Taxonomic revision of <i>Chloromonas nivalis</i> (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting <i>Chloromonas</i> species

<div><p><i>Chloromonas nivalis</i> (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting <i>Chloromonas</i> species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of <i>C</i>. <i>nivalis</i>; however, strains with such vegetative cells in snow-inhabiting species of <i>Chloromonas</i> have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several <i>C</i>. <i>nivalis</i> strains. The morphological data demonstrated that one North American strain could be identified as <i>C</i>. <i>nivalis</i>, whereas three other strains should be re-classified as <i>C</i>. <i>hoshawii</i> sp. nov. and <i>C</i>. <i>remiasii</i> sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of <i>C</i>. <i>nivalis</i> was phylogenetically separated from the Austrian and Japanese specimens previously identified as <i>C</i>. <i>nivalis</i> based on zygote morphology.</p></div

Genetic differences between <i>Chloromonas remiasii</i> Matsuzaki et al. sp. nov. and <i>C</i>. <i>chenangoensis</i> Hoham et al.

<p>(A) Comparison of the most conserved region (near the apex of helix III encompassing the YGGY motif) of nuclear rDNA ITS2 secondary structures. Open box indicates compensatory base change. Boldface marks the YGGY motif. For the complete nuclear rDNA ITS2 secondary structures, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193603#pone.0193603.s006" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193603#pone.0193603.s007" target="_blank">S7</a> Figs. (B) Nucleotide differences (%) from pairwise comparisons in four genes. Black: nuclear-encoded 1,748 bases of 18S ribosomal DNA (rDNA). Green: nuclear-encoded 2,020 bases of 26S rDNA. Red: chloroplast-encoded 1,128 bases of ATP synthase beta subunit gene (<i>atp</i>B). Blue: chloroplast-encoded 1,392 bases of P700 chlorophyll <i>a</i> apoprotein A2 gene (<i>psa</i>B). Note that the sequences from <i>Chloromonas remiasii</i> strains CCCryo 005–99 and CCCryo 047–99 were identical. The nucleotide differences between snow-inhabiting and mesophilic sister species [<i>C</i>. <i>hohamii</i> H.U. Ling et Seppelt vs. <i>C</i>. <i>tenuis</i> Matsuzaki et Nozaki; and <i>C</i>. <i>chlorococcoides</i> (H. Ettl et K. Schwarz) Matsuzaki et al. vs. <i>C</i>. <i>reticulata</i> (Goroschankin) Gobi] are according to the previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193603#pone.0193603.ref005" target="_blank">5</a>].</p

Vegetative cells of the three snow-inhabiting <i>Chloromonas</i> species: Line drawings.

<p>Identical magnification throughout. Left, optical section. Right, surface view. (A) <i>C</i>. <i>nivalis</i> (Chodat) Hoham et Mullet. (B) <i>C</i>. <i>hoshawii</i> Matsuzaki et al. sp. nov. (C) <i>C</i>. <i>remiasii</i> Matsuzaki et al. sp. nov.</p

Morphological characteristics of the three snow-inhabiting <i>Chloromonas</i> species.

<p>Morphological characteristics of the three snow-inhabiting <i>Chloromonas</i> species.</p

Vegetative cells of the three snow-inhabiting <i>Chloromonas</i> species: Transmission electron micrographs.

<p>Abbreviations: c, chloroplast; e, eyespot; G, Golgi body; m, mitochondrion; n, nucleus; v, vacuole with crystalline content. (A, B) <i>C</i>. <i>nivalis</i> (Chodat) Hoham et Mullet strain UTEX SNO71. (A) Longitudinal cell section. (B) Tangential cell section. (C, D) <i>C</i>. <i>hoshawii</i> Matsuzaki et al. sp. nov. strain UTEX SNO66. (C) Longitudinal cell section. (D) Tangential cell section. (E-G) <i>C</i>. <i>remiasii</i> Matsuzaki et al. sp. nov. strain CCCryo 005–99. (E) Longitudinal cell section. (F) Tangential cell section. (G) Eyespot composed of a single layer of electron-dense globules.</p

Vegetative cells of the three snow-inhabiting <i>Chloromonas</i> species: Light micrographs.

<p>Identical magnification throughout. Abbreviations: e, eyespot; n, nucleus. (A-D) <i>C</i>. <i>nivalis</i> (Chodat) Hoham et Mullet strain UTEX SNO71. (A) Optical section. (B) Epifluorescence image of (A). (C) Surface view. (D) Epifluorescence image of (C). (E-H) <i>C</i>. <i>hoshawii</i> Matsuzaki et al. sp. nov. strain UTEX SNO66. (E) Optical section. (F) Epifluorescence image of (E). (G) Surface view. (H) Epifluorescence image of (G). (I-L) <i>C</i>. <i>remiasii</i> Matsuzaki et al. sp. nov. strain CCCryo 005–99. (I) Optical section. (J) Epifluorescence image of (I). (K) Surface view. (L) Epifluorescence image of (K).</p

A Case Study for Effects of Operational Taxonomic Units from Intracellular Endoparasites and Ciliates on the Eukaryotic Phylogeny: Phylogenetic Position of the Haptophyta in Analyses of Multiple Slowly Evolving Genes

<div><p>Recent multigene phylogenetic analyses have contributed much to our understanding of eukaryotic phylogeny. However, the phylogenetic positions of various lineages within the eukaryotes have remained unresolved or in conflict between different phylogenetic studies. These phylogenetic ambiguities might have resulted from mixtures or integration from various factors including limited taxon sampling, missing data in the alignment, saturations of rapidly evolving genes, mixed analyses of short- and long-branched operational taxonomic units (OTUs), intracellular endoparasite and ciliate OTUs with unusual substitution etc. In order to evaluate the effects from intracellular endoparasite and ciliate OTUs co-analyzed on the eukaryotic phylogeny and simplify the results, we here used two different sets of data matrices of multiple slowly evolving genes with small amounts of missing data and examined the phylogenetic position of the secondary photosynthetic chromalveolates Haptophyta, one of the most abundant groups of oceanic phytoplankton and significant primary producers. In both sets, a robust sister relationship between Haptophyta and SAR (stramenopiles, alveolates, rhizarians, or SA [stramenopiles and alveolates]) was resolved when intracellular endoparasite/ciliate OTUs were excluded, but not in their presence. Based on comparisons of character optimizations on a fixed tree (with a clade composed of haptophytes and SAR or SA), disruption of the monophyly between haptophytes and SAR (or SA) in the presence of intracellular endoparasite/ciliate OTUs can be considered to be a result of multiple evolutionary reversals of character positions that supported the synapomorphy of the haptophyte and SAR (or SA) clade in the absence of intracellular endoparasite/ciliate OTUs.</p> </div

List of the volvocalean strains and their presence or absence of rickettsial endosymbionts examined in this study.

a<p>Microbial Culture Collection at the National Institute for Environmental Studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031749#pone.0031749-Kasai1" target="_blank">[24]</a>.</p>b<p>Culture Collection of Algae at the University of Texas at Austin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031749#pone.0031749-Starr1" target="_blank">[50]</a>.</p>c<p>Determined in this study.</p>d<p>Based on TEM by Nozaki et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031749#pone.0031749-Nozaki4" target="_blank">[22]</a>.</p>e<p>Based on TEM and DAPI-staining by Nozaki et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031749#pone.0031749-Nozaki2" target="_blank">[20]</a>.</p

FISH identification of rickettsiacean endosymbionts in <i>Carteria cerasiformis</i> cells.

<p><b>A–C.. </b><i>C. cerasiformis</i> NIES-425. <b>D–F.. </b><i>C. cerasiformis</i> NIES-424. Horizontal panels show the same cells, composed of Nomarski differential interference images (<b>A, D</b>), epifluorescence images with DAPI staining (<b>B, E</b>) and epifluorescence images with the volv-835 probe specific for the endosymbiont of <i>C. cerasiformis</i> NIES-425 (<b>C</b>, <b>F</b>; for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031749#s4" target="_blank">Materials and Methods</a>). Arrowheads point to the signals from the endosymbionts. The green signals (<b>C</b>) represent endosymbiont-specific probes and the yellow background (<b>C</b>, <b>F</b>) is autofluorescence. All are shown at the same magnification. The ‘n’ indicates host cell nuclei.</p

Eukaryotic phylogeny based on nuclear-encoded protein sequences plus nucleotide sequences of 18S rRNA genes using M 10:16 (modified from the data matrix by Parfrey et al.[<b>29</b>]), including (A) and excluding (B) intracellular endoparasite/ciliate OTUs.

<p>The analysis is based on the concatenated dataset of slowly evolving nuclear proteins (15 proteins; 5710 amino acid positions) and 18S rRNA genes (868 nucleotide positions). The tree was prepared using RAxML with the WAG+4G model (for amino acid positions) and the GTR+4G model (for nucleotide positions). Numbers at the left or right side at the branches represent BV (≥50%) obtained using 1,000 replicates with the RAxML or MP analysis, respectively. Asterisks at the branches indicate 100% BV by all three methods.</p