Mitochondrial and Plastid Genomes of the Colonial Green Alga Gonium pectorale Give Insights into the Origins of Organelle DNA Architecture within the Volvocales

Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.     

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

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 Tetrabaena socialis, which is basal to the colonial and multicellular volvocines.

Tetrabaena socialis has a circular-mapping mitochondrial genome, contrasting with the linear mtDNA architecture of its relative Chlamydomonas reinhardtii. 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 T. socialis 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.     

Cleavage, incomplete inversion, and cytoplasmic bridges in Gonium pectorale (Volvocales, Chlorophyta)

Multicellularity arose several times in evolution of eukaryotes. The volvocine algae have full range of colonial organization from unicellular to colonies, and thus these algae are well-known models for examining the evolution and mechanisms of multicellularity. Gonium pectorale is a multicellular species of Volvocales and is thought to be one of the first small colonial organisms among the volvocine algae. In these algae, a cytoplasmic bridge is one of the key traits that arose during the evolution of multicellularity. Here, we observed the inversion process and the cytoplasmic bridges in G. pectorale using time-lapse, fluorescence, and electron microscopy. The cytoplasmic bridges were located in the middle region of the cell in 2-, 4-, 8-, and 16-celled stages and in inversion stages. However, there were no cytoplasmic bridges in the mature adult stage. Cytoplasmic bridges and cortical microtubules in G. pectorale suggest that a mechanism of kinesin-microtubule machinery similar to that in other volvocine algae is responsible for inversion in this species.     

Stable nuclear transformation of Gonium pectorale

Background  

Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri.     

FLAGELLAR APPARATUS STRUCTURE IS SIMILAR BUT NOT IDENTICAL IN VOLVULINA STEINII,EUDORINA ELEGANS,AND PLEODORINA ILLINOISENSIS (CHLOROPHYTA): IMPLICATIONS FOR THE “VOLVOCINE EVOLUTIONARY LINEAGE”1

The colonial and multicellular members of the Volvocales can be arranged in order of increasing size and complexity as the “volvocine series.” This series is often assumed to reflect an evolutionary progression. The flagellar apparatuses of previously examined algae are not consistent with a simple lineage. The flagellar apparatuses of Astrephomene gubernaculifera Pocock, Gonium pectorale Müller, Platydorina caudata Kofoid, Volvox rousseletii G. S. West, and V. carteri f. weismannia (Powers) Iyengar differ from one another, and there is no apparent progression inflagellar apparatus features from the simple to complex colonial forms. We examined the flagellar apparatuses of Volvulina steinii Playfair, Eudorina elegans Ehr., and Pleodorina illinoisensis Kofoid and found them to be similar to one another. The basal bodies are connected by a distal fiber that is offset to the anti side of the cell. Two microtubular rootlets originate on the inside of the basal bodies and extend toward the syn side. The other two rootlets are oriented perpendicular to the first two and are anti-parallel to each other. A coarsely striated component underlies the four-membered rootlets and extends to the basal bodies. A proximal fiber complex connects the two basal bodies. This complex consists of a branched striated component on the cis side of each basal body. One part extends toward the anti side of the cell, while the other extends into a fibrous component that runs between basal bodies. An additional structure extends in the anti direction from the trans side of each basal body. A fibrous component extends past one basal body in all four species. This component goes past the trans basal body in Volvulina steinii and the cis basal body in E. elegans and P. illinoisensis. The flagellar apparatuses of these organisms are similar to those of G. pectorale and Volvox carteri but different from the other colonial volvocalean algae examined. The algae examined in this study plus G. pectorale and V. carteri probably share a common evolutionary history that postdates the transition from the unicellular to colonial habit. Such a shared evolutionary history is a requirement of the volvocine hypothesis. However, we have not observed progressive changes in the flagellar apparatus correlated with increasing cell number, differentiation, and sexual specialization. Thus, it is possible, but not certain, that G. pectorale, Volvulina steinii, E. elegans, P. illinoisensis, and Volvox carteri may form part of a volvocine lineage.     

Evolution of reproductive development in the volvocine algae

The evolution of multicellularity, the separation of germline cells from sterile somatic cells, and the generation of a male–female dichotomy are certainly among the greatest innovations of eukaryotes. Remarkably, phylogenetic analysis suggests that the shift from simple to complex, differentiated multicellularity was not a unique progression in the evolution of life, but in fact a quite frequent event. The spheroidal green alga Volvox and its close relatives, the volvocine algae, span the full range of organizational complexity, from unicellular and colonial genera to multicellular genera with a full germ–soma division of labor and male–female dichotomy; thus, these algae are ideal model organisms for addressing fundamental issues related to the transition to multicellularity and for discovering universal rules that characterize this transition. Of all living species, Volvox carteri represents the simplest version of an immortal germline producing specialized somatic cells. This cellular specialization involved the emergence of mortality and the production of the first dead ancestors in the evolution of this lineage. Volvocine algae therefore exemplify the evolution of cellular cooperation from cellular autonomy. They also serve as a prime example of the evolution of complex traits by a few successive, small steps. Thus, we learn from volvocine algae that the evolutionary transition to complex, multicellular life is probably much easier to achieve than is commonly believed.     

A posttranslationally regulated protease, VheA, is involved in the liberation of juveniles from parental spheroids in Volvox carteri

The lineage of volvocine algae includes unicellular Chlamydomonas and multicellular Volvox in addition to their colonial relatives intermediate in size and cell number. In an asexual life cycle, daughter cells of Chlamydomonas hatch from parental cell walls soon after cell division, while Volvox juveniles are released from parental spheroids after the completion of various developmental events required for the survival of multicellular juveniles. Thus, heterochronic change in the timing of hatching is considered to have played an important role in the evolution of multicellularity in volvocine algae. To study the hatching process in Volvox carteri, we purified a 125-kD Volvox hatching enzyme (VheA) from a culture medium with enzymatic activity to degrade the parental spheroids. The coding region of vheA contains a prodomain with a transmembrane segment, a subtilisin-like Ser protease domain, and a functionally unknown domain, although purified 125-kD VheA does not contain a prodomain. While 143-kD VheA with a prodomain is synthesized long before the hatching stage, 125-kD VheA is released into the culture medium during hatching due to cleavage processing at the site between the prodomain and the subtilisin-like Ser protease domain, indicating that posttranslational regulation is involved in the determination of the timing of hatching.     

Analysis of mitochondrial and chloroplast genomes in two volvocine algae: Eudorina elegans and Eudorina cylindrica (Volvocaceae,Chlorophyta)

The colonial volvocine algae span the full range of organizational complexity, from four-celled species to multicellular species, and this group of algae is often used for the study of evolution. In recent years, many organelle genomes have been sequenced using the application of next generation sequencing technology; however, only a few organelle genomes have been reported in colonial volvocine algae. In this study, we determined the organelle genomes of Eudorina elegans and Eudorina cylindrica and analysed the organelle genome size, structure and gene content between these volvocine species. This provided useful information to help us understand the composition of colonial volvocine organelle genomes. Based on the chloroplast genome protein-coding genes, we conducted a phylogenomics analysis of the volvocine algae. The result revealed an unexpected phylogenetic relationship, namely, E. elegans is more closely related to Pleodorina starrii than to E. cylindrica. The substitution rate of volvocine algae was then calculated based on organelle genome protein-coding genes; our analysis suggested the possibility that the two Eudorina species may be under similar evolutionary pressure. Lastly, the synteny analysis of the mitochondrial genome showed that gene arrangements and contents are highly conserved in the family Volvocaceae, and the synteny analysis of the chloroplast genome indicated that the genus Eudorina may have experienced genomic changes.     

Genetic basis for soma is present in undifferentiated volvocine green algae

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co‐opted to produce soma. Determining that the reg cluster was co‐opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.     

Sexual reproduction and sex determination in green algae

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt⁺) and mating type minus (mt^?), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt⁺ and mt^? mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.     

Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

Regulation of sedimentation rate shapes the evolution of multicellularity in a close unicellular relative of animals

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma-close unicellular relatives of animals with brief transient multicellular life stages-we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least 2 distinct physical mechanisms. First, we find extensive (>300×) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular Sphaeroforma arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal “clumping” multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in 4 additional species from the Sphaeroforma genus, suggesting that variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodeling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.

The transition to multicellularity is associated with the emergence of new features, including an increase in sedimentation rate, but how does such a key transition first occur? An experimental evolution study of ichthyosporeans, close unicellular relatives of animals, shows how cellular control of density and size drive sedimentation rate variation, likely shaping the evolution of multicellularity.     

Chloroplast phylogenomics of unicellular and colonial Volvocales provides perspectives on the evolution of morphological characters

Volvocales forms a species-rich clade with wide morphological variety and is regarded as an ideal model for tracing the evolutionary transitions in multicellularity. The phylogenetic relationships among the colonial volvocine algae and its relatives are important for investigating the origin of multicellularity in the clade Reinhardtinia. Therefore, a robust phylogenetic framework of the unicellular and colonial volvocine algae with broad taxon and gene sampling is essential for illuminating the evolution of multicellularity. Recent chloroplast phylogenomic studies have uncovered five major orders in the Chlorophyceae, but the family-level relationships within Sphaeropleales and Volvocales remain elusive due to the uncertain positions of some incertae sedis taxa. In this study, we contributed six newly sequenced chloroplast genomes in the Volvocales and analyzed a dataset with 91 chlorophycean taxa and 58 protein-coding genes. Conflicting phylogenetic signals were detected among chloroplast genes that resulted in discordant tree topologies among different analyses. We compared the phylogenetic trees inferred from original nucleotide, RY-coding, codon-degenerate, and amino acid datasets, and improved the robustness of phylogenetic inference in the Chlorophyceae by reducing base compositional bias. Our analyses indicate that the unicellular Chlamydomonas and Vitreochlamys are close to or nested within the colonial taxa, and all the incertae sedis taxa are nested within the monophyletic Sphaeropleales s.l. We propose that the colonial taxa in the Reinhardtinia are paraphyletic and multicellularity evolved once in the volvocine green algae and might be lost in Chlamydomonas and Vitreochlamys.     

Philosophical foundations for the hierarchy of life

We review Evolution and the Levels of Selection by Samir Okasha. This important book provides a cohesive philosophical framework for understanding levels-of-selections problems in biology. Concerning evolutionary transitions, Okasha proposes that three stages characterize the shift from a lower level of selection to a higher one. We discuss the application of Okasha’s three-stage concept to the evolutionary transition from unicellularity to multicellularity in the volvocine green algae. Okasha’s concepts are a provocative step towards a more general understanding of the major evolutionary transitions; however, the application of certain ideas to the volvocine model system is not straightforward.     

Sex-Specific Posttranslational Regulation of the Gamete Fusogen GCS1 in the Isogamous Volvocine Alga Gonium pectorale

Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to male) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (females) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor.