Kazuyuki Miyaji

PHYLOGENETIC RELATIONSHIPS WITHIN CLADOPHORALES (ULVOPHYCEAE, CHLOROPHYTA) INFERRED FROM 18S rRNA GENE SEQUENCES, WITH SPECIAL REFERENCE TO AEGAGROPILA LINNAEI 1

The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.

Ultrastructure of the Dermal Chromatophores in a Lizard (Scincidae: Plestiodon latiscutatus) with Conspicuous Body and Tail Coloration

Abstract Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.

Taxonomic re‐examination of 17 species of Nitella subgenus tieffallenia (Charales, Charophyceae) based on internal morphology of the oospore wall and multiple DNA marker sequences (Vol. 41:195–211)

In an attempt to reconstruct the natural taxonomic system for Nitella, 17 species of Nitella subgenus Tieffallenia were reexamined using SEM observations of the internal morphology of the oospore wall (IMOW) and phylogenetic analyses of 4553 base pairs from multiple DNA markers (atpB, rbcL, psaB, and ITS‐5.8S rRNA genes). Our SEM observations identified three types of IMOW: homogeneous (HG), weakly spongy (W‐SG), and strongly spongy (S‐SG) types. Based on differences in the IMOW, species with reticulate or tuberculate oospore wall ornamentation in the external morphology of the oospore wall (EMOW) were subdivided into two distinct groups (characterized by the HG or S‐SG types of IMOW, respectively), which were robustly separated from each other in our molecular phylogenetic analyses. In our molecular phylogeny, the subgenus Tieffallenia consisted of four robust monophyletic groups—three clades of the HG type and a spongy (S‐SG and W‐SG) type clade—that were characterized by differences in the IMOW and EMOW. In addition, our SEM observations and sequence data verified the distinct status of five species (N. japonica Allen, N. oligospira A. Braun, N. vieillardii stat. nov., N. imperialis stat. nov., and N. morongii Allen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested that N. megaspora (J. Groves) Sakayama originally identified by LM includes at least two distinct species, characterized by W‐SG and S‐SG types of IMOW, respectively.

Carotenoid compositions of Cladophora balls (Aegagropila linnaei) and some members of the Cladophorales (Ulvophyceae, Chlorophyta): Their taxonomic and evolutionary implication

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α‐carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila‐clade (clade 2), and divided clade 1 into subclade 1‐1 and subclade 1‐2. All the examined species positioned in the Aegagropila‐clade and those of the subclade 1‐1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1‐2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1‐2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus.

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.

Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions.

There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT(+), cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT(+) cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different.

A taxonomic study of Eudorina unicocca (Volvocaceae, Chlorophyceae) and related species, based on morphology and molecular phylogeny

Colonial volvocacean algae engage in two types of sexual reproduction: isogamy and anisogamy/oogamy with sperm packets. This difference is an important generic diagnosis within the Volvocaceae. Although Yamagishiella differs from the anisogamous genus Eudorina in its isogamous sexual reproduction, the vegetative morphology and asexual reproduction characteristics of the two genera are indistinguishable, especially between Eudorina unicocca G. M. Smith and Yamagishiella unicocca (Rayburn et Starr) Nozaki. We re-examined morphological characteristics of E. unicocca and related species, using multiple strains of E. unicocca and Y. unicocca and molecular phylogenetic analyses. Strains from two Japanese lakes, which produced aplanospores and were solely asexual, could be assigned to either E. unicocca or Y. unicocca, based on traditional morphological diagnoses. However, a new morphological diagnosis (the difference in the distribution and number of contractile vacuoles on the cell surface) and molecular phylogenetic analyses demonstrated that all were E. unicocca. Furthermore, E. unicocca can be divided into two species on the basis of the presence or absence of individual cellular sheaths in the colonial gelatinous matrix, which are observable with methylene blue staining. These two species, E. peripheralis (Goldstein) T. K. Yamada stat. nov. (= E. unicocca var. peripherialis Goldstein) and E. unicocca (including the Japanese aplanosporic strains), formed two robust monophyletic groups, based on chloroplast gene sequences for the large RuBisCO subunit and internal transcribed spacer regions of nuclear ribosomal DNA.

Comparing the low‐salinity tolerance of Ulva species distributed in different environments

The green macroalgal genus Ulva (Ulvales, Ulvophyceae, Chlorophyta) is distributed worldwide from marine to freshwater environments. Comparative analyses of hyposalinity tolerance among marine, brackish, and freshwater Ulva species were performed by fluorescein diacetate viability counts. The subtidal marine species Ulva sp., collected from a depth of 30 m, showed the poorest tolerance to low salinity. This species died in 5 practical salinity units (PSU) artificial seawater or freshwater within 1 day. Its closely related species U. linza L. (an intertidal species) and U. prolifera Müller (a brackish species) showed varying tolerances to low salinity. After 7 days of freshwater exposure, the viability of U. linza L. decreased to approximately 20%, while U. prolifera Müller showed nearly 100% viability. The freshwater species U. limnetica Ichihara et Shimada, not yet found in coastal areas, was highly viable in seawater.

Pigment cell mechanisms underlying dorsal color-pattern polymorphism in the Japanese four-lined snake.

To provide histological foundation for studying the genetic mechanisms of color‐pattern polymorphisms, we examined light reflectance profiles and cellular architectures of pigment cells that produced striped, nonstriped, and melanistic color patterns in the snake Elaphe quadrivirgata. Both, striped and nonstriped morphs, possessed the same set of epidermal melanophores and three types of dermal pigment cells (yellow xanthophores, iridescent iridophores, and black melanophores), but spatial variations in the densities of epidermal and dermal melanophores produced individual variations in stripe vividness. The densities of epidermal and dermal melanophores were two or three times higher in the dark‐brown‐stripe region than in the yellow background in the striped morph. However, the densities of epidermal and dermal melanophores between the striped and background regions were similar in the nonstriped morph. The melanistic morph had only epidermal and dermal melanophores and neither xanthophores nor iridophores were detected. Ghost stripes in the shed skin of some melanistic morphs suggested that stripe pattern formation and melanism were controlled independently. We proposed complete‐ and incomplete‐dominance heredity models for the stripe‐melanistic variation and striped, pale‐striped, and nonstriped polymorphisms, respectively, according to the differences in pigment‐cell composition and its spatial architecture. J. Morphol. 274:1353–1364, 2013.

Systematics of Rhizoclonium-like algae (Cladophorales, Chlorophyta) from Japanese brackish waters, based on molecular phylogenetic and morphological analyses

Ichihara K., Shimada S. and Miyaji K. 2013. Systematics of Rhizoclonium-like algae (Cladophorales, Chlorophyta) from Japanese brackish waters, based on molecular phylogenetic and morphological analyses. Phycologia 52: 398–410. DOI: 10.2216/12–102.1 Analyses of partial small subunit (SSU) and large subunit (LSU) rRNA genes of Rhizoclonium-like filamentous green algae, collected from brackish waters in Japan, revealed the existence of five genetically distinct species. Four species (Rhizoclonium spp. 1–4) were closely related to Rhizoclonium hieroglyphicum. The phylogenetic position of the Rhizoclonium-like sp. 5 remained largely unresolved, although LSU analyses suggested a relationship with Chaetomorpha ligustica. Rhizoclonium spp. 1 and 4 were identified as Rhizoclonium riparium and Rhizoclonium lubricum, respectively. Rhizoclonium spp. 2 and 3 were morphologically distinguishable from other Rhizoclonium species and minute Chaetomorpha species. Rhizoclonium sp. 2 exhibited a characteristically small L/D (cell length/cell diameter) ratio and large number of nuclei (3–7 per cell), while Rhizoclonium sp. 3 was characterized by unique spindle or band-like, densely arranged chloroplasts; therefore, we described them as new species, Rhizoclonium breve and Rhizoclonium confertum. Rhizoclonium-like sp. 5 was characterized by perforated chloroplasts and polypyramidal pyrenoids and was clearly distinct from known Rhizoclonium or Chaetomorpha species. Based on the phylogenetic position and morphological features of Rhizoclonium-like sp. 5, we described it as a new species of Chaetomorpha, Chaetomorpha tokyoensis.