A conserved RWP-RK transcription factor VSR1 controls gametic differentiation in volvocine algae.

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 default plus 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.

Reorganization of the ancestral sex-determining regions during the evolution of trioecy in Pleodorina starrii.

The coexistence of three sexual phenotypes (male, female and bisexual) in a single species, 'trioecy', is rarely found in diploid organisms such as flowering plants and invertebrates. However, trioecy in haploid organisms has only recently been reported in a green algal species, Pleodorina starrii. Here, we generated whole-genome data of the three sex phenotypes of P. starrii to reveal a reorganization of the ancestral sex-determining regions (SDRs) in the sex chromosomes: the male and bisexual phenotypes had the same "male SDR" with paralogous gene expansions of the male-determining gene MID, whereas the female phenotype had a "female SDR" with transposition of the female-specific gene FUS1 to autosomal regions. Although the male and bisexual sex phenotypes had the identical male SDR and harbored autosomal FUS1, MID and FUS1 expression during sexual reproduction differed between them. Thus, the coexistence of three sex phenotypes in P. starrii is possible.

Three sex phenotypes in a haploid algal species give insights into the evolutionary transition to a self-compatible mating system.

Mating systems of haploid species such as fungi, algae, and bryophytes are either heterothallic (self-incompatible) with two sex phenotypes (male and female, or mating type minus and plus in isogamous species) or homothallic (self-compatible) with only a bisexual phenotype producing zygotes within a clone. The anisogamous volvocine green alga Pleodorina starrii is a haploid species previously reported to have a heterothallic mating system. Here, we found that two additional culture strains originating from the same water system of P. starrii were taxonomically identified as P. starrii and produced male and female gametes and zygotes within a clone (bisexual). Sequences of rapidly evolving plastid genome regions were identical between the bisexual and unisexual (male or female) P. starrii strains. Intercrossings between the bisexual and unisexual strains demonstrated normal thick-walled zygotes and high survivability of F1 strains. Thus, these strains belong to the same biological species. Pleodorina starrii has a new haploid mating system that is unique in having three sex phenotypes, namely, male, female, and bisexual. Genetic analyses suggested the existence of autosomal "bisexual factor" locus independent of volvocine male and female determining regions. The present findings increase our understanding of the initial evolutionary step of transition from heterothallism to homothallism.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism.

Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

Anisogamy evolved with a reduced sex-determining region in volvocine green algae.

Male and female gametes differing in size-anisogamy-emerged independently from isogamous ancestors in various eukaryotic lineages, although genetic bases of this emergence are still unknown. Volvocine green algae are a model lineage for investigating the transition from isogamy to anisogamy. Here we focus on two closely related volvocine genera that bracket this transition-isogamous Yamagishiella and anisogamous Eudorina. We generated de novo nuclear genome assemblies of both sexes of Yamagishiella and Eudorina to identify the dimorphic sex-determining chromosomal region or mating-type locus (MT) from each. In contrast to the large (>1 Mb) and complex MT of oogamous Volvox, Yamagishiella and Eudorina MT are smaller (7-268 kb) and simpler with only two sex-limited genes-the minus/male-limited MID and the plus/female-limited FUS1. No prominently dimorphic gametologs were identified in either species. Thus, the first step to anisogamy in volvocine algae presumably occurred without an increase in MT size and complexity.

Exploring the Limits and Causes of Plastid Genome Expansion in Volvocine Green Algae.

Plastid genomes are not normally celebrated for being large. But researchers are steadily uncovering algal lineages with big and, in rare cases, enormous plastid DNAs (ptDNAs), such as volvocine green algae. Plastome sequencing of five different volvocine species has revealed some of the largest, most repeat-dense plastomes on record, including that of Volvox carteri (∼525 kb). Volvocine algae have also been used as models for testing leading hypotheses on organelle genome evolution (e.g., the mutational hazard hypothesis), and it has been suggested that ptDNA inflation within this group might be a consequence of low mutation rates and/or the transition from a unicellular to multicellular existence. Here, we further our understanding of plastome size variation in the volvocine line by examining the ptDNA sequences of the colonial species Yamagishiella unicocca and Eudorina sp. NIES-3984 and the multicellular Volvox africanus, which are phylogenetically situated between species with known ptDNA sizes. Although V. africanus is closely related and similar in multicellular organization to V. carteri, its ptDNA was much less inflated than that of V. carteri. Synonymous- and noncoding-site nucleotide substitution rate analyses of these two Volvox ptDNAs suggest that there are drastically different plastid mutation rates operating in the coding versus intergenic regions, supporting the idea that error-prone DNA repair in repeat-rich intergenic spacers is contributing to genome expansion. Our results reinforce the idea that the volvocine line harbors extremes in plastome size but ultimately shed doubt on some of the previously proposed hypotheses for ptDNA inflation within the lineage.

Establishment of a Clonal Culture of Unicellular Conjugating Algae.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

Molecular evolutionary analysis of a gender-limited MID ortholog from the homothallic species Volvox africanus with male and monoecious spheroids.

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.

Multiple Independent Changes in Mitochondrial Genome Conformation in Chlamydomonadalean Algae.

Chlamydomonadalean green algae are no stranger to linear mitochondrial genomes, particularly members of the Reinhardtinia clade. At least nine different Reinhardtinia species are known to have linear mitochondrial DNAs (mtDNAs), including the model species Chlamydomonas reinhardtii. Thus, it is no surprise that some have suggested that the most recent common ancestor of the Reinhardtinia clade had a linear mtDNA. But the recent uncovering of circular-mapping mtDNAs in a range of Reinhardtinia algae, such as Volvox carteri and Tetrabaena socialis, has shed doubt on this hypothesis. Here, we explore mtDNA sequence and structure within the colonial Reinhardtinia algae Yamagishiella unicocca and Eudorina sp. NIES-3984, which occupy phylogenetically intermediate positions between species with opposing mtDNA mapping structures. Sequencing and gel electrophoresis data indicate that Y. unicocca has a linear monomeric mitochondrial genome with long (3 kb) palindromic telomeres. Conversely, the mtDNA of Eudorina sp., despite having an identical gene order to that of Y. unicocca, assembled as a circular-mapping molecule. Restriction digests of Eudorina sp. mtDNA supported its circular map, but also revealed a linear monomeric form with a matching architecture and gene order to the Y. unicocca mtDNA. Based on these data, we suggest that there have been at least three separate shifts in mtDNA conformation in the Reinhardtinia, and that the common ancestor of this clade had a linear monomeric mitochondrial genome with palindromic telomeres.

Identification and Characterization of a cis-Regulatory Element for Zygotic Gene Expression in Chlamydomonas reinhardtii.

Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.

The Gonium pectorale genome demonstrates co-option of cell cycle regulation during the evolution of multicellularity.

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.

Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

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.

Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle.

The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.

Two different rickettsial bacteria invading Volvox carteri.

BACKGROUND: Bacteria of the family Rickettsiaceae are principally associated with arthropods. Recently, endosymbionts of the Rickettsiaceae have been found in non-phagotrophic cells of the volvocalean green algae Carteria cerasiformis, Pleodorina japonica, and Volvox carteri. Such endosymbionts were present in only C. cerasiformis strain NIES-425 and V. carteri strain UTEX 2180, of various strains of Carteria and V. carteri examined, suggesting that rickettsial endosymbionts may have been transmitted to only a few algal strains very recently. However, in preliminary work, we detected a sequence similar to that of a rickettsial gene in the nuclear genome of V. carteri strain EVE. METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the origin of the rickettsial gene-like sequences in the endosymbiont-lacking V. carteri strain EVE, by performing comparative analyses on 13 strains of V. carteri. By reference to our ongoing genomic sequence of rickettsial endosymbionts in C. cerasiformis strain NIES-425 cells, we confirmed that an approximately 9-kbp DNA sequence encompassing a region similar to that of four rickettsial genes was present in the nuclear genome of V. carteri strain EVE. Phylogenetic analyses, and comparisons of the synteny of rickettsial gene-like sequences from various strains of V. carteri, indicated that the rickettsial gene-like sequences in the nuclear genome of V. carteri strain EVE were closely related to rickettsial gene sequences of P. japonica, rather than those of V. carteri strain UTEX 2180. CONCLUSION/SIGNIFICANCE: At least two different rickettsial organisms may have invaded the V. carteri lineage, one of which may be the direct ancestor of the endosymbiont of V. carteri strain UTEX 2180, whereas the other may be closely related to the endosymbiont of P. japonica. Endosymbiotic gene transfer from the latter rickettsial organism may have occurred in an ancestor of V. carteri. Thus, the rickettsiae may be widely associated with V. carteri, and likely have often been lost during host evolution.

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.

Distribution of the sex-determining gene MID and molecular correspondence of mating types within the isogamous genus Gonium (Volvocales, Chlorophyta).

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.

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.

The evolution of male-female sexual dimorphism predates the gender-based divergence of the mating locus gene MAT3/RB.

The molecular bases for the evolution of male-female sexual dimorphism are possible to study in volvocine algae because they encompass the entire range of reproductive morphologies from isogamy to oogamy. In 1978, Charlesworth suggested the model of a gamete size gene becoming linked to the sex-determining or mating type locus (MT) as a mechanism for the evolution of anisogamy. Here, we carried out the first comprehensive study of a candidate MT-linked oogamy gene, MAT3/RB, across the volvocine lineage. We found that evolution of anisogamy/oogamy predates the extremely high male-female divergence of MAT3 that characterizes the Volvox carteri lineage. These data demonstrate very little sex-linked sequence divergence of MAT3 between the two sexes in other volvocine groups, though linkage between MAT3 and the mating locus appears to be conserved. These data implicate genetic determinants other than or in addition to MAT3 in the evolution of anisogamy in volvocine algae.