Sexual Spheroids Research Articles

Morphology, mating system and taxonomy of Volvox africanus (Volvocaceae, Chlorophyceae) from Thailand

BackgroundThe oogamous green algal genus Volvox exhibits extensive diversity in mating systems, including heterothallism and homothallism with unisexual (male and/or female) and/or bisexual spheroids. Although four mating systems have been recognized worldwide in strains identified as “Volvox africanus”, most of these strains are extinct. However, we previously rediscovered two types of the four mating systems (heterothallic, and homothallic with male and bisexual spheroids within a clone) from an ancient Japanese lake, Lake Biwa.ResultsHere, we obtained strains exhibiting the third mating system (homothallic with unisexual male and female spheroids within a clone) from a freshwater area of Kalasin Province, Thailand. When sexual reproduction was induced in the present Thai strains, both male and female unisexual spheroids developed to form smooth-walled zygotes within a clonal culture. Phylogenetic analyses of the internal transcribed spacer region-2 of nuclear ribosomal DNA sequences from all four mating systems, including the extinct strains, resolved the third mating system is basal or paraphyletic within the homothallic clade.ConclusionsThe present morphological and molecular data of the Thai strains indicate that they belong to the homothallic species V. africanus. The phylogenetic results suggested that third mating system (homothallic with separate male and female sexual spheroids) may represent an initial evolutionary stage of transition from heterothallism to homothallism within Volvox africanus. Further field collections in geologically stable intracontinental regions may be fruitful for studying diversity and taxonomy of the freshwater green algal genus Volvox.

A new preferentially outcrossing monoicous species of Volvox sect. Volvox (Chlorophyta) from Thailand.

Volvox sect. Volvox is an interesting group of green algae; it comprises mostly monoicous species, but evidence suggests an evolution towards dioicy. Based on cultured strains originating from Thailand, we describe Volvox longispiniferus, a novel species in Volvox sect. Volvox. This species is distinguished from others in the section by the large number of sperm packets in its monoicous sexual spheroids and by the long spines on its zygote wall. Phylogenetic analyses indicate that V. longispiniferus is distinct from the other species of two monophyletic groups within Volvox sect. Volvox. In addition, the novel species produces more zygotes when different cultures are combined compared with a single culture, suggesting a preference for outcrossing.

Morphological and molecular identification of the dioecious "African species Volvox rousseletii (Chlorophyceae) in the water column of a Japanese lake based on field-collected and cultured materials.

Volvox rousseletii is a dioecious species belonging to Volvox sect. Volvox that has previously only been found in Africa. During field surveys in a large dam lake (Lake Sagami) in Kanagawa Prefecture, central Japan, we encountered a Volvox sect. Volvox species that produces dioecious sexual spheroids in the water column. Although sexual induction of this species in culture did not produce adequately well-developed sexual spheroids for species identification, molecular data directly obtained from field-collected sexual spheroids verified the identity of field-collected male and female sexual spheroids as well as cultured materials. Based on molecular and morphological data, the species was identified as V. rousseletii. This is the first record of a dioecious species of Volvox sect. Volvox in Japan.

Morphology, reproduction and taxonomy of Volvox dissipatrix (Chlorophyceae) from Thailand, with a description of Volvox zeikusii sp. nov.

ABSTRACTA strain of Volvox collected from Thailand produced a very large asexual spheroid with maximum diameter over 2 mm long. This Thai alga was identified as Volvox dissipatrix based on the delicate cytoplasmic bridges between somatic cells, gelatinous matrix structure, monoecious sexual spheroids and the original description reporting the large asexual spheroids. Comparative analyses of light and electron microscopic features and internal transcribed spacer 2 sequences of the nuclear ribosomal DNA of Thai and Australian monoecious strains of V. dissipatrix and their closely related heterothallic strains demonstrated that the latter represents a new species, Volvox zeikusii Nozaki.

Morphology, taxonomy and mating-type loci in natural populations of Volvox carteri in Taiwan

BackgroundVolvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the production of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a > 1 Mbp linear chromosome. Fifteen sex-specific genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identified in natural populations of this species.ResultsDuring a recent fieldwork in Taiwan, we encountered natural populations of V. carteri that had not previously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identified as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to > 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specific genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specific gene HMG1f was not amplified by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain.ConclusionsMale spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon.

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

Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.

A New Morphological Type of Volvox from Japanese Large Lakes and Recent Divergence of this Type and V. ferrisii in Two Different Freshwater Habitats.

Volvox sect. Volvox is characterized by having unique morphological characteristics, such as thick cytoplasmic bridges between adult somatic cells in the spheroids and spiny zygote walls. Species of this section are found from various freshwater habitats. Recently, three species of Volvox sect. Volvox originating from rice paddies and a marsh were studied taxonomically based on molecular and morphological data of cultured materials. However, taxonomic studies have not been performed on cultured materials of this section originating from large lake water bodies. We studied a new morphological type of Volvox sect. Volvox (“Volvox sp. Sagami”), using cultured materials originating from two large lakes and a pond in Japan. Volvox sp. Sagami produced monoecious sexual spheroids and may represent a new morphological species; it could be clearly distinguished from all previously described monoecious species of Volvox sect. Volvox by its small number of eggs or zygotes (5–25) in sexual spheroids, with short acute spines (up to 3 μm long) on the zygote walls and elongated anterior somatic cells in asexual spheroids. Based on sequences of internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA; ITS-1, 5.8S rDNA and ITS-2) and plastid genes, however, the Volvox sp. Sagami lineage and its sister lineage (the monoecious species V. ferrisii) showed very small genetic differences, which correspond to the variation within a single biological species in other volvocalean algae. Since V. ferrisii was different from Volvox sp. Sagami, by having approximately 100–200 zygotes in the sexual spheroids and long spines (6–8.5 μm long) on the zygote walls, as well as growing in Japanese rice paddies, these two morphologically distinct lineages might have diverged rapidly in the two different freshwater habitats. In addition, the swimming velocity during phototaxis of Volvox sp. Sagami spheroids originating from large lakes was significantly higher than that of V. ferrisii originating from rice paddies, suggesting adaptation of Volvox sp. Sagami to large water bodies.

Delineating a New Heterothallic Species of Volvox (Volvocaceae, Chlorophyceae) Using New Strains of "Volvox africanus".

The volvocine algae represent an excellent model lineage in which to study evolution of female and male genders based on comparative analyses of related species. Among these species, Volvox carteri has been extensively studied as a model of an oogamous and complex organism. However, it may have unique derived features that are not present in other species of Volvox. Therefore, information regarding the characteristics of sexual reproduction of other species of Volvox is also important. In 1971, Starr studied four types of sexuality in several global strains identified as Volvox africanus; however, further taxonomic studies of these strains have been lacking, and strains of three of the four sexual types are not available. Here, we studied the morphology, sexual reproduction, and taxonomy of two V. africanus-like species isolated recently from Lake Biwa, Japan. These two species were very similar to two sexual types described by Starr in 1971: one producing dioecious sexual spheroids in heterothallic strains and the other forming both male spheroids and monoecious spheroids in a single strain. The former species produced zygotes with a reticulate cell wall, whereas a smooth zygote wall was observed in the latter species as in V. africanus previously reported from various localities around the world. Our multigene phylogenetic analysis demonstrated that these are sister species to each other. However, the presence of a compensatory base change in the most conserved region of the secondary structure of nuclear ribosomal DNA internal transcribed spacer-2, hybrid inviability demonstrated by intercrossing experiments, and morphological differences in the density of abutment between the gelatinous material of adjacent cells (individual sheaths) in the spheroid supported the recognition of the two species, V. africanus having a smooth zygote wall and V. reticuliferus Nozaki sp. nov. having a reticulate zygote wall.

Morphology and reproduction of Volvox capensis (Volvocales, Chlorophyceae) from Montana, USA

:Volvox capensis was recorded previously only from South Africa. Here we established culture strains of this species from a sample collected in Montana, USA. Morphological details of asexual and sexual spheroids and molecular phylogeny of these strains were studied. The present alga was identified as V. capensis on the basis of morphological characteristics of asexual spheroids and zygotes. However, differences between the Montana and South African materials were recognized in number of sperm packets in a sexual, monoecious spheroid as well as in mode of gametic union between sperm and eggs. Possible polyspermy was observed in eggs of V. capensis by 4′-6-diamidino-2-phenylidole staining. Genetic difference between these two entities was small based on sequences of internal transcribed spacer 2 region of nuclear ribosomal DNA.

DESCRIPTION OF TWO NEW MONOECIOUS SPECIES OF VOLVOX SECT. VOLVOX (VOLVOCACEAE, CHLOROPHYCEAE), BASED ON COMPARATIVE MORPHOLOGY AND MOLECULAR PHYLOGENY OF CULTURED MATERIAL(1).

Species of Volvox sect. Volvox (Volvocaceae, Chlorophyceae) are unique because they have thick cytoplasmic bridges between somatic cells and spiny-walled zygotes. This section is taxonomically important because the genus Volvox is polyphyletic. However, taxonomic studies of species in Volvox sect. Volvox have not been carried out on cultured material. Here, we performed a taxonomic study of monoecious species of Volvox sect. Volvox based on the comparative morphology and molecular phylogeny of chloroplast genes and the internal transcribed spacer (ITS) regions of nuclear rDNA using various strains originating from Japan and two preserved strains from the USA. The strains were clearly divided into four species, V.globator L., V.barberi W. Shaw, V.kirkiorum sp. nov., and V.ferrisii sp. nov., on the basis of differences in numbers of zygotes (eggs) in the sexual spheroids, form of zygote wall, and somatic cell shape. Sequences for ITS of nuclear rDNA resolved that the two new species have phylogenetic positions separated from V.globator, V.barberi, V.capensis F. Rich et Pocock, and V.rousseletii G. S. West UTEX 1862 within Volvox sect. Volvox.

Characterization of a sperm lysin of Volvox carteri

A cell-wall degrading enzyme has been isolated from mature sperm packets of the green flagellate Volvox carteri (Poona strain). This sperm lysin (S-lysin) is a Ca2+-dependent protease of 34 kDa with an essential serine group in its active centre. Neither SH group-blocking reagents nor transition metal chelators inhibit its action. S-lysin degrades the hydroxyproline-rich glycoprotein structures of the cell walls of sheath cells and gonidia (eggs) of vegetative and sexual spheroids in a characteristic manner. In asexual spheroids the somatic envelope is totally disintegrated, whereas in sexual spheroids pores are formed by local lysis at sites of adjacent eggs. Although S-lysin is very similar to the G-lysin of the closely related Chlamydomonads, it is species specific and does not attack the mother or daughter cell walls of Chlamydomonas reinhardtii. S-lysin resembles the aerosin of animal sperm cells in some aspects of its action.

Early event of sexual induction in Volvox: Chemical modification of the extracellular matrix

The sexual pheromone of Volvox carteri elicits drastic changes in the synthesis of extracellular sulfated glycoproteins. Synthesis of at least two sulfated glycoproteins is turned on. Induction of this synthetic capacity is as sensitive to the pheromone (∼10 −16 M) as the overall process of sexual induction. The earliest response, detectable a few minutes after the application of the pheromone, is the synthesis of a tyrosine sulfate-containing glycoprotein (SG 70). SG 70 is a short-lived molecule (half-life ∼ 20 min) and serves as a precursor for an insoluble extracellular matrix structure. The pheromone-induced sulfated glycoproteins described are exclusively synthesized by somatic cells, rather than by reproductive cells, the ultimate recipients of the pheromone's message. Contrary to earlier reports in the literature, it is demonstrated that isolated reproductive cells remain responsive to the sexual pheromone and develop to sexual spheroids. In the light of this finding together with the site of their synthesis the role of these pheromone-induced glycoproteins is discussed.

L-Glutamic acid as a mediator of sexual morphogenesis in Volvox capensis

In Volvox capensis the development of sexual individuals is in response to low concentrations (68 nM) of L-glutamic acid rather than to such species-specific glycoproteins as have been isolated in Volvox carteri or are believed to exist in a number of other species. V. capensis grows equally as well in light and in darkness in a medium supplemented with sodium acetate; however, L-glutamic acid is active as an inducer of the sexual form only in populations grown in the light. The site of action of L-glutamic acid and its biochemical role in the sexual response are unknown. Attempts to induce the sexual response by using the other L-amino acids, various analogs of glutamic acid, compounds of similar structure (e.g., gamma-aminobutyric acid), and intermediates of biochemical pathways known to involve L-glutamic acid (e.g., alpha-ketoglutarate or pyroglutamic acid) have been unsuccessful. L-Glutamic acid is produced by V. capensis as a natural product of the digestion of the glycoproteinaceous parental matrix at the time young spheroids escape. As a population increases, so does the level of L-glutamic acid produced at each succeeding generation until the threshold of sensitivity is reached and the induction of sexual forms is effected. This serves as a mechanism for ensuring the production of sexual spheroids and their zygotes, the only phase in the life cycle resistant to drying. Thus, Volvox is especially adapted to an existence in ephemeral pools of water resulting from seasonal rains.

The Genetics and Physiology of Non-inducibility in Volvox carteri f. nagariensis I YGENGAR

Summary In Volvox carteri f. nagariensis addition of the sex hormone produced by male strains to asexual populations of either the standard male (69-1 b) of female (HK10) strains results in 100% induction of sexual spheroids in the next generation. A mutant strain has been found in which sexual spheroid formation is impaired, such that 100% induction is not achieved in any concentration of the sex inducer. The factor effecting this non-inducibility phenotype is inherited at a single gene locus, but the different degrees of induction given among strains carrying non-inducibility suggests more than one locus is involved. Genetic analysis indicates suppressor factors carried in parental strains recombine randomly with the non-inducibility locus and, as a consequence, produce diverse expressions of non-inducibility among the siblings strains. Induction in strains carrying non-inducibility is related to light intensity and CO2 concentration in a way that suggests non-inducibility may be the result of a photosynthetic defect. However, a comparison of photosynthetic 14CO2 fixation in female strains carrying non-inducibility and the standard female strain shows that photosynthesis is not altered by the non-inducibility locus. Furthermore, studies of induction influenced by the photosynthetic inhibitor DCMU suggest that the effect of CO2 concentration on induction (CO2 enhancement) is not entirely related to photosynthetic CO2 uptake. These results indicate maximum induction in strains carrying non-inducibility 1) requires a metabolic rate substantially higher than that needed for 100% induction in the standard strains and 2) involves at least one process not needed for induction in the standard strains. The efficiency by which sexual developmental processes are accomplished is discussed as a function on the non-inducibility locus.