Transfer of the Thecate Amoeba Lecythium mutabilis to a Novel Genus Omnivora (Fiscullidae, Thecofilosea, Cercozoa).

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. As attempts to establish a culture of Lecythium mutabilis repeatedly failed, it was not yet investigated by molecular means. We isolated single cells of L. mutabilis directly from their habitat and successfully sequenced the V4 region of their SSU rDNA. Phylogenetic analyses showed that L. mutabilis is not directly related to the genus Lecythium and instead branches within the Fiscullidae (Tectofilosida, Thecofilosea). Accordingly, we transfer the species L. mutabilis to a novel genus Omnivora gen. nov.

Marteilia cochillia is released into seawater via cockle Cerastoderma edule faeces.

Outbreaks of Marteilia cochillia have caused massive mortalities of common cockle, Cerastoderma edule, in some natural beds in Galicia (NW Spain) since 2012. The life cycle of Marteilia spp. is still unresolved and the most accepted hypothesis suggests that an additional host is involved. Researchers have assumed that sporangia are shed into the environment in the faeces, but details about this process have not been reported previously. Here, we report the massive liberation of Marteilia cochillia sporangia through the exhalant siphon into the environment, packaged as faeces. Using light microscopy observations on fresh samples, imprints and histology, we also describe a thick (ca. 5 µm) transparent envelope covering the sporangia that has not been reported previously. The massive release of encapsulated sporangia reported here ensures that millions of infective stages of M. cochillia cycle through the environment and become available for infection. The elucidation of the role played by the sporangia envelope would be of utmost importance for the understanding M. cochillia life cycle.

Mikrocytos mytilicoli n.sp. (Cercozoa, Mikrocytida, Mikrocytiidae) infecting the copepod Mytilicola intestinalis (Arthropoda, Cyclopoida, Mytilicolidae), a symbiont of Mytilus galloprovincialis in Galicia (NW Spain).

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host.

Gazelletta kashiwaensis sp. nov. (Medusettidae, Phaeodaria, Cercozoa), Its Morphology, Phylogeny, Distribution, and Feeding Behavior.

A phaeodarian morphotype, characterized by the feet surrounded with forked pedal spines with anchor-like structures, was collected in the subtropical North Pacific. Considering the morphological and phylogenetic uniqueness, this morphotype is described as Gazelletta kashiwaensis sp. nov. The distribution of this new species is possibly affected by the Kuroshio Current. The feeding behavior of living phaeodarians was first filmed: the present new species floated in the water column stretching "protoplasmic webs" and collected diatoms by repeating the expansion and retraction of "pseudopodium-like tentacles".

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Phytocercomonas venanatans, a New Species of Cercozoa Associated with Chlorotic Streak of Sugarcane.

Chlorotic streak is a global disease of commercial sugarcane (Saccharum spp. hybrids). The disease is transmitted by wet soil, water, as well as in diseased planting material. Although first recognized almost 90 years ago and despite significant research effort, the identity of the causal agent has been elusive. Metagenomic high throughput sequencing (HTS) facilitated the discovery of novel protistan ribosomal and nuclear genes in chlorotic streak-infected sugarcane. These sequences suggest a possible causal agent belonging to the order Cercomonadida (Rhizaria, phylum Cercozoa). An organism with morphological features similar to cercomonads (=Cercomonadida) was isolated into pure axenic culture from internal stalk tissues of infected sugarcane. The isolated organism contained DNA sequences identical to those identified in infected plants by HTS. The DNA sequences and the morphology of the organism did not match any known species. Here we present a new genus and species, Phytocercomonas venanatans, which is associated with chlorotic streak of sugarcane. Amplicon sequencing also supports that P. venanatans is associated with this disease. This is the first reported member from Cercomonadida showing a probable pathogenic association with higher plants.

Natural selection drove metabolic specialization of the chromatophore in Paulinella chromatophora.

BACKGROUND: Genome degradation of host-restricted mutualistic endosymbionts has been attributed to inactivating mutations and genetic drift while genes coding for host-relevant functions are conserved by purifying selection. Unlike their free-living relatives, the metabolism of mutualistic endosymbionts and endosymbiont-originated organelles is specialized in the production of metabolites which are released to the host. This specialization suggests that natural selection crafted these metabolic adaptations. In this work, we analyzed the evolution of the metabolism of the chromatophore of Paulinella chromatophora by in silico modeling. We asked whether genome reduction is driven by metabolic engineering strategies resulted from the interaction with the host. As its widely known, the loss of enzyme coding genes leads to metabolic network restructuring sometimes improving the production rates. In this case, the production rate of reduced-carbon in the metabolism of the chromatophore. RESULTS: We reconstructed the metabolic networks of the chromatophore of P. chromatophora CCAC 0185 and a close free-living relative, the cyanobacterium Synechococcus sp. WH 5701. We found that the evolution of free-living to host-restricted lifestyle rendered a fragile metabolic network where >80% of genes in the chromatophore are essential for metabolic functionality. Despite the lack of experimental information, the metabolic reconstruction of the chromatophore suggests that the host provides several metabolites to the endosymbiont. By using these metabolites as intracellular conditions, in silico simulations of genome evolution by gene lose recover with 77% accuracy the actual metabolic gene content of the chromatophore. Also, the metabolic model of the chromatophore allowed us to predict by flux balance analysis a maximum rate of reduced-carbon released by the endosymbiont to the host. By inspecting the central metabolism of the chromatophore and the free-living cyanobacteria we found that by improvements in the gluconeogenic pathway the metabolism of the endosymbiont uses more efficiently the carbon source for reduced-carbon production. In addition, our in silico simulations of the evolutionary process leading to the reduced metabolic network of the chromatophore showed that the predicted rate of released reduced-carbon is obtained in less than 5% of the times under a process guided by random gene deletion and genetic drift. We interpret previous findings as evidence that natural selection at holobiont level shaped the rate at which reduced-carbon is exported to the host. Finally, our model also predicts that the ABC phosphate transporter (pstSACB) which is conserved in the genome of the chromatophore of P. chromatophora strain CCAC 0185 is a necessary component to release reduced-carbon molecules to the host. CONCLUSION: Our evolutionary analysis suggests that in the case of Paulinella chromatophora natural selection at the holobiont level played a prominent role in shaping the metabolic specialization of the chromatophore. We propose that natural selection acted as a "metabolic engineer" by favoring metabolic restructurings that led to an increased release of reduced-carbon to the host.

A New Phaeodarian Species Discovered from the Japan Sea Proper Water, Auloscena pleuroclada sp. nov. (Aulosphaeridae, Phaeosphaerida, Phaeodaria).

A new phaeodarian species, characterized by the presence of long developed side branches recurved proximally and distally on the surface of its radial tube, was described as Auloscena pleuroclada. This new species was only collected from the layers below the 250 m depth in the Sea of Japan. They have never been found in the shallower layers (above 250 m) of this sea or in other investigated areas. The distribution of the present new species is presumably restricted within the deep water of this area, and this species could be a specific phaeodarian adapted to the deep-sea environment.

Prospective function of FtsZ proteins in the secondary plastid of chlorarachniophyte algae.

BACKGROUND: Division of double-membraned plastids (primary plastids) is performed by constriction of a ring-like division complex consisting of multiple plastid division proteins. Consistent with the endosymbiotic origin of primary plastids, some of the plastid division proteins are descended from cyanobacterial cell division machinery, and the others are of host origin. In several algal lineages, complex plastids, the "secondary plastids", have been acquired by the endosymbiotic uptake of primary plastid-bearing algae, and are surrounded by three or four membranes. Although homologous genes for primary plastid division proteins have been found in genome sequences of secondary plastid-bearing organisms, little is known about the function of these proteins or the mechanism of secondary plastid division. RESULTS: To gain insight into the mechanism of secondary plastid division, we characterized two plastid division proteins, FtsZD-1 and FtsZD-2, in chlorarachniophyte algae. FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal. Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans. The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes. Furthermore, gene expression analysis confirmed that transcripts of ftsZD genes were periodically increased soon after cell division during the B. natans cell cycle, which is not consistent with the timing of plastid division. CONCLUSIONS: Our findings suggest that chlorarachniophyte FtsZD proteins are involved in partial constriction of the inner pair of plastid membranes, but not in the whole process of plastid division. It is uncertain how the outer pair of plastid membranes is constricted, and as-yet-unknown mechanism is required for the secondary plastid division in chlorarachniophytes.

Detailed process of shell construction in the photosynthetic testate amoeba Paulinella chromatophora (euglyphid, Rhizaria).

Most euglyphids, a group of testate amoebae, have a shell that is constructed from numerous siliceous scales. The euglyphid Paulinella chromatophora has photosynthetic organelles (termed cyanelles or chromatophores), allowing it to be cultivated more easily than other euglyphids. Like other euglyphids, P. chromatophora has a siliceous shell made of brick-like scales. These scales are varied in size and shape. How a P. chromatophora cell makes this shell is still a mystery. We examined shell construction process in P. chromatophora in detail using time-lapse video microscopy. The new shell was constructed by a specialized pseudopodium that laid out each scale into correct position, one scale at a time. The present study inferred that the sequence of scale production and secretion was well controlled.

Evolving a photosynthetic organelle.

The evolution of plastids from cyanobacteria is believed to represent a singularity in the history of life. The enigmatic amoeba Paulinella and its 'recently' acquired photosynthetic inclusions provide a fascinating system through which to gain fresh insight into how endosymbionts become organelles.The plastids, or chloroplasts, of algae and plants evolved from cyanobacteria by endosymbiosis. This landmark event conferred on eukaryotes the benefits of photosynthesis--the conversion of solar energy into chemical energy--and in so doing had a huge impact on the course of evolution and the climate of Earth 1. From the present state of plastids, however, it is difficult to trace the evolutionary steps involved in this momentous development, because all modern-day plastids have fully integrated into their hosts. Paulinella chromatophora is a unicellular eukaryote that bears photosynthetic entities called chromatophores that are derived from cyanobacteria and has thus received much attention as a possible example of an organism in the early stages of organellogenesis. Recent studies have unlocked the genomic secrets of its chromatophore 23 and provided concrete evidence that the Paulinella chromatophore is a bona fide photosynthetic organelle 4. The question is how Paulinella can help us to understand the process by which an endosymbiont is converted into an organelle.

Esquamula lacrimiformis n. g., n. sp., a new member of thaumatomonads that lacks siliceous scales.

We report a new naked cercozoan flagellate, Esquamula lacrimiformis n. g., n. sp., collected from a sandy beach in Japan. Its cells were 4.5-11.3 µm in length and 3.9-8.8 µm in width and possess two unequal flagella. Cells move in a smooth gliding motion and have a trailing long posterior flagellum. Phylogenetic analyses with small and large subunit ribosomal RNA genes revealed that E. lacrimiformis forms a novel lineage within the Thaumatomonadida, the members of which are flagellates with siliceous scales. However, our light and electron microscopic observations indicated that E. lacrimiformis cells do not possess any siliceous structures. Furthermore, other morphological characteristics, such as the shape of the extrusomes and the structural arrangement of the microbody, were clearly different from those of previously described thaumatomonads. On the basis of a combination of these morphological observations and our phylogenetic analyses, we conclude that E. lacrimiformis should be treated as a new species of a new genus and placed into a new family, Esquamulidae n. fam., under Thaumatomonadida.

Nucleomorph ribosomal DNA and telomere dynamics in chlorarachniophyte algae.

Chlorarachniophytes are enigmatic marine unicellular algae that acquired photosynthesis by secondary endosymbiosis. Chlorarachniophytes are unusual in that the nucleus of the engulfed algal cell (a green alga) persists in a miniaturized form, termed a nucleomorph. The nucleomorph genome of the model chlorarachniophyte, Bigelowiella natans CCMP621, is 373 kilobase pairs (kbp) in size, the smallest nuclear genome characterized to date. The B. natans nucleomorph genome is composed of three chromosomes, each with canonical eukaryotic telomeres and sub-telomeric ribosomal DNA (rDNA) operons transcribed away from the chromosome end. Here we present the complete rDNA operon and telomeric region from the nucleomorph genome of Lotharella oceanica CCMP622, a newly characterized chlorarachniophyte strain with a genome ∼610 kbp in size, significantly larger than all other known chlorarachniophytes. We show that the L. oceanica rDNA operon is in the opposite chromosomal orientation to that of B. natans. Furthermore, we determined the rDNA operon orientation of five additional chlorarachniophyte strains, the majority of which possess the same arrangement as L. oceanica, with the exception of Chlorarachnion reptans and those very closely related to B. natans. It is thus possible that the ancestral rDNA operon orientation of the chlorarachniophyte nucleomorph genome might have been the same as in the independently evolved, red algal-derived, nucleomorph genomes of cryptophytes. A U2 small nuclear RNA gene was found adjacent to the telomere in Gymnochlora stellata CCMP2057 and Chlorarachnion sp. CCMP2014. This feature may represent a useful evolutionary character for inferring the relationships among extant lineages.

Shell Colour in Cercozoa; a Simple Trait to Distinguish Thecofilosea from Imbricatea?

Rigid and persistent shells of microeukaryotes are widely used as bioindicators in ecological and paleontological studies. Drawing conclusions on ecological or evolutionary patterns depends strongly on the right taxonomic assignment of the observed species, however confusion is common. Especially in filose shelled amoebae it is often unclear whether species belong to the Imbricatea or Thecofilosea when only morphological data are collected. Molecular surveys shed light on their evolutionary relationship; based on these we propose a hypothesis how to differentiate doubtful species even light microscopically.

Phylogeny and Classification of Novel Diversity in Sainouroidea (Cercozoa, Rhizaria) Sheds Light on a Highly Diverse and Divergent Clade.

Sainouroidea is a molecularly diverse clade of cercozoan flagellates and amoebae in the eukaryotic supergroup Rhizaria. Previous 18S rDNA environmental sequencing of globally collected fecal and soil samples revealed great diversity and high sequence divergence in the Sainouroidea. However, a very limited amount of this diversity has been observed or described. The two described genera of amoebae in this clade are Guttulinopsis, which displays aggregative multicellularity, and Rosculus, which does not. Although the identity of Guttulinopsis is straightforward due to the multicellular fruiting bodies they form, the same is not true for Rosculus, and the actual identity of the original isolate is unclear. Here we isolated amoebae with morphologies like that of Guttulinopsis and Rosculus from many environments and analyzed them using 18S rDNA sequencing, light microscopy, and transmission electron microscopy. We define a molecular species concept for Sainouroidea that resulted in the description of 4 novel genera and 12 novel species of naked amoebae. Aggregative fruiting is restricted to the genus Guttulinopsis, but other than this there is little morphological variation amongst these taxa. Taken together, simple identification of these amoebae is problematic and potentially unresolvable without the 18S rDNA sequence.

Taxonomic Implications of Morphological Complexity Within the Testate Amoeba Genus Corythion from the Antarctic Peninsula.

Precise and sufficiently detailed morphological taxonomy is vital in biology, for example in the accurate interpretation of ecological and palaeoecological datasets, especially in polar regions, where biodiversity is poor. Testate amoebae on the Antarctic Peninsula (AP) are well-documented and variations in their population size have recently been interpreted as a proxy for microbial productivity changes in response to recent regional climate change. AP testate amoeba assemblages are dominated by a small number of globally ubiquitous taxa. We examine morphological variation in Corythion spp. across the AP, finding clear evidence supporting the presence of two morphospecies. Corythion constricta (Certes 1889) was identified on the AP for the first time and has potentially been previously misidentified. Furthermore, a southerly trend of decreasing average test size in Corythion dubium (Taránek 1881) along the AP suggests adaptive polymorphism, although the precise drivers of this remain unclear, with analysis hindered by limited environmental data. Further work into morphological variation in Corythion is needed elsewhere, alongside molecular analyses, to evaluate the potential for (pseudo)cryptic diversity within the genus. We advocate a parsimonious taxonomical approach that recognises genetic diversity but also examines and develops accurate morphological divisions and descriptions suitable for light microscopy-based ecological and palaeoecological studies.

Shedding Light on the Polyphyletic Thecate Amoeba Genus Plagiophrys: Transition of Some of its Species to Rhizaspis (Tectofilosida, Thecofilosea, Cercozoa) and the Establishment of Sacciforma gen. nov. and Rhogostomidae fam. nov. (Cryomonadida, Thecofilosea, Cercozoa).

For over a century testate amoebae have been a favoured group of interest for protistologists, however there is still an endless amount of unanswered questions. The genus Plagiophrys, Claparède and Lachmann 1859, is still one of the unresolved mysteries as it comprises species with high morphological diversity of which no molecular data are available. To shed light on the phylogeny and taxonomy of Plagiophrys we (a) cultured four isolates of three Plagiophrys morphospecies and provided morphological observations (b) obtained three new SSU RNA gene sequences and conducted phylogenetic analyses of the Thecofilosea and (c) did intensive literature research, showing that Plagiophrys is polyphyletic. We partially untangle this polyphyly by combining several of its species with the genus Rhizaspis, Skuja 1948. Furthermore, we establish Sacciforma gen. nov. to accommodate P. sacciformis as it groups within the formerly known Rhizaspididae, which do not comprise our isolates of Rhizaspis (and therefore were renamed Rhogostomidae) since Rhizaspis it groups with maximum support as a sister-group to the Pseudodifflugiidae.

Diversity of the Photosynthetic Paulinella Species, with the Description of Paulinella micropora sp. nov. and the Chromatophore Genome Sequence for strain KR01.

The thecate filose amoeba Paulinella chromatophora is a good model organism for understanding plastid organellogenesis because its chromatophore was newly derived from an alpha-cyanobacterium. Paulinella chromatophora was the only known photosynthetic Paulinella species until recent studies that suggested a species level of diversity. Here, we described a new photosynthetic species P. micropora sp. nov. based on morphological and molecular evidence from a newly established strain KR01. The chromatophore genome of P. micropora KR01 was fully determined; the genome was 976,991bp in length, the GC content was 39.9%, and 908 genes were annotated. A pairwise comparison of chromatophore genome sequences between strains KR01 and FK01, representing two different natural populations of P. micropora, showed a 99.85% similarity. Differences between the two strains included single nucleotide polymorphisms (SNPs) in CDSs, which resulted in 357 synonymous and 280 nonsynonymous changes, along with 245 SNPs in non-coding regions. Indels (37) and microinversions (14) were also detected. Species diversity for photosynthetic Paulinella was surveyed using samples collected from around the world. We compared our new species to two photosynthetic species, P. chromatophora and P. longichromatophora. Phylogenetic analyses using four gene markers revealed three distinct lineages of photosynthetic Paulinella species including P. micropora sp. nov.

Polyphyly in the Thecate Amoeba Genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa), Redescription of its Type Species L. hyalinum, Description of L. jennyae sp. nov. and the Establishment of Fisculla gen. nov. and Fiscullidae fam. nov.

Although testate amoebae have attracted great interest of protistologists for more than a century, some groups, especially those with a hyaline, organic test (=theca) are still poorly known. One of those is the genus Lecythium Hertwig et Lesser, 1874. Only recently Lecythium spp. were characterized by morphological and molecular means, but data on the type species Lecythium hyalinum Hertwig et Lesser, 1874, was still lacking. In this study, we screened for L. hyalinum in freshwater samples of Germany and the Netherlands. Four different isolates of L. hyalinum and one novel species were cultured and characterized by light microscopy. Phylogenetic analyses based on the small ribosomal subunit (SSU) RNA gene show that the genus Lecythium forms two robust clades, one forming a sister group to the Rhizaspididae/Pseudodifflugiidae clade (Tectofilosida), the other branching within 'Novel Clade 4' (Tectofilosida). We untangle this polyphyly by establishing Fisculla gen. nov. and the Fiscullidae fam. nov. for the former of these two clades.