Paphiopedilum× yingjiangense,a New Hybrid of Orchidaceae from China

Paphiopedilum× yingjiangense Z . J . Liu et S . C . Chen , a new hybrid from China, is described and illustrated. This entity may be a natural hybrid between Paphiopedilum villosum and P . wardii . It differs from the former by having obscurely deep and light green nets on upper surface of leaves, a pale yellow-green flower with fine dark purple spots on petals and lower part of dorsal sepal and broadly ovate-orbicular staminode without a central glossy knob; from the latter by longer leaves not obviously mottled pale and dark bluish green above nor densely spotted purple below, a much larger flower, and a broadly ovate-orbicular staminode .     

盈江兜兰, 中国兰科一新杂种

对我国兰科新杂种盈江兜兰( ^{Paphiopedilum}× yingjiangense) 进行了描述和绘图。该植物可能是紫毛兜兰( P. villosum) 与彩云兜兰( P. wardii ) 之间的天然杂种。它与前者的区别在于叶上面有轻微的深浅绿色网斑, 花淡黄绿色并在花瓣上和中萼片下半部有黑紫色细斑点, 以及退化雄蕊宽卵圆形, 其中央不具脐状突起; 与后者的区别在于叶较长, 上面不具明显的深浅蓝绿色斑, 背面亦无密集的紫色斑点, 花明显较大, 以及退化雄蕊为宽卵圆形。     

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Polyphasic characterization of Plectosphaerella oligotrophica,a new oligotrophic species from China

Oligotrophic fungi can grow on carbon-free media. Most of the currently known oligotrophic fungi were reported from soil. Plectosphaerella oligotrophica sp. nov. was recently isolated from soil in China using a low carbon medium. The new species is morphologically characterized by pale yellow colonies on PDA, phialidic conidiogenous cells, and hyaline, ellipsoidal and 0–1-septate conidia. Phylogenetic analysis based on internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences confirmed the placement of P. oligotrophica as a new species in the genus Plectosphaerella. The new species is compared to the morphologically and phylogenetically closely related species. Beta-tubulin fragments from the new species were also sequenced and deposited in GenBank. A key to the currently accepted species of the genus is provided.     

翡翠兜兰,中国云南兰科一新种

对兰科新种翡翠兜兰(Paphiopedilum smaragdinum)作了描述与绘图。新种产云南西部高黎贡山,与虎斑兜兰有亲缘关系,但花淡黄绿色无斑点和斑纹,退化雄蕊矩圆形,先端有短尖,短尖长1～1．5mm,易于区别。     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

Secondary structure model for the ITS-2 precursor rRNA of strongyloid nematodes of equids: implications for phylogenetic inference

In order to maximise the positional homology in the primary sequence alignment of the second internal transcribed spacer for 30 species of equine strongyloid nematodes, the secondary structures of the precursor ribosomal RNA were predicted using an approach combining an energy minimisation method and comparative sequence analysis. The results indicated that a common secondary structure model of the second internal transcribed spacer of these nematodes was maintained, despite significant interspecific differences (2–56%) in primary sequences. The secondary structure model was then used to refine the primary second internal transcribed spacer sequence alignment. The “manual” and “structure” alignments were both subjected to phylogenetic analysis using three different tree-building methods to compare the effect of using different sequence alignments on phylogenetic inference. The topologies of the phylogenetic trees inferred from the manual second internal transcribed spacer alignment were usually different to those derived from the structure second internal transcribed spacer alignment. The results suggested that the positional homology in the second internal transcribed spacer primary sequence alignment was maximised when the secondary structure model was taken into consideration.     

Multilocus phylogenetic analyses, pullulan production and xylanase activity of tropical isolates of Aureobasidium pullulans

Aureobasidium pullulans is the source of the commercially valuable polysaccharide pullulan and the enzyme xylanase. Isolates are typically off-white to pale pink or black on solid media, while some tropical isolates have been described as ‘color variants’ with bright pigments of red, yellow or purple. We sequenced 5 loci (internal transcribed spacer, intergenic spacer 1, translation elongation factor-1 alpha, beta tubulin, and RNA polymerase II) from 45 new isolates from Thailand. Based on the phylogenetic analyses, isolates were classified into 12 clades. Each clade showed different colors on different culture media including two clades with ‘color variants’ and some clades exhibited high levels of pullulan production or xylanase activity. Colony characteristics do not correlate perfectly with DNA sequence phylogeny or the physiological characters, but DNA sequence differences rapidly identify isolates with genetic novelty.     

Characterisation of Ascaris from human and pig hosts by nuclear ribosomal DNA sequences.

The sequences of the nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S rRNA gene and the second internal transcribed spacer were determined for Ascaris samples from pigs and humans from different geographical regions. The sequences of the 5.8S gene and the second internal transcribed spacer were the same for all samples examined, whereas all Ascaris samples from humans had six (1.3%) nucleotide differences in the first internal transcribed spacer compared with those from pigs. These differences provided some support for the existence of separate species of Ascaris or population variation within this genus. Using a nucleotide difference within a site for the restriction enzyme HaeIII, a PCR-linked restriction fragment length polymorphism method was established which allowed the delineation of the Ascaris samples from pigs and humans used herein. Exploiting the sequence differences in the first internal transcribed spacer, a PCR-based single-strand conformation polymorphism method was established for future analysis of the genetic structure of pig and human Ascaris populations in sympatric and allopatric zones.     

A phylogeny of the ITS and ETS for Montanoa (Asteraceae: Heliantheae)

A phylogeny of the genus Montanoa based on the internal transcribed spacer (ITS) and the external transcribed spacer (ETS) is presented. Each of the two clades revealed by the Bayesian and parsimony analyses has approximately half of the number of species in the genus. One lineage is composed mostly of central and southern Mexican species whereas the other lineage contains those species endemic to Mesoamerica and South America. The molecular phylogeny is compared to previous phylogenetic hypotheses based on morphological characters. Key features in the structure of the capitulum such as pale morphology, heavily used in the past to construct hypotheses of relationship within the genus, are viewed as of minimal value to circumscribe natural groups. The relationships of Montanoa to other genera in the Heliantheae are briefly discussed.     

根茎兜兰——缅甸兰科一新种

基于采自缅甸北部的栽培植物,对兰科新种根茎兜兰（Paphiopedilum rhizomatosum)作了描述和绘图。此新种具长8－10cm、粗8－12mm的根状茎与较坚硬的根;与我们所知的种类有极明显的不同。它在花的色泽与形态方面与小叶兜兰（P.barbigerum)相近,区别点还在于叶要大得多,宽达2.5-3.5cm,退化雄蕊先端近截形并有短尖头。     

Mycorrhizal specificity, preference, and plasticity of six slipper orchids from South Western China

Mycorrhizal fungi of six endangered species, Paphiopedilum micranthum, Paphiopedilum armeniacum, Paphiopedilum dianthum, Cypripedium flavum, Cypripedium guttatum, and Cypripedium tibeticum, from two closely related genera in the Orchidaceae from Southwestern China, were characterized using the nuclear internal transcribed spacer (ITS) and part of the large subunit gene of mitochondrial rDNA (mtLSU) sequences. The most frequently detected fungi belonged to the Tulasnellaceae. These fungi were represented by 25 ITS sequence types and clustered into seven major clades in the phylogenetic analysis of 5.8S sequences. Species of Paphiopedilum and Cypripedium shared no fungal ITS sequence types in common, but their fungal taxa sometimes occurred in the same major clade of the 5.8S phylogenetic tree. Although it had several associated fungal ITS sequence types in a studied plot, each orchid species had in general only a single dominant type. The fungal sequence type spectra of different species of Paphiopedilum from similar habitats sometimes overlapped; however, the dominant sequence types differed among the species and so did the sequence-type spectra within Cypripedium. Orchids of P. micranthum and P. armeniacum transplanted from the field and grown in two greenhouses had a greater number of mycorrhizal associations than those sampled directly from the field. Root specimens from P. micranthum taken from the greenhouses were preferably associated with mycobionts of the Tulasnella calospora complex, while those from the field had mycorrhizal associations of other tulasnelloid taxa. Such plasticity in mycorrhizal associations makes ex situ conservation or even propagation by means of mycorrhization of axenically grown seedlings possible.     

Endophytic Phomopsis species: host range and implications for diversity estimates

Foliar endophyte assemblages of teak trees growing in dry deciduous and moist deciduous forests of Nilgiri Biosphere Reserve were compared. A species of Phomopsis dominated the endophyte assemblages of teak, irrespective of the location of the host trees. Internal transcribed spacer sequence analysis of 11 different Phomopsis isolates (ten from teak and one from Cassia fistula) showed that they fall into two groups, which are separated by a relatively long branch that is strongly supported. The results showed that this fungus is not host restricted and that it continues to survive as a saprotroph in teak leaf, possibly by exploiting senescent leaves as well as the litter. Although the endophyte assemblage of a teak tree growing about 500 km from the forests was also dominated by a Phomopsis sp., it separated into a different group based on internal transcribed spacer sequence analysis. Our results with an endophytic Phomopsis sp. reinforce the earlier conclusions reached by others for pathogenic Phomopsis sp., i.e., that this fungus is not host specific, and the species concept of Phomopsis needs to be redefined.     

Ribosomal DNA evidence and disjunctions of western American Portulacaceae

Phylogenetic analysis of ribosomal DNA internal transcribed spacer sequences from 35 members of western American Portulacaceae plus seven Portulacaceae outgroups generally supports morphologically based interpretations of multiple intercontinental disjunctions. The data neither support nor refute monophyly of the western American group but strongly support a group comprising the western American taxa plus Phemeranthus, the only strictly American genus of the morphology-based eastern American/African group of Portulacaceae, along with the Australian genus Parakeelya. Support is strong for the monophyly of Calandrinia, Montiopsis, Lewisia, Claytonia, and Montia, along with a sister relationship of the last two. The data neither strongly support nor refute the morphologically based diagnosis of Cistanthe, but they strongly support a clade including the North American Cistanthe section Calyptridium and the South American Cistanthe sections Amarantoideae and Philippiamra. The internal transcribed spacer data fail to resolve the phylogenetic relationships among most of the western American lineages, suggesting either rapid radiation or, alternatively, erratic evolution of the internal transcribed spacer. The internal transcribed spacer and morphological evidence together suggest that in this group there have been 8-13 dispersal and colonization events across >2000 km (1 for every 15-26 extant species in this group). The internal transcribed spacer data document complex molecular evolutionary patterns, including strong substitution biases, among-site rate heterogeneity, positional bias for deamination-type substitutions, nonstationarity, and variable rates of insertion/deletion. Our phylogenetic conclusions, however, do not appear to be sensitive to these patterns.     

ITS2 sequences of Dictyocaulus species from cattle, roe deer and moose in Sweden: molecular evidence for a new species.

Total DNA was isolated from adult lungworms of the genus Dictyocaulus, collected from cattle, moose (Alces alces) and roe deer (Capreolus capreolus) in Sweden. The second ribosomal internal transcribed spacer was amplified with PCR, and DNA sequences were determined from nine individual worms that all came from different hosts in order to avoid analysis of siblings. The sequence data obtained were aligned and compared with similar data derived from German lungworm isolates from cattle and fallow deer (Cervus dama). These analyses clearly showed that specimens of the cattle lungworm, Dictyocaulus viviparus, were almost identical irrespective of their geographical origin. However, when the second internal transcribed spacer sequence of D. viviparus was compared with that of lungworms from moose and roe deer, major differences were noticed. Although lungworms collected from these cervids had identical second internal transcribed spacer sequences, they proved to be genetically different from Dictyocaulus eckerti of German fallow deer, displaying a 66.5% similarity. In an evolutionary tree, inferred by maximum likelihood analysis, the Dictyocaulus species from cattle and wild cervids clustered as compared with Dictyocaulus filaria from sheep. The study has thus demonstrated that A. alces and C. capreolus in Sweden are parasitised with a Dictyocaulus species that is different from D. viviparus and D. eckerti, indicating that we are dealing with a new species in moose and roe deer.     

Morphology evolution and molecular phylogeny of Pestalotiopsis (Coelomycetes) based on ITS2 secondary structure

The internal transcribed spacer 2 (ITS2) located between the 5.8S and 28S genes of the nuclear ribosomal gene cistron is conserved at the level of secondary structure rather than primary sequence. Within the fungal genus Pestalotiopsis, there were two types of ITS2 sequence patterns, and hence secondary structures, which were supported by high bootstrap values in phylogenies based on the ProfDist distance and Profile neighbor-joining algorithms. Pestalotiopsis consists of two groups that differ in color intensity of the spore as measured by optical density (OD) in three median cells of conidia comprised of five cells with one basal and two to four apical appendages. OD was quantified using a novel method with the publicly available software, Image J. OD values of species within one clade were high (dark OD >0.6), while OD values of species in the other clade were low (pale OD <0.6). However, knobbed-tipped appendages, which have been used to classify species of Pestalotiopsis, were observed in both clades. In the dark clade, knobbed-tipped appendage strains aggregated in one subclade, but in the pale group, these strains did not aggregate.     

Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates

The evolutionary conservation and versatility of a new set of nuclear primers for the amplification of the ribosomal internal transcribed spacer regions in insects and other invertebrates have been studied. These primers, conserved across Insecta and other invertebrates, are based on a comprehensive taxonomic survey of the current DNA sequence databases. Their versatility was demonstrated by extensive polymerase chain reaction assays in 16 species from two arachnid orders, eight insect orders, three invertebrate and vertebrate chordate orders and by direct sequencing of the amplified products. The availability of these primers to the insect research community should facilitate the use of internal transcribed spacer regions in intraspecific studies as well as phylogenetic analysis of closely related taxa.     

Fungal Olecranon Bursitis in an Immunocompetent Patient by <Emphasis Type="Italic">Knoxdaviesia dimorphospora</Emphasis> sp. nov.: Case Report and Review

Bursitis is a common medical condition that can occur either with or without infection. We present a case of fungal olecranon bursitis in an immunocompetent individual caused by the new species Knoxdaviesia dimorphospora. It is a dematiaceous filamentous fungus characterized by the production of two different conidia: hyaline and cylindrical, which rise up from phialidic conidiogenous cells located in the upper part of differentiated and unbranched conidiophores, and pale brown and ellipsoidal conidia produced by phialidic conidiogenous cells which are born directly on hyphae. In addition to its morphological peculiarities, the novelty of the fungus was confirmed by sequence analysis of the internal transcribed spacer (ITS) regions and D1/D2 domains of the 28S of the nuclear rRNA gene. The fungal infection was confirmed by cytological examination and repeated cultures. The infection was resolved by surgical debridement and drainage, and the patient presented a complete functional recovery 3 months later. The in vitro antifungal susceptibility to this new human opportunist is provided, terbinafine being the drug with the most potent activity.     

Internal transcribed spacer rDNA can be used to infer the phylogenetic relationships of species within the genus Nematodirus (Nematoda: molineoidea)

Sequences of the first internal transcribed spacer rDNA were characterised for four veterinary important species of gastrointestinal nematodes from the genus Nematodirus. The sequence data were combined with previously published data of the second internal transcribed spacer to determine whether these rDNA regions provided a suitable number of informative characters to determine the phylogenetic relationships of species within the genus. A total of 32 alignment positions of the first internal transcribed spacer data set and 33 characters from the second internal transcribed spacer data set were informative in phylogenetic analyses. Irrespective of whether the data from each spacer were analysed separately or combined, only one most parsimonious tree was produced, with the relationships of the four species fully resolved. In addition, several regions of conservatism in the first internal transcribed spacer sequence among the four Nematodirus species suggests that this rDNA region may also provide phylogenetic information for higher taxonomic levels within the Molineoidea.