Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP- RK family transcription factor (TF) MID is encoded by the minus mating type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the defaultplus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP- RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho- group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating- type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID- VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.

The evolution of germ-soma cellular differentiation represents a key step in the evolution of multicellular individuality. Volvox carteri and its relatives, the volvocine green algae, provide a model system for studying the evolution of cellular differentiation. In V. carteri, the regA gene controls somatic cell differentiation and is found in a group of paralogs called the reg cluster, along with rlsA, rlsB, and rlsC. However, the developmental program of V. carteri is derived compared to other volvocine algae. Here we examine Volvox powersii which possesses an ancestral developmental program and independent evolution of the Volvox body plan. We sequenced the reg cluster from V. powersii wild-type and a mutant with fewer cells and altered germ-soma ratio. We found that the mutant strain's rlsB gene has a deletion predicted to cause a truncated protein product. We developed a genetic transformation procedure to insert wild-type rlsB into the mutant strain. Transformation did not result in phenotypic rescue, suggesting the rlsB mutation is insufficient for generating the mutant phenotype. The transformation techniques and sequences described here provide essential tools to study V. powersii, a species well suited for studying the evolution of cellular differentiation and convergent evolution of Volvox morphology.

Hypothesis: Genetic recombination underlies the frequent origins of self-fertilization and rare reversions to outcrossing. Organisms: Volvox perglobator, Gonium pectorale, volvocine green algae. Methods: We report the rediscovery of a volvocine alga, obligate outcrossing V. perglobator, including morphological characteristics and molecular phylogeny. We use ancestral-state reconstruction and phylogenetic simulation to demonstrate multiple transitions between selffertilization and outcrossing. We sequence sex-determining genes in multiple volvocine algae. Results: We find significant support for numerous origins of self-fertilization and multiple reversions to outcrossing. Volvox perglobator is one of these reversions to outcrossing. Combination of ancestrally sex-restricted and sex-determining genes into the same genotype correlates with the evolution of self-fertilization. Re-segregation of these same sex-determining loci into separate sexes correlates with the evolution of outcrossing. These results suggest that recombination underlies the evolutionary transitions between self-fertilization and outcrossing in the volvocine algae.

Multicellularity is the premier example of a major evolutionary transition in individuality and was a foundational event in the evolution of macroscopic biodiversity. The volvocine chlorophyte lineage is well suited for studying this process. Extant members span unicellular, simple colonial, and obligate multicellular taxa with germ-soma differentiation. Here, we report the nuclear genome sequence of one of the most morphologically simple organisms in this lineage-the 4-celled colonial Tetrabaena socialis and compare this to the three other complete volvocine nuclear genomes. Using conservative estimates of gene family expansions a minimal set of expanded gene families was identified that associate with the origin of multicellularity. These families are rich in genes related to developmental processes. A subset of these families is lineage specific, which suggests that at a genomic level the evolution of multicellularity also includes lineage-specific molecular developments. Multiple points of evidence associate modifications to the ubiquitin proteasomal pathway (UPP) with the beginning of coloniality. Genes undergoing positive or accelerating selection in the multicellular volvocines were found to be enriched in components of the UPP and gene families gained at the origin of multicellularity include components of the UPP. A defining feature of colonial/multicellular life cycles is the genetic control of cell number. The genomic data presented here, which includes diversification of cell cycle genes and modifications to the UPP, align the genetic components with the evolution of this trait.

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.

Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.

Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT–, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale–V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.

To understand the hierarchy of life in evolutionary terms, we must explain why groups of one kind of individual, say cells, evolve into a new higher level individual, a multicellular organism. A fundamental step in this process is the division of labor into nonreproductive altruistic soma. The regA gene is critical for somatic differentiation in Volvox carteri, a multicellular species of volvocine algae. We report the sequence of regA-like genes and several syntenic markers from divergent species of Volvox. We show that regA evolved early in the volvocines and predict that lineages with and without soma descended from a regA-containing ancestor. We hypothesize an alternate evolutionary history of regA than the prevailing "proto-regA" hypothesis. The variation in presence of soma may be explained by multiple lineages independently evolving soma utilizing regA or alternate genetic pathways. Our prediction that the genetic basis for soma exists in species without somatic cells raises a number of questions, most fundamentally, under what conditions would species with the genetic potential for soma, and hence greater individuality, not evolve these traits. We conclude that the evolution of individuality in the volvocine algae is more complicated and labile than previously appreciated on theoretical grounds.

It has been argued that for certain lineages, noncoding DNA expansion is a consequence of the increased random genetic drift associated with long-term escalations in organism size. But a lack of data has prevented the investigation of this hypothesis in most plastid-bearing protists. Here, using newly sequenced mitochondrial and plastid genomes, we explore the relationship between organelle DNA noncoding content and organism size within volvocine green algae. By looking at unicellular, colonial, and differentiated multicellular algae, we show that organelle DNA complexity scales positively with species size and cell number across the volvocine lineage. Moreover, silent-site genetic diversity data suggest that the volvocine species with the largest cell numbers and most bloated organelle genomes have the smallest effective population sizes. Together, these findings support the view that nonadaptive processes, like random genetic drift, promote the expansion of noncoding regions in organelle genomes.

Background Isogamous organisms lack obvious cytological differences in the gametes of the two complementary mating types. Consequently, it is difficult to ascertain which of the two mating types are homologous when comparing related but sexual isolated strains or species. The colonial volvocalean algal genus Gonium consists of such isogamous organisms with heterothallic mating types designated arbitrarily as plus or minus in addition to homothallic strains. Homologous molecular markers among lineages may provide an “objective” framework to assign heterothallic mating types. Methodology/Principal Findings Using degenerate primers designed based on previously reported MID orthologs, the “master regulator” of mating types/sexes in the colonial Volvocales, MID homologs were identified and their presence/absence was examined in nine strains of four species of Gonium. Only one of the two complementary mating types in each of the four heterothallic species has a MID homolog. In addition to heterothallic strains, a homothallic strain of G. multicoccum has MID. Molecular evolutionary analysis suggests that MID of this homothallic strain retains functional constraint comparable to that of the heterothallic strains. Conclusion/Significance We coordinated mating genotypes based on presence or absence of a MID homolog, respectively, in heterothallic species. This scheme should be applicable to heterothallic species of other isogamous colonial Volvocales including Pandorina and Yamagishiella. Homothallism emerged polyphyletically in the colonial Volvocales, although its mechanism remains unknown. Our identification of a MID homolog for a homothallic strain of G. multicoccum suggests a MID-dependent mechanism is involved in the sexual developmental program of this homothallic species.

Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT-. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT--gene conversion in the rearranged domains, and crossover exchanges in flanking domains--both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+ ×MT+ crosses, it was still suppressed in MT- ×MT- crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.

Gametes were induced separately in cultures of each mating type of the heterothallic, isogamous colonial volvocalean Gonium pectorale O. F. Müll. to examine the tubular mating structure (TMS) of both mating types plus and minus ( plus and minus ), referred to as “bilateral mating papillae.” Addition of dibutyryl cyclic adenosine monophosphate (DcAMP or db‐cAMP) and 3‐isobutyl‐1‐methylxanthine (IBMX) to approximately 3‐week‐old cultures of each mating type induced immediate release of naked gametes from the cell walls. Both plus and minus gametes formed a TMS in the anterior region of the protoplasts. Accumulation of actin was visualized by antibody staining in the TMS of both mating types as occurs in the TMS (fertilization tubule) of the plus gametes of the unicellular volvocalean Chlamydomonas reinhardtii P. A. Dang. Induction of naked gametes with a TMS in each mating type will be useful for future cell biological and evolutionary studies of the isogametes of colonial volvocalean algae.

Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non-sex-related genes.

The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

Gonium pectorale O. F. Müll. (Volvocales, Chlorophyta), a colonial 8‐ or 16‐cellular alga, is phylogenetically important as an intermediate form between isogametic unicellular Chlamydomonas and oogamous Volvox . We identified the mating‐type specific gene GpMTD1, from G . pectorale , the first homologue of Chlamydomonas reinhardtii MTD1 ( CrMTD1 ). The GpMTD1 gene was found to be present only in the minus mating‐type locus and was expressed specifically in the gametic phase as is the case for CrMTD1 , suggested to participate in development of the minus gametes. This gene is useful as a probe in analyzing the bacterial artificial chromosome (BAC) library for resolving genomic structures of the mating‐type loci in isogamous and oogamous colonial volvocaleans.