Molecular phylogeny and morphology of <Emphasis Type="Italic">Ophiocordyceps unituberculata</Emphasis> sp. nov. (Ophiocordycipitaceae), a pathogen of caterpillars (Noctuidae,Lepidoptera) from Yunnan,China

Ophiocordyceps unituberculata, a new insect pathogenic fungus from southwestern China, is described using molecular phylogenetic and morphological data. This fungus differs from other Ophiocordyceps species by its enormously long monophialidic conidiogenous cells and a large periclinal protuberance often growing near the apex of conidiogenous cells, single conidia (lanceolate to fusiform) embedded in mucous sheath, and much larger conidial dimensions. Four-locus (nrSSU, nrLSU, tef-1α and rpb1) and ITS data phylogenetic analyses show that O. unituberculata belongs to the Hirsutella nodulosa clade within the genus Ophiocordyceps of Ophiocordycipitaceae and is a separate clade from other allied species. Molecular phylogeny and morphology both strongly support the distinctiveness of this taxon. The interspecific relationships in the H. nodulosa clade are discussed.     

Polycephalomyces lianzhouensis sp. nov., a new species,co-occurs with Ophiocordyceps crinalis

A new species from Guangdong Province in south China, Polycephalomyces lianzhouensis, is described and illustrated here; this species was found to co-occur with Ophiocordyceps crinalis. The phylogenetic position of this species was investigated according to morphological features and phylogenetic inferences based on the nrSSU, nrLSU, tef1, rpb1, and ITS gene sequences. Results demonstrated that P. lianzhouensis was highly associated with P. ramosopulvinatus but could be distinguished from the latter using the sequences of rpb1, nrLSU, and ITS gene and the features of much smaller stromata and perithecia and longer part-spores.     

Melampsora salicis-sinicae (Melampsoraceae,Pucciniales), a new rust fungus found on willows in China

Based on morphological characteristics and molecular phylogeny, a rust fungus on Salix hsinganica, S. sinica, S. starkeana, S. wallichiana and S. xerophila in China is described as a new species, Melampsora salicis-sinicae. This new species morphologically resembles M. capraearum and M. epiphylla in having subcuticular teliospores with a thickened apical wall, but it differs from these two species in its amphigenous telia. In addition, this new species differs from M. capraearum in its slender teliospores and thinner apical thickness of teliospores, and it also differs from M. epiphylla in the dimension of urediniospores, the position of teliospores and the apical thickness of teliospores. Phylogenetic analyses using partial sequences of the D1/D2 region of nuclear large subunit and the internal transcribed spacer (ITS1 and ITS2) regions including 5.8S of ribosomal RNA gene and the translation elongation factor 1 alpha (EF-1α) gene revealed that this rust fungus was distinct from its morphologically similar species, M. capraearum and M. epiphylla.     

On the reliability of DNA sequences of Ophiocordyceps sinensis in public databases

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5 %), 197 representing protein-coding genes (22.5 %), 92 representing random markers with unknown functions (10.5 %), and 30 representing microsatellite loci (3.5 %). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 β-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.     

New species and distinctive geographical divergences of the genus Sparassis (Basidiomycota): evidence from morphological and molecular data

Species of the genus Sparassis in East Asia were investigated using morphology and DNA sequences data. Phylogenetic analyses inferred from sequences of the internal transcribed spacer (ITS), the nuclear gene coding for the ribosomal large subunit (nLSU) and partial gene coding RNA polymerase subunit II (rpb2) strongly supported lineages corresponding to morphological features. Three taxa, S. subalpina, S. cystidiosa f. flabelliformis and S. latifolia were recognized from East Asia, and the former two taxa are new to science. The occurrence of S. latifolia in Japan and in the Russian Far East was confirmed. Geographical divergences of Sparassis in the Holarctic were observed. Most species have relatively narrow distribution ranges, and taxa with intercontinental distributions were not detected. Divergence of species in the Northern Hemisphere in different clades appears to have taken place at different times: the S. latifolia-crispa-radicata species complex appears to have undergone a recent radiation, while the S. subalpina-brevipes-spathularia species complex represents a relatively ancient speciation.     

Ophiocordyceps sessilis sp. nov., a new species of Ophiocordyceps on Camponotus ants in Japan

Ophiocordyceps sessilis, a new species of Ophiocordycipitaceae, Hypocreales, was found on Camponotus obscuripes ants, where the ants were primarily infected by another close relative, O. pulvinata. Morphological observation clearly indicated that O. sessilis and O. pulvinata are distinct species. Ophiocordyceps sessilis exhibited superficial development of perithecia and ascospore disarticulation into part-spores, not known in O. pulvinata. Nucleotide sequence data suggested that O. sessilis belongs in Ophiocordyceps, Ophiocordycipitaceae. Molecular data also indicated that O. sessilis is a close relative of O. cuboidea, O. prolifica, O. paracuboidea, and O. ryogamiensis, which are all known to produce part-spores. Since O. sessilis is always associated with ants infected by O. pulvinata, O. sessilis may be a hyperparasite of O. pulvinata.     

Sexual Reproduction as the Cause of Heat Resistance in the Food Spoilage Fungus Byssochlamys spectabilis (Anamorph Paecilomyces variotii)

Paecilomyces variotii is a common cosmopolitan species that is able to spoil various food- and feedstuffs and is frequently encountered in heat-treated products. However, isolates from heat-treated products rarely form ascospores. In this study we examined by using molecular techniques and mating tests whether this species can undergo a sexual cycle and form ascospores. The population structure of this species was examined by analyzing the nuclear ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S rRNA gene, as well as partial β-tubulin, actin, and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species. Partition homogeneity tests revealed that P. variotii has a recombining population structure. In addition to sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of Talaromyces spectabilis and that it has a biallelic heterothallic mating system. Since Paecilomyces sensu stricto anamorphs group within Byssochlamys, a new combination Byssochlamys spectabilis is proposed.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.     

Stachybotrys from soil in China, identified by morphology and molecular phylogeny

Four Stachybotrys strains were isolated from soil in China. One was identified as a novel species by morphological characters of phialides and conidia. It produced cylindrical conidia with irregular striations and smooth, hyaline conidiophores. Phylogenetic analysis of three DNA markers, the internal transcribed spacer region of rDNA (ITS1–5.8S–ITS2), the translation elongation factor 1 alpha (tef1) and RNA polymerase II subunit (rpb2), supported the morphological results. The correlation between morphological and molecular-based clustering demonstrated that the studied isolate was a new species. Two other isolates were identified as S. cf. elegans.     

Alpova komoviana (Boletales, Paxillaceae), a new sequestrate fungus from Montenegro, with a revised phylogeny of the genus in Europe

A new ectomycorrhizal species, Alpova komoviana, is described from several collections from Montenegro (south-eastern Europe), in association with Alnus incana ssp. incana (Betulaceae). Its interesting basal position in the strictly Alnus-associated Alpova lineage is discussed through morphology and phylogenetic analyses based on ITS, gpd and rpb2 nuclear DNA. ITS sequences of two other taxa are included in the analysis: Alpova rubescens and A. rubescens var. obscuratus; their identity and systematic positions are discussed.     

Salinomyces polonicus: A moderately halophilic kin of the most extremely halotolerant fungus Hortaea werneckii

A clade where the most halotolerant fungus in the world – Hortaea werneckii, belongs (hereafter referred to as Hortaea werneckii lineage) includes five species: Hortaea werneckii, H. thailandica, Stenella araguata, Eupenidiella venezuelensis, and Magnuscella marina, of which the first species attracts increasing attention of mycologists. The species diversity and phylogenetic relationships within this lineage are weakly known. In this study two moderately halophilic black yeast strains were isolated from brine of graduation tower in Poland. Molecular phylogenetic analyses based on the rDNA ITS1-5.8S-ITS2 (=ITS), rDNA 28S D1–D2 (=LSU), and RNA polymerase II (rpb2) sequences showed that the two strains belong to Hortaea werneckii lineage but cannot be assigned to any described taxa. Accordingly, a new genus and species, Salinomyces and Salinomyces polonicus, are described for this fungus. Furthermore, molecular phylogenetic analyses have revealed that Hortaea thailandica is more closely related to S. polonicus than to H. werneckii. A new combination Salinomyces thailandicus is proposed for this fungus.     

Characterization and Sequence Variation in the rDNA Region of Six Nematode Species of the Genus Longidorus (Nematoda)

Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.     

冬虫夏草菌3个细胞核蛋白编码基因的分子进化

【目的】分析3个细胞核蛋白编码基因(csp1、MAT1-1-1和MAT1-2-1)在不同冬虫夏草菌株间的分子进化。【方法】从125个冬虫夏草样品中分别扩增csp1、MAT1-1-1和MAT1-2-1基因序列,比较外显子和内含子间以及2个交配型基因间的序列变异程度,比较基于不同基因或基因区域所构建的系统发育树拓扑结构的差异,分析3个基因承受的选择压力和DNA重组情况。【结果】3个蛋白编码基因外显子区的长度在不同菌株间高度保守,具有4.5%?5.7%的变异位点;内含子区的长度在不同菌株间相同或不同,具有1.8%?22%的变异位点。对于2个交配型基因,MAT1-1-1的碱基变异率低于MAT1-2-1。基于外显子与内含子构建的系统发育树的拓扑结构,以及基于2个交配型基因外显子构建的系统发育树的拓扑结构都存在明显差异。3个蛋白编码基因都经历着净化选择作用。基因内部的不同DNA位点间有重组,但3个基因片段之间没有明显的重组发生。【结论】由于冬虫夏草菌不同基因以及基因的不同区域表现出进化上的差异,所以在开展冬虫夏草菌进化相关的研究时,应该联合使用多个不同的基因片段。     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

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.