to the extreme microclimatic conditions, caused by diurnal fluctuations and seasonal changes. In this study we investigated seasonality patterns of protists in tree canopies of a temperate floodplain forest via high-throughput sequencing with group-specific primers for the phyla Cercozoa and Endomyxa. We observed consistent seasonality and identified divergent spring and autumn taxa. Tree crowns were characterized by a dominance of bacterivores and omnivores, while eukaryvores gained a distinctly larger share in litter and soil communities on the ground. Seasonality was largest among communities detected on the foliar surface. Higher variance within alpha diversity of foliar communities in spring indicated greater heterogeneity during community assembly. However, communities underwent distinct changes during the aging of leaves in autumn, reflecting recurring phenological changes during microbial colonization of leaves. Surprisingly, endomyxan root pathogens appeared to be exceptionally abundant across tree canopies during autumn season, demonstrating a potential role of the canopy surface as an important reservoir for wind-dispersed propagules. Overall, about 80% of detected OTUs could not be assigned to known species – representing only a fraction of dozens of microeukaryotic taxa whose canopy inhabitants are waiting to be discovered.
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 an order of naked amoebae of considerable genetic diversity. Three families have been well defined (Vampyrellidae, Leptophryidae, Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterised. 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 known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behaviour 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'.
Keywords: Algae Diatomeae Endomyxa amoebae diatoms vampyrellids.