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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.

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

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.

Seven species of Volvocaceae were recorded in the lower Vistula River and its oxbow lakes, including Pleodorina californica for the first time in Poland. Three species – Eudorina cylindrica, E. illinoisensis and E. unicocca – were found in the Polish Vistula River in the 1960s and 1970s, as well as at present. They are rare species in the Polish aquatic ecosystems. Three species are common both in the oxbow lakes and in the Vistula River: Eudorina elegans, Pandorina morum and Volvox aureus. New and rare Volvocaceae species were described in terms of morphology and ecology; also photographic documentation (light microscope microphotographs) was completed.

This review covers essentially all aspects of the organisms in the green algal family Volvocaceae and suggests the genetic history of the various steps in their evolution from their unicellular ancestors.

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.

Steering their swimming direction toward the light is crucial for the viability of Volvox colonies, the larger members of the volvocine algae. While it is known that this phototactic steering is achieved by a difference in behavior of the flagella on the illuminated and shaded sides, conflicting reports suggest that this asymmetry arises either from a change in beating direction or a change in beating frequency. Here, we report direct observations of the flagellar behavior of various Volvox species with different phyletic origin in response to light intensity changes and thereby resolve this controversy: Volvox barberi W. Shaw from the section Volvox sensu Nozaki (2003) changes the direction of the flagellar beating plane, while species encompassed in the group Eudorina (Volvox carteri F. Stein, Volvox aureus Ehrenb., and Volvox tertius Art. Mey.) decrease the flagellar beating frequency, sometimes down to flagellar arrest.

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...

The volvocacean genus Pleodorina has been morphologically characterized as having small somatic cells in spheroidal colonies and anisogamous sexual reproduction with sperm packets. In this study we examined two new species that can be assigned to the genus Pleodorina based on morphology: P. starrii H. Nozaki et al. sp. nov. and P. thompsonii F. D. Ott et al. sp. nov. P. starrii was collected from Japan and had 32- or 64-celled colonies with anterior somatic cells and spheroidal individual cellular sheaths that were weakly attached to each other within the colonial envelope. P. thompsonii from Texas (USA) exhibited four or 12 somatic cells in the anterior pole of 16- or 32-celled colonies, respectively, and had a single large pyrenoid in the chloroplast of mature reproductive cells. The chloroplast multigene phylogeny placed P. starrii and P. indica (Iyenger) H. Nozaki in a clade that was robustly separated from the type species P. californica Shaw and P. japonica H. Nozaki. Pleodorina thompsonii was resolved as a basal branch within a large monophyletic group (Eudorina group) composed of Eudorina, Pleodorina and Volvox (excluding section Volvox). Thus, Pleodorina was found among three separate lineages within the Eudorina group in which Eudorina and Volvox were also resolved as nonmonophyletic. The DNA sequences from additional species/strains as well as recognition of morphological attributes that characterize the monophyletic groups within the Eudorina group are needed to construct a natural generic classification within these members of the Volvocaceae.

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.

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.

Mating affinity, nuclear rDNA internal transcribed spacer (ITS) sequence, and geographic distribution of more than 100 isolates of Pandorina, Volvulina, and Yamagishiella were determined. Comparative analysis of ITS sequences reaffirmed the unity of Pandorina morum and its separation from all other species of Volvocaceae except those of Volvulina. This latter genus, represented by four species, appears to represent only morphological variants of several P. morum subclades. The P. morum clade (including also additional species such as P. smithii and P. colemaniae) encompasses an evolutionary span, as determined by comparison of ITS, greater than either of the multispecies genera Gonium or Eudorina. There are at least 30 Pandorina/Volvulina syngens, sexually isolated groups, so far as can be determined, among the current collection of strains. In addition, as in other volvocacaean genera, two clones are homothallic, capable of forming zygotes within a genetic clone. The existence of so many syngens suggests that considerable evolutionary diversification of the genes controlling gamete compatibility and intercross survival has occurred, unaccompanied by significant morphological change. Within each syngen, genetic distance increases with geographic distance between collection sites. At least half of the isolates studied must have been introduced northward since the Pleistocene. Although we probably know more about characters in this group of algae than any other algal group, in part because they are so easy to culture, we are still largely ignorant of what circumscribes their niche in nature. The study of all these organisms, distributed throughout the world presumably by the activities of shorebirds, emphasizes the evolutionary role of mating genes in the inevitable formation of new genetically isolated subclades in these eukaryotes and provides initial data on their rate of appearance versus their rate of distribution over the earth.

Phylogenetic analyses of 19 strains representing five species of Eudorina, one strain of Pleodorina indica, and seven strains of Yamagishiella unicocca were carried out by sequencing the internal transcribed spacer region (ITS 1 and ITS 2) of the nuclear ribosomal DNA (rDNA) repeats. The sequence data resolved five phylogenetic groups, one consisting of Y. unicocca and the other four encompassing all the Eudorina species. Two isolates, Eudorina sp. (ASW 05157) and Pleodorina indica (ASW 05153), were of uncertain affiliation. Whereas one monophyletic group included strains of E. elegans only, the other strains of E. elegans appeared alongside E. cylindrica, E. illinoisensis, and E. unicocca var. unicocca in the other Eudorina clades. The distribution pattern of the carotenoid loroxanthin ([3R,3′R,6′R]-β,e-carotene-3,19,3′-triol), a systematically useful biochemical marker within chlorophycean flagellates, was shown to match the evaluated molecular data. Whereas it was either totally absent or universally present in six of the deduced phylogenetic lines, it occurred randomly in the E. elegans clade containing only E. elegans isolates. The results substantiated the current hypothesis that the unique vegetative morphology of E. elegans has independently arisen at various times during evolution and that it is not a marker of a monophyletic group.

The chloroplast-encoded atpB gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbcL gene sequence data.1128 base pairs in the coding regions of the atpB gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera—Eudorina, Pleodorina, and Volvox, excluding the section Volvox (=Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbcL gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbcL gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbcL gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atpB gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atpB and rbcL genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.

Species and varieties in the genus Eudorina Ehrenberg (Volvocaceae, Chlorophyta) were evaluated on the basis of phylogenetic analyses of the large subunit ofribulose-1,5-bis-phosphate carboxylase/oxygenase (rbcL) gene sequences from 14 strains of four Eudorina species, as well as from nine species of Pleodorina and Volvox. The sequence data suggested that 10 of the 14 Eudorina strains form three separate and robust monophyletic groups within the nonmonophyletic genus Eudorina. The first group comprises all three strains of E. unicocca G. M. Smith; the second group consists of one of the E. elegans Ehrenberg var. elegans strains, the E. cylindrica Korshikov strain, and both E. illinoisensis (Kofoid) Pascher strains; and the third group consists of two monoecious varieties of E. elegans [two strains of E. elegans var. synoica Goldstein and one strain of E. elegans var. carteri (G. M. Smith) Goldstein]. In addition, E. illinoisensis represents a poly- or paraphyletic species within the second group. The remaining four strains, all of which are assigned to E. elegans var. elegans, are nonmonophyletic. Although their position in the phylogenetic trees is more or less ambiguous, they are ancestral to other taxa in the large anisogamous/oogamous monophyletic group including Eudorina, Pleodorina, and Volvox (except for sect. Volvox). Thus, the four Eudorina groups resolved in the present molecular phylogeny do not correspond with the species concepts of Eudorina based on vegetative morphology, but they do reflect the results of the previous intercrossing experiments and modes of monoecious and dioecious sexual reproduction.

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.

The chloroplast-encoded large subunit of the ribulose-1, 5-bisphosphate carboxylase / oxygenase (rbcL) gene was sequenced from 20 species of the colonial Volvocales (the Volvacaceae, Goniaceae, and Tetrabaenaceae) in order to elucidate phylogenetic relationships within the colonial Volvocales. Eleven hundred twenty-eight base pairs in the coding regions of the (rbcL) gene were analyzed by the neighbor-joining (NJ) method using three kinds of distance estimations, as well as by the maximum parsimony (MP) method. A large group comprising all the anisogamous and oogamous volvocacean species was resolved in the MP tree as well as in the NJ trees based on overall and synonymous substitutions. In all the trees constructed, Basichlamys and Tetrabaena (Tetrabaenaceae) constituted a very robust phylogenetic group. Although not supported by high bootstrap values, the MP tree and the NJ tree based on nonsynonymous substitutions indicated that the Tetrabaenaceae is the sister group to the large group comprising the Volvocaceae and the Goniaceae. In addition, the present analysis strongly suggested that Pandorina and Astrephomene are monophyletic genera whereas Eudorina is nonmonophyletic. These results are essentially consistent with the results of the recent cladistic analyses of morphological data. However, the monophyly of the Volvocaceae previously supported by four morphological synapomorphies is found only in the NJ tree based on nonsynonymous substitutions (with very low bootstrap values). The genus Volvox was clearly resolved as a polyphyletic group with V. rousseletii Pocock separated from other species of Volvox in the rbcL gene comparisons, although this genus represents a monophyletic group in the previous morphological analyses. Furthermore, none of the rbcL gene trees supported the monophyly of the Goniaceae; Astrephomene was placed in various phylogenetic positions.

A cladistic analysis was used to deduce the phylogenetic relationships within the colonial Volvocales. Forty-one pairs of characters related to gross morphology and ultrastructure of vegetative colonies as well as asexual and sexual reproduction were analyzed based on parsimony, using the PAUP 3.0 computer program, for 25 species belonging to nine volvocacean and goniacean genera of the colonial Volvocales. Chlamydomonas reinhardtii Dangeard was the outgroup. The strict consensus tree indicated the presence of two monophyletic groups, one composed of all the volvocacean species analyzed in this study and the other containing the goniacean species except for the four-celled species Gonium sociale (Dujardin) Warming. In addition, these two groups constitute a large monophyletic group, to which G. sociale is a sister group. A new combination Tetrabaena socialis (Dujardin) Nozaki et Itoh and a new family Tetrabaenaceae Nozaki et Itoh are thus proposed for G. sociale. In addition, the analysis suggests that the volvocacean genera Eudorina and Pleodorina are paraphyletic groups, respectively, and that the monotypic genus Yamagishiella has no autapomorphic characters and represents primitive features of the anisogamous and oogamous genera of the Volvocaceae. Phylogenetic relationships within the Volvocaceae and the Goniaceae, as well as the various modes of sexual reproduction exhibited by these organisms, are discussed on the basis of the analysis.

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 .

Vegetative colonies of Pandorina unicocca Rayburn et Starr, four taxa of Eudorina [E. elegans Ehrenberg (type species), E. illinoisensis (Kofoid) Pascher, E. unicocca G.M. Smith var. unicocca andE. unicocca var. peripheralis Goldstein] and two species of Pleodorina [Po californicaShaw (type species) and P. indica (Iyengar) Nozaki] were examined with electron microscopy in order to characterize the structure of the extracellular matrix. Each cell of the colonies of all the taxa examined was tightly enclosed by a dense fibrillar zone of the extracellular matrix (cellular envelope) with sparse fibrillar material filling the space outside the cellular envelopes within the tripartite colonial boundary of the matrix. This arrangement is essentially different from that of Pandorina morum (O.F. Miiller) Bory (type species) and P. colemaniae Nozaki. As Eudorina and Pleodorina both have anisogamous sexual reproduction with sperm packets (bundles of male gametes), a new genus, Yamagishiella Nozaki, is propo...

Comparative ultrastructural data have shown that at least two distinct groups exist within Carteria. Similarly, interpretations of variation in gross morphological features have led to the discovery of morphologically distinct groups within the genus. Partial sequences from the nuclear-encoded small- and large-subunit ribosomal RNA molecules of selected Carteria taxa were studied as a means of 1) testing hypotheses that distinct groups of species exist within the genus and 2) assessing monophyly of the genus. Parsimony analysis of the sequence data suggests that three Carteria species, C. lunzensis, C. crucifera, and C. olivieri, form a monophyletic group that is the basal sister group to all other ingroup flagellate taxa (including species of Chlamydomonas, Haematococcus, Stephanosphaera, Volvox, and Eudorina). Two other Carteria taxa, C. radiosa and Carteria sp. (UTEX isolate LB 762), form a clade that is the sister group to a clade that includes Haematococcus spp., Chlamydomonas spp., and Stephanosphaera. Thus, the sequence data support the interpretations of ultrastructural evidence that described two distinct Carteria lineages. Moreover, the sequence data suggest that these two Carteria groups do not form a monophyletic assemblage. Parsimony analysis of a suite of organismal (morphological, ultra-structural, life history, and biochemical) character data also suggest two distinct lineages among the five Carteria taxa; however, the organismal data are ambiguous regarding monophyly of these Carteria taxa. When the two independent data sets are pooled, monophyly of Carteria is not supported; therefore, the weight of available evidence, both molecular and organismal, fails to support the concept of Carteria as a natural genus.

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.

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.

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.

Traditionally, the volvocacean genera Pandorina and Eudorina are distinguished by vegetative morphology: Eudorina has hollow coenobia with loosely arranged spherical to ovoid cells, whereas Pandorina coenobia are compact with pear- to keystoneshaped cells contiguous in the centre. However, sometimes intermediate forms are found which cannot be identified to genus by traditional morphological characters. Especially the degree of compactness of Pandorina coenobia may be highly variable. Such abberant forms must be taken into culture in order to establish their identity, by studying (1) their life cycles, both sexual and asexual processes, and (2) the influence of varied culture conditions (temperature, daylenght, nutrients) on their morphology. In these ways, the taxonomic position may be clarified.

The existence of various microorganisms, eukaryote in structure, that lack certain characteristics expressed in other Kingdoms (animals, plants, and fungi) has resulted in the appearance of the Protoctista Kingdom. This Kingdom comprises a diverse group of eukaryote microorganisms, grouped in various Phyla, among which there are certain classes of algae, such as Chlorophyta. The order of volvocales, formed of colonies of spherical cells (from 8, 16, 32, 64 up to 128) embedded in a clear gelatinous matrix, belongs to this kingdom. Each triangular-round cell contains a nucleus, more or less central, a pyrenoid, and a contractile vacuole into which are inserted two flagella. Freshwater colonies are widely distributed in pools, ditches, reservoirs, and lakes. We describe in a sputum smear stained with the Papanicolaou technique, and corresponding to a 68-yr-old-man who presented various episodes of hemoptysis and with COPD clinical antecedents, the presence of a contaminant, a protoctista structure, catalogued as a Chlorophyte alga (presumably Eudorina sp.). The moruliform disposition of the cells, with very striking nuclei and the nonvisualization of the gelatinous capsule (Fig. C-1A), resembles certain types of Adenocarcinoma. Under greater magnification, the existence of a capsule and the presence of flagella in two cells (Fig. C-1B) are suggested. To confirm the existence of the capsule, the preparation was discolored and restained using the Peryodic Acid Schiff (PAS) technique, evidencing the existence of a PAS (+) structure surrounding all the cells in the form of a capsule (Fig. C-2). To catalogue this structure as a contaminant, the presence of inexistent structures in human cells such as contractile vacuole, flagella, and a gelatinous capsule were considered. Furthermore, with a careful microscopic examination of two consecutive samples corresponding to the same patient, similar structures were not found. We postulate that this peculiar contaminant came from tap water used during staining procedure or due to a contamination of the sputum in the oral cavity. This case highlights the importance of considering uncommon findings in cytological smears (artefacts and contaminants) to avoid false positives and, with a careful evaluation, establish a correct cytodiagnosis.

Two comparable methods were used to study the feeding of four species of Daphnia on large spherical particles which differed in size and hardness. The first method used gut analysis to estimate the selectivities of daphnids feeding in a broad size range of a single particle type, including polystyrene beads (4–60 µm diameter) in the laboratory and Eudorina colonies (10–90 µm) in the field. In the second method, Daphnia of different sizes fed in a mixture of 6.5 µm Chlamydomonas and one of eight test particles. Smaller daphnids were less effective in feeding on large test particles. Nonlinear regression was therefore used to estimate the Daphnia body size at which the clearance rate on a test particle was reduced to 50% of that for Chlamydomonas. The results of both methods show that prey size and hardness are both very important in determining daphnid feeding selectivity. For a given particle size, ‘soft’ algae (naked and gelatinous flagellates) are more readily ingested than ‘hard’ algae (diatoms and dinoflagellates), and ‘hard’ algae are more readily ingested than polystyrene beads. Daphnia can feed effectively on algae that are 2–5 times larger than the largest ingestible bead.

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.

Publisher Summary This chapter presents a summary of the morphological and biochemical evidence that supports the volvocine-lineage hypothesis. It describes attempts to evaluate the validity of this hypothesis objectively with molecular phylogenetic methods. The chapter also reviews advances that have occurred in three areas of Volvox research:(1) the genetic and molecular control of germ or soma differentiation, (2) the mechanism of action of the sexual pheromone, and (3) the comparative anatomy and evolution of selected structural genes of Volvox and Chlamydomonas. The volvocine algae (Chlamydomonas and the members of the family Volvocaceae) all have a fundamentally similar and distinctive pattern of subcellular organization. However, the volvocine algae can be conceptually lined up in a series (Chlamydomonas, Gonium, Pandorina, Eudorina, Pleodorina, and Volvox), in which there is a progressive increase in each of the parameters—cell number, organismic size, tendency for some cells to differentiate terminally as somatic cells, and ratio of somatic-cell number to reproductive-cell number. The volvocine lineage hypothesis postulates that this conceptual series may actually reflect the evolutionary history of the group. The aspect of Volvox carteri development that is most interesting from an evolutionary genetic standpoint is the aspect that most clearly differentiates it from other genera of volvocine algae, and from most other species of Volvox, i.e., the early and complete segregation of separate germ and somatic-cell lineages.

A new method was utilized to study species-specific responses of phytoplankton to phosphorus limitation in a nutrient enrichment experiment. A substrate, ELF, produces a fluorescent precipitate at the sites of alkaline phosphatase (AP), which makes it possible to visually detect phosphorus (P) limitation in individual cells of multiple species. Lake water was incubated in the laboratory to induce nitrogen (N) or P limitation. Initially, little or no ELF labeling was observed for any of the phytoplankton species, indicating a general lack of P limitation. This observation was supported by low bulk AP activity in the initial field samples. During the experiment, several chlorophyte taxa (Coelastrum, Eudorina, a solitary spiny coccoid) were driven to P limitation, as evidenced by a high percentage of cells displaying ELF labeling when inorganic N was added. Taxa such as Actinastrum and Dictyosphaerium, on the contrary, were never P limited. Little or no ELF was observed in cyanobacterial species, suggesting that P limitation was not achieved in these organisms. Using traditional bulk AP activity, significantly higher levels of AP activity were observed in treatments with inorganic N additions, compared to those with phosphate additions. ELF labeling generally followed the trend of bulk AP, except in species that did not dominate the biomass. Finally, we noted that all species observed were ELF labeled at least on one occasion, except for fragile flagellates which did not withstand the labeling procedure.

The morphology, reproduction and taxonomy of Pleodorina japonica Nozaki sp. nov. collected in Japan were studied using clonal cultures. This taxon is distinguished from the other species of Pleodorina and Eudorina by its unique vegetative colonies, in which only 20–27% of the cells are somatic and anterior, and in which the gelatinous matrix does not form individual sheaths. Zygote germination was observed for the first time in Pleodorina. A single biflagellate gone-cell moved out from the zygote wall. In addition, abundant bacteria-like endosymbionts in the cytoplasm of somatic and reproductive cells as well as in the male and female gametes and zygotes were revealed by means of 4'-6-diamidino-2-phenylindole (DAPI) staining and electron microscopy. The endosymbiont was rigidly rod-shaped, measured 1.5–1.9 μm long and emitted strong blue-white fluorescence under the epifluorescence microscope after DAPI staining, and it was surrounded by electron transparent areas. The generic delimitation betwee...

Reversion of an acetate requiring strain and the induction of sectored colonies are used to establish optimal conditions for nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans Ehrenberg. Nitrosoguanidine is more effective in causing reversion of the acetate requiring strain and inducing auxotrophs. Morphogenetic mutants are more readily induced by ultraviolet light. The effectiveness of ultraviolet light as a mutagen is cell cycle dependent whereas the mutagenic action of nitrosoguanidine is not.

SUMMARY Analysis of synchronous populations of Eudorina elegans Ehrenberg reveals that DNA replication occurs coincidentally with the very rapid sequence of cell divisions. The average doubling time for DNA in such synchronous populations is 40 min. The basal amount of DNA/E. elegans cell is estimated to be 0.5 × 10−12 g.

Colonies of Eudorina elegans Ehrenberg were treated with a 0.2% colchicine solution for periods of up to 48 h, and a number of alterations were observed in dividing colonies. Nuclear alterations were observed after 20 min of treatment, due to the inhibition of spindle microtubule polymerization. This inhibition resulted in increased ploidy levels, and permanent diploid colonies were obtained. The inhibition of cytoplasmic microtubule polymerization resulted in a number of structural alterations including: unequal cytokinesis of plakeal cells, the partial or complete inhibition of cytokinesis (30 min treatment), production of “stellate” cells (90 min treatment), and the subsequent formation of extra-cytoplasmic particles around the plakeal cells (3 h treatment). A possible cytoskeletal function of peripherally orient ed microtubules, and the role of the phycoplast microtubules is discussed. In addition, colchicine treatment caused an inhibition of inversion (60 min treatment), an increase in golgi-associated vesicles, and an excessive production of colonial envelope material (3 h treatment). The latter resulted in the formation of flattened Gonium-like colonies. The process of inversion is discussed in light of the above results. Chloroplast microtubules, however, were unaffected by colchicine treatment.

Several nuclear events seen during the cleavage period in Eudorina suggest that chromosome endoreduplication, proportional to the number of cells to be produced, may occur in the gonidia prior to cleavage. Presumably the DNA concentration is reduced to the haploid level during rapid, successive divisions of the cleavage period. To test this hypothesis, I determined DNA content of gonidia as they grew from 4 mum to 38 mum in diameter between cleavage periods. During growth from 4 mum to 8 mum in diameter, the DNA concentration remained at the haploid level of 0.17 pg/cell. As gonidia in 64 cell colonies continued to grow from 8 mum to 33 mum in diameter, their DNA concentrations increased 60-fold. Analysis of the Eudorina DNA by equilibrium centrifugation in CsCl showed only 2 bands with buoyant densities of 1.721 g/cm3 and 1.699 g/cm3, presumed to be nuclear and chloroplast, respectively, on the basis of labelling with 3H-thymidine and 3H-adenine. The 8:2 ratio of the two bands did not change with increase in cell size and no other bands were detected, suggesting that both nuclear and chloroplast DNAs were synthesised proportionately prior to the cleavage period.

The colonial matrices of the volvocacean algae were examined for the presence of sulfated and carboxylated polysaccharides. These results were compared to a similar examination of the single-celled Chlamydomonas reinhardtii Dang. The colonial algae examined were Pandorina morum Bory, Eudorina elegans Ehr., Platydorina caudata Kofoid, Pleodorina californica Shaw, Pleodorina illinoisensis Kofoid and Volvox carteri var. nagariensis Iyengar. Alcian blue staining of whole colonies at pH 0.5 and 2.5 showed evidence for the presence of both sulfated and carboxylated polysaccharides in the extracellular matrix. Quantitative measurement of alcian blue bound to solubilized matrices supported the in vivo results. There was a trend toward an increase in sulfated polysaccharides in the more evolutionary advanced forms with the exception of Pleodorina. This trend was readily seen in the sulfate: carboxyl ratios: Pandorina morum—0.4, Eudorina elegans—1.0, Platydorina caudata—2.1 and Volvox carteri—2.2. The acidic nature of the Pleodorina matrix with a sulfate: carboxyl ratio of 0.2 appeared to be more like that of Pandorina rather than that of the more advanced Volvox.

The temporal relationship of DNA replication and nuclear division has been examined by Feulgen microspectro-photometry in the colonial alga Eudorina elegans. DNA replication was found to precede each nuclear division and to occupy the entire 10–15 min interphase between divisions. Conventional G1 and G2 phases are absent or are very brief in Eudorina. Nuclear growth occurred during the interval of rapid divisions compensating, to a large extent, for the decrease in nuclear size resulting from the division sequences.

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.

New records of the fungus have been obtained from bodies of water in England, Scotland, Switzerland, Italy, Canada and the U.S.S.R. Additional information regarding certain stages in the life history is described and illustrated. Noteworthy are: the presence, within the motile zoospore, of two minute contractile-type vacuoles; and the development, by the zoospore, of short rod-like processes. There is slight evidence that these processes become attached to host flagella and so initiate the first stage in colonization. The hitherto unknown presence of a bunch of finger-like rhizoids within infected host cells is demonstrated. Sporangia may be spherical as well as elongate. Resting spore formation was identical to that already described. As well as spiny-walled spores, those with a smooth wall and with very strongly developed spines giving the surface a bullate appearance were found. On germination, the spore buds out a sporangium. The host range is extended to include Eudorina unicocca and Pandorina morum.

Three nitrate reductase mutants were independently isolated and characterized in the colonial alga, Eudorina elegans Ehrenberg. nar-1 is a leaky mutant, deficient in the production of nitrate reductase. nar-2 and nar-3 both lack the ability to produce nitrate reductase. However, nar-2 grows and nar-3 does not grow when hypoxanthine is the sole nitrogen source. The specific activity of the next enzyme, in the pathway, nitrite reductase is increased in nar-3 when compared to wild-type, nar-1 and nar-2.

The nuclear DNA content during normal vegetative growth and division has been examined in three species of Volvocales, Chlamydomonas reinhardtii Dangeard, Pandorina morum Bory, and Volvox carteri f. nagariensis Iyengar. The results are consistent with the nuclear cycle reported in the literature for Eudorina. Nuclear DNA content does not increase during the prolonged cell growth phase. At the time of colony formation, nuclear DNA doubles, the nucleus divides, and this alternation continues until the final 2n complement of progeny nuclei is formed. The 4- and 8-nucleate stages of dividing gonidia of V. carteri have a nuclear DNA content in the same range as the somatic cells; they are not polyploid or polytene. Four normal clones of Pandorina, having 2, 5 or 12 chromosomes, all had similar amounts of DNA per nucleus, suggesting that the species has a nuclear genome of fairly constant size rather than consisting of many strains representing a polyploid series. One unique clone, a hybrid with double the chromosome number of either its parents, had twice as much DNA as the normal clones. The Feulgen spectrophotometric method is sufficiently sensitive to detect 2-fold differences in DNA content at the level of 2 × 10−13 g of DNA /nucleus, and its use avoids the complications associated with the presence of organelle DNA.

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.

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.

Electron microscopy of the eyespot of the coenobic yolvocacean Eudorina illinoiensis (Kofoid) Pascher shows that besides the well-documented anterior-posterior decrease in size, congruity of structure is involved in eyespot differentiation. In the anterior cells the eyespot lies adjacent to the plasmalemma with the thylakoid underlayers parallel to it, while in the posterior cells eyespot placement and orientation may vary considerably. Deep-sited eyespots of congruent organisation probably represent the mother-cell eyespot; discongruent ones are typical of posterior cells, but the smallest ones may be random associations of osmiophilic globules. Eyespot structure is discussed in relation to photoreception and phototaxis.

A new pattern of colony differentiation was observed in a portion of a clone (5%) in the normally undifferentiated species, Eudorina elegans. The new form possesses somatic and generative cells arranged on opposite sides of the anterior–posterior axis of the colony and the somatic cells die gradually during colony maturation. The development and ultimate pattern of colony differentiation is described and compared with known patterns of differentiation in Eudorina illinoisensis and Eudorina (Pleodorina) californica. A possible mechanism to account for colony differentiation in Eudorina is discussed.