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.

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.

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.

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.

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.

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".

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.

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.

Rhogostomidae (Cercozoa) from soils, roots and plant leaves (Arabidopsis thaliana): Description of Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov.

Cercozoa are a highly diverse protist phylum in soils and in the phyllosphere of plants. Many families are still poorly described and the vast majority of species are still unknown. Although testate amoebae are among the better-studied protists, only little quantitative information exists on the morphology, phylogeny and ecology of cercozoan Rhogostomidae. We cultured four different strains of Rhogostoma spp. isolated from Arabidopsis leaves, agricultural soil and rhizosphere soil of Ocimum basilicum and Nicotiana sp. We describe Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov. and present their morphology, studied their food spectra in food range experiments and obtained two SSU rDNA gene sequences resulting in an updated thecofilosean phylogeny. Short generation times, desiccation resistance and the ability to prey on a wide range of algae and yeasts from the phyllosphere were seen as crucial traits for the phyllosphere colonization by Rhogostoma. In contrast, the soil-dwelling R. cylindrica did not feed on eukaryotes in our experiment.

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.

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.

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.

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.

The Cytoskeleton Architecture of Algivorous Protoplast Feeders (Viridiraptoridae, Rhizaria) Indicates Actin-Guided Perforation of Prey Cell Walls.

Several distantly related, phagotrophic microeukaryotes share the remarkable ability to perforate foreign cell walls in a well-defined pattern to acquire protoplast material as food. The underlying cellular processes, especially the local application of cell wall degrading agents, are still unexplored. We examined the distribution of F-actin and alpha-tubulin in the algivorous, viridiraptorid amoeboflagellates Orciraptor agilis and Viridiraptor invadens over their life histories using phalloidin conjugates and immunolocalization. During attack, both species form distinctive, F-actin-rich structures at the contact zone to the algal prey cell, which exactly match the species-specific cell wall perforations and resemble invadopodia and podosome rosettes of mammalian cells to a certain extent. Furthermore, F-actin is involved in the extraction of plastid material by Orciraptor and in prey cell invasion by Viridiraptor (here, F-actin localizes to a characteristic hyaline channel, which surrounds the streaming cytoplasm). The digestive-reproductive stages of viridiraptorids display a highly ordered microtubular cytoskeleton, whereas distinct phalloidin-positive actin structures could not be detected. We discuss the presumed function of F-actin during perforation and penetration of the algal cell wall by viridiraptorids, and compare the cytoskeleton architecture of these protoplast feeders to amoeboflagellates from different eukaryotic supergroups.

Placement of the unclassified Cyranomonas australis Lee 2002 within a novel clade of Cercozoa.

Two heterotrophic flagellate strains were isolated from marine sediment samples off eastern Canada and Korea. These new isolates are indistinguishable by light microscopy from the unclassified protist Cyranomonas australis. The organisms are ovoid-shaped cells, 3.5-6µm long, laterally compressed, and somewhat flexible. They have two unequal flagella, about 1.1-2.5 times body length. Typically, the cells show a gliding motility and do not exhibit any amoeboid form or pseudopodia. 18S rDNA phylogenies clearly indicate that the isolates can be assigned to the taxon Filosa, within Cercozoa. The isolates are closest to an environmental sequence (CYSGM-16; 99% identity). Cyranomonas, CYSGM-16, and uncultured eukaryote RM1-SGM46 form a clade with strong statistical supports, here called novel clade CU (Cyranomonas plus Uncultured eukaryotes). This clade may be sister to the order Marimonadida. The novel clade CU and the Marimonadida have been detected only in marine habitats. Our findings suggest that C. australis may not belong to any previously described family within Filosa and Cercozoa.

Local and intercontinental comparisons of test morphology in the little-known testate amoeba Cyphoderia laevis Penard.

The morphology of tests ("shells") representing populations of the small testate amoeba Cyphoderia laevisPenard, 1902 (Cercozoa, Euglyphida) from Canada and Hungary was quantified. Despite often considerable variation the cross-sectional shape of tests, multivariate statistical analysis confirmed the general uniformity of test morphology within populations and between populations at both local (regional, inter- and intra-watershed) scales and over inter-continental scales (Canada and Hungary). Corrections are suggested to recently published misidentifications of C. laevis as Corythionella georgiana Nicholls. The differences between these two taxa are subtle, but significant, and are emphasized here to help preclude similar errors in the future. Schaudinnula nanaBadewitz, 2003 is shown to be a superfluous synonym for Cyphoderia laevis. Cyphoderia truncataSchulze, 1875 has some similarities to C. laevis, but its status remains unresolved. Some preliminary reservations are expressed about the classification of Cyphoderia laevis within the genus Cyphoderia owing mainly to its Corythionella-like scale structure and other characteristics of its test that appear to be outside the "usual" range found in other Cyphoderia species.

A Novel Lineage of 'Naked Filose Amoebae'; Kraken carinae gen. nov. sp. nov. (Cercozoa) with a Remarkable Locomotion by Disassembly of its Cell Body.

The term 'filose amoebae' describes a highly polyphyletic assemblage of protists whose phylogenetic placement can be unpredictable based on gross morphology alone. We isolated six filose amoebae from soils of two European countries and describe a new genus and species of naked filose amoebae, Kraken carinae gen. nov. sp. nov. We provide a morphological description based on light microscopy and small subunit rRNA gene sequences (SSU rDNA). In culture, Kraken carinae strains were very slow-moving and preyed on bacteria using a network of filopodia. Phylogenetic analyses of SSU sequences reveal that Kraken are core (filosan) Cercozoa, branching weakly at the base of the cercomonad radiation, most closely related to Paracercomonas, Metabolomonas, and Brevimastigomonas. Some Kraken sequences are >99% similar to an environmental sequence obtained from a freshwater lake in Antarctica, indicating that Kraken is not exclusively soil dwelling, but also inhabits freshwater habitats.

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.

Paulinella longichromatophora sp. nov., a New Marine Photosynthetic Testate Amoeba Containing a Chromatophore.

The freshwater testate filose amoeba Paulinella chromatophora is the sole species in the genus to have plastids, usually termed "chromatophores", of a Synechococcus/Prochlorococcus-like cyanobacterial origin. Here, we report a new marine phototrophic species, Paulinella longichromatophora sp. nov., using light and electron microscopy and molecular data. This new species contains two blue-green U-shaped chromatophores reaching up to 40 µm in total length. Further, the new Paulinella species is characterized by having five oral scales surrounding the pseudostomal aperture. All trees generated using three nuclear rDNA datasets (18S rDNA, 28S rDNA, and the concatenated 18S + 28S rDNA) demonstrated that three photosynthetic Paulinella species (two freshwater species, P. chromatophora and Paulinella strain FK01, and one marine species, P. longichromatophora) congruently formed a monophyletic group with strong support (≥ 90% of ML and ≥ 0.90 of PP), but their relationship to each other within the clade remained unresolved in all trees. P. longichromatophora, nevertheless, clustered consistently together with Paulinella strain FK01 with very low support, but the clade received strong support in plastid phylogenies. Phylogenetic analyses inferred from plastid-encoded 16S rDNA and a concatenated dataset of plastid 16S+23S rDNA demonstrated that chromatophores of all photosynthetic Paulinella species were monophyletic. The monophyletic group fell within a cyanobacteria clade having a close relationship to an α-cyanobacterial clade containing Prochlorococcus and Synechococcus species with very robust support (100% of ML and 1.0 of PP). Additionally, phylogenetic analyses of nuclear 18S rDNA and plastid 16S rDNA suggested divergent evolution within the photosynthetic Paulinella population after a single acquisition of the chromatophore. After the single acquisition of the chromatophore, ancestral photosynthetic Paulinella appears to have diverged into at least two distinct clades, one containing the marine P. longichromatophora and freshwater Paulinella strain FK01, the other P. chromatophora CCAC 0185.

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.