Cellular aggregation in <i>Chlamydomonas</i> (Chlorophyceae) is chimaeric and depends on traits like cell size and motility

<p>How the variation in phenotypic traits like cell size and motility impacts predator-induced cellular aggregation is not known. Furthermore the genetic composition of cell groups in mixed populations of <i>Chlamydomonas</i> has not been investigated. An examination of these two questions will not only enhance our understanding of <i>Chlamydomonas</i> ecology, but also shed light on the primordial steps before integrated multicellular groups were established. Group living comes with viability and reproductive costs and it is not known how these are shared if groups are genetically heterogeneous. We observed that the natural predator <i>Peranema trichophorum</i> (Euglenoidea) induced clumping in <i>Chlamydomonas</i>. When co-cultured with <i>P. trichophorum</i> cells protected themselves by forming facultative groups (reverting back to a unicellular lifestyle once predators were removed). The dynamics of group formation in different <i>Chlamydomonas</i> species and strains correlated with cell size and swimming speed. Small or less motile strains aggregated more readily than large, fast-swimming ones. Interestingly, <i>Chlamydomonas</i> groups were both intra-species and inter-species chimaeric. This suggests that the predator-induced group formation in <i>Chlamydomonas</i> involved cells coming together rather than staying together and during aggregation cells showed little or no discrimination between self and non-self. These data demonstrate that the dynamics of cell aggregation, in unicellular volvocines at least, depends on phenotypic traits like cell size and motility and high genetic relatedness is not mandatory at this initial stage. These findings further our understanding of aggregation in mixed <i>Chlamydomonas</i> populations and have implications for understanding the very first steps on the road to simple multicellularity.</p

count data

Cell count raw data for Fig 2 in main tex

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

Supplementary Figures providing detailed experimental protocols and methodologies from Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

The supplementary figures provide details of the experimental protocols and methodologies as well as the raw data for ligation reaction

Supplementary Tables of sequence data, meme analyses and RNA structures from Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

The supplementary tables provide the raw data for oligonucleotides, sequence data, meme analyses and secondary structures of RNA molecule

A Role for Programmed Cell Death in the Microbial Loop

<div><p>The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others' fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD) on a population's growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, ¹⁴C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by <i>Dunalliela salina</i> cells undergoing PCD complements the nutritional needs of other non-PCD <i>D. salina</i> cells. This occurs <i>in vitro</i> in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph <i>Halobacterium salinarum</i> re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in <i>D. salina</i> causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. <i>H. salinarum</i> also re-mineralizes the dissolved material promoting algal growth. PCD in <i>D. salina</i> was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism's own population growth and in the exchange of nutrients in the microbial loop.</p></div

Initial and fitted parameter values for model of growth for pure <i>D. salina</i> and <i>D. salina</i> + <i>H</i>. <i>salinarum</i> co-cultures.

<p>“±CI_95%” are parameter 95% confidence intervals such that the lower and upper bound of estimated values are X-CI_95% and X+CI_95%, respectively.</p

Diurnally synchronized syntrophic interaction with <i>H.salinarum</i> increases productivity of <i>D. salina</i>.

<p>(A) Intra- and (B) extra-cellular glycerol concentrations in <i>D. salina</i> culture individually (blue) or with <i>H. salinarum</i> (red) over several day: night cycles, dash lines represent +/− standard deviation. (C) Radiolabel incorporation and tracing shows daytime uptake and nighttime release of ¹⁴C by <i>D. salina</i>. Uptake of ¹⁴C by <i>D. salina</i> at night is enhanced two-fold in co-cultures relative to pure cultures indicating nighttime assimilation of ¹⁴C in presence of <i>H. salinarum</i>. (D) Simultaneous tracing of C within <i>H. salinarum</i> cells demonstrates uptake and processing of ¹⁴C in sync with the diurnal cycle.</p

Dissolved organic material (DOM or photosynthate) released by <i>D.salina</i> fully complements nutritional requirements of <i>H. salinarum</i>.

<p>Supernatant of <i>D. salina</i> culture in artificial seawater (MM1) supported <i>H. salinarum</i> growth at a level that was comparable to its growth in MM1 supplemented with amino acids at naturally occurring concentrations.</p

Cell death is triggered at nighttime as part of the diurnal synchronized program of <i>D.salina</i>.

<p>(A) Cell numbers for <i>D. salina</i> in pure and co-cultures with <i>H. salinarum</i> over several diurnal cycles. Live cell concentration measured using flow cytometry are indicated with blue (pure culture) and red (co-culture) points while lines are fitted model simulations. Green boxed region indicates time frame reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062595#pone-0062595-g003" target="_blank"><b>Fig. 3D</b></a> over which caspase-3 activity was assayed. (B) <i>H. salinarum</i> induces cell death in <i>D. salina</i> under continuous light regime. Intracellular glycerol within <i>D. salina</i> was stained with quinacrine and quantified with flow cytometry. Decrease of intracellular glycerol proportionally with higher cell density of <i>H. salinarum</i>. Unstimulated (pure <i>D. salina</i> culture and dark shifted samples are shown as controls. (L/L>L, cultures grown on a 24 h constant light regime maintained in the light during the measurements, LL>D, cultures grown in constant light shifted to dark conditions (0 µmoles m²s⁻¹). (C) The decrease in <i>D. salina</i> cell number in the model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062595#pone-0062595-g004" target="_blank"><b>Fig. 4A</b></a> (blue line) due to cell death is supported by the time course of annexin V labeled cells (blue line) indicating percentage of cells exhibiting externalization of PS and SYTOX® blue stained cells indicating the percent dead cells (red line). (D) The decrease in cell number in the model (blue dotted line) due to cell death is also supported by higher levels of caspase-3 during nighttime. Red line is Savitsky-Golay smoothed (span of 5) average of two replicate measurements for each time point.</p