Molecular phylogeny and taxonomy of the genus Chaetophora (Chlorophyceae,Chlorophyta), including descriptions of Chaetophoropsis aershanensis gen. et sp. nov.

The broadly defined genus Chaetophora consisted of species with minute, uniseriate branching filaments enveloped in soft or firm mucilage forming macroscopic growths that are spherical, hemispherical, and tubercular or arbuscular, growing epiphytically on freshwater aquatic plants and other submerged surfaces in standing or fast‐flowing water. Recent molecular analyses clearly showed that this genus was polyphyletic. In this study, eight strains of Chaetophora and three strains of Stigeoclonium were identified and successfully cultured. In combination with the morphological data, a concatenated data set of four markers (18S + 5.8S + ITS2+ partial 28S rDNA) was also used to determine their taxonomic relationships and phylogenetic positions. The molecular analysis resolved the broadly defined Chaetophora to at least two genera. Species with a globose thallus of genus Chaetophora formed a separate monophyletic clade, which clearly separated from, a type of lobe‐form Chaetophora species. Therefore, we propose to erect a new genus, Chaetophoropsis, which includes all globose species of the Chaetophora. Chaetophoropsis aershanensis was determined to be a new species, based on its special characteristic of profuse long rhizoids. Stigeoclonium polyrhizum, as the closest relative to Chaetophoropsis, revealed its distant relationships to other species of Stigeoclonium. A globose thallus with a thick, soft mucilage matrix, and special rhizoidal branches lent further support to the placement of S. polyrhizum in the genus Chaetophoropsis and had the closest relationship to C. aershanensis. Taxonomic diversity was proven by distinctive morphological differences and by phylogenetic divergence in the broadly defined Chaetophora identified herein.     

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales,Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400–600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy—in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria—and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup “Pleiochloris,” was included in the genus Ixipapillifera as I. deasonii comb. nov.     

Ultrastructure and LSU rDNA-based phylogeny of Peridinium lomnickii and description of Chimonodinium gen. nov. (Dinophyceae)

Several populations of Peridinium lomnickii were examined by SEM and serial section TEM. Comparison with typical Peridinium, Peridiniopsis, Palatinus and Scrippsiella species revealed significant structural differences, congruent with phylogenetic hypotheses derived from partial LSU rDNA sequences. Chimonodinium gen. nov. is described as a new genus of peridinioids, characterized by the Kofoidian plate formula Po, cp, x, 4', 3a, 7', 6c, 5s, 5', 2', the absence of pyrenoids, the presence of a microtubular basket with four or five overlapping rows of microtubules associated with a small peduncle, a pusular system with well-defined pusular tubes connected to the flagellar canals, and the production of non-calcareous cysts. Serial section examination of Scrippsiella trochoidea, here taken to represent typical Scrippsiella characters, revealed no peduncle and no associated microtubular strands. The molecular phylogeny placed C. lomnickii comb. nov. as a sister group to a clade composed of Thoracosphaera and the pfiesteriaceans. Whereas the lack of information on fine structure of the swimming stage of Thoracosphaera leaves its affinities unexplained, C. lomnickii shares with the pfiesteriaceans the presence of a microtubular basket and the unusual connection between two plates on the left side of the sulcus, involving extra-cytoplasmic fibres.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

Molecular phylogeny of Cercomonadidae and kinetid patterns of Cercomonas and Eocercomonas gen. nov. (Cercomonadida, Cercozoa)

Cercomonads are among the most abundant and widespread zooflagellates in soil and freshwater. We cultured 22 strains and report their complete 18S rRNA sequences and light microscopic morphology. Phylogenetic analysis of 51 Cercomonas rRNA genes shows in each previously identified major clade (A, B) two very robust, highly divergent, multi-species subclades (A1, A2; B1, B2). We studied kinetid ultrastructure of five clade A representatives by serial sections. All have two closely associated left ventral posterior microtubular roots, an anterior dorsal root, a microtubule-nucleating left anterior root, and a cone of microtubules passing to the nucleus. Anterior centrioles (=basal bodies, kinetosomes) of A1 have cartwheels; the posterior centriole does not, suggesting it is older, and implying flagellar transformation similar to other bikonts. Strain C-80 (subclade A2) differs greatly, having a dorsal posterior microtubule band, but lacking the A1-specific fibrillar striated root, nuclear extension to the centrioles, centriolar diaphragm, extrusomes; both mature centrioles lack cartwheels. For clade A2 we establish Eocercomonas gen. n., with type Eocercomonas ramosa sp. n., and for clade B1 Paracercomonas gen. n. (type Paracercomonas marina sp. n.). We establish Paracercomonas ekelundi sp. n. for culture SCCAP C1 and propose a Cercomonas longicauda neotype and Cercomonas (=Neocercomonas) jutlandica comb. n. and Paracercomonas (=Cercomonas) metabolica comb. n.     

Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae)

The little known Rubiaceae genus Villaria is endemic mostly to the coastal forests of the Philippines. Traditionally, it has been placed in the tribe Gardenieae. Later it was transferred to Octotropideae sensu Robbrecht and Puff. Villaria was placed among the ??primitive?? genera of the tribe, which are essentially characterized by large fruits, horizontal ovules and numerous seeds. Parsimony and Bayesian analyses of the combined plastid (rps16 and trnT-F) dataset strongly support the inclusion of Villaria in Octotropideae as well as monophyly of the genus. However, our molecular results do not conform to the current informal groups of the tribe delimited by fruit size, ovule position, number of seeds and exotesta thickenings. Instead, a close relationship between Villaria and two ??central genera?? (Hypobathrum and Pouchetia) is revealed for the first time. This clade is sister to a group comprising ??primitive?? (Fernelia), ??advanced?? (Kraussia and Polysphaeria) and ??central?? (Feretia) representatives. In addition, our combined tree strongly supports a sister taxa relationship between Canephora and Paragenipa. Villaria is characterized by unilocular ovaries, parietal placentation and strictly horizontal ovules. These features are unique within the Octotropideae. We recognize a total of five Villaria species, one new species (V. leytensis) is described here, and two species (V. philippinensis and V. rolfei) are transferred into synonymy with V. odorata. Each species is fully described, and a key to the species, a distribution map and illustrations are provided.     

Taxonomic revision of the Agaraceae with a description of Neoagarum gen. nov. and reinstatement of Thalassiophyllum

We confirmed the monophyly of the Agaraceae based on phylogenetic analyses of six mitochondrial and six chloroplast gene sequences from Agarum, Costaria, Dictyoneurum, and Thalassiophyllum species, as well as representative species from other laminarialean families. However, the genus Agarum was paraphyletic, comprising two independent clades, A. clathratum/A. turneri and A. fimbriatum/A. oharaense. The latter clade was genetically most closely related to Dictyoneurum spp., and morphologically, the species shared a flattened stipe bearing fimbriae (potential secondary haptera) in the mid‐ to upper portion. The phylogenetic position of Thalassiophyllum differed between the two datasets: in the chloroplast gene phylogeny, Thalassiophyllum was included in the A. clathratum/A. turneri clade, but in the mitochondrial gene phylogeny, it formed an independent clade at the base of the Agaraceae, the same position it took in the phylogeny when the data from both genomes were combined despite a larger number of bp being contributed by the chloroplast gene sequences. Considering the remarkable morphological differences between Thalassiophyllum and other Agaraceae, and the molecular support, we conclude that Thalassiophyllum should be reinstated as an independent genus. Dictyoneurum reticulatum was morphologically distinguishable from D. californicum due to its midrib, but because of their close genetic relationship, further investigations are needed to clarify species‐level taxonomy. In summary, we propose the establishment of a new genus Neoagarum to accommodate A. fimbriatum and A. oharanese and the reinstatement of the genus Thalassiophyllum.     

Molecular phylogeny and taxonomic revision of the genus Wittrockiella (Pithophoraceae,Cladophorales), including the descriptions of W. australis sp. nov. and W. zosterae sp. nov.

Wittrockiella is a small genus of filamentous green algae that occurs in habitats with reduced or fluctuating salinities. Many aspects of the basic biology of these algae are still unknown and the phylogenetic relationships within the genus have not been fully explored. We provide a phylogeny based on three ribosomal markers (ITS, LSU, and SSU rDNA) of the genus, including broad intraspecific sampling for W. lyallii and W. salina, recommendations for the use of existing names are made, and highlight aspects of their physiology and life cycle. Molecular data indicate that there are five species of Wittrockiella. Two new species, W. australis and W. zosterae, are described, both are endophytes. Although W. lyallii and W. salina can be identified morphologically, there are no diagnostic morphological characters to distinguish between W. amphibia, W. australis, and W. zosterae. A range of low molecular weight carbohydrates were analyzed but proved to not be taxonomically informative. The distribution range of W. salina is extended to the Northern Hemisphere as this species has been found in brackish lakes in Japan. Furthermore, it is shown that there are no grounds to recognize W. salina var. kraftii, which was described as an endemic variety from a freshwater habitat on Lord Howe Island, Australia. Culture experiments indicate that W. australis has a preference for growth in lower salinities over full seawater. For W. amphibia and W. zosterae, sexual reproduction is documented, and the split of these species is possibly attributable to polyploidization.     

Hemichloris antarctica, gen. et sp. nov. (Chlorococcales, Chlorophyta), a cryptoendolithic alga from Antarctica

Hemichloris antarctica gen. et sp. nov. (Oocystaceae, Chlorococcales) is characterized by a single, articulated, pyrenoid-less, thick saucer-shaped chloroplast, which generally fills less than half of the cell periphery. Multiplication is only by autospores. The species is psychrophilic and is damaged at temperatures above 20 degree C. Hemichloris antarctica is a member of the cryptoendolithic microbial community living in porous sandstone rocks of the Antarctica cold desert. It inhabits the zone below that of cryptoendolithic lichens and survives at extremely low light intensities. In the natural habitat, morphology is somewhat different from that in culture, as chloroplasts are smaller and without articulation, and the cells develop a gelatinous sheath.     

Characterization and phylogeny of a novel methanotroph, Methyloglobulus morosus gen. nov., spec. nov

A novel methanotrophic gammaproteobacterium, strain KoM1, was isolated from the profundal sediment of Lake Constance after initial enrichment in opposing gradients of methane and oxygen. Strain KoM1 grows on methane or methanol as its sole source of carbon and energy. It is a Gram-negative methanotroph, often expressing red pigmentation. Cells are short rods and occur sometimes in pairs or short chains. Strain KoM1 grows preferably at reduced oxygen concentrations (pO₂ = 0.05–0.1 bar). It can fix nitrogen, and grows at neutral pH and at temperatures between 4 and 30 °C. Phylogenetically, the closest relatives are Methylovulum miyakonense and Methylosoma difficile showing 91% 16S rRNA gene sequence identity. The only respiratory quinone is ubiquinone Q8; the main polar lipids are phosphatidyl ethanolamine and phosphatidyl glycerol. The major cellular fatty acids are summed feature 3 (presumably C16:1ω7c) and C16:1ω5c, and the G + C content of the DNA is 47.7 mol%. Strain KoM1 is described as the type strain of a novel species within a new genus, Methyloglobulus morosus gen. nov., sp. nov.     

A taxonomic revision of a Hypnea charoides-valentiae complex (Rhodophyta, Gigartinales) in Japan, with a description of Hypnea flexicaulis sp. nov.

Morphology of a Hypnea charoides‐valentiae complex (Rhodophyta, Gigartinales) in Japan is investigated on the basis of field‐collected and cultured plants. We can recognize two species, Hypnea charoides Lamouroux and H. flexicaulis Yamagishi et Masuda, sp. nov., which are distinguished by: (i) conspicuous percurrent main axes growing straight in the former versus less conspicuous percurrent main axes growing flexuosely in the latter; (ii) narrow branching angles (less than 90°) in the former versus wide branching angles (up to 150°) in the latter; (iii) the presence of branches and branchlets showing abrupt adaxial bending in the former versus those showing pronounced abaxial bending, some growing into hooked branchlets in the latter; and (iv) the absence (in the former) or presence (in the latter) of antler‐like branches. Nucleotide sequences of the plastid‐encoded large subunit of the ribulose‐1,5‐bisphosphate carboxylase/oxygenase gene (rbcL) support the differentiation of these two entities: the pairwise distance between them was 83 b.p. (6.2%).     

Lithoautotrophic growth of sulfate-reducing bacteria,and description of Desulfobacterium autotrophicum gen. nov., sp. nov.

The capacity of mesophilic sulfate-reducing bacteria to grow lithoautotrophically with H₂, sulfate and CO₂ was investigated with enrichment cultures and isolated species. (a) Enrichments in liquid mineral media with H₂, sulfate and CO₂ consistently yielded mixed cultures of nonautotrophic, acetate-requiring Desulfovibrio species and autotrophic, acetate-producing Acetobacterium species (cell ratio approx. 20:1). (b) By direct dilution of mud samples in agar, various non-sporing sulfate reducers were isolated in pure cultures that did grow autotrophically. Two oval cell types (strains HRM2, HRM4) and one curved cell type (strain HRM6) from marine sediment were studied in detail. The strains grew in mineral medium supplemented only with vitamins (biotin, p-aminobenzoate, nicotinate). Carbon autotrophy was evident (i) from comparative growth experiments with non-autotrophic, acetate-requiring species, (ii) from high cell densities ruling out a cell synthesis from organic impurities in the mineral media, and (iii) by demonstrating that 96–99% of the cell carbon was derived from ¹⁴C-labelled CO₂. Autotrophic growth occurred with a doubling time of 16–20 h at 24–28°C. Formate, fatty acids up to palmitate, ethanol, lactate, succinate, fumarate, malate and other organic acids were also used and completely oxidized. The three strains possessed cytochromes of the b-and c-type, but no desulfoviridin. Strain HRM2 is described as a new species of a new genus, Desulfobacterium autotrophicum. (c) The capacity for autotrophic growth was also tested with sulfate-reducing bacteria that originally had been isolated on organic substrates. The incompletely oxidizing, non-sporing types such as Desulfovibrio and Desulfobulbus species and Desulfomonas pigra were confirmed to be obligate heterotrophs that required acetate for growth with H₂ and sulfate. In contrast, several of the completely oxidizing sulfate reducers were facultative autotrophs, such as Desulfosarcina variabilis, Desulfonema limicola, Desulfococcus niacini, and the newly isolated Desulfobacterium vacuolatum and Desulfobacter hydrogenophilus. The only incompletely oxidizing sulfate reducer that could grow autotrophically was the sporing Desulfotomaculum orientis, which obtained 96% of its cell carbon from ¹⁴C-labelled CO₂. Desulfovibrio baarsii and Desulfococcus multivorans may also be regarded as types of facultative autotrophs; they could not oxidize H₂, but grew on sulfate with formate as the only organic substrate.     

Molecular phylogeny and developmental studies of Apoglossum and Paraglossum (Delesseriaceae,Rhodophyta) with a description of Apoglosseae trib. nov.

Our morphological and molecular studies indicate that species from the southern hemisphere previously placed in Delesseria belong in Paraglossum and that Paraglossum and Apoglossum comprise a separate tribe, the Apoglosseae, S.-W. Lin, Fredericq & Hommersand, trib. nov., within the family Delesseriaceae. From a vegetative perspective the Apoglosseae is readily recognized because some or all fourth-order cell rows are formed on the inner sides of third-order cell rows. All fourth-order cell rows grow adaxially in Apoglossum, whereas both adaxial and abaxial cell rows are present in Paraglossum. Periaxial cells do not divide in Apoglossum, whereas they divide transversely in Paraglossum in the same way as in Delesseria. Major branches are formed mainly from the margins of midribs in the Apoglosseae. The procarp consists of a straight carpogonial branch and two sterile cells, with the second formed on the same side as the first. The carpogonium cuts off two connecting cells in tandem from its apical end, the terminal cell being nonfunctional and the subterminal cell typically fusing with the auxiliary cell. Gonimoblast filaments radiate in all directions from the gonimoblast initials and produce carposporangia terminally in branched chains, with pit connections between the inner gonimoblast cells broadening and enlarging. The auxiliary cell, supporting cell, and sterile cells unite into a fusion cell, which remains small in Apoglossum but incorporates the branched inner gonimoblast filaments and cells in the floor of the cystocarp in Paraglossum. Elongated inner cortical cells seen in mature cystocarps in the Delesserieae are absent in the Apoglosseae. Phylogenetic studies based on rbcL (RuBisCO large subunit gene) sequence analyses strongly support the recognition of the Apoglosseae within the subfamily Delesserioideae of the Delesseriaceae, in agreement with our previous observations based primarily on analyses of large subunit ribosomal DNA (LSU).     

Molecular phylogeny,systematics, and revision of the type species of Lobomonas,L. francei (Volvocales,Chlorophyta) and closely related taxa

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field‐collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the “Chlamydomonas/Volvox‐clade.” Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.