Fig 1.
Nomarski interference microscopy of Volvox reticuliferus Nozaki sp. nov.
(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.
Fig 2.
Phylogenetic positions of Volvox reticuliferus Nozaki sp. nov. and V. africanus G. S. West within the advanced members of the Volvocaceae (Eudorina group [8, 23]), as inferred from 6021 base pairs of five chloroplast genes.
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 Volvox sect. Merrillosphaera sensu Smith [2].
Fig 3.
Phylogenetic relationships of various strains of Volvox reticuliferus Nozaki sp. nov. and V. africanus G. S. West, as inferred from internal transcribed spacer region 2 (ITS-2) of nuclear ribosomal DNA.
The tree was constructed by the maximum likelihood (ML) method (based on T92+G model). Branch lengths are proportional to the genetic distances, which are indicated by the scale bar above the tree. Numbers on the left or right side at the branches represent bootstrap values (≥50%, based on 1,000 replicates) obtained with the ML calculation and maximum parsimonious analysis (using a branch-and-bound search), respectively. Sexual types (S1 Table) are shown right to the strain designations.
Fig 4.
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 Volvox reticuliferus Nozaki sp. nov., see Discussion of the main text), lineage AF (including V. africanus G. S. West) and “V. africanus” strain UTEX 1889 (Fig 3).
Note the modified YGGY motif (boldface). For secondary structures of ITS-2, see S5 Fig. Compensatory base changes between lineages RT/AF and “V. africanus” strain UTEX 1889 are marked with dotted open boxes, and those between lineages RT and AF are indicated by open boxes.