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.

[1]  K. Brandt Über die morphologische und physiologische Bedeutung des Chlorophylls bei Thieren , 1883 .

[2]  J. B. Loefer Isolation and Growth Characteristics of the "Zoochlorella" of Paramecium bursaria , 1936, The American Naturalist.

[3]  H. Skuja Taxonomie des Phytoplanktons einiger Seen in Uppland, Schweden , 1948 .

[4]  R. Krauss,et al.  Chlorella, physiology and taxonomy of forty-one isolates , 1965 .

[5]  John J. Lee,et al.  The taxonomic identity and physiological ecology of Chlamydomonas hedleyi sp. nov. algal flagellate symbiont from the foraminifer Archaias angulatus , 1974 .

[6]  T. M. Sonneborn The Paramecium aurelia Complex of Fourteen Sibling Species , 1975 .

[7]  F. Schuster,et al.  Symbiosis and the evolution of larger foraminifera , 1979 .

[8]  J. J. Lee,et al.  Symbiotic Chlorella Species from Larger Foraminifera , 1982 .

[9]  J. Komárek,et al.  Chlorophyceae (Grünalgen) ; Ordnung: Chlorococcales , 1983 .

[10]  W. Reisser,et al.  Autotrophic Eukaryotic Freshwater Symbionts , 1984 .

[11]  W. Reisser The taxonomy of green algae endosymbiotic in ciliates and a sponge , 1984 .

[12]  T. Kalina,et al.  Taxonomy of the subfamily Scotiellocystoideae Fott 1976 (Chlorellaceae, Chlorophyceae) , 1987 .

[13]  W. Reisser,et al.  Taxonomic studies on endocytobiotic chlorophycean algae isolated from different american and european strains of Paramecium bursaria , 1988 .

[14]  J. V. Van Etten,et al.  A comparison of viruses infecting two different Chlorella-like green algae. , 1988, Virology.

[15]  M. Raht,et al.  Symbiococcum hydrae gen. et sp. nov. (Chlorosarcinales, Chlorophyta): an endosymbiotic green alga from cells of the Japanese Hydra magnipapillata (Coelenterata) , 1989 .

[16]  V. Huss,et al.  Biochemical Taxonomy of Symbiotic Chlorella Strains from Paramecium and Acanthocystis , 1990 .

[17]  V. Reich,et al.  Zoospore formation by Symbiococcum hydrae (Chlorosarcinales, Chlorophyta), an alga endosymbiotic in Hydra magnipapillata (Coelenterata) , 1991 .

[18]  M. Rahat,et al.  THERE IS AN ECOLOGICAL BASIS FOR HOST/SYMBIONT SPECIFICITY IN CHLORELLA/HYDRA SYMBIOSES , 1993 .

[19]  W. Reisser Enigmatic chlorophycean algae forming symbiotic associations with ciliates , 1994 .

[20]  V. Huss,et al.  Picoplanktonic Choricystis species (Chlorococcales, Chlorophyta) and problems surrounding the morphologically similar ‘Nannochloris-like algae’ , 1996 .

[21]  A. Coleman,et al.  Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness. , 1997, Journal of molecular evolution.

[22]  U. Schlösser Additions to the Culture Collection of Algae since 1994 , 1997 .

[23]  David Posada,et al.  MODELTEST: testing the model of DNA substitution , 1998, Bioinform..

[24]  Paul C. Silva 1379) Proposal to Conserve the Name Chlorella against Zoochlorella (Chlorophyceae) , 1999 .

[25]  U. Schlosser Additions to the culture collection of algae at Gottingen since 1997 , 2000 .

[26]  M. Melkonian,et al.  Molecular phylogeny and taxonomic revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and description of Oogamochlamys gen. nov. and Lobochlamys gen. nov. , 2001, Protist.

[27]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[28]  D. Swofford PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4.0b10 , 2002 .

[29]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[30]  Miho Nakahara,et al.  Choricystis minor as a new symbiont of simultaneous two-species association with Paramecium bursaria and implications for its phylogeny , 2004 .

[31]  R. Hoshina,et al.  Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan. , 2004, Plant biology.

[32]  E. Pringsheim Die Sammlung von Algen-, Flagellaten- und Mooskulturen am Botanischen Institut der Universität Cambridge , 1951, Archiv für Mikrobiologie.

[33]  M. Wolf,et al.  Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae) , 2004 .

[34]  D. Posada,et al.  Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. , 2004, Systematic biology.

[35]  J. V. Van Etten,et al.  Chlorovirus: a genus of Phycodnaviridae that infects certain chlorella-like green algae. , 2005, Molecular plant pathology.

[36]  W. Reisser The endosymbiotic unit ofStentor polymorphus andChlorella sp. morphological and physiological studies , 1981, Protoplasma.

[37]  R. Hoshina,et al.  Genetic evidence of "American" and "European" type symbiotic algae of Paramecium bursaria Ehrenberg. , 2005, Plant biology.

[38]  A. Coleman,et al.  Paramecium aurelia Revisited , 2005, The Journal of eukaryotic microbiology.

[39]  T. Pröschold,et al.  Effect of external pH on the growth, photosynthesis and photosynthetic electron transport of Chlamydomonas acidophila Negoro, isolated from an extremely acidic lake (pH 2.6) , 2005 .

[40]  Tobias Müller,et al.  4SALE – A tool for synchronous RNA sequence and secondary structure alignment and editing , 2006, BMC Bioinformatics.

[41]  K. Kvitko,et al.  Genomic dactyloscopy of Chlorella sp., symbionts of Paramecium bursaria , 2006 .

[42]  半田 信司,et al.  タンスイカイメンから得られた共生藻 Choricystis minor (トレボウクシア藻鋼,緑色植物門) , 2006 .

[43]  Lisa A. Fitzgerald,et al.  Phycodnaviruses: a peek at genetic diversity. , 2006, Virus research.

[44]  T. Pröschold,et al.  Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae). , 2006, Protist.

[45]  半田 信司,et al.  Choricystis minor (Trebouxiophyceae, Chlorophyta) as a symbiont of several species of freshwater sponge , 2006 .

[46]  R. Sommaruga,et al.  An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella. , 2007, Environmental microbiology.

[47]  T. Pröschold,et al.  Systematics of the green algae: Conflict of classic and modern approaches , 2007 .

[48]  R. Hoshina,et al.  Multiple origins of the symbioses in Paramecium bursaria. , 2008, Protist.

[49]  R. Sommaruga,et al.  CILIATE‐SYMBIONT SPECIFICITY OF FRESHWATER ENDOSYMBIOTIC CHLORELLA (TREBOUXIOPHYCEAE, CHLOROPHYTA) 1 , 2008, Journal of phycology.

[50]  Thomas Dandekar,et al.  Synchronous visual analysis and editing of RNA sequence and secondary structure alignments using 4SALE , 2008, BMC Research Notes.

[51]  A. Coleman,et al.  Ribosomal DNA and ITS-2 sequence comparisons as a tool for predicting genetic relatedness , 1997, Journal of Molecular Evolution.

[52]  C. Bock,et al.  Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae). , 2010, Plant biology.

[53]  U. Karsten,et al.  Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta) , 2010 .

[54]  R. Hoshina,et al.  Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae, Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year , 2010 .

[55]  Prasanta K. Jana,et al.  A Monograph on , 2011 .