The Sphaeropleales are a dominant group of green algae, which contain species important to freshwater ecosystems and those that have potential applied usages. In particular, Raphidocelis subcapitata is widely used worldwide for bioassays in toxicological risk assessments. However, there are few comparative genome analyses of the Sphaeropleales. To reveal genome evolution in the Sphaeropleales based on well-resolved phylogenetic relationships, nuclear, mitochondrial, and plastid genomes were sequenced in this study. The plastid genome provides insights into the phylogenetic relationships of R. subcapitata, which is located in the most basal lineage of the four species in the family Selenastraceae. The mitochondrial genome shows dynamic evolutionary histories with intron expansion in the Selenastraceae. The 51.2 Mbp nuclear genome of R. subcapitata, encoding 13,383 protein-coding genes, is more compact than the genome of its closely related oil-rich species, Monoraphidium neglectum (Selenastraceae), Tetradesmus obliquus (Scenedesmaceae), and Chromochloris zofingiensis (Chromochloridaceae); however, the four species share most of their genes. The Sphaeropleales possess a large number of genes for glycerolipid metabolism and sugar assimilation, which suggests that this order is capable of both heterotrophic and mixotrophic lifestyles in nature. Comparison of transporter genes suggests that the Sphaeropleales can adapt to different natural environmental conditions, such as salinity and low metal concentrations.

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The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.

A major shell matrix protein originally obtained from a freshwater snail is a molluscan homologue of Dermatopontins, a group of Metazoan proteins also called TRAMP (tyrosine-rich acidic matrix protein). We sequenced and identified 14 molluscan homologues of Dermatopontin from eight snail species belonging to the order Basommatophora and Stylommatophora. The bassommatophoran Dermatopontins fell into three types, one is suggested to be a shell matrix protein and the others are proteins having more general functions based on gene expression analyses. N-glycosylation is inferred to be important for the function involved in shell calcification, because potential N-glycosylation sites were found exclusively in the Dermatopontins considered as shell matrix proteins. The stylommatophoran Dermatopontins fell into two types, also suggested to comprise a shell matrix protein and a protein having a more general function. Phylogenetic analyses using maximum likelihood and Bayesian methods revealed that gene duplication events occurred independently in both basommatophoran and stylommatophoran lineages. These results suggest that the dermatopontin genes were co-opted for molluscan calcification at least twice independently after the divergence of basommatophoran and stylommatophoran lineages, or more recently than we have expected.

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A new flagellate of the Raphidophyceae, Chlorinimonas sublosa gen. et sp. nov., collected from Wakayama Prefecture, Japan is described based on morphological observations, microspectrophotometry of chloroplasts, and phylogenetic analysis of SSU rDNA sequences. The cell was usually elliptical, sometimes spherical, oval or slender, and possessed two subequal heterodynamic flagella emerging from a subapical pit. Greenish yellow discoidal chloroplasts, 15-25 per cell, were situated at the periphery of the cell. The alga is very similar to the genus Heterosigma, but distinct in that there is no invagination of thylakoids into the pyrenoids and no typical girdle lamella in the chloroplast, and the chloroplasts are greenish yellow. Phylogenetic analysis of SSU rDNA revealed that this alga forms a sister clade with the clade of Chattonella and Heterosigma. Based on these results, we propose a new genus Chlorinimonas with Chlorinimonas sublosa as the type species. In addition, this paper is the first report of molecular data covering all genera of the Raphidophyceae. The phylogenetic analysis suggests that the intrusion to freshwater habitat has occurred only once in the Raphidophyceae.

The genus Nephroselmis (Nephroselmidophyceae), which had been placed in the Prasinophyceae, is one of the primitive green flagellates that are important to our understanding of the early evolution of green plants. We studied a new species of Nephroselmis isolated from Japan, Fiji and South Africa. This species has been known for a long time as undescribed species 'N. viridis.' N. viridis possesses some ultrastructural characters shared with only the freshwater type species N. olivacea, including a disc-like structure beneath the pyrenoid and bipolar spiny body scales with 1-5-8-5-1 spines. Molecular phylogenetic analysis based on 18S rDNA also supports a sister relationship between N. viridis and N. olivacea. However, N. viridis is distinguishable from N. olivacea by the shape of its starch sheath, its scales, its pigment composition and its habitat. In this paper, we designate the formal description of N. viridis sp. nov. We also describe variability in the 18S rDNA introns of various N. viridis strains. This detailed study of N. viridis provides some insights into the evolution of Nephroselmis.

Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. Currently, it is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. However, despite this organism’s, our knowledge of its cell biology—for example, the patterns of nuclear and cytoplasmic division in the mitotic stage—is limited. Although it has been reported that P. subcapitata proliferates by popularity forming four daughter cells (autospores) through multiple fission after two nuclear divisions, here, we report two additional reproductive patterns by which two autospores are formed by binary fission (“two-autospore type”) and eight autospores are formed by multiple fission (“eight-autospore type”). Moreover, we found that cell reproductive patterns differed markedly with the culture conditions or with exposure to either of two typical toxicants, potassium dichromate (K2Cr2O7) and 3,5-dichlorophenol (3,5-DCP). The eight-autospore type occurred at the highest frequency in the early phase of culture, but it disappeared under 3,5-DCP at 2.0 mg/L. Under 0.3 mg/L K2CrO7 (Cr(VI)) the eight-autospore type took substantially longer to appear than in control culture. The two-autospore type occurred only in the late phase of culture. To our knowledge, this is the first detailed evaluation of the reproductive patterns of P. subcapitata, which changed dramatically in the presence of toxicants. These findings suggest that observation of the reproductive patterns of P. subcapitata will help to elucidate different cell reactions to toxicants.