ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR‐ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM,HORMIDIELLA, AND ENTRANSIA1

Nuclear‐encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well‐supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as “Klebsormidium” emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384‐1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst’s morphology‐based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.     

Biodiversity of green algae covering artificial hard substrate surfaces in a suburban environment: a case study using molecular approaches

In Middle European suburban environments green algae often cover open surfaces of artificial hard substrates. Microscopy reveals the Apatococcus/Desmococcus morphotype predominant over smaller coccoid forms. Adverse conditions such as limited water availability connected with high PAR and UV irradiance may narrow the algal diversity to a few specialists in these subaerial habitats. We used rRNA gene cloning/sequencing from both DNA extracts of the biofilms without culturing as well as cultures, for the unambiguous determination of the algal composition and to assess the algal diversity more comprehensively. The culture independent approach revealed mainly just two genera (Apatococcus, Trebouxia) for all study sites and five molecular operational taxonomic units (OTUs) for a particular study site, which based on microscopic observation was the one with the highest morphological diversity. The culture approach, however, revealed seven additional OTUs from five genera (Chloroidium, Coccomyxa, Coenochloris, Pabia, Klebsormidium) and an unidentified trebouxiophyte lineage for that same site; only two OTUs were shared by both approaches. Two OTUs or species were recovered for which references have been isolated only from Antarctica so far. However, the internal transcribed spacer (ITS) sequence differences among them supported they are representing distinct populations of the same species. Within Apatococcus five clearly distinct groups of ITS sequences, each putatively representing a distinct species, were recovered with three or four such ITS types co‐occurring at the same study site. Except for the streptophyte Klebsormidium only members of Trebouxiophyceae were detected suggesting these algae may be particularly well‐adapted to subaerial habitats.     

Dehydration,temperature, and light tolerance in members of the aeroterrestrial green algal genus Interfilum (Streptophyta) from biogeographically different temperate soils

Unicellular green algae of the genus Interfilum (Klebsormidiales, Streptophyta) are typical components of biological soil crusts. Four different aeroterrestrial Interfilum strains that have previously been molecular‐taxonomically characterized and isolated from temperate soils in Belgium, Czech Republic, New Zealand, and Ukraine were investigated. Photosynthetic performance was evaluated under different controlled abiotic conditions, including dehydration, as well as under a light and temperature gradient. For standardized desiccation experiments, a new methodological approach with silica gel filled polystyrol boxes and effective quantum yield measurements from the outside were successfully applied. All Interfilum isolates showed a decrease and inhibition of the effective quantum yield under this treatment, however with different kinetics. While the single cell strains exhibited relatively fast inhibition, the cell packet forming isolates dried slower. Most strains fully recovered effective quantum yield after rehydration. All Interfilum isolates exhibited optimum photosynthesis at low photon fluence rates, but with no indication of photoinhibition under high light conditions suggesting flexible acclimation mechanisms of the photosynthetic machinery. Photosynthesis under lower temperatures was generally more active than respiration, while the opposite was true for higher temperatures. The presented data provide an explanation for the regular occurrence of Interfilum species in soil habitats where environmental factors can be particularly harsh.     

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg²⁺ or Ca²⁺, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales,Streptophyta) with special reference to cell division and thallus formation

Representatives of the closely related genera, Interfilum and Klebsormidium, are characterized by unicells, dyads or packets in Interfilum and contrasting uniseriate filaments in Klebsormidium. According to the literature, these distinct thallus forms originate by different types of cell division, sporulation (cytogony) versus vegetative cell division (cytotomy), but investigations of their morphology and ultrastructure show a high degree of similarity. Cell walls of both genera are characterized by triangular spaces between cell walls of neighbouring cells and the parental wall or central space among the walls of a cell packet, exfoliations and projections of the parental wall and cap-like and H-like fragments of the cell wall. In both genera, each cell has its individual cell wall and it also has part of the common parental wall or its remnants. Therefore, vegetative cells of Interfilum and Klebsormidium probably divide by the same type of cell division (sporulation-like). Various strains representing different species of the two genera are characterized by differences in cell wall ultrastructure, particularly the level of preservation, rupture or gelatinization of the parental wall surrounding the daughter cells. The differing morphologies of representatives of various lineages result from features of the parental wall during cell separation and detachment. Cell division in three planes (usual in Interfilum and a rare event in Klebsormidium) takes place in spherical or short cylindrical cells, with the chloroplast positioned perpendicularly or obliquely to the filament (dyad) axis. The morphological differences are mainly a consequence of differing fates of the parental wall after cell division and detachment. The development of different morphologies within the two genera mostly depends on characters such as the shape of cells, texture of cell walls, mechanical interactions between cells and the influence of environmental conditions.     

MYCOSPORINE‐LIKE AMINO ACIDS AND PHYLOGENIES IN GREEN ALGAE: PRASIOLA AND ITS RELATIVES FROM THE TREBOUXIOPHYCEAE (CHLOROPHYTA)1

A UV‐absorbing mycosporine‐like amino acid (324 nm‐MAA), so far only known from the green macroalgal genus Prasiola (Trebouxiophyceae), was also identified in other morphologically diverse green algae closely related to Prasiola spp. in 18S rDNA phylogenies. Using HPLC, a second UV‐absorbing compound was found only in Myrmecia incisa Reisigal among all studied strains. This substance showed an absorption maximum at 322 nm and hence was designated as putative 322 nm‐MAA. Preliminary UV‐exposure experiments indicated that all species containing one or the other MAA showed a strong accumulation of the respective compound, thus supporting their function as putative UV sunscreen. Both UV‐absorbing substances were only identified in the studied members of the Trebouxiophyceae but were absent in members of the Ulvophyceae and Chlorophyceae. When mapped on an 18S rDNA phylogeny, the distribution of 324 nm‐MAA was found to be scattered within the Trebouxiophyceae but was consistent with a distribution that follows phylogenetic patterns rather than ecological adaptations. The 324 nm‐MAA was also detected in two phylogenetically related species from freshwater as well as from subaerial habitats, Watanabea reniformis Hanagata et al. and isolate UR7/5, which were phylogenetically independent of Prasiola and its closer allies. MAAs were absent in another Trebouxiophyceae clade comprising lichen photobionts (Coccomyxa pringsheimii Jaag) as well as freshwater picoplanktonic algae (Choricystis minor (Skuja) Fott). The data presented suggest a chemotaxonomic value of the 324 nm‐MAA in green algal taxonomy. To address the paraphyly of the genus Myrmecia Printz as presently circumscribed, we propose the new combination Lobosphaera incisa.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

Stenomitos terricola sp. nov. (Leptolyngbyaceae,Cyanobacteria) from the moist soil of Mt. Gwanggyo,Republic of Korea

Stenomitos terricola FBCC-A190 was collected from soils around the trees of Mt. Gwanggyo, located in Yeongtong-gu, Suwon-si, Gyeonggi-do. S. terricola FBCC-A190 is a thin and simple filament with a cell length that is longer than its width. It has a thin and firm sheath, exhibiting a blue-green color. Species belonging to genus Stenomitos is semi-cryptic species with slight morphological differences from each other. They were confirmed as Stenomitos species by analysis using 16S rRNA and 16S–23S ITS. A monophyletic cluster was formed with the previously reported genus Stenomitos, with 16S rRNA gene sequences sharing similarities of 95.9–97.9% except for S. pantisii TAU-MAC 4318. In addition, 16S–23S ITS gene sequencing showed tRNA^Ala, tRNA^Ile and V2, similar to the previously reported genus Stenomitos. From these results, Stenomitos terricola sp. nov. was proposed as a new species belonging to genus Stenomitos.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach

The genera Elliptochloris and Pseudochlorella were erected for Chlorella‐like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS‐2 Barcodes using the ITS‐2/CBC approach.     

Morphology,Phylogeny and Culture Characteristics of <i>Ganoderma gibbosum</i> Collected from Kunming,Yunnan Province,China

Ganoderma is a genus of medicinally and economically important mushrooms in the family Ganodermataceae. Ganoderma species are popular medicinal mushrooms and their health benefits are well-documented. Ganoderma is a cosmopolitan genus that is widely distributed in both tropical and temperate regions. This genus is characterized by its unique laccate or non-laccate species with double-walled basidiospores. Here, we report on eight collections of G. gibbosum collected during surveys in Kunming, Yunnan Province, China. The specimens are described and illustrated based on macro- and micro-morphological characteristics. Total DNA of the eight G. gibbosum strains were extracted using the Biospin Fungal Extraction Kit following manufacturer protocol. Amplification of the Internal Transcribed Spacer (nrITS) region was carried out using ITS5/ITS4 primers and LROR/LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU). Phylogenetic analysis with closely related species to G. gibbosum showed that all eight collections grouped with G. gibbosum with 100% bootstrap support. Phylogenetic similarity and morphological variations within the eight collections of G. gibbosum are discussed.     

A new species of the genus Trematosphaeria from China

A new species of the genus Trematosphaeria was isolated and identified from the soil in Jiuzhaigou Nature Reserve, southwestern China. The new taxon was studied based on morphological characters and phylogenetic analyses of three nuclear loci, i.e., the internal transcribed spacer rDNA operon (ITS), and large and small subunit nuclear ribosomal DNA (nuc28S rDNA, nuc18S rDNA). The analyzed Trematosphaeria species were clustered in several clades within the Pleosporales and the genus was shown to be polyphyletic. The new species is introduced as Trematosphaeria terricola, and it is characterized by short-papillate ascomata with suborbicular ostiolum, 3-septate, cinnamon-brown, fusiform ascospores with narrowly rounded ends, verruculose at maturity. Trematosphaeria terricola is compared with morphologically similar species T. confusa, T. mangrovis, and T. pertusa, which differ from the former species in the number of septa, and the shape and size of the ascospores with or without a sheath. This is the first report of a Trematosphaeria isolate from alpine soil in China.     

共球藻纲丝状藻中国新记录属种——洪泛拟寇丽藻

从甘肃省张掖市黑河流域分离了一株不分枝的丝状绿藻,对其进行纯培养并保藏于中国科学院淡水藻种库,编号FACHB 2451。形态观察表明,该藻株具有相对较长的营养细胞 (细胞长为宽的3~8倍)、双叶形的片状叶绿体、末端细胞顶端钝圆或略尖细等特征。结合形态比较和基于18S rDNA 基因序列的系统发育分析,确定该藻株为中国淡水共球藻纲新记录属种——洪泛拟寇丽藻 (Koliellopsis inundata Lokhorst)。分子系统发育研究结果表明,所分离的藻株与分离自比利时与荷兰边境农地的洪泛拟寇丽藻模式藻株序列相似度极高,且拟寇丽藻属与寇丽藻属、针丝藻属和拟针丝藻属等不分枝丝状拟寇丽藻科成员亲缘关系密切,可以较好地聚成一个亚支。     

Atypical non-lipid-dependent strains of <Emphasis Type="Italic">Malassezia furfur</Emphasis>

Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern — such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species level via their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to be M. furfur.     

BUILDING A SPECIES PHYLOGENY FOR THE GENUS COLEOCHAETE (COLEOCHAETALES) USING THE GENES RBCL AND ATPB

The freshwater green alga Coleochaete Breb. (Coleochaetaceae; Coleochaetales) is a key streptophyte genus and is important to the understanding of the evolutionary origin of embryophytes (land plants). To date only a few species have been available from public culture collections. To facilitate research on this genus we have isolated 17 previously uncultured species of Coloechaete from material collected in the United States, Puerto Rico, and the Dominican Republic. Sequences for the genes rbcL and atpB were determined for these new isolates of Coleochaete (and for existing cultures) and combined with sequences from representative other streptophytes. Phylogenetic analyses indicate that Coleochaete, along with Chaetosphaeridium and Chara, are closely related to embryophytes and constitute a ‘higher streptophyte’ clade. At least four well‐supported lineages exist within Coleochaete. Characteristic growth forms have been identified for these four lineages, with important characters including aspects of thallus establishment, thallus habit, zygote development and hair sheath position. These data provide an improved understanding of species diversity and character evolution in the genus Coleochaete, and facilitate examination of hypotheses concerning character evolution in the streptophytes.     

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera. The GenBank accession numbers for DNA sequences of the partial 16S rDNA with ITS region in each isolate determined in this study were: GP30 DQ003219; GP127 DQ003220; GP83 DQ003221; GP42, DQ003222; GP59 DQ003223; GP50 DQ003224; GP36 DQ003225; GP110 DQ003226; GP26 DQ003227; GP37 DQ003228; GP60 DQ003229; GP31 DQ003230; GP57 DQ003231; GP75 DQ003232; GP115 DQ003233; GP65 DQ003234; GP32 DQ003235; GP76 DQ003236; GP78 DQ003237.     

A beauvericin hot spot in the genus Isaria

Beauvericin is a naturally occurring cyclohexadepsipeptide originally described from Beauveria bassiana but also reported from several Fusarium species as well as members of the genus Isaria. Twenty-six isolates of Isaria species and its Cordyceps teleomorph, and ten taxonomically close strains including Beauveria, Nomuraea and Paecilomyces species were sequenced and tested for beauvericin production. Trees using ITS rDNA and β-tubulin sequence data were constructed and used to infer the phylogenetic distribution of beauvericin production. A group comprising Isaria tenuipes and its known teleomorph Cordyceps takaomontana, Isaria cicadae and its Cordyceps teleomorph, Isaria japonica and Isaria fumosorosea, showed positive beauvericin production which correlated well with combined ITS rDNA and β-tubulin phylogenies. The results suggested that beauvericin can serve as a chemotaxonomic marker for these limited species of the I. tenuipes complex.