Inflated organelle genomes and a circular-mapping mtDNA probably existed at the origin of coloniality in volvocine green algae

<p>The volvocine lineage is a monophyletic grouping of unicellular, colonial and multicellular algae, and a model for studying the evolution of multicellularity. In addition to being morphologically diverse, volvocine algae boast a surprising amount of organelle genomic variation. Moreover, volvocine organelle genome complexity appears to scale positively with organismal complexity. However, the organelle DNA architecture at the origin of colonial living is not known. To examine this issue, we sequenced the plastid and mitochondrial DNAs (ptDNA and mtDNA) of the 4-celled alga <i>Tetrabaena socialis</i>, which is basal to the colonial and multicellular volvocines.</p> <p><i>Tetrabaena</i><i>socialis</i> has a circular-mapping mitochondrial genome, contrasting with the linear mtDNA architecture of its relative <i>Chlamydomonas reinhardtii</i>. This suggests that a circular-mapping mtDNA conformation emerged at or near the transition to group living in the volvocines, or represents the ancestral state of the lineage as a whole. The <i>T. socialis</i> ptDNA is very large (>405 kb) and dense with repeats, supporting the idea that a shift from a unicellular to a colonial existence coincided with organelle genomic expansion, potentially as a result of increased random genetic drift. These data reinforce the idea that volvocine algae harbour some of the most expanded plastid chromosomes from the eukaryotic tree of life. Circular-mapping mtDNAs are turning out to be more common within volvocines than originally thought, particularly for colonial and multicellular species. Altogether, volvocine organelle genomes became markedly more inflated during the evolution of multicellularity, but complex organelle genomes appear to have existed at the very beginning of colonial living.</p

Additional file 5: Figure S1. of Alternative evolution of a spheroidal colony in volvocine algae: developmental analysis of embryogenesis in Astrephomene (Volvocales, Chlorophyta)

Morphological observation of vegetative colonies in Astrephomene gubernaculifera strain 2014-1002-YkAs8. Figure S2. Maximum-likelihood tree of Astrephomene and Gonium based on rbcL genes. Figure S3. Diagram of making preparations for light microscopy time-lapse imaging of Astrephomene embryogenesis. Figure S4. Western blot of two species with anti-CrSAS-6 antibody. (PDF 688 kb

Additional file 2 of Evolution of cytokinesis-related protein localization during the emergence of multicellularity in volvocine green algae

Figure S1. Alignment of DRP1A from Arabidopsis thaliana (At) and DRP1 from Chlamydomonas reinhardtii (Cr), Tetrabaena socialis (Ts), Gonium pectorale (Gp) and Volvox carteri (Vc). Black and gray background indicates identical or similar amino acid, respectively. GTPase domain, dynamin middle domain, and GTPase effector domain are indicated by pink, green, and yellow background color, respectively. The region corresponding to the antigen for an anti-TsDRP1 antibody is showed under the alignment (gray bar). Figure S2. Specificity of the affinity-purified anti-TsDRP1 antibody. The specificity of the anti-TsDRP1 antibody was validated in three volvocine algae by western blotting. A single band was detected in each lane (~75 kDa) with the antibody that was incubated with acetone powder of E. coli with the empty vector (left) while no signal was detected with the antibody that was incubated with acetone powder of E. coli expressing TsDRP1 (middle). For details of the methods, see Information S1 (Additional file 2). Figure S3. Western blot analyses of DRP1 proteins of Chlamydomonas reinhardtii (CrDRP1), Tetrabaena socialis (TsDRP1), and Gonium pectorale (GpDRP1) using anti-TsDRP1 antibody. Time-course of synchronous culture and western blot (WB) of C. reinhardtii, T. socialis, and G. pectorale are shown in a, b, and c, respectively. Time-course samples were obtained from five points (arrows in each line graph): the greatest number of dividing cells (0), three (−3) and six (−6) hours before 0 point, and three (+3) and six (+6) hours after 0 point. Coomassie brilliant blue (CBB) staining of a duplicate gel shows the equal protein loading in each lane. Information S1. Methods for specificity of the affinity-purified anti-TsDRP1 antibody (Additional file 2: Figure S2). (PDF 1785 kb

The Simplest Integrated Multicellular Organism Unveiled

<div><p>Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular <i>Chlamydomonas</i> to >500-celled <i>Volvox</i>. Multicellular volvocalean species including <i>Gonium pectorale</i> and <i>Volvox carteri</i> generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species <i>Tetrabaena socialis</i> that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of <i>T. socialis</i> and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature <i>T. socialis</i> colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like <i>G. pectorale</i> and <i>V. carteri</i>. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of <i>T. socialis</i> even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. <i>T. socialis</i> is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.</p></div

List of three volvocine algae used in this study.

<p>List of three volvocine algae used in this study.</p

Light and transmission electron microscopy of vegetative colonies of <i>Tetrabaena socialis</i> NIES-571.

<p>(A–C) Three light microscopic views of four-celled vegetative colony, showing positions of flagella and eyespots. Note that flagella (asterisks) and eyespots (arrowheads) are arranged in symmetric pattern in the whole colony. (D, E) Transmission electron microscopy. ECM, extracellular matrix; ce, chloroplast envelope; ch, chloroplast; cm, cell membrane; n, nucleus; p, pyrenoid. (D) Longitudinal section of vegetative colony. (E) Eyespot composed three layers of globules.</p

Transmission electron microscopy of cytokinesis of four-celled embryos of <i>Tetrabaena socialis</i> NIES-571.

<p>(A) Transverse section of almost central part of four daughter protoplasts before formation of new flagella. Note a daughter protoplast connected to two neighbors by cytoplasmic bridges (arrowheads). (B) Semi-longitudinal section of daughter protoplasts after formation of new flagella (large frame) within parental extracellular matrix (ECM) (asterisks). Note two protoplast connected to each other by cytoplasmic bridges (small frame). (C, D) Enlarged images of two frames in (B). (C) New flagella (arrows) within parental ECM (asterisks). (D) Cytoplasmic bridges (arrowhead) connecting two daughter protoplasts.</p

Rough outline of phylogenetic relationships in volvocine green algae [9], [10], [15].

<p>Rough outline of phylogenetic relationships in volvocine green algae <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081641#pone.0081641-Nozaki1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081641#pone.0081641-Herron1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081641#pone.0081641-Nozaki3" target="_blank">[15]</a>.</p

Images and diagrams of microtubular rootlet (MTR) and basal bodies (BB)/pro-basal bodies (pBB).

<p>(A–I) Immunofluorescence microscopy. (A–C) Double stained fluorescence of acetylated tubulin and CrSAS-6 showing MTR and BB/pBB, respectively. Each scale bar represents 5 µm. (A) <i>Chlamydomonas reinhardtii</i>. (B) <i>Tetrabaena socialis</i>. (C) <i>Gonium pectorale</i>. (D–F) Fluorescence of acetylated tubulin. Each white arrowhead or asterisk indicates distal end of MTR or flagellum, respectively. Each scale bar represents 1 µm. Upper sides of panels E and F represent the directions of center in the flattened colonies. (D) <i>C. reinhardtii</i>. (E) <i>T. socialis.</i> (F) <i>G. pectorale</i>. (G–I) Fluorescence of CrSAS-6. Each arrow or arrowhead indicates BB or pBB, respectively. Each scale bar represents 1 µm. Upper sides of panels H and I represent the directions of center in the flattened colonies. (G) <i>C. reinhardtii.</i> (H) <i>T. socialis.</i> (I) <i>G. pectorale.</i> (J–L) Diagrams of MTR and BB/pBB arrangements. Upper sides of panels K and L represent the directions of center in the flattened colonies. (J) <i>C. reinhardtii.</i> (K) <i>T. socialis.</i> (L) <i>G. pectorale.</i> (M, N) Transmission electron microscopy of <i>T. socialis</i>. ECM, extracellular matrix; cm, cell membrane; df, distal fiber; pf, proximal fiber; asterisk, flagellar proper. (M) Longitudinal section of anterior end of cell showing BB and distal fiber. Note proximal ends of the two BB (white arrows) are separated from each other. (N) Longitudinal section of anterior end of cell showing BB with proximal fiber.</p

Time course of synchronous culture of <i>Tetrabaena socialis</i> NIES-571.

<p>Light-dark cycle (light:dark = 12 h:12 h) were indicated on the horizontal axis, percentages of cells during cytokinesis were indicated on a vertical line of left side with a pink line, and number of cells were indicated on a right side with a green line. Each error bars shows standard deviation (n = 3).</p