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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.

Eudorina elegans was used to examine the response of an easily manipulated, multicellular organism to ultraviolet irradiation. The results indicate that E. elegans possesses an efficient photoreversal process. It is capable of complete reversal of ultraviolet induced damage sufficient to inactivate 99.99% of the colony-forming ability of the organism. Eudorina loses the ability to respond to visible light reversal of ultraviolet-induced damage exponentially with time. The half-life of this loss is about 10 h at 32° and about 20 h at 22°. Postultraviolet temperature of incubation influences the surviving fraction with fewer survivors at 22° than at 32°. The survival patterns of E. elegans suggest that a specific dark repair of ultraviolet-induced lesions may not occur, but that some repair processes take place during cellular deoxyribonucleic acid (DNA) synthesis.

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

The fine structure of the colonial volvocacean alga Eudorina illinoiensis (Kofoid) Pascher is described in detail, excepting the eye spot. The structure conforms closely to the Chlamydomonas type and helps confirm that certain ultrastructural features are peculiar to certain taxonomic groups, the characteristic structure of the transitional region of the flagellum being an example in this case. A spiral filament around the outer doublets of the axoneme is newly reported. Particular attention is given to the flagellar apparatus and to the structure of the chloroplast in relation to the pyrenoid. Small stacks of thylakoids pass between the starch plates to enter the pyrenoid where they assume a tubular form. The spatial re-organisations required to achieve this are described.

SYNOPSIS. A comparative morphological study was made of 73 clones of Eudorina isoated from 44 natural populations and grown under controlled environmental conditions. Utilizing the information obtained in this study in association with the existing taxonomic and experimental literature concerning Eudorina a monographic treatment of the genus was prepared. Nine species and 4 varieties are now recognized. Sexual compatibility was investigated with 22 heterothallic pairs, 3 male strains and one female strain representing 4 species and one variety of Eudorina. Five partially or completely sexually-isolated groups were found: 3 groups containing strains of a single species, a fourth group composed of strains of another species, and a fifth group made up of strains of 3 different species. Strains with different zygote arrangements were found generally not to intercross. Intraspecific crosses indicated that a single pair of alleles controls the inheritance of mating types and that gene flow occurs between strains representing different natural populations. Interspecific crosses resulted in the formation of polyploid or aneuploid F1 offspring which shewed an abnormal segregation of the mating types. Abnormal mating type segregation was also obtained in F2 crosses involving certain of the hybrid polyploid offspring.

Fifteen of 16 species of aquatic insects studied, including 5 aquatic Hemiptera, 5 caddisflies, 1 stonefly, 2 dobsonflies, and 2 mayflies, were found to be transporting 27 genera of viable small aquatic organisms. Three corixids carried 5 genera of blue-green algae, 11 green algae, 1 yellow-green alga, 2 protozoans, and fungi. Eudorina, representing colonial volvocalean algae, was found for the 1st time on field-exposed aquatic insects. The suitability of the Corixidae and Notonectidae as passive dispersal vehicles for small organisms is discussed. Only 1 of 6 caddisflies studied carried algae and protozoans, but that species, Hydropsyche orris Ross, carried 10 genera. Organisms taken in casual samplings of Plecoptera, Megaloptera, and Ephemeroptera are indicated. Excluding fungi, Chlorella was the most common viable form being carried, followed by Bodo, Nannochloris Chloroccoccum, and Microcystis, in order of frequency of occurrence. Unidentified fungi and Fusarium were carried by more than 75% of the 16 insects sampled and occurred in more than 50% of the 112 culture samples.

SUMMARY Male and female strains of Eudorina elegans Ehrenberg were examined by serological methods for flagellar and cellular antigenic differences. No significant differences could be detected between the strains by using agglutination, immobilization and diffusion tests. However, differentiated sperm were neither agglutinated nor immobilized by anti-vegetative cell sera.

Multicellular animals and plants are larger and longer-lived than protists, and long-term isolate culture demands a great deal of patience. There are very few cases in which asexual metazoans or metaphytes have been taken through more than a hundred generations in the laboratory. The available literature is summarized in Table 6. Plants I have already mentioned Hartmann's culture of the colonial green alga Eudorina , which went through over 228 generations with no loss of vigour (see Table 2). Uspenski and Uspenskaya (1927) cultured the related Volvox aureus through 47 asexual generations in 15 months, but do not give details of the rate of reproduction. These algae are haplonts. A particularly vivid instance of senescence and rejuvenation was described by Mather and Jinks (1958) in the fungus Aspergillus . Continued propagation by asexual spores led to a rather steep decline in perithecial production within 10 or so generations, but vitality was completely restored by a single sexual episode. This seems to implicate a cytoplasmic deterioration analogous to macronuclear senescence in ciliates. Although vascular plants are generally too long-lived for isolate culture to be feasible, it is a commonplace of horticulture that some vegetative clones are very stable and can be transmitted by tubers (e.g. potatoes) or grafts (e.g. pear-trees) for many generations without deterioration. There is, of course, powerful artificial selection against any inferior lines that do emerge. More generally, the aging of meristems is also a commonplace, in terms of the size, shape and physiology of leaves borne at successive nodes. However, the only laboratory study of meristematic aging concerns the greatly-reduced aquatic form Lemna (Ashby et al. 1948).

Since the classical investigations on phototaxis and photoaccumulation of Euglena, Phacus, and Trachelomonas by Engelmann (1892), Oltmanns (1917) and Mast (1917), who observed maximal activity at wavelengths between 470 and 490 nm and, in addition, at 405 nm (Oltmanns 1917), phototactic reactions of flagellates are regarded as typical blue light responses, comparable to the phototropism. This has been confirmed by studies on the spectral phototactic sensitivity of Eudorina, Volvox and Chlamydomonas carried out by Luntz (1931). He observed phototactic reactions in the range between 360 and 578 nm with a maximum at 494 nm (Fig. 1). According to Oltmanns and Mast, however, the maximum of Chlamydomonas lies between 500 and 510 nm.

A study of nuclear structure and chromosome number in the colonial Volvocales has led to the development of an aceto-carmine technic particularly applicable to this group of plants. The genera investigated are Gonium, Volvulina, Eudorina, Pleodorina, and Volvox. Another genus, as yet undescribed in the literature, has also been investigated successfully by this method.

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.

SUMMARY A culture strain, CCFA 646, originating from the People's Republic of China and designated as ‘Eudorina sp.’ in the Culture Collection of Freshwater Algae at the Institute of Hydrobiology, Chinese Academy of Sciences, was re-examined taxonomically based on light and electron microscopy and molecular phylogenetic analyses. Vegetative colonies of CCFA 646 were sub-spherical or ovoid and contained 16 or 32 cells of identical size. The cells had a cup-shaped chloroplast with a single basal pyrenoid. During asexual reproduction, each daughter colony developed within a transparent vesicle inside the parental colony. Under the electron microscope, the vegetative colony exhibited a tripartite colonial boundary of the extracellular matrix. Inside the boundary, each cell was tightly enclosed by a dense fi-brillar layer of the matrix (cellular envelope). These morphological characteristics suggested that CCFA 646 is assignable to Eudorina unicocca G. M. Smith or Ya-magishiella unicocca (Rayburn et Starr) Nozaki. In addition, 1128 base pairs of the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL) gene were sequenced from CCFA 646 and NIES-578 of Y. unicocca. Phylogenetic trees were constructed based on these sequence data as well as on the previously published rbcL gene sequences from five strains of Y. unicocca, six strains of four Eudorina species and 28 related species. The sequence data suggested with high bootstrap values that CCFA 646 and all six strains of Yama-gishiella form a monophyletic group positioned outside the large monophyletic group comprising Eudorina, Pleo-dorina and Volvox (except for sect. Volvox). Therefore, CCFA 646 can be assigned to Y. unicocca, which has not previously been reported from P. R. China.

The fluorescent dye 2,5-bis (4′-aminophenyl (1′))-1,3,4-oxidiwle (BAO) was compared to the Feulgen procedure as a method for quantitative comparison of DNA levels in two species of Eudorina. The BAO procedure proved to be specific and as quantitative as the Feulgen procedure, and was more convenient and rapid. The DNA level of the nuclei of gonidial cells of Eudorina elegans Ehrenberg and E. californica (Shaw) Goldstein doubled prior to each division. The DNA level of the somatic, usually non-dividing nuclei of E. californica did not vary from the IC (haploid) level.