Morphological and molecular characterization of two ITS groups of <Emphasis Type="Italic">Erysiphe</Emphasis> (Erysiphales) occurring on <Emphasis Type="Italic">Syringa</Emphasis> and <Emphasis Type="Italic">Ligustrum</Emphasis> (Oleaceae)

ITS sequences determined for 53 Erysiphe specimens on Syringa and Ligustrum collected in Europe, East Asia, and North and South America were divided into two ITS groups, S and K types. Phylogenetic analysis showed that these two ITS types do not share a common ancestor and form separate clades. The K type on Ligustrum was identified as Erysiphe ligustri based on the three-dimensional branching pattern of appendages. Morphological observations showed that there are some morphological differences—pigmentation of appendages and number of ascospores per ascus—between the S and K types on Syringa. Based on these morphological observations, the S and K types on Syringa were identified as E. syringae and E. syringae-japonicae, respectively. The recent abundant production of chasmothecia by lilac powdery mildew in Europe was caused by E. syringae-japonicae introduced from East Asia. DNA sequence analyses of the rDNA ITS region and the 28S rDNA, tub2, CYP51, and Chs1 genes did not support an interspecific hybrid origin for E. syringae-japonicae. Haplotype analysis suggested that E. syringae originated in North America and independently migrated to East Asia and Europe/South America.     

Teleomorph-anamorph relationships and reclassification of <Emphasis Type="Italic">Cordyceps cuboidea</Emphasis> and its allied species

Based on morphological characteristics and molecular phylogeny, we reclassified Cordyceps cuboidea and allied species C. alboperitheciata, C. prolifica, and Ophiocordyceps ryogamiensis. We investigated their teleomorph-anamorph relationships and revealed that these four species have Hirsutella-like anamorphs with morphological differences between them. By analyzing their molecular phylogeny, inferred from DNA sequences of internal transcribed spacer (ITS) and large subunit (LSU) D1/D2 region of rDNA, they were separated into four close-knit clades. Although C. prolifica and O. ryogamiensis formed their own clades, isolates of C. cuboidea separated into two clades, i.e., a true C. cuboidea clade and one resembling a new species, the O. paracuboidea clade. The latter two species are distinguished by the fruiting region of the stroma. In addition, C. alboperitheciata is regarded as a synonym of C. cuboidea. From the morphology, teleomorph-anamorph relationships, and molecular phylogeny, we concluded these species should be assigned to the genus Ophiocordyceps.     

Chromosome phylogeny of <Emphasis Type="Italic">Zamia</Emphasis> and <Emphasis Type="Italic">Ceratozamia</Emphasis> by means of Robertsonian changes detected by fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization (FISH) technique of rDNA

 Appearance and location of 45S rDNA and 5S rDNA signals were compared in chromosomes of nine species of the aneuploid Zamia and their taxonomically and phylogenetically closely related Ceratozamia mexicana. The 45S rDNA signal was detected in the proximal region of six chromosomes in Zamia angustifolia, Z. integrifolia, Z. pumila and Z. pygmaea (all 2n=16); in the proximal region of 6–14 chromosomes in Z. furfuracea, Z. loddigesii, Z. skinneri and Z. vazquezii (all 2n=18); and on the proximal region of 20 chromosomes in Z. muricata (2n=23). The 5S rDNA signals were commonly seen near the terminal region of the short arm of two metacentric chromosomes in the four species with 2n=16 and Z. furfuracea, Z. loddigesii and Z. vazquezii with 2n=18. Other 5S rDNA signals were seen near the terminal region of two terminal-centromeric chromosomes in Z. skinneri and near the terminal region of a metacentric and a telocentric chromosomes in Z. muricata. In contrast, those with 45S and 5S rDNA signals were exhibited in chromosomes of Ceratozamia mexicana in a different manner from those in the nine species of Zamia; the 45S rDNA signal in the terminal region of four metacentric and two submetacentric chromosomes and the 5S rDNA signal near the proximal region of two metacentric chromosomes. Received November 1, 1999 Accepted January 10, 2001     

The crustacean parasites <Emphasis Type="Italic">Ellobiopsis</Emphasis> Caullery, 1910 and <Emphasis Type="Italic">Thalassomyces</Emphasis> Niezabitowski, 1913 form a monophyletic divergent clade within the Alveolata

The Ellobiopsidae are enigmatic parasites of crustaceans that have been grouped together exclusively on the basis of morphological similarities. Ultrastructural studies have revealed their affiliation within the alveolates, which was confirmed by the phylogenetic analysis of the ribosomal RNA gene (SSU rDNA) sequences of two species of Thalassomyces Niezabitowski, 1913. However, their precise systematic position within this group remains unresolved, since they could not be definitively allied with any particular alveolate group. To better determine the systematic position of ellobiopsids by molecular phylogeny, we sequenced the SSU rDNA from the type-species of the Ellobiopsidae, Ellobiopsis chattoni Caullery, 1910. We found E. chattoni infecting various copepod hosts, Acartia clausi Giesbrecht, Centropages typicus Kröyer and Clausocalanus sp., in the Bay of Marseille, NW Mediterranean Sea, which allowed us to study several stages of the parasite development. A single unicellular multinucleate specimen provided two different sequences of the SSU rDNA gene, indicating the existence of polymorphism at this locus within single individuals. Ellobiopsis Caullery, 1910 and Thalassomyces formed a very divergent and well-supported clade in phylogenetic analyses. This clade appears to be more closely related to the dinoflagellates (including the Syndiniales/Marine Alveolate Group II and the Dinokaryota) and Marine Alveolate Group I than to the other alveolates (Ciliophora, Perkinsozoa and Apicomplexa).     

Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.     

A novel clade of sporocarp-forming species of glomeromycotan fungi in the <Emphasis Type="Italic">Diversisporales</Emphasis> lineage

In the early times of taxonomy of arbuscular mycorrhizal fungi (Glomeromycota), exclusively sporocarpic species were described. Since then the focus has mainly shifted to species forming spores singly. For many of the sporocarpic species, no molecular data have been made available, and their phylogenetic position has remained unclear. We obtained small subunit ribosomal rDNA and internal transcribed spacer data from specimens of glomeromycotan sporocarps from tropical areas that were assigned to three morphospecies. The complete sequence of the 18S small rDNA subunit sequence, internal transcribed spacers (ITS) 1 and 2 and 5.8S rDNA subunit, was determined from a sporocarp of Glomus fulvum. Partial sequences of the small subunit and the other regions were obtained from Glomus pulvinatum and the newly described species Glomus megalocarpum. Molecular phylogenetic analyses placed all species analyzed as a monophyletic sister group to the Diversispora spurca/Glomus versiforme clade group (“Glomus group C”) within the Diversisporales. The phylogenetic divergence from other known species suggests that this clade may constitute a new genus. These findings will have important consequences for taxon definition in the Diversisporales. They will facilitate identification of these fungi using rDNA sequences within colonized roots or the environment. Taxonomic novelties: Glomus megalocarpum D. Redecker     

Taxonomy and phylogeny of the xerophilic genus <Emphasis Type="Italic">Wallemia</Emphasis> (Wallemiomycetes and Wallemiales,cl. et ord. nov.)

The genus Wallemia comprises xerophilic species. Based on parenthesome ultrastructure it has been linked to the Filobasidiales (basidiomycetes). Species show a unique type of conidiogenesis, including basauxic development of fertile hyphae, segregation of conidial units more or less basipetally, and disarticulation of conidial units into mostly four arthrospore-like conidia. Wallemia is known from air, soil, dried food (causing spoilage), and salt. It can be isolated from hypersaline water of man-made salterns on different continents. Based on analyses of the nuclear small subunit ribosomal DNA (SSU rDNA) Wallemia has been placed into a highly supported clade together with Ustilaginomycetes and Hymenomycetes (Basidiomycota). Within this clade, it possesses an isolated position distantly related to the Filobasidiales and was characterized by numerous nucleotide substitutions not shared by any other fungus. Tests on xerotolerance indicated that Wallemia presents one of the most xerophilic fungal taxa. Xerotolerance is otherwise rare in the Basidiomycota. To acknowledge its unique morphology, evolution, and xerotolerance, a new basidiomycetous class Wallemiomycetes covering an order Wallemiales, is proposed. Based on differences in conidial size, xerotolerance, and sequence data of the rDNA internal transcribed spacer regions (ITS rDNA), at least three Wallemia species are segregated, identified as Wallemia ichthyophaga, Wallemia sebi, and Torula epizoa var. muriae, for which the combination Wallemia muriae is proposed. The three species are neotypified. Wallemia ichthyophaga differs from W. sebi and W. muriae in numerous nucleotides of the SSU and ITS rDNA. This high variation within Wallemia indicates existence of at least two cryptic genera not distinguishable by morphological characters.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

A new name in <Emphasis Type="Italic">Pavetta</Emphasis> L. (<Emphasis Type="Italic">Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.     

A monograph of <Emphasis Type="Italic">Octoknema</Emphasis> (Octoknemaceae — Olacaceae <Emphasis Type="Italic">s.l.</Emphasis>)

A revision of Octoknema Pierre is provided, based on morphological data gathered from a study of herbarium specimens and observations in the field. Fourteen species of Octoknema are recognised including six new species: O. bakossiensis Gosline & Malécot, O. belingensis Gosline & Malécot, O. chailluensis Malécot & Gosline, O. kivuensis Gosline & Malécot, O. mokoko Gosline & Malécot and O. ogoouensis Malécot & Gosline. Data are given for four additional poorly known taxa (Octoknema species A, B, C and D).     

Phylogeny of Chinese <Emphasis Type="Italic">Polystichum</Emphasis> (Dryopteridaceae) based on chloroplast DNA sequence data (<Emphasis Type="Italic">trnL-F</Emphasis> and <Emphasis Type="Italic">rps4-trnS</Emphasis>)

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Target site selection by the <Emphasis Type="Italic">mariner</Emphasis>-like element, <Emphasis Type="Italic">Mos1</Emphasis>

The eukaryotic transposon Mos1 is a class-II transposable element that moves using a “cut-and-paste” mechanism in which the transposase is the only protein factor required. The formation of the excision complex is well documented, but the integration step has so far received less investigation. Like all mariner-like elements, Mos1 was thought to integrate into a TA dinucleotide without displaying any other target selection preferences. We set out to synthesize what is currently known about Mos1 insertion sites, and to define the characteristics of Mos1 insertion sequences in vitro and in vivo. Statistical analysis can be used to identify the TA dinucleotides that are non-randomly targeted for transposon integration. In vitro, no specific feature determining target choice other than the requirement for a TA dinucleotide has been identified. In vivo, data were obtained from two previously reported integration hotspots: the bacterial cat gene and the Caenorhabditis elegans rDNA locus. Analysis of these insertion sites revealed a preference for TA dinucleotides that are included in TATA or TA × TA motifs, or located within AT-rich regions. Analysis of the physical properties of sequences obtained in vitro and in vivo do not help to explain Mos1 integration preferences, suggesting that other characteristics must be involved in Mos1 target choice.     

Numerical Taxonomy of <Emphasis Type="Italic">Vibrionaceae</Emphasis> Isolated from Cultured Amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S_SM and S_J coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S_SM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish. Received: 27 March 2002 / Accepted: 24 May 2002     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Out of Alaska: morphological diversity within the genus <Emphasis Type="Italic">Eurytemora</Emphasis> from its ancestral Alaskan range (Crustacea,Copepoda)

The copepod genus Eurytemora occupies a wide range of habitat types throughout the Northern Hemisphere, with among the broadest salinity ranges of any known copepod. The epicenter of diversity for this genus lies along coastal Alaska, where several species are endemic. Systematic analysis has been difficult, however, because of a tendency toward morphological stasis in this genus, despite large genetic divergences among populations and species. The goals of this study were to (1) analyze patterns of morphological variation and divergence within this genus, focusing on Eurytemora species that occur in North America, and (2) determine patterns of geographic and salinity distribution of Eurytemora species within the ancestral range in Alaska. We applied a comparative multivariate morphological analysis using 16–26 characters from 125 specimens from 20 newly collected sites in Alaska and 15 existing samples predominantly from North America. Results from principal component and hierarchical cluster analyses identified seven distinct morphological species of Eurytemora in North America (E. affinis, E. americana, E. canadensis, E. composita, E. herdmani, E. pacifica, and E. raboti), and identified diagnostic characters that distinguish the species (forming the basis for a new identification key). Several previously named species were regarded as synonyms. The sites we sampled in Alaska were remarkable in the high levels of sympatry of Eurytemora species, to a degree not seen outside of Alaska. Future studies of Eurytemora should shed light on patterns of habitat invasions and physiological evolution within the genus, and yield insights into mechanisms leading to its remarkably broad geographic and habitat range.     

Chemical composition and antimicrobial activity of <Emphasis Type="Italic">Thymus pannonicus</Emphasis> All. (<Emphasis Type="Italic">Lamiaceae</Emphasis>) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 μ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 μ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 μ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.     

A new peronosporomycete, <Emphasis Type="Italic">Halioticida noduliformans</Emphasis> gen. et sp. nov., isolated from white nodules in the abalone <Emphasis Type="Italic">Haliotis</Emphasis> spp. from Japan

Four strains belonging to the Peronosporomycetes (formerly Oomycetes) were isolated from white nodules found on the mantle of three species of abalone. In artificial seawater, the four isolates formed fragments such as in the genus Haliphthoros, but the protoplasm constriction was weaker, and fragments were longer, with smaller spaces between them, than those of Haliphthoros. The four strains form one or more discharge tubes from each zoosporangium. The four strains were similar, but not identical, to the genus Haliphthoros based on morphological characteristics. As a result, the four isolates were classified in a new genus and species, Halioticida noduliformans gen. et sp. nov. Phylogenetic analysis of the D1/D2 region of the large subunit ribosomal RNA gene (LSU rDNA) was performed, and the four isolates showed 100%–99.8% concordance. In the phylogenetic tree, the four isolates were not classified in the subclass Peronosporomycetidae, Saprolegniomycetidae, or Rhipidiomycetidae. However, the four isolates formed a new clade with genera Haliphthoros and Halocrusticida in Peronosporomycetes. Within this new clade, the four isolates, Haliphthoros spp. and Halocrusticida spp., were grouped in their respective independent subclades. These results showed that these were the new genus and species from the morphological characteristics.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

The neglected hypogeous fungus <Emphasis Type="Italic">Hydnotrya bailii</Emphasis> Soehner (1959) is a widespread sister taxon of <Emphasis Type="Italic">Hydnotrya tulasnei</Emphasis> (Berk.) Berk. &; Broome (1846)

The neglected false truffle species Hydnotrya bailii Soehner (Ascomycetes, Discinaceae) is re-described and separated from its sister taxon Hydnotrya tulasnei by morphological and phylogenetic analyses based on internal transcribed spacer rDNA sequences. The most distinct morphological and ecological characters are small globose, rather than kidney-like, ascomata as known from the sister taxon H. tulasnei, strictly monoseriate ascospores and montane habitats. Phylogenetic analyses resulted in two clearly separated clusters that revealed the ectomycorrhizal specificity of H. bailii to Picea abies and that H. tulasnei is preferably associated to Fagus sylvatica. We also show that H. bailii was already present in mycorrhizal samples but until now could not be correctly assigned. Our analyses also indicate cryptic diversity within H. cerebriformis and other, morphologically not yet characterized, Hydnotrya groups. An emended determination key for all Hydnotrya species known from Central Europe is provided.