Fossil-calibrated molecular clock data enable reconstruction of steps leading to differentiated multicellularity and anisogamy in the Volvocine algae.

BACKGROUND: Throughout its nearly four-billion-year history, life has undergone evolutionary transitions in which simpler subunits have become integrated to form a more complex whole. Many of these transitions opened the door to innovations that resulted in increased biodiversity and/or organismal efficiency. The evolution of multicellularity from unicellular forms represents one such transition, one that paved the way for cellular differentiation, including differentiation of male and female gametes. A useful model for studying the evolution of multicellularity and cellular differentiation is the volvocine algae, a clade of freshwater green algae whose members range from unicellular to colonial, from undifferentiated to completely differentiated, and whose gamete types can be isogamous, anisogamous, or oogamous. To better understand how multicellularity, differentiation, and gametes evolved in this group, we used comparative genomics and fossil data to establish a geologically calibrated roadmap of when these innovations occurred. RESULTS: Our ancestral-state reconstructions, show that multicellularity arose independently twice in the volvocine algae. Our chronograms indicate multicellularity evolved during the Carboniferous-Triassic periods in Goniaceae + Volvocaceae, and possibly as early as the Cretaceous in Tetrabaenaceae. Using divergence time estimates we inferred when, and in what order, specific developmental changes occurred that led to differentiated multicellularity and oogamy. We find that in the volvocine algae the temporal sequence of developmental changes leading to differentiated multicellularity is much as proposed by David Kirk, and that multicellularity is correlated with the acquisition of anisogamy and oogamy. Lastly, morphological, molecular, and divergence time data suggest the possibility of cryptic species in Tetrabaenaceae. CONCLUSIONS: Large molecular datasets and robust phylogenetic methods are bringing the evolutionary history of the volvocine algae more sharply into focus. Mounting evidence suggests that extant species in this group are the result of two independent origins of multicellularity and multiple independent origins of cell differentiation. Also, the origin of the Tetrabaenaceae-Goniaceae-Volvocaceae clade may be much older than previously thought. Finally, the possibility of cryptic species in the Tetrabaenaceae provides an exciting opportunity to study the recent divergence of lineages adapted to live in very different thermal environments.

Phylotranscriptomics points to multiple independent origins of multicellularity and cellular differentiation in the volvocine algae.

BACKGROUND: The volvocine algae, which include the single-celled species Chlamydomonas reinhardtii and the colonial species Volvox carteri, serve as a model in which to study the evolution of multicellularity and cellular differentiation. Studies reconstructing the history of this group have by and large relied on datasets of one to a few genes for phylogenetic inference and ancestral character state reconstruction. As a result, volvocine phylogenies lack concordance depending on the number and/or type of genes (i.e., chloroplast vs nuclear) chosen for phylogenetic inference. While multiple studies suggest that multicellularity evolved only once in the volvocine algae, that each of its three colonial families is monophyletic, and that there have been at least three independent origins of cellular differentiation in the group, other studies call into question one or more of these conclusions. An accurate assessment of the evolutionary history of the volvocine algae requires inference of a more robust phylogeny. RESULTS: We performed RNA sequencing (RNA-seq) on 55 strains representing 47 volvocine algal species and obtained similar data from curated databases on 13 additional strains. We then compiled a dataset consisting of transcripts for 40 single-copy, protein-coding, nuclear genes and subjected the predicted amino acid sequences of these genes to maximum likelihood, Bayesian inference, and coalescent-based analyses. These analyses show that multicellularity independently evolved at least twice in the volvocine algae and that the colonial family Goniaceae is not monophyletic. Our data further indicate that cellular differentiation arose independently at least four, and possibly as many as six times, within the volvocine algae. CONCLUSIONS: Altogether, our results demonstrate that multicellularity and cellular differentiation are evolutionarily labile in the volvocine algae, affirming the importance of this group as a model system for the study of major transitions in the history of life.

Cryopreservation of clonal and polyclonal populations of Chlamydomonas reinhardtii.

Long-term preservation of laboratory strains of Chlamydomonas reinhardtii has historically involved either liquid nitrogen cryopreservation, which is expensive and labor intensive, or storage on agar plates, which requires frequent transfer to new plates, and which may leave samples susceptible to contamination as well as genetic drift and/or selection. The emergence of C. reinhardtii as a model organism for genetic analysis and experimental evolution has produced an increasing demand for an efficient method to cryopreserve C. reinhardtii populations. The GeneArt™ Cryopreservation Kit for Algae provides the first method for algal storage at -80°C; however, little is known about how this method affects recovery of different clones, much less polyclonal populations. Here, we compare postfreeze viability of clonal and genetically mixed samples frozen at -80°C using GeneArt™ or cryopreserved using liquid nitrogen. We find that the GeneArt™ protocol yields similar percent recoveries for some but not all clonal cultures, when compared to archiving via liquid N2. We also find that relative frequency of different strains recovered from genetically mixed populations can be significantly altered by cryopreservation. Thus, while cryopreservation using GeneArt™ is an effective means for archiving certain clonal populations, it is not universally so. Strain-specific differences in freeze-thaw tolerance complicate the storage of different clones, and may also bias the recovery of different genotypes from polyclonal populations.