Akinori Yabuki

Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised.

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.

Palpitomonas bilix gen. et sp. nov.: A novel deep-branching heterotroph possibly related to Archaeplastida or Hacrobia.

We describe the ultrastructure and putative molecular phylogenetic position of a free-living heterotrophic flagellate Palpitomonas bilix gen. et sp. nov. This flagellate is 3-8mum in size, and possesses two subequal flagella, approximately 20mum long. Electron microscopical observations revealed that the flagellar apparatus of P. bilix resembles that of members of the green algal class, Charophyceae, while the mastigonemes of the P. bilix flagellum share some characteristics with those found in cryptophytes and telonemids. In order to better understand the phylogenetic position of P. bilix, we sequenced six commonly used phylogenetic marker genes encoding the small and large subunits of ribosomal RNA, alpha-tubulin, beta-tubulin, 90kDa heat shock protein, and translation elongation factor 2. Depending on the genes analyzed, P. bilix shows a generally weak phylogenetic affinity to either the newly erected Hacrobia, which includes cryptophytes and haptophytes, or to Archaeplastida. Since the current study identified no clear close relative of P. bilix, this novel biflagellate was classified into a new genus Palpitomonas with the higher-level classification being unclear. On the basis of the results of both morphological and molecular studies, we discuss the possibility that P. bilix might provide key information on the early evolution of major groups of photosynthetic eukaryotes.

A wide diversity of previously undetected free‐living relatives of diplomonads isolated from marine/saline habitats

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free-living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas-like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small-subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad-retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation - it is likely that undersampling at the level of major lineages is still widespread for protists.

Phylogeny and nucleomorph karyotype diversity of chlorarachniophyte algae

ABSTRACT. Chlorarachniophytes are flagellated and/or reticulopod‐forming marine algae with chlorophyll a‐ and b‐containing plastids of secondary endosymbiotic origin. They are one of only two algal groups known to possess a “nucleomorph” (i.e. the remnant nucleus of the eukaryotic endosymbiont that donated the plastid). Apart from the recently sequenced nucleomorph genome of Bigelowiella natans, little is known about the size, structure, and composition of chlorarachniophyte nucleomorph genomes. Toward the goal of better understanding nucleomorph genome diversity, as well as establishing a phylogenetic framework with which to interpret variation in chlorarachniophyte morphology, ultrastructure, and life cycle, we are studying a wide range of chlorarachniophyte strains from public culture collections and natural habitats. We have obtained 22 new chlorarachniophyte nuclear and nucleomorph 18S rRNA gene (18S rDNA) sequences and nucleomorph genome size estimates for 14 different strains. Consistent with previous studies, all of the chlorarachniophytes examined appear to possess three nucleomorph chromosomes. However, our results suggest considerable variation in nucleomorph genome size and structure, with individual chromosome sizes ranging from ∼90 to ∼210 kbp, and total genome sizes between ∼330 kbp in Lotharella amoebiformis and ∼610 kbp in unidentified chlorarachniophyte strain CCMP622. The significance of these phylogenetic and nucleomorph karyotype data is discussed.

Tsukubamonas globosa n. gen., n. sp., a novel excavate flagellate possibly holding a key for the early evolution in "Discoba".

ABSTRACT. We report the ultrastructure and phylogenetic position of a free‐living heterotrophic flagellate, Tsukubamonas globosa n. gen., n. sp. This flagellate was isolated from a pond in the University of Tsukuba, Japan. Under light microscopy, the spherical vegetative cells were naked and highly vacuolated, and always swam with rotating motion. Electron microscopic observations revealed that T. globosa possessed a ventral feeding groove, which is one of the hallmark characteristics of the supergroup Excavata. The position of T. globosa was unresolved in the small subunit ribosomal RNA phylogeny. On the other hand, a multigene phylogeny using α‐tubulin, β‐tubulin, actin, heat shock protein 90, and translation elongation factor 2 robustly united T. globosa with members of the “Discoba” clade of Excavata, composed of jakobids, euglenozoans, and heteroloboseans, although the precise position of T. globosa in this clade remained unresolved. Our detailed morphological comparisons elucidated that T. globosa possessed a novel set of morphological features, and could not be classified into any taxa in the Discoba clade. Instead we classified T. globosa into Tsukubamonadidae n. fam. under Tsukubamonadida n. ord.

Rigifila ramosa n. gen., n. sp., a filose apusozoan with a distinctive pellicle, is related to Micronuclearia.

We report the ultrastructure, 18S and 28S rDNA sequences, and phylogenetic position of a distinctive free-living heterotrophic filose protist, Rigifila ramosa n. gen., n. sp., from a freshwater paddyfield. Rigifila lacks cilia and has a semi-rigid, radially symmetric, well-rounded, partially microtubule-supported, dorsal pellicle, and flat mitochodrial cristae. From a central aperture in a ventral depression emerges a protoplasmic stem that branches into several branching filopodia that draw bacteria to it. Electron microscopy reveals a general cell structure similar to Micronuclearia, the only non-flagellate previously known in Apusozoa; the large basal vacuole is probably an unusual giant contractile vacuole. Phylogenetic analysis of concatenated rDNA sequences groups Rigifila and Micronuclearia as sisters with maximal statistical support. However, novel morphological differences unique to Rigifila, notably a double (not single) proteinaceous layer beneath the cell membrane, and cortical microtubules, lead us to place it in a new family Rigifilidae. Our morphological and molecular analyses show that Rigifila is the closest known relative of Micronuclearia. Therefore we group Micronucleariidae and Rigifilidae as a new order Rigifilida within the existing class Hilomonadea, which now excludes planomonads. Rigifilida groups weakly with Collodictyon (Diphyllatea). We discuss the possible relationships of Rigifilida to other Apusozoa and Diphyllatea.

Lateral transfer of tetrahymanol-synthesizing genes has allowed multiple diverse eukaryote lineages to independently adapt to environments without oxygen

Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes.ReviewersThis article was reviewed by Eric Bapteste and Eugene Koonin.

Partenskyella glossopodia gen. et sp. nov., the First Report of a Chlorarachniophyte that Lacks a Pyrenoid

A new chlorarachniophyte, Partenskyella glossopodia gen. et sp. nov., is described from a culture isolated from the Mediterranean Sea pelagic waters and maintained as strain RCC365 at the Roscoff Culture Collection (France). Vegetative cells of P. glossopodia are non-motile naked spherical cells. However, flagellate and amoeboid stages are also present in its life cycle. The cells are 2-4mum in diameter containing a pale-green, cup-shaped chloroplast, 1-2 mitochondria, a nucleus, and a Golgi apparatus. Vesicles containing storage product-like material are also present. The chloroplast is surrounded by four membranes possessing a nucleomorph in the periplastidal compartment. The minute cell size and the absence of a pyrenoid at any stage of the life cycle are unique characteristics among the chlorarachniophytes, which justifies our proposition for a new genus for strain RCC365.

Mataza hastifera n. g., n. sp.: a possible new lineage in the Thecofilosea (Cercozoa).

ABSTRACT. A new cercozoan flagellate Mataza hastifera n. g., n. sp. is described from a surface seawater sample collected in Tokyo Bay. Cells are 3–5 μm in diameter and have two flagella. The cells alternate between swimming and stationary states in culture. Swimming cells have a nodding motion. Phylogenetic analyses using small subunit rDNA sequences demonstrate that M. hastifera belongs to the clade comprised of only environmental sequences closely related to thecofilosean cercozoans. Ultrastructural observations reveal that M. hastifera is quite similar to members of Cryomonadida, an order in Thecofilosea, and especially to Cryothecomonas spp. The cell of M. hastifera is covered with a thin double‐layered theca and possesses a cylinder‐shaped extrusome, as reported from cryomonads. On the other hand, the funnel that is characteristic of cryomonads was not found in the flagellar pit of M. hastifera. Combining both morphological and molecular analyses, we conclude that M. hastifera is a new lineage in Thecofilosea and suggest that Thecofilosea may be a larger group than previously thought.

Phylogeny and Reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996

Hemistasia phaeocysticola is a marine flagellate that preys on diatoms and dinoflagellates among others. Although its morphology and ultrastructure were previously observed and characterized, its phylogenetic position has not been analyzed using molecular sequence data. This flagellate was classified as a kinetoplastid on the basis of the presence of a kinetoplast in the mitochondrion. However, several morphological characteristics similar to those of diplonemids, a sister group of kinetoplastids, have also been noted. Herein, we report that H. phaeocysticola branches within the diplonemid clade in the phylogenetic tree reconstructed by analyzing 18S rRNA gene sequences. Its systematic placement based on this finding is also discussed.