The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae, Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica.

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.

Chlorella: 125 years of the green survivalist.

Chlorella, the archetype of unicellular green algae, is a high-performance primary producer in aquatic and terrestrial ecosystems. Under the simple spherical morphology of Chlorella, many other 'green balls' unfolded as independent phylogenetic lineages as a result of convergent evolution. By contrast, green algae with strikingly different phenotypes were unmasked as close relatives of Chlorella by modern molecular techniques. Here, we point to the increasing impact of these diverse protists on ecology, evolution, and biotechnology in the light of integrative taxonomy.

Presence of potential toxin-producing cyanobacteria in an oligo-mesotrophic lake in Baltic Lake District, Germany: an ecological, genetic and toxicological survey.

Massive developments of potentially toxic cyanobacteria in Lake Stechlin, an oligo-mesotrophic lake in the Baltic Lake District of Germany raised concerns about toxic contamination of these important ecosystems. Field samples in the phase of mass developments of cyanobacteria were used for genetic and toxicological analyses. Microcystins and microcystin genes were detected in field samples of the lake for the first time. However, the toxins were not produced by the dominant taxa (Dolichospermum circinale and Aphanizomenon flos-aquae) but by taxa, which were present only in low biomass in the samples (Microcystis cf. aeruginosa and Planktothrix rubescens). The phytoplankton successions during the study period revealed an increase of cyanobacterial populations. The findings contribute to the changes that have been investigated in Lake Stechlin since the mid-1990s. The possible reasons behind these developments may be climate change, special weather conditions and an increased nutrient pool.

Cyanobacterial diversity in the hot spring, pelagic and benthic habitats of a tropical soda lake.

Hot springs and saline-alkaline lakes of East Africa are extreme habitats regarding temperature, or salinity and pH, respectively. This study examines whether divergent habitats of Lake Bogoria, Kenya, impacts cyanobacterial community structure. Samples from the hot springs, pelagic zone and sediment were analysed by light microscopy, multilocus 454-amplicons sequencing and metagenomics to compare the cyanobacterial diversity. Most of the phylogenetic lineages of Cyanobacteria occurred exclusively in the Bogoria hot springs suggesting a high degree of endemism. The prevalent phylotypes were mainly members of the Oscillatoriales (Leptolyngbya, Spirulina, Oscillatoria-like and Planktothricoides). The Chroococcales were represented by different clades of Synechococcus but not a single phylotype clustered with any of the lineages described earlier from different continents. In contrast, we found that the pelagic zone and the sediments were inhabited by only a few taxa, dominated by Arthrospira and Anabaenopsis. Arthrospira, the main food base of Lesser Flamingo, was detected in all three habitats by amplicons pyrosequencing, indicating its resilience and key role as a primary producer. Despite the close connection between the three habitats studied, the cyanobacterial communities in the hot springs and lake differed considerably, suggesting that they are unable to adapt to the extreme conditions of the neighbouring habitat.

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia, Tetrastrum spp. Crucigenia tetrapedia, and C. lauterbornii (Trebouxiophyceae, Chlorophyta)(1).

The subfamily Crucigenioideae was traditionally classified within the well-characterized family Scenedesmaceae (Chlorophyceae). Several morpho-logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer-mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.

The systematics of Zoochlorella revisited employing an integrative approach.

Symbiosis of green algae with protozoa and invertebrates has been studied for more than 100 years. Endosymbiotic green algae are widely distributed in ciliates (e.g. Paramecium, Stentor, Climacostomum, Coleps, Euplotes), heliozoa (e.g. Acanthocystis) and invertebrates (e.g. Hydra, Spongilla), and have traditionally been identified as named or unnamed species of Chlorella Beij. or Zoochlorella K. Brandt or referred to as Chlorella-like algae or zoochlorellae. We studied 17 strains of endosymbionts isolated from various hosts and geographical localities using an integrative approach (nuclear encoded small subunit and internal transcribed spacer regions of rRNA gene sequences including their secondary structures, morphology, physiology and virus sensitivity). Phylogenetic analyses have revealed them to be polyphyletic. The strains examined belong to five independent clades within the Trebouxiophyceae (Choricystis-, Elliptochloris-, Auxenochlorella- and Chlorella-clades) and Chlorophyceae (Scenedesmus-clade). The most studied host organism, Paramecium bursaria, harbours endosymbionts representing at least five different species. On the basis of our results, we propose a taxonomic revision of endosymbiotic 'Chlorella'-like green algae. Zoochlorella conductrix K. Brandt is transferred to Micractinium Fresen. and Zoochlorella parasitica K. Brandt to Choricystis (Skuja) Fott. It was shown that Choricystis minor (Skuja) Fott, the generitype, is a later heterotypic synonym of Choricystis parasitica (K. Brandt) comb. nov. A new species, Chlorella heliozoae, is proposed to accommodate the endosymbiont of Acanthocystis turfacea.

SSU rRNA GENE PHYLOGENY OF MORPHOSPECIES AFFILIATED TO THE BIOASSAY ALGA "SELENASTRUM CAPRICORNUTUM" RECOVERED THE POLYPHYLETIC ORIGIN OF CRESCENT-SHAPED CHLOROPHYTA(1).

The generic concept of coccoid green algae exhibiting a crescent-shaped morphotype is evaluated using SSU rRNA gene sequence analyses and light and electron microscopical observations. These common chlorophytes evolved polyphyletically in 10 different clades of the Chlorophyceae and three clades of the Trebouxiophyceae. Six clades are assigned to known genera of Selenastraceae: Kirchneriella, Nephrochlamys, Raphidocelis, Rhombocystis, Selenastrum, and Tetranephris. Four other clades, named following their present genus designation as Ankistrodesmus-like I and II and Monoraphidium-like I and II, require further investigation. One crescent-shaped morphotype, which evolved within the Trebouxiophyceae, is designated as Neocystis mucosa sp. nov. The other two lineages containing trebouxiophycean algae with this morphotype are the Elliptochloris and the Watanabea clades. The taxonomic placement of the widely used bioassay strain "Selenastrum capricornutum" NIVA-CHL 1 in the genus Raphidocelis (species name Raphidocelis subcapitata) is indicated by molecular data.

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1.

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS-1, and ITS-2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium-like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.

Molecular detection of uncultured cyanobacteria and aminotransferase domains for cyanotoxin production in sediments of different Kenyan lakes.

PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments was used to identify the cyanobacterial phylotypes in sediments and plankton of saline-alkaline and freshwater lakes of Kenya. The detection of the aminotransferase domain located on modules mcyE and ndaF using specific molecular markers confirmed the presence of potential toxin-producing cyanobacteria. The eight nucleotide sequences obtained from DGGE bands were placed in three divergent cyanobacterial clusters. Five nucleotide sequences were close to members of the genera Anabaenopsis and Umezakia (Nostocales), two sequences fell in the cluster with Arthrospira sp. (Oscillatoriales) and one sequence was related to Chroococcidiopsis sp. (Pleurocapsales). The presence of the latter taxon was demonstrated de novo in the investigated lakes. All nine attained nucleotide sequences of the aminotransferase region belonged to the mcyE module. Five sequences of the aminotransferase domain were included in the cluster having the nucleotide sequence of Anabaena sp. but showed a separate lineage. Other four aminotransferases were placed in the cluster represented by nucleotide sequence of Microcystis aeruginosa. To our knowledge, this is the first report on molecular detection of cyanobacterial phylotypes in sediments of African lakes and aminotransferase domains for cyanotoxin production from sediment samples in general.

TAXONOMIC REEXAMINATION OF CHARA GLOBULARIS (CHARALES, CHAROPHYCEAE) FROM JAPAN BASED ON OOSPORE MORPHOLOGY AND rbcL GENE SEQUENCES, AND THE DESCRIPTION OF C. LEPTOSPORA SP. NOV.(1).

Chara globularis Thuillier (=f. globularis sensu R. D. Wood) is a widespread species of the genus and inhabits fresh- and brackish-water environments. In an attempt to reexamine the taxonomic status of C. globularis collected from Japan, we reassessed vegetative and oospore morphology of Japanese material and herbarium specimens originating from Europe (including the type specimen) and conducted molecular phylogenetic analyses based on rbcL gene sequences. Although the other vegetative morphologies were consistent with the description of C. globularis f. globularis sensu R. D. Wood, we identified two types of branchlets within the Japanese materials: one has elongate end segments (EL type), and the other has short end segments (SH type) corresponding to the type material. Moreover, the oospore wall of the EL type was different from that in the SH type. The oospores of the EL type were dark brown to reddish brown and had a spongy pattern with the pusticular elevations on the fossa wall, whereas the fossa wall of the SH type was black with a granulate to papillate or fine pusticular pattern. In addition, our sequence data demonstrated that these two types are separated phylogenetically from each other. Therefore, we describe the EL type as a new species, C. leptospora sp. nov.

Morphological and phylogenetic analysis of Anabaenopsis abijatae and Anabaenopsis elenkinii (nostocales, cyanobacteria) from tropical inland water bodies.

Anabaenopsis spp. are heterocytous cyanobacteria commonly found in tropical, subtropical, and temperate water bodies. So far, the knowledge about the phylogeny of this genus is poor. Therefore, we have isolated 15 Anabaenopsis spp. strains from Kenyan and Mexican alkaline and saline water bodies and from a Ugandan freshwater body and studied the morphology and phylogeny in a polyphasic approach. Morphologically, the investigated strains could be discriminated in two groups. One group was containing six Anabaenopsis abijatae and A. cf. abijatae strains with up to more than 500 vegetative cells in one filament, mostly single intercalary heterocyte formation, and the ability to branch out. The other group comprised nine strains of Anabaenopsis elenkinii with short filaments with up to 38 vegetative cells, intercalary heterocytes in pairs, and no ability to branch out. The morphological differences were reflected in the two distinct clusters, which were found in the phylogenetic trees of 16S rDNA and PC-IGS. While the high 16S rDNA similarity values > 97.5% found between all investigated A. abijatae and A. elenkinii strains support the assignment of these two species to one single genus, the morphological differences and the low similarity values (< 87.3) found in PC-IGS sequences between the two clusters indicate two separate genera. A close morphological and phylogenetic relationship was found for A. abijatae and Anabaenopsis (Cyanospira) rippkae.

Toxic cyanobacteria and their toxins in standing waters of Kenya: implications for water resource use.

Phytoplankton biodiversity studies in Kenya's standing waters were carried out between 2001 and 2003. Toxin producing cyanobacteria were recorded in twelve water bodies. Microcystis and Anabaena were the most common species in freshwaters while Anabaena and Anabaenopsis were common in alkaline saline lakes. Seven lakes with cyanobacteria blooms and a hot spring had detectable levels of microcystins and anatoxin-a. Cell bound microcystins (LR equivalents) concentration ranged from 1.6-19800 microgg(-1) Dry Weight (DW) while anatoxin-a varied from below the limit of detection to 1260 microgg(-1) DW. In alkaline-saline lakes, microcystins and anatoxin-a were also present in stomach contents and liver samples of dead flamingos. Monoculture strains of A. fusiformis from Lakes Sonachi and Bogoria had detectable levels of microcystins while anatoxin-a was present in strains isolated from Lakes Sonachi, Bogoria and Nakuru. Two freshwater sites, Nyanza Gulf (L. Victoria) and Lake Baringo recorded cyanotoxin concentration exceeding WHO'S upper limit of 1.0 microgl(-1) for drinking water. The results confirm that cyanotoxins could have played a role in the mortality of flamingos in Lakes Bogoria and Nakuru. The implications of these findings on water resource use, measures to be taken to reduce the risk of exposure and eutrophication control steps to reduce cyanobacteria bloom formation are considered in this paper.

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae).

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.

Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya.

Cyanobacterial mats at hot springs on the shore of the alkaline Lake Bogoria, Kenya, were investigated regarding species community and cyanobacterial toxin content. The hepatotoxins microcystin-LR, -RR, -LF and -YR, and the neurotoxin anatoxin-a were present. The mats were dominated by Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. The concentration of microcystins in mat samples, ranged from 221 to 845 microg microcystin-LR equivalents g(-1) DW of mat. Anatoxin-a concentrations ranged from 10 to 18 microg g(-1) DW of mat. A contribution of the cyanobacterial toxins from the hot spring mats to the mass mortalities of Lesser Flamingos is suggested by: (a), the presence of hot spring cyanobacterial cells and cell fragments, and high concentrations of the cyanobacterial hepato- and neurotoxins in flamingo stomach contents and faecal pellets; (b), observations of neurological signs of bird poisoning at the lake. Cyanobacterial toxins in stomach contents, intestine and fecal pellets were 0.196 microg g(-1) fresh weight (FW) for the microcystins and 4.34 microg g(-1) FW for anatoxin-a. Intoxication with cyanobacterial toxins could occur by uptake of detached cyanobacterial cells from the mats, as the flamingos need to drink fresh or brackish water, and to wash their feathers daily, which they do in the vicinity of the hot springs, where salinity is lower than in the main body of water of the lake.

PHYLOGENETIC POSITION OF THE OOCYSTACEAE (CHLOROPHYTA).

The complete 18S rRNA gene sequences of three Oocystis A. Braun species (Oocystaceae) and three other chlorococcal algae, Tetrachlorella alternans (G. M. Smith) Kors. (Scenedesmaceae), Makinoella tosaensis Okada (Scenedesmaceae), and Amphikrikos cf. nanus (Fott & Heynig) Hind. (Chlorellaceae) were determined and subjected to four different phylogenetic analysis algorithms. Independent of the reconstruction method, these taxa clustered together as a monophyletic group (Oocystaceae) within the Trebouxiophyceae. This result was supported by high bootstrap values. A comparison of morphological data with the phylogenetic reconstructions indicated that the evolution of Oocystaceae was accompanied by a reduction in the number of plastids. This study fully supports the taxonomic assignment of the Oocystaceae as a distinct family. The diacritic criterion that the cell walls are composed of several cellulose layers with perpendicular fibril orientations is in accordance with the molecular data.