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Significance The physiological and ecological importance of natural products often remains obscured. Here, we report that Streptomyces-derived marginolactones, a distinct group of soil-borne natural products, specifically trigger the formation of gloeocapsoids, previously undescribed protective aggregate structures produced by the unicellular green alga Chlamydomonas reinhardtii. Gloeocapsoids are distinct palmelloids differing in their protective capability toward azalomycin F. The presence of marginolactone biosynthesis gene clusters in numerous streptomycetes, their ubiquity in soil, and our observation that three different members of this natural product group trigger the formation of gloeocapsoids suggest a cross-kingdom competition with ecological relevance. In the course of evolution, the polysaccharide matrix may have developed from a transient protective feature into the foundation of true multicellularity because of sustained marginolactone stress., Photosynthetic microorganisms including the green alga Chlamydomonas reinhardtii are essential to terrestrial habitats as they start the carbon cycle by conversion of CO2 to energy-rich organic carbohydrates. Terrestrial habitats are densely populated, and hence, microbial interactions mediated by natural products are inevitable. We previously discovered such an interaction between Streptomyces iranensis releasing the marginolactone azalomycin F in the presence of C. reinhardtii. Whether the alga senses and reacts to azalomycin F remained unknown. Here, we report that sublethal concentrations of azalomycin F trigger the formation of a protective multicellular structure by C. reinhardtii, which we named gloeocapsoid. Gloeocapsoids contain several cells which share multiple cell membranes and cell walls and are surrounded by a spacious matrix consisting of acidic polysaccharides. After azalomycin F removal, gloeocapsoid aggregates readily disassemble, and single cells are released. The presence of marginolactone biosynthesis gene clusters in numerous streptomycetes, their ubiquity in soil, and our observation that other marginolactones such as desertomycin A and monazomycin also trigger the formation of gloeocapsoids suggests a cross-kingdom competition with ecological relevance. Furthermore, gloeocapsoids allow for the survival of C. reinhardtii at alkaline pH and otherwise lethal concentrations of azalomycin F. Their structure and polysaccharide matrix may be ancestral to the complex mucilage formed by multicellular members of the Chlamydomonadales such as Eudorina and Volvox. Our finding suggests that multicellularity may have evolved to endure the presence of harmful competing bacteria. Additionally, it underlines the importance of natural products as microbial cues, which initiate interesting ecological scenarios of attack and counter defense.

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.

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.

The biodiversity and taxonomy of colonial volvocine green algae are important in ancient lakes in tropical regions. However, few taxonomic studies of these algae have been conducted in African ancient lakes. Here, we describe two species of colonial volvocine green algae in cultures originating from water samples from Lake Victoria, an ancient lake in Africa. One was identified as an undescribed morphological species of Eudorina; E. compacta sp. nov. This new species can be distinguished from other Eudorina species by its compactly arranged vegetative cells that form a hollow ellipsoidal colony. The other was identified as Colemanosphaera charkowiensis. The genus Colemanosphaera is new to Africa.

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.

In situ growth rates of the typical dominant algae species in Pengxi River were investigated using two kinds of in situ incubation devices(incubation cage and incubation bucket) for field incubation experiment in Lake Gaoyang of Pengxi River.Adequate nutrients,superior solar-thermal and relatively stable hydrodynamic conditions are the main characteristic of the habitat during the study.The selected environment was suitable for the algal growth and the period chosen was sensitive for the occurrence of algal blooms.During this period,in situ growth rates of Cyllotella and Cryptomonas representing phytoplankton with CR and CRS growth strategies,respectively,were both gradually declined,and those of Anabaena and Eudorina which both represent the CS growth strategies increased firstly and then declined.The in situ growth rate of Microcystis which represents the S growth strategy was negative in early days,however,the general trend of growth rate was increased.The maximum specific growth rate of each species in the incubation cage and incubation bucket were Cyllotella hubeiana 0.31,0.21 d-1,Microcystis aeruginosa 0.09,0.03 d-1,Eudorina elegans 0.16,0.42 d-1,Anabaena flos-aquae 0.30,0.26 d-1,Cryptomonas ovata 0.49,0.95 d-1,respectively.In this study,changes in the algae growth rates of the different growth strategies to a certain extent reflected the different growth trend.In addition,comprehensive analysis of the results of the performance test and in situ incubation experiment suggested that both of the two devices were able to meet the testing requirements of in situ growth rate of algae in natural water.There were potential impacts of the incubation devices on the in situ growth of phytoplankton.

Colonial volvocacean algae engage in two types of sexual reproduction: isogamy and anisogamy/oogamy with sperm packets. This difference is an important generic diagnosis within the Volvocaceae. Although Yamagishiella differs from the anisogamous genus Eudorina in its isogamous sexual reproduction, the vegetative morphology and asexual reproduction characteristics of the two genera are indistinguishable, especially between Eudorina unicocca G. M. Smith and Yamagishiella unicocca (Rayburn et Starr) Nozaki. We re-examined morphological characteristics of E. unicocca and related species, using multiple strains of E. unicocca and Y. unicocca and molecular phylogenetic analyses. Strains from two Japanese lakes, which produced aplanospores and were solely asexual, could be assigned to either E. unicocca or Y. unicocca, based on traditional morphological diagnoses. However, a new morphological diagnosis (the difference in the distribution and number of contractile vacuoles on the cell surface) and molecular phylo...

It is widely accepted that alkaline phosphatase activity (APA) is an efficient indicator of phosphate limitation in freshwater phytoplankton communities. In this study, we investigated whether the response in APA to phosphate limitation differs among the taxa in a mixed phytoplankton assemblage. We used the new enzyme-labeled fluorescence (ELF) technique, which allows microscopic detection of phosphate limitation in individual cells of multiple species. The most prominent findings of this study were that alkaline phosphatase (AP) was induced in many, but not all taxa and that different taxa, as well as different cells within a single taxon, experienced different degrees of phosphate stress under the same environmental conditions. Our approach was to manipulate the limiting nutrient in a natural freshwater phytoplankton community by incubating lake water in the laboratory. We induced nitrogen (N) or phosphate limitation through additions of inorganic nutrients. Both the ELF assay and bulk APA indicated that the lake phytoplankton were not phosphate limited at the start of the experiment. During the experiment, several chlorophyte taxa (e.g., Eudorina and an unidentified solitary spiny coccoid) were driven to phosphate limitation when inorganic N was added, as evidenced by a higher percentage of ELF-labeled cells relative to controls, whereas other chlorophyte taxa such as Actinastrum and Dictyosphaeriumwere not phosphate stressed under these conditions. In the phosphate-limited treatments, little or no ELF labeling was observed in any cyanobacterial taxa. Furthermore, all taxa observed after the ELF labeling procedure ( .10-mm fraction) were labeled with ELF at least on one occasion, demonstrating the wide applicability of the ELF method. By using ELF labeling in tandem with bulk APA, the resolution and analysis of phosphate limitation was increased, allowing the identification of specific phosphate-stressed taxa.

Internal transcribed spacer (ITS) regions of nuclear ribosomal repeats were compared among 50 Eudorina and Pleodorina isolates and two Volvox species known to clade with Eudorina species. Of the six major subclades found, four containing Eudorina and Pleodorina illinoisensis isolates, one containing Eudorina and Pleodorina indica, and one containing Volvox gigas and V. powersii, the basal branching order remains uncertain, but the positioning of isolates known to mate was always as nearest neighbors on the terminal branches of the tree. Four hybrid clones from a cross of E. elegans with P. illinoisensis, known from chromosome counts to be products of the failure of meiosis at zygote germination, contain both parental ITS repeat regions, as expected. However, they have in addition both crossover and other variant ITS cistrons among their many repeats of ITS. Such variation is limited to terminal regions of helices, as recognized from knowledge of RNA transcript secondary structure. Proper alignment then utilizes all of the nucleotide positions; the hybrid variants appear in positions intermediate between their parents in the tree. In fact, such variants seem to be hallmarks of recent hybridization events, since they were not found in any of the other 50 isolates.

From a small ephemeral basin in Plovdiv (Bulgaria) were found and described a new species of the genus Eudorina ( Chlorophyta , Volvcoales) - E. plurivacuolata sp. nov. The closes t to the newly described species are Eudorina unicocca and Eudorina elegans .

Background The evolution of oogamy from isogamy, an important biological event, can be summarized as follows: morphologically similar gametes (isogametes) differentiated into small “male” and large “female” motile gametes during anisogamy, from which immotile female gametes (eggs) evolved. The volvocine green algae represent a model lineage to study this type of sex evolution and show two types of gametic unions: conjugation between isogametes outside the parental colonies (external fertilization during isogamy) and fertilization between small motile gametes (sperm) and large gametes (eggs) inside the female colony (internal fertilization during anisogamy and oogamy). Although recent cultural studies on volvocine algae revealed morphological diversity and molecular genetic data of sexual reproduction, an intermediate type of union between these two gametic unions has not been identified. Results We identified a novel colonial volvocine genus, Colemanosphaera, which produces bundles of spindle-shaped male gametes through successive divisions of colonial cells. Obligately anisogamous conjugation between male and female motile gametes occurred outside the female colony (external fertilization during anisogamy). This new genus contains 16- or 32-celled spheroidal colonies similar to those of the volvocine genera Yamagishiella and Eudorina. However, Colemanosphaera can be clearly distinguished from these two genera based on its sister phylogenetic position to the enigmatic flattened colonial volvocine Platydorina and external fertilization during anisogamy. Two species of Colemanosphaera were found in a Japanese lake; these species are also distributed in European freshwaters based on a published sequence of an Austrian strain and the original description of Pandorina charkowiensis from Ukraine. Conclusions Based on phylogeny and morphological data, this novel genus exhibits a missing link between Platydorina and the typical spheroidal colonial volvocine members such as Pandorina or Yamagishiella. Considering the external obligate anisogamy, oogamy evolution may have been preceded by the transition from external to internal fertilization during anisogamy within the volvocine green algae.

Sequence analysis based on multiple isolates representing essentially all genera and species of the classic family Volvocaeae has clarified their phylogenetic relationships. Cloned internal transcribed spacer sequences (ITS-1 and ITS-2, flanking the 5.8S gene of the nuclear ribosomal gene cistrons) were aligned, guided by ITS transcript secondary structural features, and subjected to parsimony and neighbor joining distance analysis. Results confirm the notion of a single common ancestor, and Chlamydomonas reinharditii alone among all sequenced green unicells is most similar. Interbreeding isolates were nearest neighbors on the evolutionary tree in all cases. Some taxa, at whatever level, prove to be clades by sequence comparisons, but others provide striking exceptions. The morphological species Pandorina morum , known to be widespread and diverse in mating pairs, was found to encompass all of the isolates of the four species of Volvulina . Platydorina appears to have originated early and not to fall within the genus Eudorina , with which it can sometimes be confused by morphology. The four species of Pleodorina appear variously associated with Eudorina examples. Although the species of Volvox are each clades, the genus Volvox is not. The conclusions confirm and extend prior, more limited, studies on nuclear SSU and LSU rDNA genes and plastid-encoded rbcL and atpB. The phylogenetic tree suggests which classical taxonomic characters are most misleading and provides a framework for molecular studies of the cell cycle-related and other alterations that have engendered diversity in both vegetative and sexual colony patterns in this classical family.

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.

The growth of Eudorina unicocca in laboratory culture is shown to conform to a model based upon the in situ formation of daughter colonies from the cells in mother colonies and upon their subsequent recruitment to the population. It is shown that this mode of growth enables the alga to maintain a rapid rate of increase (ca. 0·6 day-1, equivalent to approx. 0·9 cell divisions day-1) even though only 4% of the colonies divide each day. Net growth is possible even at high intensities of filter-feeding by zooplankton.

The present communication deals with the details of sexual reproduction including the process of fusion of gametes in Eudorina californica (Shaw) Goldstein (earlier described as E. indica lyengar). The present observations are compared with those on other species in which the process of fusion was recorded.

PREVIOUS DESCRIPTIONS of mitosis in those colonial forms ascribed by Smith (1950) to the Volvocaceae present a very confusing picture. Considerable variation has been reported from genus to genus, especially with respect to the centrioles, persistence of the nuclear membrane, and nucleolus. These variations may be due to the fact that, with the exception of Pleodorina illinoisensis Kofoid, which has been investigated twice (Merton, 1908; Hovasse, 1937), the investigators were dealing with different species; or they may be due to the cytological methods used, or to differences in interpretation of the structures observed. Since, of necessity, one is usually dealing with fixed material in the study of mitosis, different interpretations of the chronology of events would lead to entirely different pictures of the process. Dangeard (1900), Merton (1908), and Metzner (1945) were unable to see centrioles or centrosomes in Pandorina morum Bory, Pleodorina illinoisensis Kofoid, and Volvox Carteri Stein, respectively, On the other hand, in Eudorina elegans Ehr. Hartmann (1921) described a small granule adjacent to the nucleolus (Binnenk6rper) which divided in prophase. The division products moved apart to form the spindle and could be seen as centrioles at the poles in metaphase. Zimmermann (1921) found a small body (Randk6rper) attached to the nucleolus (Binnenkorper) in the metabolic nucleus of Volvox aureus Ehr. He considered it to be an ergastic structure since it disappeared at prophase, and thought the centriole of Hartmann was just such a body. Zimmermann stated that centrioles were often seen at the poles of the intranuclear spindle. Doraiswami (1940) pictured a dark body near the nucleolus at prophase in Eudorina indica Iyengar. Although he did not see it divide, he found centriole-like structures at the poles at metaphase and thought it possible that they arose from division of the body observed at prophase. Chatton (1910) made similar observations on Pleodorina californica Shaw, and in addition, showed the centrioles connected by a "centrodesmose axiale." In Eudorina (Pleodorina) illinoisensis (Kofoid) Pascher, Hovasse (1937) termed the small body seeen in prophase a "satellite du caryosome." Since sometimes several bodies were present, he considered them merely the chromatic granules of the prophase chromosomes. The centrosomes which he noted throughout the mitotic cycle arose from the basal granules (grains basaux) of the flagella. In Vol

A formal genetic analysis of the heterothallic, colonial green algaEudorina eleganshas been described. Wild-type strains were found to be sensitive to different drugs when grown on minimal agar containing very low concentrations of these drugs. Mutant strains resistant to high concentrations of drugs have been isolated. These aremsr-500(resistant to 500 μg/ml of DL-methionine-DL-sulfoximine),ery-r-100(resistant to 100 μg/ml of erythromycin) andsr-100(resistant to 100 μg/ml of streptomycin). The wild-type phenotypes sensitive to these drugs have been designated asmss-500,ery-s-100andss-100respectively. Thesr-100also showed cross-resistance to other antibiotics belonging to the streptomycin group.On genetic analysis, themsr-500andery-s-100were found to be inherited in a Mendelian way. These alleles are not linked to each other or to the mating type locus. The inheritance of mating type was found to be due to a single gene difference.The inheritance ofss-100/sr-100was found to be non-chromosomal and was characteristically uniparental, always transmitted through the female parent. The evidence for the non-chromosomal gene (NC genes) controllingsr-100/ss-100phenotypes in this organism has been derived from the exceptional zygotes in which the male parent apparently transmits streptomycin resistance to the progeny. Although ultraviolet or gamma-radiation resulted in normal survival curves of the exposed cells, no mutant deficient in any nutritional requirement was isolated.

Background Volvocine algae, which range from the unicellular Chlamydomonas to the multicellular Volvox with a germ–soma division of labor, are a model for the evolution of multicellularity. Within this group, the spheroidal colony might have evolved in two independent lineages: Volvocaceae and the goniacean Astrephomene. Astrephomene produces spheroidal colonies with posterior somatic cells. The feature that distinguishes Astrephomene from the volvocacean algae is lack of inversion during embryogenesis; the volvocacean embryo undergoes inversion after successive divisions to orient flagella toward the outside. The mechanisms of inversion at the molecular and cellular levels in volvocacean algae have been assessed in detail, particularly in Volvox carteri. However, embryogenesis in Astrephomene has not been subjected to such investigations. Results This study relied on light microscopy time-lapse imaging using an actively growing culture of a newly established strain to conduct a developmental analysis of Astrephomene as well as to perform a comparison with the similar spheroidal volvocacean Eudorina. During the successive divisions involved in Astrephomene embryogenesis, gradual rotation of daughter protoplasts resulted in movement of their apical portions toward the embryonic posterior, forming a convex-to-spheroidal cell sheet with the apical ends of protoplasts on the outside. Differentiation of the posterior somatic cells from the embryo periphery was traced based on cell lineages during embryogenesis. In contrast, in Eudorina, the rotation of daughter protoplasts did not occur during successive cell divisions; however, inversion occurred after such divisions, and a spheroidal embryo was formed. Indirect immunofluorescence microscopy of basal bodies and nuclei verified this difference between Astrephomene and Eudorina in the movement of embryonic protoplasts. Conclusions These results suggest different tactics for spheroidal colony formation between the two lineages: rotation of daughter protoplasts during successive cell divisions in Astrephomene, and inversion after cell divisions in Eudorina. This study will facilitate further research into the molecular and genetic mechanisms of the parallel evolution of the spheroidal colony in volvocine algae. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0794-x) contains supplementary material, which is available to authorized users.

A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available. Here, we describe the stable nuclear transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable nuclear transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.