Hisayoshi Nozaki

• The University of Tokyp

Species of the green algal genus Mychonastes have been recognized as ubiquitous ultrasmall components of freshwater communities worldwide, mainly based on molecular data. However, detailed taxonomic identification of Japanese members of Mychonastes has not yet been conducted. We taxonomically identified species of 13 Mychonastes strains in the Micr...

This review describes the development of evolutionary studies of sex based on the volvocine lineage of green algae, which was facilitated by whole-genome analyses of both model and non-model species. Volvocine algae, which include Chlamydomonas and Volvox species, have long been considered a model group for experimental studies investigating the ev...

Volvox sect. Volvox is a group of green algae with unique morphological features (thick cytoplasmic bridges between somatic cells and spiny zygote walls) and a worldwide distribution. Despite research interest in the diversity of organisms in ancient lakes, Volvox sect. Volvox from ancient lakes worldwide has not been identified to the species leve...

Eukaryotic flagella collectively form metachronal waves that facilitate the ability to cause flow or swim. Among such flagellated and planktonic swimmers, large volvocine genera such as Eudorina, Pleodorina and Volvox form bundles of small male gametes (sperm) called “sperm packets” for sexual reproduction. Although these sperm packets reportedly h...

Mitochondrial biogenesis relies on hundreds of proteins that are derived from genes encoded in the nucleus. According to characteristic properties of N-terminal targeting peptides (TP) and multi-step authentication by the protein translocase called the TOM complex, nascent polypeptides satisfying the requirements are imported into mitochondria. How...

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome...

The diversity and the evolution of mating systems have been studied in various eukaryotes. Volvox sect. Volvox of the volvocine green algae is suitable for evolutionary studies of the transition from homothallic to heterothallic mating systems because this transition is recognized in distal lineages within Volvox sect. Volvox . MID homologs are imp...

The coexistence of three sexual phenotypes (male, female and bisexual) in a single species, 'trioecy', is rarely found in diploid organisms such as flowering plants and invertebrates. However, trioecy in haploid organisms has only recently been reported in a green algal species, Pleodorina starrii. Here, we generated whole-genome data of the three...

Eukaryotic flagella collectively form metachronal waves and exhibit the ability to swim or cause flow. Large volvocine genera such as Eudorina , Pleodorina , and Volvox form bundles of small male gametes (sperm) called “sperm packets” for sexual reproduction. Although sperm packets have been reported to have flagella and the ability to swim, there...

Male and female genotypes in heterothallic (self-incompatible) species of haploid organisms, such as algae and bryophytes, are generally determined by male and female sex-determining regions (SDRs) in the sex chromosomes. To resolve the molecular genetic basis for the evolution of homothallic (bisexual and self-compatible) species from a heterothal...

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmal...

Background Astrephomene is an interesting green algal genus that, together with Volvox , shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two...

Background The 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. Howeve...

Background: Astrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two...

Characteristics of transverse walls in filaments, and modes of conjugation are essential for species identification in the genus Spirogyra. Although most Spirogyra species have plane transverse walls, species with other types of transverse walls (replicate, semi-replicate and colligate types) have also been described. Recent molecular phylogenetic...

Following the publication of the original paper [1], the authors spotted error in Additional file 1 (Table S3). Corrected file is captured as supplementary file of this article.

Abstract Background Colonial and multicellular volvocine green algae have been extensively studied recently in various fields of the biological sciences. However, only one species (Pandorina morum) has been cryopreserved in public culture collections. Results Here, we investigated conditions for cryopreservation of the multicellular volvocine alga...

Background Pyrenoids are protein microcompartments composed mainly of Rubisco that are localized in the chloroplasts of many photosynthetic organisms. Pyrenoids contribute to the CO 2 -concentrating mechanism. This organelle has been lost many times during algal/plant evolution, including with the origin of land plants. The molecular basis of the e...

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene , exhibit convergent evolution of germ-soma differentiation. The com...

Photo-induced behavioral responses (photobehaviors) are crucial to the survival of motile phototrophic organisms in changing light conditions. Volvocine green algae are excellent model organisms for studying the regulatory mechanisms of photobehavior. We recently reported that unicellular Chlamydomonas reinhardtii and multicellular Volvox rousselet...

Here we report the complete organellar genome sequences of Medakamo hakoo , a green alga identified in freshwater in Japan. It has 90.8-kb plastid and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein coding genes, respectively, representing the smallest organellar genome among currently known core Trebouxiophyceae.

Mating systems of haploid species such as fungi, algae, and bryophytes are either heterothallic (self-incompatible) with two sex phenotypes (male and female, or mating type minus and plus in isogamous species) or homothallic (self-compatible) with only a bisexual phenotype producing zygotes within a clone. The anisogamous volvocine green alga Pleod...

Chionaster nivalis is frequently detected in thawing snowpacks and glaciers. However, the taxonomic position of this species above the genus level remains unclear. We herein conducted molecular analyses of C. nivalis using the ribosomal RNA operon sequences obtained from more than 200 cells of this species isolated from a field-collected material....

Significance Evolutionary transitions between species with separate sexes and species in which individuals have both sex functions have wide-ranging biological implications. It is largely unknown how such transitions occur in systems with haploid male- and female-determining chromosomes in algae and bryophytes. We investigated such a transition in...

Snow algae are microalgae, growing in melting snowpacks, and are thought to act as primary producers in the snow ecosystem. Chloromonas (Volvocales, Chlorophyceae) contains more than 15 snow-inhabiting species. Although vegetative cells and zygotes, or asexual cysts, of snow species of the genus are frequently collected in the field, sexual reprodu...

Conjugating algae perform characteristic sexual reproduction through conjugation by gametes without flagella. These algae sexually reproduce through the formation of zygospores from single clones (homothallic) and through the formation of zygospores between different clones (heterothallic). The conjugating algae genus Spirogyra is generally found i...

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 n...

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...

[This corrects the article DOI: 10.1371/journal.pone.0221632.].

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 o...

The genus Spirogyra is abundant in freshwater habitats worldwide, and comprises approximately 380 species. Species assignment is often difficult because identification is based on the characteristics of sexual reproduction in wild-collected samples and spores produced in the field or laboratory culture. We developed an identification procedure base...

A 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...

Recent molecular data has strongly suggested that field-collected cysts of snow algae that are morphologically identifiable as the zygotes of Chloromonas nivalis are composed of multiple species. Motile vegetative cells, however, have not been directly obtained from these cysts because of the difficulties involved in inducing their germination. Rec...

Fifty “Chloromonas nivalis zygotes” assigned to the specimen Hakkoda-Green. Identical magnification throughout. Scale bar = 10 μm. (JPG)

Secondary structure of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) transcript of Chloromonas muramotoi sp. nov. strain HkCl-57. The 3' end of the 5.8S ribosomal RNA (rRNA) and the 5' end of the large subunit (LSU) rRNA are shown (DDBJ/ENA/GenBank accession number: LC438455). The sequences of this region are identical among the strain...

Nucleotide differences (%) in nuclear ribosomal DNA internal transcribed spacer 2. (1) Chloromonas muramotoi strain HkCl-57 vs. a specimen of “C. nivalis zygotes,” Tateyama-Green. (2) Strain HkCl-57 vs. a specimen of “C. nivalis zygotes,” Tateyama-Orange. (3) Strain HkCl-57 vs. a specimen of “C. nivalis zygotes,” Hakkoda-Green, (4) C. reticulata st...

Substitution models applied to the respective data matrices of the present phylogenetic analyses (Figs 2 and 3). (DOCX)

Morphological characteristics of field-collected “Chloromonas nivalis zygotes” examined by scanning electron microscopy. (DOCX)

Fifty “Chloromonas nivalis zygotes” assigned to the specimen Tateyama-Orange. Identical magnification throughout. Scale bar = 10 μm. (JPG)

Nucleotide differences (%) from pairwise comparisons in four genes. Black: nuclear-encoded 1,748 bases of the small subunit (SSU) ribosomal DNA (rDNA). Green: nuclear-encoded 2,017 bases of the large subunit (LSU) rDNA. Red: chloroplast-encoded 1,128 bases of ATP synthase beta subunit gene (atpB). Blue: chloroplast-encoded P700 chlorophyll a apopro...

Primers for amplification and sequencing of the ATP synthase beta subunit and the P700 chlorophyll a apoprotein A2 genes. (DOCX)

Fifty “Chloromonas nivalis zygotes” assigned to the specimen Tateyama-Green. Identical magnification throughout. Scale bar = 10 μm. (JPG)

Strain/specimens examined in this study. (DOCX)

Taxa/specimens/strains used for our molecular analyses (Figs 2 and 3; S6 Fig) and DDBJ/ENA/GenBank accession numbers for the five genes. (DOCX)

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 tha...

From the male peacock's tail plumage to the floral displays of flowering plants, traits related to sexual reproduction are often complex and exaggerated. Why has sexual reproduction become so compli-cated? Why have such exaggerated sexual traits evolved? Early work posited a connection between multicellularity and sexual traits such as anisogamy (i...

Background: Volvox 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 packe...

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we...

Strains examined in this study. (DOCX)

Vegetative cell of the strain UTEX SNO74. Abbreviations: c, chloroplast; n, nucleus; p, pyrenoid. (A) Optical section focused on a pyrenoid. (B) Surface view. The strain [formerly designated as Chloromonas nivalis (Chodat) Hoham et Mullet] was not used in course of this study since the strain might be replaced with contamination by the species of t...

Taxa/specimens/strains in the present molecular analyses (Figs 4 and 5; S4 and S5 Figs) and DDBJ/ENA/GenBank accession numbers of the five genes. (DOCX)

Primers for amplification and sequencing of P700 chlorophyll a apoprotein A2 gene from Chloromonas remiasii strains. (DOCX)

Substitution models applied to respective data matrices of the present phylogenetic analyses (Fig 4; S4 and S5 Figs). (DOCX)

Cell aggregates in cultures of Chloromonas remiasii Matsuzaki et al. sp. nov. Aggregates result from repeated divisions of daughter cells retained in parental cell walls (double arrowhead). Open arrowhead indicates a daughter cell wall surrounding offspring of a daughter cell. All at the identical magnification. (A) Strain CCCryo 005–99 after 7 day...

Bayesian phylogenetic tree of snow-inhabiting Chloromonas spp. based on 18S ribosomal DNA sequences. C. nivalis zygote specimens (Field-collected samples) are underlined, and the Austrian C. nivalis zygote specimen (P24/DR4 [19]) and the Slovak C. nivalis subsp. tatrae zygote specimen (LP01 [20]) are shadowed in black. Groups A–D are as indicated i...

Secondary structure of nuclear ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) transcript of Chloromonas remiasii Matsuzaki et al. sp. nov. strain CCCryo 005–99. The 3′ end of the 5.8S ribosomal RNA (rRNA) and the 5′ end of the 26S rRNA are shown (DDBJ/ENA/GenBank accession number: HQ404862). The sequence from C. remiasii strains CCCryo 0...

Asexual reproduction of three snow-inhabiting Chloromonas species. All at identical magnification. Arrows in A, C, E indicate position of each contractile vacuole originating from the parent cell. (A, B) C. nivalis (Chodat) Hoham et Mullet strain UTEX SNO71. (A) Immediately prior to the first transverse division. (B) Sixteen daughter cells within t...

Bayesian phylogenetic tree of snow-inhabiting Chloromonas spp. based on the large subunit of RuBisCO gene sequences. C. nivalis zygote specimens (Field-collected samples) are underlined, and the Austrian and Japanese C. nivalis zygote specimens examined in the previous studies (P24/DR4 [19,20], and Gassan-NIV1 and Gassan-NIV2 [41], respectively) an...

Secondary structure of nuclear ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) transcript of Chloromonas chenangoensis strain UTEX SNO150. The 3′ end of the 5.8S ribosomal RNA (rRNA) and the 5′ end of the 26S rRNA are shown (DDBJ/ENA/GenBank accession number: LC360497). Note U-U mismatch in helix II (arrowheads) and the YGGY motif on the...

BLASTn results using two gene sequences of the four strains as queries against nucleotide collection. (DOCX)

Morphological characteristics of 13 snow-inhabiting species having elongate or ellipsoidal vegetative cells with a rounded posterior end, in the genus Chloromonas sensu Ettl. (DOCX)

Taxonomic treatment of the strain UTEX SNO74. (DOCX)

Key to vegetative cells of snow-inhabiting species of Chloromonas sensu Ettl. (DOCX)

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 tha...

Outcrossing and self-fertilization are fundamental strategies of sexual reproduction, each with different evolutionary costs and benefits. Self-fertilization is thought to be an evolutionary “dead-end” strategy, beneficial in the short term but costly in the long term, resulting in self-fertilizing species that occupy only the tips of phylogenetic...

Multicellularity is the premier example of a major evolutionary transition in individuality and was a foundational event in the evolution of macroscopic biodiversity. The volvocine chlorophyte lineage is well suited for studying this process. Extant members span unicellular, simple colonial, and obligate multicellular taxa with germ-soma differenti...

Volvocine algae constitute a green algal lineage comprising unicellular Chlamydomonas, four-celled Tetrabaena, eight to 32-celled Gonium, and others up to Volvox spp., which consist of up to 50,000 cells. These algae proliferate by multiple fissions with cellular growth up to several fold in size and subsequent successive cell divisions. Chlamydomo...

Background: The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sis...

Significance Extremely acidic environments are scattered worldwide, and their ecosystems are supported by acidophilic microalgae as primary producers. To understand how acidophilic algae evolved from their respective neutrophilic ancestors, we determined the draft genome sequence of the acidophilic green alga Chlamydomonas eustigma and performed co...

Pleodorina sphaerica Iyengar was considered to be a phylogenetic link between Volvox and the type species Pleodorina californica Shaw because it has small somatic cells distributed from the anterior to posterior poles in 64- or 128-celled vegetative colonies. However, cultural studies and molecular and ultrastructural data are lacking in P. sphaeri...

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 domina...

Alignment of seven MID homologs from the Volvocaceae (Volvox africanus, V. reticuliferus, V. ferrisii, V. carteri, Eudorina sp., Pleodorina starrii, and Yamagishiella unicocca). Black or gray back colors indicate over 70% of identity or similarity, respectively. (DOCX)

Alternative splicing variants of the Volvox africanus MID ortholog (VaMID). Variants 2–4 are intron retention. (DOCX)

Degenerate primers used in this study. (DOCX)

Gene specific primers used in this study. (DOCX)

The four sexual types of Volvox africanus-like algae recognized by Starr (1971, Sexual reproduction in Volvox africanus. Contributions in Phycology. Allen Press, pp. 59–66). (DOCX)

Light microscopic images of Volvox africanus (homothallic, monoecious with males type) and V. reticuliferus (heterothallic, dioecious type). Scale bars = 50 μm. sp: sperm packet, e: egg. A-C. V. africanus strain 2013-0703-VO4. A. Asexual spheroid. B. Monoecious spheroid. C. Male spheroid. D, E. V. reticuliferus. D. Male spheroid in male strain VO12...

Results of genomic PCR for parental strains and four F1 progeny strains of Volvox reticuliferus (Table 1). Parental strains are 2013-0703-VO2 (VO-2) and 2013-0703-VO3 (VO-3). F1 progeny strains are VO123-F1-6 (F1-6), VO123-F1-7 (F1-7), VO123-F1-9 (F1-9), VO123-F1-10 (F1-10). (F): Female, (M):Male strain. (DOCX)

DAPI staining for estimating comparative genome size in Volvox africanus and V. reticuliferus. A-C. Mean gray value of ten nuclei with imageJ at 0.5, 0.67, 1.0, 1.5, 2.0, 2.5 s exposure time. Bars show means and standard deviations. ctrl: V. carteri EVE strain in the same slide for control. A. V. africanus, B. V reticuliferus male strain, C. V reti...

Southern blot analysis of Volvox reticuliferus and V. africanus. Restriction enzyme digested genomic DNA was electrophoresed on an agarose gel and stained with ethidium bromide. The corresponding Southern blot data are shown in the upper panels. A. Southern blotting using a VrMID fragment, located in exon1-exon3 as shown in Fig 2B. B. Southern blot...

List of Volvocales included in the phylogenetic analyses of MID sequences and DDBJ/EMBL/GENBANK accession numbers. (DOCX)

Conditions for PCR cycles and primers used in semi-quantitative RT- PCR analyses (Fig 5). (DOCX)

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behavior. The volvocine green algae provide an excellent mode...

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 rec...

We were the first group to successfully sequence the 100%-complete entire eukaryotic genome in 2007, mainly using automated Sanger sequencing of the unicellular, ultrasmall red algal species Cyanidioschyzon merolae 10D. This world record was principally based on the ultrasmall size of the C. merolae genome (ca. 16 megabase pairs) as well as on thre...

Cyanidioschyzon merolae is a unicellular alga without a cell wall that inhabits sulfuric hot springs. It is a member of the red algae Cyanidiales that diverged from other red algal lineages approximately 1.3–1.4 billion years ago. Cyanidiales are the only eukaryotes and phototrophic organisms found in sulfuric hot springs, but some rare species hav...

This comprehensive book highlights the importance of Cyanidioschyzon merolae (C. merolae), an ultrasmall unicellular red alga, as a model eukaryote organism. The chapters introduce recent studies on C. merolae, from culture, synchronization and isolation methods of nucleic acids, proteins and organelles for molecular biological and cytological anal...

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...