Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

New species Micractinium kostikovii (Chlorellaceae,Trebouxiophyceae) from Russia

Correctly identifying species of Chlorella-like microalgae is difficult because of their morphological simplicity and high phenotypic plasticity. The use of molecular tools has revolutionized research on algal diversity, enabling such advances as the discovery of numerous new taxa. This article presents the results of a study of strains that are newly isolated from a freshwater Lake Prudovikov (Samara region, Russian Federation). These strains had the typical Chlorella morphology, exhibiting spherical cells and a cup-shaped parietal chloroplast. The chloroplast contained a single pyrenoid enveloped by starch grains. However, 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strains are strongly allied with the well-supported genus Micractinium. Based on the results of a comparative analysis of the morphological, molecular and environmental characteristics of the studied strains (employing a polyphasic approach), we propose that they are new species of Micractinium: Micractinium kostikovii sp. nov.     

Kalinella bambusicola gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid Chlorella-like subaerial alga from Southeast Asia

The traditional green algal genus Chlorella , which comprised coccoid algae surrounded by a smooth cell wall and reproducing solely by autosporulation, has proved to be polyphyletic and extremely diverse in phylogenetic terms. We studied a new subaerial Chlorella -like strain CAUP H7901 and morphological, ultrastructural, and molecular phylogenetic investigations indicated that it represents a new lineage of the trebouxiophycean Watanabea clade, dissimilar from other members of this group. The alga has globular coccoid cells with a single parietal pyrenoid-bearing chloroplast. The pyrenoid is transected by multiple radial thylakoid bands. The alga reproduces exclusively by means of asexual autospores of unequal size. In 18S rDNA sequence phylogenies, it was nested within the Watanabea clade close to lineages containing Chlorella saccharophila , Chlorella luteoviridis , Heveochlorella hainangensis , and two uncharacterized strains, but alternative positions within the Watanabea clade could not be rejected by an approximately unbiased (AU) test. Here we describe this organism as a new genus and species Kalinella bambusicola gen. et sp. nov. Furthermore, we describe Heterochlorella gen. nov. to accommodate a species previously referred to as Chlorella luteoviridis .     

Tetraflagellochloris mauritanica gen. et sp. nov. (Chlorophyceae), a New Flagellated Alga from the Mauritanian Desert: Morphology,Ultrastructure, and Phylogenetic Framing

Morphological, ultrastructural, and molecular‐sequence data were used to assess the phylogenetic position of a tetraflagellate green alga isolated from soil samples of a saline dry basin near F'derick, Mauritania. This alga can grow as individual cells or form non‐coenobial colonies of up to 12 individuals. It has a parietal chloroplast with an embedded pyrenoid covered by a starch sheath and traversed by single parallel thylakoids, and an eyespot located in a parietal position opposite to the flagellar insertion. Lipid vacuoles are present in the cytoplasm. Microspectroscopy indicated the presence of chlorophylls a and b, with lutein as the major carotenoid in the chloroplast, while the eyespot spectrum has a shape typical of green‐algal eyespots. The cell has four flagella, two of them long and two considerably shorter. Sequence data from the 18S rRNA gene and ITS2 were obtained and compared with published sequences for green algae. Results from morphological and ultrastructural examinations and sequence analysis support the placement of this alga in the Chlorophyceae, as Tetraflagellochloris mauritanica L. Barsanti et A. Barsanti, gen. et sp. nov.     

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300‰ salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30–35°C. The isolated HNF grew optimally at 150‰ salinity and tolerated up to 363‰ salinity, but it failed to grow below 75‰ salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.     

A new class of the stramenopiles,Placididea Classis nova: description of Placidia cafeteriopsis gen. et sp. nov

A marine flagellate resembling Cafeteria roenbergensis (bicosoecids, stramenopiles) in cell shape and behavior of the cell while attached to substratum was collected from the coast of Japan. The flagellate was examined by light and electron microscopy, and the 18S rDNA was sequenced to elucidate its taxonomic and phylogenetic position. Ultrastructural features suggested that the flagellate is not a bicosoecid, but a relative of the recently described stramenopile, Wobblia lunata. 18S rDNA phylogenetic trees also revealed that the flagellate forms a monophyletic clade with W. lunata and that it is distantly related to Cafeteria and other bicosoecids. The flagellate differs from W. lunata due to its lack of wobbling motion as well as intracellular features such as the number of mitochondria, flagellar apparatus architecture, the presence of a paranuclear body and cytoplasmic microtubules. The similarity of 18S rDNA sequences was 81% between the flagellate and W. lunata. This new flagellate was described as Placidia cafeteriopsis gen. et sp. nov. Because the phylogenetic lineage comprised of W. lunata and P. cafeteriopsis was one of the major, deep-branching clades of the stramenopiles, the class Placididea (= Placidiophyceae) classis nova was proposed.     

A new aerial alga, Stichococcus ampulliformis sp. nov. (Trebouxiophyceae, Chlorophyta) from Japan

A new aerial alga, Stichococcus ampulliformis S. Handa sp. nov. (Trebouxiophyceae, Chlorophyta) is described based on a clone isolated from the bark of Cephalotaxus harringtonia (Knight ex Forbes) K. Koch collected from Taishaku‐kyo Gorge, Hiroshima Prefecture, south‐west Japan. This alga was examined by light microscopy and transmission electron microscopy and subjected to molecular phylogenetic analysis. Based on its morphological features and life‐cycle, especially short filament formation, the alga was assigned to the genus Stichococcus Nägeli. However, this alga differs from other described Stichococcus species in that it reproduces by a form of ‘budding’, producing two daughter cells of different sizes. The larger cell, enclosed within the mother cell wall, soon reaches the size of a normal vegetative cell. The smaller cell is extruded and takes longer to reach full vegetative size. A phylogenetic tree constructed using 18S rRNA sequences indicated that, within the Trebouxiophyceae, S. ampulliformis is closely related to S. bacillaris Nägeli and some species of Prasiola Meneghini.     

Hemichloris antarctica, gen. et sp. nov. (Chlorococcales, Chlorophyta), a cryptoendolithic alga from Antarctica

Hemichloris antarctica gen. et sp. nov. (Oocystaceae, Chlorococcales) is characterized by a single, articulated, pyrenoid-less, thick saucer-shaped chloroplast, which generally fills less than half of the cell periphery. Multiplication is only by autospores. The species is psychrophilic and is damaged at temperatures above 20 degree C. Hemichloris antarctica is a member of the cryptoendolithic microbial community living in porous sandstone rocks of the Antarctica cold desert. It inhabits the zone below that of cryptoendolithic lichens and survives at extremely low light intensities. In the natural habitat, morphology is somewhat different from that in culture, as chloroplasts are smaller and without articulation, and the cells develop a gelatinous sheath.     

Endozoicomonas elysicola gen. nov., sp. nov., a gamma-proteobacterium isolated from the sea slug Elysia ornata

A Gram-negative, strictly aerobic, rod-formed bacterium, strain MKT110(T), was isolated from a mollusk, the sea slug Elysia ornata collected in seawater off the coast of Izu-Miyake Island, Japan at a depth of 15m. Comparative 16S rRNA gene sequences analysis indicated that the isolate MKT110(T) constituted a novel lineage in gamma-proteobacteria related to the genera Zooshikella, Oceanospirillum, Microbulbifer, Marinobacter, Saccharospirillum and Pseudomonas. The strain MKT110(T) was closely related to the clones from marine sponge Halichondria okadai (AB054136, AB054161) and the coral Pocillopora damicornis (AY700600, AY700601). The phylogenetic tree based on the 16S rRNA gene sequences showed that MKT110(T) and four clones formed a sub-lineage related to the genus Zooshikella, with a bootstrap value of 100%. MKT110(T) required salt for its growth and was mesophilic. The bacterium contained 16:1omega7c, 16:0 and 14:0 as major cellular fatty acids, and 3-OH 14:0, 3-OH 10:0 and 3-OH 12:0 as major hydroxy fatty acids. The DNA base composition of the isolate was 50.4 mol% G+C. The major quinone was Q-9. The bacterium is distinguished from currently recognized bacterial genera based on phylogenetic and phenotypic features and should be classified in a novel genus for which the name Endozoicomonas elysicola gen. nov., sp. nov. is proposed. (type strain MKT110(T)=IAM 15107(T)=KCTC 12372(T); GenBank accession no. AB196667).     

Lunachloris lukesovae gen. et sp. nov. (Trebouxiophyceae,Chlorophyta), a novel coccoid green alga isolated from soil in South Bohemia,Czech Republic

Culture collections of microorganisms can still hold undiscovered biodiversity; with molecular techniques, considerable progress has been made in characterizing microalgae which were isolated in the past and misidentified due to a lack of morphological features. However, many strains are still awaiting taxonomic reassessment. Here we analysed the phylogenetic position, morphology and ultrastructure of the strain CCALA 307 previously identified as Coccomyxa cf. gloeobotrydiformis Reysigl isolated in 1987 from field soil in South Bohemia, Czech Republic. Molecular phylogenetic analyses based on SSU rDNA and the plastid rbcL gene revealed that the strain CCALA 307 formed a distinct sister lineage to Neocystis and Prasiola clades within the Trebouxiophyceae. We describe this strain as a new genus and species, Lunachloris lukesovae. Multiple conserved nucleotide positions identified in the secondary structures of the highly variable ITS2 rDNA barcoding marker provide further evidence of the phylogenetic position of Lunachloris. Minute vegetative cells of this newly recognized species are spherical or ellipsoid, with a single parietal chloroplast without a pyrenoid. Asexually, it reproduces by the formation of 2–6 autospores. Since the majority of recent attention has been paid to algae from the tropics or extreme habitats, the biodiversity of terrestrial microalgae in temperate regions is still notably unexplored and even a ‘common’ habitat like agricultural soil can contain new, as yet unknown species. Moreover, this study emphasizes the importance of culture collections of microorganisms even in the era of culture-independent biodiversity research, because they may harbour novel and undescribed organisms as well as preserving strains for future studies.     

Coccomyxa actinabiotis sp. nov. (Trebouxiophyceae,Chlorophyta), a new green microalga living in the spent fuel cooling pool of a nuclear reactor

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h^?1 to several thousands of Gy · h^?1, the microalga revealed to be able to survive intense gamma‐rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA‐Internal Transcribed Spacer (ITS) 1‐5.8S rRNA‐ITS2‐28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov.     

New genus Koliellopsis (Trebouxiophyceae, Chlorophyta): its phylogenetic position inferred from ultrastructure and nuclear ribosomal DNA sequences

Koliellopsis inundata Lokhorst gen. & sp. nov. (Trebouxiophyceae) is described from periodically flooded agricultural fields in the borderland of Belgium and the Netherlands. This new, unbranched, filamentous alga is typified by relatively long vegetative cells, which have a bilobed, laminate chloroplast with a nucleus positioned in its median constriction. Its filaments lack a distinct basal‐distal differentiation and both ends terminate in about equally shaped, rounded or, more often (slightly) tapering cells. Despite its semiterres‐trial occurrence the new alga does not attach to hard substrate, presumably owing to the lack of end cells to produce mucilage and to function as a holdfast. The systematic position of Koliellopsis among the green algae is inferred from ultrastructural examinations of the cell division patterns and from phylogenetic analyses of partial 18S rRNA gene sequences.     

The Morphology,Ultrastructure and SSU rRNA Gene Sequence of a New Freshwater Flagellate,Neobodo borokensis n. sp. (Kinetoplastea,Excavata)

A small free‐living freshwater bacteriotrophic flagellate Neobodo borokensis n. sp. was investigated by electron microscopy and analysis of its SSU ribosomal RNA gene. This protist has paraxonemal rods of typical bodonid structure in the flagella, mastigonemes on the proximal part of the posterior flagellum, two nearly parallel basal bodies, a compact kinetoplast, and discoid mitochondrial cristae. The flagellar pocket is supported by three microtubular roots (R1, R2 and R3) originating from the kinetosome. The cytopharynx is supported by the root R2, a microtubular prism, cytopharynx associated additional microtubules (CMT) and cytostome associated microtubules (FAS) bands. Symbiotic bacteria and small glycosomes were found in the cytoplasm. Cysts have not been found. The flagellate prefers freshwater habitats, but tolerates salinity up to 3–4‰. The overall morphological and ultrastructural features confirm that N. borokensis represents a new species of the genus Neobodo. Phylogenetic analysis of SSU rRNA genes is congruent with the ultrastructure and strongly supports the close relationship of N. borokensis to Neobodo saliens, N. designis, Actuariola, and a misidentified sequence of “Bodo curvifilus” within the class Kinetoplastea.