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

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

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

Delineating a New Heterothallic Species of <i>Volvox</i> (Volvocaceae, Chlorophyceae) Using New Strains of “<i>Volvox africanus</i>”

<div><p>The volvocine algae represent an excellent model lineage in which to study evolution of female and male genders based on comparative analyses of related species. Among these species, <i>Volvox carteri</i> has been extensively studied as a model of an oogamous and complex organism. However, it may have unique derived features that are not present in other species of <i>Volvox</i>. Therefore, information regarding the characteristics of sexual reproduction of other species of <i>Volvox</i> is also important. In 1971, Starr studied four types of sexuality in several global strains identified as <i>Volvox africanus</i>; however, further taxonomic studies of these strains have been lacking, and strains of three of the four sexual types are not available. Here, we studied the morphology, sexual reproduction, and taxonomy of two <i>V</i>. <i>africanus</i>-like species isolated recently from Lake Biwa, Japan. These two species were very similar to two sexual types described by Starr in 1971: one producing dioecious sexual spheroids in heterothallic strains and the other forming both male spheroids and monoecious spheroids in a single strain. The former species produced zygotes with a reticulate cell wall, whereas a smooth zygote wall was observed in the latter species as in <i>V</i>. <i>africanus</i> previously reported from various localities around the world. Our multigene phylogenetic analysis demonstrated that these are sister species to each other. However, the presence of a compensatory base change in the most conserved region of the secondary structure of nuclear ribosomal DNA internal transcribed spacer-2, hybrid inviability demonstrated by intercrossing experiments, and morphological differences in the density of abutment between the gelatinous material of adjacent cells (individual sheaths) in the spheroid supported the recognition of the two species, <i>V</i>. <i>africanus</i> having a smooth zygote wall and <i>V</i>. <i>reticuliferus</i> Nozaki sp. nov. having a reticulate zygote wall.</p></div

Phylogenetic positions of <i>Volvox reticuliferus</i> Nozaki sp. nov. and <i>V</i>. <i>africanus</i> G. S. West within the advanced members of the Volvocaceae (<i>Eudorina</i> group [8, 23]), as inferred from 6021 base pairs of five chloroplast genes.

<p>The tree was constructed by Bayesian inference unlinked GTR+I+G, F81+I+G and GTR+I+G models for the first, second and third codon positions in the five concatenated genes, respectively. Branch lengths are proportional to the genetic distances, which are indicated by the scale bar above the tree. Numbers on the left, middle, or right side at the branches represent posterior probabilities (PP) of BI (≥0.95), bootstrap values (≥50%, based on 1,000 replicates) obtained with the maximum likelihood and maximum parsimony analyses, respectively. Asterisks at the branches indicate 1.00 PP and 100% bootstrap values by the two methods. For details of the methods, see the text. Longitudinal dashed lines associated with “M” represent species of <i>Volvox</i> sect. <i>Merrillosphaera</i> sensu Smith [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142632#pone.0142632.ref002" target="_blank">2</a>].</p

Comparison of the most conserved region (near the YGGY motif of helix III [39]) of nuclear rDNA ITS-2 secondary structure between lineage RT (corresponding to <i>Volvox reticuliferus</i> Nozaki sp. nov., see Discussion of the main text), lineage AF (including <i>V</i>. <i>africanus</i> G. S. West) and “<i>V</i>. <i>africanus</i>” strain UTEX 1889 (Fig 3).

<p>Note the modified YGGY motif (boldface). For secondary structures of ITS-2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142632#pone.0142632.s005" target="_blank">S5 Fig</a>. Compensatory base changes between lineages RT/AF and “<i>V</i>. <i>africanus</i>” strain UTEX 1889 are marked with dotted open boxes, and those between lineages RT and AF are indicated by open boxes.</p

Nomarski interference microscopy of <i>Volvox reticuliferus</i> Nozaki sp. nov.

<p>(A) Surface view of asexual spheroids showing small somatic cells and larger reproductive cells (gonidia). (B, C) Somatic cells in the anterior portion of asexual spheroid showing lack of cytoplasmic bridges between cells. (B) Surface view showing stigma. (C) Optical section showing pyrenoid. (D, E) Asexual spheroid stained with dilute aniline blue, showing a broad secondary boundary layer (asterisk) of the gelatinous matrix surrounding each somatic cell. (D) Front view of somatic cells. (E) Side view of somatic cells. (F) Surface view of chloroplast of gonidium of asexual spheroid. Note presence of radial striations in the chloroplast surface. (G) Pre-inversion stage. Note morphological differentiation of gonidia of the next generation. (H, I) Mature male spheroids with sperm packets. (J) Female spheroid with eggs. (K, L) Two views of mature zygotes with a reticulate wall. Abbreviations: e, egg; g, gonidium; p, pyrenoid; s, stigma; sp, sperm packet. (A-C, H, I) Strain VO123-F1-7. (D-G) Strain VO123-F1-6. (D-G) Strain 2013-0703-VO2. (K, L) Strains 2013-0703-VO2x3.</p