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.     

Origin and evolution of the colonial volvocales (Chlorophyceae) as inferred from multiple, chloroplast gene sequences

A combined data set of DNA sequences (6021 bp) from five protein-coding genes of the chloroplast genome (rbcL, atpB, psaA, psaB, and psbC genes) were analyzed for 42 strains representing 30 species of the colonial Volvocales (Volvox and its relatives) and 5 related species of green algae to deduce robust phylogenetic relationships within the colonial green flagellates. The 4-celled family Tetrabaenaceae was robustly resolved as the most basal group within the colonial Volvocales. The sequence data also suggested that all five volvocacean genera with 32 or more cells in a vegetative colony (all four of the anisogamous/oogamous genera, Eudorina, Platydorina, Pleodorina, and Volvox, plus the isogamous genus Yamagishiella) constituted a large monophyletic group, in which 2 Pleodorina species were positioned distally to 3 species of Volvox. Therefore, most of the evolution of the colonial Volvocales appears to constitute a gradual progression in colonial complexity and in types of sexual reproduction, as in the traditional volvocine lineage hypothesis, although reverse evolution must be considered for the origin of certain species of Pleodorina. Data presented here also provide robust support for a monophyletic family Goniaceae consisting of two genera: Gonium and Astrephomene.     

Molecular phylogeny of the volvocine flagellates.

Phylogenetic studies of approximately 2,000 bases of sequence from the large and small nuclear-encoded ribosomal RNAs are used to investigate the origins of the genus Volvox. The colonial and multicellular genera currently placed in the family Volvocaceae form a monophyletic group that is significantly closer phylogenetically to Chlamydomonas reinhardtii than it is to the other unicellular green flagellates that were tested, including Chlamydomonas eugametos, Chlorella pyrenoidosa, and Haematococcus lacustris. Statistical analysis of 251 phylogenetically informative nucleotide positions rejects the "volvocine lineage" hypothesis, which postulates a monophyletic evolutionary progression from unicellular organisms (such as Chlamydomonas), through colonial organisms (e.g., Gonium, Pandorina, Eudorina, and Pleodorina) demonstrating increasing size, cell number, and tendency toward cellular differentiation, to multicellular organisms having fully differentiated somatic and reproductive cells (in the genus Volvox). The genus Volvox appears not to be monophyletic. Volvox capensis falls outside a lineage containing other representatives of Volvox (V. aureus, V. carteri, and V. obversus), and both of these Volvox lineages are more closely related to certain colonial genera than they are to each other. This implies either a diphyletic origin of Volvox from different colonial volvocacean ancestors, a phylogenetic derivation of some of the colonial genera from a multicellular (i.e., Volvox) ancestor, or both. Considered together with previously published observations, these results suggest that the different levels of organizational and developmental complexity found in the Volvocaceae represent alternative stable states, among which evolutionary transitions have occurred several times during the phylogenetic history of this group.     

A twelve-step program for evolving multicellularity and a division of labor

The volvocine algae provide an unrivalled opportunity to explore details of an evolutionary pathway leading from a unicellular ancestor to multicellular organisms with a division of labor between different cell types. Members of this monophyletic group of green flagellates range in complexity from unicellular Chlamydomonas through a series of extant organisms of intermediate size and complexity to Volvox, a genus of spherical organisms that have thousands of cells and a germ-soma division of labor. It is estimated that these organisms all shared a common ancestor about 50 +/- 20 MYA. Here we outline twelve important ways in which the developmental repertoire of an ancestral unicell similar to modern C. reinhardtii was modified to produce first a small colonial organism like Gonium that was capable of swimming directionally, then a sequence of larger organisms (such as Pandorina, Eudorina and Pleodorina) in which there was an increasing tendency to differentiate two cell types, and eventually Volvox carteri with its complete germ-soma division of labor.     

Species and Population Level Molecular Profiling Reveals Cryptic Recombination and Emergent Asymmetry in the Dimorphic Mating Locus of C. reinhardtii

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.     

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.     

Photosynthetic characteristics of a multicellular green alga Volvox carteri in response to external CO2 levels possibly regulated by CCM1/CIA5 ortholog

When CO(2) supply is limited, aquatic photosynthetic organisms induce a CO(2)-concentrating mechanism (CCM) and acclimate to the CO(2)-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO(2), we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO(2)-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO(2)-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO(2) grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein-protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.     

Volvocine cell walls and their constituent glycoproteins: An evolutionary perspective

Summary Similarities in the composition of the extracellular matrix suggest that only some species of the unicellularChlamydomonas are closely related to the colonial and multicellular flagellated members of the family Volvocaceae. The cell walls from all of the algae in this volvocine group contain a crystalline layer. This lattice structure can be used as a phylogenetic marker to divideChlamydomonas species into distinct classes, only one of which includes the volvocacean algae. Similarly, not all species ofChlamydomonas are sensitive to each other's cell wall lytic enzymes, implying divergence of the enzyme's inner wall substrate. Interspecific reconstitution of the crystalline layer is possible betweenC. reinhardtii and the multicellularVolvox carteri, but not betweenC. reinhardtii andC. eugametos. The hydroxyproline-rich glycoproteins (HRGPs) which make up the crystalline layer in genera which have a similar crystal structure exhibit many homologies. Interestingly, the evolutionarily distant cell walls ofC. reinhardtii andC. eugametos also contain some HRGPs displaying a few morphological and amino acid sequence homologies. The morphological similarities between the flagellar agglutinins (HRGPs responsible for sexual recognition and adhesion during the mating reaction) and the cell wall HRGPs leads to the proposal of a superfamily from which novel HRGPs (designed for self-assembly/recognition) can constantly evolve. Just as variations in the wall HRGPs can lead to unique wall structures, new agglutinins facilitate sexual isolation of new species. Thus, the HRGPs could emerge as valuable phylogenetic markers.Abbreviations GLE gametic lytic enzyme - GP glycoprotein - HRGP hydroxyproline-rich glycoprotein - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - VLE vegetative lytic enzyme - VSP vegetative serine/proline-rich - WP wall protein - ZSP zygotic serine/proline-rich     

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.     

Identification of the minus-dominance gene ortholog in the mating-type locus of Gonium pectorale

The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT- locus. 3'- and 5'-RACE RT-PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT-PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.     

Volvox: Simple steps to developmental complexity?

Volvox, Chlamydomonas, and their close relatives - collectively the volvocine green algae - comprise an excellent system for investigating the origins of developmental complexity. Over a relatively short period of time Volvox evolved an impressive suite of developmental traits, including asymmetric cell division, multicellularity with germ-soma division of labor, embryonic morphogenesis, and oogamy. Recent molecular genetic analyses of important developmental genes and comparative analyses of the fully sequenced Volvox and Chlamydomonas genomes have provided important insights into how these and other traits came to be. Surprisingly, the acquisition of much of the developmental innovation in this family seems to have involved relatively minor tinkering with the ancestral unicellular blueprint.     

The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales

Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the 'cellular zone' of the V. carteri ECM. Pherophorin-V1 and -V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae.     

THE CULTURE COLLECTION OF ALGAE AT INDIANA UNIVERSITY

Starr , Richard C. (Indiana U., Bloomington.) The Culture Collection of Algae at Indiana University. Amer. Jour. Bot. 47(1) : 67–86. 1960.–A list is presented of cultures of algae available for purposes of research and teaching. The list includes 592 Chlorophyta; 77 Chrysophyta; 56 Cyanophyta; 55 Euglenophyta; 6 Rhodophyta; 1 Phaeophyta; 2 residual flagellates. Formulae of culture media and directions for maintaining the cultures are included as are instructions for evoking sexual reproduction in species of Chlamydomonas, Pandorina, Eudorina, Volvox, Cosmarium, Clostcrium, Oedogonium, Bulbochaete and Vaucheria.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.     

PHYLOGENETIC ANALYSIS OF YAMAGISHLELLA AND PLATYDORINA (VOLVOCACEAE,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Yamagishiella, based on Pandorina unicocca Rayburn et Starr, is distinguished from Eudorina by its isogamous sexual reproduction, whereas Platydorina exhibits anisogamous sexual reproduction. In the present study, we sequenced the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) genes from five Japanese and North American strains of Y. unicocca (Rayburn et Starr) Nozaki, two Platydorina caudata Kofoid strains, and two strains of Eudorina unicocca G. M. Smith, as well as eight related colonial and unicellular species. Phylogenetic trees were constructed based on these sequence data and on previously published rbcL gene sequences from 23 volvocalean species in order to deduce phylogenetic relationships within the colonial Volvocales, with particular regard to the phylogenetic positions and status of the genera Yamagishiella and Platydorina. Two robust monophyletic groups of the anisogamous/oogamous volvocacean species were resolved in the maximum-parsimony tree as well as in the neighbor-joining distance tree. One of the two groups comprises three species of Volvox section Volvox, whereas the other is composed of other sections of Volvox as well as of all the species of Eudorina and Pleodorina. Platydorina, however, was positioned outside these two monopliyletic groups. Therefore, derivation of the Platydorina lineage may be earlier than that of such anisogamous/oogamous groups, or orgin of “anisogamy with sperm packets” in Platydorina may be independent of sperm packet evolution in Eudorina, Pleodorina, and Volvox. It was also resolved with high bootstrap values that all of the Y. unicocca strains form a monophyletic group positioned outside the large monophyletic group including Eudorina and Pleodorina. These reject the possibility of the reverse evolution of isogamy from anisogamy to give rise to Yamagishiella within the lineage of Eudorina.     

Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma

In the filamentous ascomycete Neurospora tetrasperma, a large (approx. 7 Mbp) region of suppressed recombination surrounds the mating-type (mat) locus. While the remainder of the genome is largely homoallelic, this region of recombinational suppression, extending over 1500 genes, is associated with sequence divergence. Here, we used microarrays to examine how the molecular phenotype of gene expression level is linked to this divergent region, and thus to the mating type. Culturing N. tetrasperma on agar media that induce sexual/female or vegetative/male tissue, we found 196 genes significantly differentially expressed between mat A and mat a mating types. Our data show that the genes exhibiting mat-linked expression are enriched in the region genetically linked to mating type, and sequence and expression divergence are positively correlated. Our results indicate that the phenotype of mat A strains is optimized for traits promoting sexual/female development and the phenotype of mat a strains for vegetative/male development. This discovery of differentially expressed genes associated with mating type provides a link between genotypic and phenotypic divergence in this taxon and illustrates a fungal analogue to sexual dimorphism found among animals and plants.     

Orchestration of sexual reproduction and virulence by the fungal mating-type locus

The mating-type locus (MAT) orchestrates sexual reproduction in fungi. Sexual reproduction is related not only to fitness of an organism, but also correlated with virulence in certain pathogens. In the dandruff-associated fungus Malassesia globosa, although the sexual cycle remains to be discovered, whole genome analysis has led to the hypothesis that mating may occur on host skin. Furthermore, the MAT locus of M. globosa and U. hordei provides evidence that transitions between tetrapolar and bipolar systems have independently occurred. These results, together with studies recapitulating the ancestral tetrapolar mating system in Cryptococcus and the structure of MAT in related smut fungi, have furthered understanding on transitions between different mating systems and the evolution of MAT in the Basidiomycota.