Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism.

Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

What Happened before Losses of Photosynthesis in Cryptophyte Algae?

In many lineages of algae and land plants, photosynthesis was lost multiple times independently. Comparative analyses of photosynthetic and secondary nonphotosynthetic relatives have revealed the essential functions of plastids, beyond photosynthesis. However, evolutionary triggers and processes that drive the loss of photosynthesis remain unknown. Cryptophytes are microalgae with complex plastids derived from a red alga. They include several secondary nonphotosynthetic species with closely related photosynthetic taxa. In this study, we found that a cryptophyte, Cryptomonas borealis, is in a stage just prior to the loss of photosynthesis. Cryptomonas borealis was mixotrophic, possessed photosynthetic activity, and grew independent of light. The plastid genome of C. borealis had distinct features, including increases of group II introns with mobility, frequent genome rearrangements, incomplete loss of inverted repeats, and abundant small/medium/large-sized structural variants. These features provide insight into the evolutionary process leading to the loss of photosynthesis.

Carbon Nitride Loaded with an Ultrafine, Monodisperse, Metallic Platinum-Cluster Cocatalyst for the Photocatalytic Hydrogen-Evolution Reaction.

For the realization of a next-generation energy society, further improvement in the activity of water-splitting photocatalysts is essential. Platinum (Pt) is predicted to be the most effective cocatalyst for hydrogen evolution from water. However, when the number of active sites is increased by decreasing the particle size, the Pt cocatalyst is easily oxidized and thereby loses its activity. In this study, a method to load ultrafine, monodisperse, metallic Pt nanoclusters (NCs) on graphitic carbon nitride is developed, which is a promising visible-light-driven photocatalyst. In this photocatalyst, a part of the surface of the Pt NCs is protected by sulfur atoms, preventing oxidation. Consequently, the hydrogen-evolution activity per loading weight of Pt cocatalyst is significantly improved, 53 times, compared with that of a Pt-cocatalyst loaded photocatalyst by the conventional method. The developed method is also effective to enhance the overall water-splitting activity of other advanced photocatalysts such as SrTiO3 and BaLa4 Ti4 O15 .

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene.

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 species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections. RESULTS: To establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36-33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12-12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES. CONCLUSIONS: The optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Cryopreservation of vegetative cells and zygotes of the multicellular volvocine green alga Gonium pectorale.

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 Gonium pectorale using vegetative colonies or cells and zygotes. Rates of vegetative cell survival in a G. pectorale strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3% and 6%) of the cryoprotectant N,N-dimethylformamide (DMF) and two types of tubes (0.2-mL polymerase chain reaction tubes and 2-mL cryotubes) used for cryopreservation. Among the four conditions investigated, the highest rate of survival [2.7 ± 3.6% (0.54-10%) by the most probable number (MPN) method] was obtained when 2.0-mL cryotubes containing 1.0 mL of culture samples with 6% DMF were subjected to cryogenic treatment. Using these optimized cryopreservation conditions, survival rates after freezing in liquid nitrogen were examined for twelve other strains of G. pectorale and twelve strains of five other Gonium species. We obtained ≥ 0.1% MPN survival in nine of the twelve G. pectorale strains tested. However, < 0.1% MPN survival was detected in eleven of twelve strains of five other Gonium species. In total, ten cryopreserved strains of G. pectorale were newly established in the Microbial Culture Collection at the National Institute for Environmental Studies. Although the cryopreservation of zygotes of volvocine algae has not been previously reported, high rates (approximately 60%) of G. pectorale zygote germination were observed after thawing zygotes that had been cryopreserved with 5% or 10% methanol as the cryoprotectant during two-step cooling and freezing in liquid nitrogen. CONCLUSIONS: The present study demonstrated that cryopreservation of G. pectorale is possible with 6% DMF as a cryoprotectant and 1.0-mL culture samples in 2.0-mL cryotubes subjected to two-step cooling in a programmable freezer.

Activation of Water-Splitting Photocatalysts by Loading with Ultrafine Rh-Cr Mixed-Oxide Cocatalyst Nanoparticles.

The activity of many water-splitting photocatalysts could be improved by the use of RhIII -CrIII mixed oxide (Rh2-x Crx O3 ) particles as cocatalysts. Although further improvement of water-splitting activity could be achieved if the size of the Rh2-x Crx O3 particles was decreased further, it is difficult to load ultrafine (<2 nm) Rh2-x Crx O3 particles onto a photocatalyst by using conventional loading methods. In this study, a new loading method was successfully established and was used to load Rh2-x Crx O3 particles with a size of approximately 1.3 nm and a narrow size distribution onto a BaLa4 Ti4 O15 photocatalyst. The obtained photocatalyst exhibited an apparent quantum yield of 16 %, which is the highest achieved for BaLa4 Ti4 O15 to date. Thus, the developed loading technique of Rh2-x Crx O3 particles is extremely effective at improving the activity of the water-splitting photocatalyst BaLa4 Ti4 O15 . This method is expected to be extended to other advanced water-splitting photocatalysts to achieve higher quantum yields.

Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes.

BACKGROUND: The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. RESULTS: The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. CONCLUSIONS: Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

Onboard experiment investigating metal leaching of fresh hydrothermal sulfide cores into seawater.

We observed the initial release rate of metals from four fresh (i.e., without long time exposure to the atmosphere) hydrothermal sulfide cores into artificial seawater. The sulfide samples were collected by seafloor drilling from the Okinawa Trough by D/V Chikyu, powdered under inert gas, and immediately subjected to onboard metal-leaching experiments at different temperatures (5 °C and 20 °C), and under different redox conditions (oxic and anoxic), for 1-30 h. Zinc and Pb were preferentially released from sulfide samples containing various metals (i.e., Mn, Fe, Cu, Zn, Cd, and Pb) into seawater. Under oxic experimental conditions, Zn and Pb dissolution rates from two sulfide samples composed mainly of iron disulfide minerals (pyrite and marcasite) were higher than those from two other sulfide samples with abundant sphalerite, galena, and/or silicate minerals. Scanning electron microscopy confirmed that the high metal-releasing sample contained several galvanic couples of iron disulfide with other sulfide minerals, whereas the low metal-releasing sample contained fewer galvanic couples or were coated by a silicate mineral. The experiments overall confirmed that the galvanic effects with iron disulfide minerals greatly induce the initial release of Zn and Pb from hydrothermal sulfides into seawater, especially under warm oxic conditions.

Evaluation of the toxicity of leaches from hydrothermal sulfide deposits by means of a delayed fluorescence-based bioassay with the marine cyanobacterium Cyanobium sp. NIES-981.

The commercial use of metals such as copper, lead, and zinc has markedly increased in recent years, resulting in increased interest in deep-sea mining of seafloor hydrothermal sulfide deposits. However, the full extent of the impact of deep-sea mining at hydrothermal field deposits on the environment remains unclear. In addition to impacting the deep sea, the leaching of heavy metals from extracted sulfide mineral may also affect the upper ocean zones as the sulfide rock is retrieved from the seafloor. Here, we used a delayed fluorescence-based bioassay using the marine cyanobacterium Cyanobium sp. NIES-981 to evaluate the toxicity of three sulfide core samples obtained from three drill holes at the Izena Hole, middle Okinawa Trough, East China Sea. Leaches from two of the cores contained high concentrations of zinc and lead, and they markedly inhibited delayed fluorescence in Cyanobium sp. NIES-981 compared with control. By examining the toxicity of artificial mixed-metal solutions with metal compositions similar to those of the leaches, we confirmed that this inhibition was a result of high zinc and lead concentrations into the leaches. In addition, we conclude that this delayed fluorescence-based bioassay is a viable method for use by deep-sea mining operations because it is quicker and requires less laboratory space and equipment than the standard assay.

Identification and detection sensitivity of Microcystis aeruginosa from mixed and field samples using MALDI-TOF MS.

To verify the applicability of identifying Microcystis aeruginosa by matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS), mixed and field samples were employed to study the sensitivity and the analysis power, respectively. Series diluted samples and artificially mixed samples by the M. aeruginosa NIES-843 strain were designed to verify the sensitivity. The lowest detection limit was 1.955 × 106 cells in pure samples, while for mixed samples, the lowest detection limit and ratio of NIES-843 strain were 2.88 × 106 cells and 33.7%, respectively. The results provided a reference for the reasonable volume of the water sample in which the M. aeruginosa could be detected. Ribosomal protein biomarkers for identifying M. aeruginosa which were successfully detected from the field samples in Taihu Lake, indicated that the identification of M. aeruginosa by MALDI-TOF MS could be applied in field samples. Furthermore, different genetic types of M. aeruginosa strains were also detected at different locations in Taihu Lake, which revealed the diversity of M. aeruginosa and the detection power of MALDI-TOF MS at the strain level for the field samples. The sensitivity and detection power in the analysis of M. aeruginosa by the MALDI-TOF MS demonstrated the applicability of this method in routine environmental monitoring.

Predators regulate prey species sorting and spatial distribution in microbial landscapes.

The role of predation in determining the metacommunity assembly model of prey communities is understudied relative to that of interspecific competition among prey. Previous work on metacommunity dynamics of competing species has shown that sorting by habitat patch type and spatial patterning can be affected by disturbances. Microcosms offer a useful model system to test the effect of multi-trophic interactions and disturbance on metacommunity dynamics. Here, we investigated the potential role of predators in enhancing or disrupting sorting and spatial pattern among prey in experimental landscapes. We exposed multi-trophic protist microcosm landscapes with one predator, two competing prey, two patch resource types, and localized dispersal to three disturbance regimes (none, low, and high). Then, we used variation partitioning and spatial clustering analysis to analyse the results. In contrast with previous experiments that did not manipulate predators, we found that patch type did not structure prey communities very well. Instead, we found that it was the distribution of the predator that most strongly predicted the composition of the prey community. The predator impacted species sorting by (1) preferentially consuming one prey, thereby acting as a strong local environmental driver, and by (2) indirectly magnifying the impact of patch food resources on the less preferred prey. The predator also enhanced spatial signal in the prey community because of its limited dispersal. Our results indicate that predators can strongly influence prey species sorting and spatial patterning in metacommunities in ways that would otherwise be attributed to stochastic effects, such as dispersal limitation or demographic drift. Therefore, whenever possible, predators should be explicitly included as separate explanatory factors in variation partitioning analyses.

Rediscovery of the species of 'ancestral Volvox': morphology and phylogenetic position of Pleodorina sphaerica (Volvocales, Chlorophyceae) from Thailand.

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. sphaerica, and this species has not been recorded since 1951. Here, we performed light and electron microscopy and molecular phylogeny of P. sphaerica based on newly established culture strains originating from Thailand. Morphological features of the present Thai species agreed well with those of the previous studies of the Indian material of P. sphaerica and with those of the current concept of the advanced members of the Volvocaceae. The present P. sphaerica strains exhibited homothallic sexuality; male and facultative female colonies developed within a single clonal culture. Chloroplast multigene phylogeny demonstrated that P. sphaerica was sister to two other species of Pleodorina (P. californica and Pleodorina japonica Nozaki) without posterior somatic cells, and these three species of Pleodorina formed a robust clade, which was positioned distally in the large monophyletic group including nine taxa of Volvox sect. Merrillosphaera and Volvox (sect. Janetosphaera) aureus Ehrenberg. Based on the present phylogenetic results, evolutionary losses of posterior somatic cells might have occurred in the ancestor of P. californica and P. japonica. Thus, P. sphaerica might represent an ancestral morphology of Pleodorina, rather than of Volvox.

Unique chlorophylls in picoplankton Prochlorococcus sp. "Physicochemical properties of divinyl chlorophylls, and the discovery of monovinyl chlorophyll b as well as divinyl chlorophyll b in the species Prochlorococcus NIES-2086".

In this review, we introduce our recent studies on divinyl chlorophylls functioning in unique marine picoplankton Prochlorococcus sp. (1) Essential physicochemical properties of divinyl chlorophylls are compared with those of monovinyl chlorophylls; separation by normal-phase and reversed-phase high-performance liquid chromatography with isocratic eluent mode, absorption spectra in four organic solvents, fluorescence information (emission spectra, quantum yields, and life time), circular dichroism spectra, mass spectra, nuclear magnetic resonance spectra, and redox potentials. The presence of a mass difference of 278 in the mass spectra between [M+H]+ and the ions indicates the presence of a phytyl tail in all the chlorophylls. (2) Precise high-performance liquid chromatography analyses show divinyl chlorophyll a' and divinyl pheophytin a as the minor key components in four kinds of Prochlorococcus sp.; neither monovinyl chlorophyll a' nor monovinyl pheophytin a is detected, suggesting that the special pair in photosystem I and the primary electron acceptor in photosystem II are not monovinyl but divinyl-type chlorophylls. (3) Only Prochlorococcus sp. NIES-2086 possesses both monovinyl chlorophyll b and divinyl chlorophyll b, while any other monovinyl-type chlorophylls are absent in this strain. Monovinyl chlorophyll b is not detected at all in the other three strains. Prochlorococcus sp. NIES-2086 is the first example that has both monovinyl chlorophyll b as well as divinyl chlorophylls a/b as major chlorophylls.

Rapid ecotoxicological bioassay using delayed fluorescence in the marine cyanobacterium Cyanobium sp. (NIES-981).

The use of delayed fluorescence intensity as an endpoint for rapid estimation of the effective concentration (ECx) has been reported as an alternative to standard growth inhibition (at 72 h after exposure) in some algal species including Pseudokirchneriella subcapitata. In marine algae, although an approach of bioassaying using delayed fluorescence measurements has not been performed yet, its development would provide many benefits for marine environmental risk assessment. In this study, we selected marine cyanobacterium Cyanobium sp. (NIES-981) as our test algal species and demonstrated that this species is valid for the standard growth inhibition test based on criteria provide by Organization for Economic Co-operation and Development guidelines. Furthermore, standard inhibition tests and shorter period test using DF were performed in NIES-981 using five chemicals (3,5-DCP, simazine, diflufenican, K2Cr2O7, and CuSO4), and their EC50 and low-toxic-effect values (EC10, EC5, and NOEC) were determined from two dose-response curves. Based on comparisons of the two dose-response curves and the EC50 values, we conclude that DF intensity is useful as an endpoint for rapid estimation of EC50 in NIES-981.

Molecular diversity of endosymbiotic Nephroselmis (Nephroselmidophyceae) in Hatena arenicola (Katablepharidophycota).

Hatena arenicola (Katablepharidophycota) is a single-celled eukaryote that temporarily possesses a chlorophyte alga of the genus Nephroselmis as an intracellular symbiont. In the present study, we investigated the molecular diversity of the endosymbiont Nephroselmis in a natural population of the host H. arenicola. We sequenced the host's 18S rRNA gene and the endosymbiont's plastid-encoded 16S rRNA gene. The results indicated that almost identical strains of the host harbored at least three distinct strains of the algal endosymbiont affiliated to the clade Nephroselmis rotunda. This finding supports our previous hypothesis that H. arenicola and its symbiotic alga are in an early stage of secondary endosymbiosis.

Functional analyses of bacterial genomes found in Symbiodiniaceae genome assemblies.

Bacterial-algal interactions strongly influence marine ecosystems. Bacterial communities in cultured dinoflagellates of the family Symbiodiniaceae have been characterized by metagenomics. However, little is known about whole-genome analysis of marine bacteria associated with these dinoflagellates. We performed in silico analysis of four bacterial genomes from cultures of four dinoflagellates of the genera Symbiodinium, Breviolum, Cladocopium and Durusdinium. Comparative analysis showed that the former three contain the alphaproteobacterial family Parvibaculaceae and that the Durusdinium culture includes the family Sphingomonadaceae. There were no large genomic reductions in the alphaproteobacteria with genome sizes of 2.9-3.9 Mb, implying they are not obligate intracellular bacteria. Genomic annotations of three Parvibaculaceae detected the gene for diacetylchitobiose deacetylase (Dac), which may be involved in the degradation of dinoflagellate cell surfaces. They also had metabolic genes for dissimilatory nitrate reduction to ammonium (DNRA) in the nitrogen (N) cycle and cobalamin (vitamin B12 ) biosynthetic genes in the salvage pathway. Those three characters were not found in the Sphingomonadaceae genome. Predicted biosynthetic gene clusters for secondary metabolites indicated that the Parvibaculaceae likely produce the same secondary metabolites. Our study suggests that the Parvibaculaceae is a major resident of Symbiodiniaceae cultures with antibiotics.

Chromosome-scale genome assembly of an acidophilic microalga Tetratostichococcus sp. P1 isolated from a tropical peatland in Malaysia.

Tetratostichococcus sp. P1 shows an acidophilic phenotype which could allow mass-scale monoculture of this green microalga without severe contamination by environmental microorganisms. In this study, we report a chromosome-scale genome assembly for Tetratostichococcus sp. P1.

Expanded male sex-determining region conserved during the evolution of homothallism in the green alga Volvox.

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 heterothallic ancestor, we compared whole-genome data from Thai and Japanese genotypes within the homothallic green alga Volvox africanus. The Thai and Japanese algae harbored expanded ancestral male and female SDRs of ∼1 Mbp each, representing a direct heterothallic ancestor. Therefore, the expanded male and female ancestral SDRs may originate from the ancient (∼75 mya) heterothallic ancestor, and either might have been conserved during the evolution of each homothallic genotype. An expanded SDR-like region seems essential for homothallic sexual reproduction in V. africanus, irrespective of male or female origin. Our study stimulates future studies to elucidate the biological significance of such expanded genomic regions.

Reorganization of the ancestral sex-determining regions during the evolution of trioecy in Pleodorina starrii.

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 sex phenotypes of P. starrii to reveal a reorganization of the ancestral sex-determining regions (SDRs) in the sex chromosomes: the male and bisexual phenotypes had the same "male SDR" with paralogous gene expansions of the male-determining gene MID, whereas the female phenotype had a "female SDR" with transposition of the female-specific gene FUS1 to autosomal regions. Although the male and bisexual sex phenotypes had the identical male SDR and harbored autosomal FUS1, MID and FUS1 expression during sexual reproduction differed between them. Thus, the coexistence of three sex phenotypes in P. starrii is possible.