Ryo Matsuzaki

New "missing link" genus of the colonial volvocine green algae gives insights into the evolution of oogamy.

BackgroundThe 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.ResultsWe 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.ConclusionsBased 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.

The Evolution of Male-Female Sexual Dimorphism Predates the Gender-Based Divergence of the Mating Locus Gene MAT3/RB

The molecular bases for the evolution of male-female sexual dimorphism are possible to study in volvocine algae because they encompass the entire range of reproductive morphologies from isogamy to oogamy. In 1978, Charlesworth suggested the model of a gamete size gene becoming linked to the sex-determining or mating type locus (MT) as a mechanism for the evolution of anisogamy. Here, we carried out the first comprehensive study of a candidate MT-linked oogamy gene, MAT3/RB, across the volvocine lineage. We found that evolution of anisogamy/oogamy predates the extremely high male-female divergence of MAT3 that characterizes the Volvox carteri lineage. These data demonstrate very little sex-linked sequence divergence of MAT3 between the two sexes in other volvocine groups, though linkage between MAT3 and the mating locus appears to be conserved. These data implicate genetic determinants other than or in addition to MAT3 in the evolution of anisogamy in volvocine algae.

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.

A taxonomic revision of Chloromonas reticulata (Volvocales, Chlorophyceae), the type species of the genus Chloromonas, based on multigene phylogeny and comparative light and electron microscopy

Matsuzaki R., Hara Y. and NOZAKI H. 2012. A taxonomic revision of Chloromonas reticulata (Volvocales, Chlorophyceae), the type species of the genus Chloromonas, based on multigene phylogeny and comparative light and electron microscopy. Phycologia 51: 74–85. DOI: 10.2216/11-18.1 Chloromonas reticulata (Goroschankin) Gobi, the type species for Chloromonas, was taxonomically revised using nine strains previously assigned to the species, based on light and transmission electron microscopy and multigene phylogenetic analyses. Based on vegetative cell and papilla shape, eyespot form and position, and the ultrastructure of the eyespot and pyrenoid, the nine strains were classified into four species: C. reticulata; C. rosae (H. & O. Ettl) H. Ettl; C. chlorococcoides (H. Ettl & K. Schwarz) Matsuzaki, Y. Hara & Nozaki comb. nov.; and C. typhlos (Gerloff) Matsuzaki, Y. Hara & Nozaki comb. nov. The taxonomic revision was supported by the robust monophyly of the species resolved in phylogenetic analyses using a combined dataset that included 18S ribosomal (r) RNA, ATP synthase β-subunit, P700 chlorophyll a-apoprotein A1 and A2 genes, and nuclear rDNA internal transcribed spacer 2 (ITS2) sequences. The phylogenetic analyses demonstrated that the pyrenoid-lacking species C. reticulata and the pyrenoid-containing species C. chlorococcoides, which has a dense pyrenoid matrix associated with starch grains, formed a robust monophyletic group to which the pyrenoid-lacking species C. rosae and the pyrenoid-containing species C. typhlos were basal. Comparison of the C. reticulata and C. chlorococcoides nuclear rDNA ITS2 secondary structures revealed two compensatory base changes in the conserved helix III region, suggesting a separation between these sister species.

Revisiting the taxonomic significance of aplanozygote morphologies of two cosmopolitan snow species of the genus Chloromonas (Volvocales, Chlorophyceae)

Abstract: Chloromonas brevispina and C. nivalis (Volvocales, Chlorophyceae) are considered cosmopolitan, as the aplanozygotes assignable to the two species are distributed worldwide. Although recent phylogenetic analyses have indicated the possible polyphyly of aplanozygotes identified as C. nivalis, the analytical resolution was not robust, possibly because only single genes were analysed. Here, we obtained long sequences of multiple DNA regions from 50 identical [on the basis of light microscopy (LM)] aplanozygotes in a field-collected sample, and the phylogenetic positions of these aplanozygotes (identified as C. brevispina and C. nivalis on the basis of LM) were determined with high statistical support using a multigene phylogeny. Comparisons of the present and previously published sequence data indicated that aplanozygotes of C. nivalis originating from Japan and Austria represented at least four different lineages, all of which were robustly separated from the North American strain CU563D of C. nivalis. Molecular analyses including the comparison of highly evolving nuclear ribosomal DNA internal transcribed spacer 2 demonstrated that one Japanese lineage of C. nivalis aplanozygotes was conspecific with C. miwae strains. In addition, the present C. brevispina aplanozygote specimens from Japan were assigned to C. krienitzii sp. nov., which was herein delineated on the basis of LM and electron microscopy and the molecular phylogeny of newly established strains.

Five Cyanophora (Cyanophorales, Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.

Uniparental Inheritance of Chloroplast DNA Is Strict in the Isogamous Volvocalean Gonium

Background A problem has remained unresolved regarding the exceptions to the unilateral inheritance of chloroplast DNA (cpDNA) from MT+/female in Chlamydomonas and other volvocaleans demonstrated by the previous genetic analyses. For identification of the parental types of cpDNA, these studies used parents that have differences in restriction fragment length polymorphisms and exhibit partial sexual incompatibility. Methodology/Principal Findings In the present study, we used sexually compatible parents of the isogamous colonial volvocalean Gonium maiaprilis that seemed an ideal species to identify the pattern of cpDNA inheritance based on the length difference in the putative group I intron interrupted in the Rubisco large subunit gene and objective identification of mating types by the presence or absence of the minus-dominance (MID) gene. We examined patterns of inheritance of cpDNA and presence/absence of a MID ortholog (GmMID) in 107 F1 progeny of G. maiaprilis that were obtained by inducing germination of separated single zygotes. The results demonstrated no exception of the uniparental inheritance of cpDNA from the MT+ parent (lacking GmMID) in sexually compatible or genetically less divergent strains of G. maiaprilis. Conclusions/Significance The present data suggest that the uniparental inheritance of cpDNA is likely more strict in crossings of less diverged strains or sexually compatible parental volvocaleans, and some genetic inconsistency between the parents may cause exceptional uniparental inheritance of cpDNA.

Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales, Chlorophyceae)

A strain of Chloromonas pseudoplatyrhyncha (Pascher) P. C. Silva, which has not been studied previously using cultured material, was established from a soil sample collected in Japan and examined by light microscopy, transmission electron microscopy, and molecular phylogenetic analyses. The chloroplasts of this species showed no pyrenoids under light microscopy. However, transmission electron microscopy and the staining methods with carmine after fixation in an acidified hypochlorite solution revealed that Chloromonas pseudoplatyrhyncha actually had multiple, atypical pyrenoids (pyrenoid matrices without associated starch grains) that were angular in shape and distributed in the interior regions of the lobes of the chloroplasts. Although some other species of Chloromonas have atypical pyrenoids in the chloroplast, such angular pyrenoids have not previously been reported within the Volvocales. The present molecular phylogenetic analysis, based on 18S ribosomal RNA, adenosine triphosphate synthase β‐subunit, and P700 chlorophyll a‐apoprotein A2 gene sequences, demonstrated that Chloromonas pseudoplatyrhyncha belonged to the Chloromonas lineage or Chloromonadinia, in which it occupied a basal position outside a robust, large monophyletic group consisting of 13 species of Chloromonas and Gloeomonas.

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.

A taxonomic study of snow Chloromonas species (Volvocales, Chlorophyceae) based on light and electron microscopy and molecular analysis of cultured material

Abstract: Although zygote morphology is important in traditional taxonomic systems of snow-inhabiting species of Chloromonas (Volvocales, Chlorophyceae), induction of sexual reproduction or hypnospore formation is difficult in culture. Thus, alternative taxonomic methodologies are required when sexual reproduction does not occur in cultures. Here, we performed a taxonomic study of snow-inhabiting Chloromonas species based on culture strains with elongate or ellipsoidal vegetative cells. Our comparative light and electron microscopy demonstrated that the strains were clearly distinguished into six species based on differences in vegetative cell shape and chloroplast morphology, the number of zoospores within the parental cell and the presence or absence of cell aggregates in old cultures. The six species are Chloromonas chenangoensis, C. fukushimae sp. nov., C. hohamii, C. pichinchae, C. tenuis sp. nov. and C. tughillensis. Although four of the six species formed a small clade based on phylogenetic analysis of multiple genes, their separation was supported by comparison of secondary structures of the internal transcribed spacer 2 of nuclear ribosomal DNA and genetic differences of nuclear- and chloroplast-encoded genes. The present study demonstrated that polyphasic analyses of culture strains of snow Chloromonas species could be used for the identification of natural species.