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     

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.     

Phylogeny and taxonomy of the Asian grapevine leaf rust fungus, <Emphasis Type="Italic">Phakopsora euvitis</Emphasis>, and its allies (Uredinales)

Heteroecious Phakopsora euvitis, P. vitis, and P. ampelopsidis, autoecious P. meliosmae, and an unconnected Aecidium on Meliosma are closely allied. A total of 45 collections representing the five rust fungi from Japan, Australia, and East Timor were subjected to molecular phylogenetic analyses: the D1/D2 region of nuclear large subunit rDNA and nuclear small subunit internal transcribed spacer 2 (ITS2) region including 5.8S rDNA were analyzed. Tree topologies generated from parsimony and distance methods of the D1/D2 and ITS2 sequences were similar. The 45 collections (44 for ITS2 and 33 for D1/D2, with 32 common for both analyses) are grouped into seven clades: P. ampelopsidis, P. vitis, unconnected Aecidium, P. euvitis from Japan, P. euvitis from Australia and East Timor, P. meliosmae on M. myriantha, and P. meliosmae on M. tenuis. The results confirm the phylogenetic distinctness of P. euvitis, P. ampelopsidis, and P. vitis distributed in Japan. A grapevine leaf rust fungus in Australia and East Timor has genetically diverged from P. euvitis in Japan. The unconnected Aecidium is highly likely to be an aecial anamorph of a Phakopsora fungus. Autoecious Phakopsora fungi on M. meliosmae and M. tenuis need further host-specificity and morphological studies to confirm their taxonomic status.     

托光柄菇属一中国新记录种

[背景] 托光柄菇属（Volvopluteus）隶属于光柄菇科（Pluteaceae）,目前世界上仅有4个种。[目的] 调查我国东北地区大型真菌资源。[方法] 采集大型真菌标本,对其形态进行详细的观察和描述,提取DNA,测定rDNA ITS序列,基于最大似然法和贝叶斯法构建系统发育树。[结果] 2019–2020年在吉林省延边朝鲜族自治州敦化市采集的标本中,有6份标本为密执安托光柄菇V.michiganensis,该种真菌此前未在我国发现。在系统发育分析中,采自我国的密执安托光柄菇V.michiganensis与该种的模式标本聚为一个分支。[结论] 密执安托光柄菇V.michiganensis为中国新记录种。     

Integrating a Numerical Taxonomic Method and Molecular Phylogeny for Species Delimitation of Melampsora Species (Melampsoraceae,Pucciniales) on Willows in China

The species in genus Melampsora are the causal agents of leaf rust diseases on willows in natural habitats and plantations. However, the classification and recognition of species diversity are challenging because morphological characteristics are scant and morphological variation in Melampsora on willows has not been thoroughly evaluated. Thus, the taxonomy of Melampsora species on willows remains confused, especially in China where 31 species were reported based on either European or Japanese taxonomic systems. To clarify the species boundaries of Melampsora species on willows in China, we tested two approaches for species delimitation inferred from morphological and molecular variations. Morphological species boundaries were determined based on numerical taxonomic analyses of morphological characteristics in the uredinial and telial stages by cluster analysis and one-way analysis of variance. Phylogenetic species boundaries were delineated based on the generalized mixed Yule-coalescent (GMYC) model analysis of the sequences of the internal transcribed spacer (ITS1 and ITS2) regions including the 5.8S and D1/D2 regions of the large nuclear subunit of the ribosomal RNA gene. Numerical taxonomic analyses of 14 morphological characteristics recognized in the uredinial-telial stages revealed 22 morphological species, whereas the GMYC results recovered 29 phylogenetic species. In total, 17 morphological species were in concordance with the phylogenetic species and 5 morphological species were in concordance with 12 phylogenetic species. Both the morphological and molecular data supported 14 morphological characteristics, including 5 newly recognized characteristics and 9 traditionally emphasized characteristics, as effective for the differentiation of Melampsora species on willows in China. Based on the concordance and discordance of the two species delimitation approaches, we concluded that integrative taxonomy by using both morphological and molecular variations was an effective approach for delimitating Melampsora species on willows in China.     

采自吉林省敦化市的2个蘑菇目(Agaricales)新记录种

【背景】在中国吉林省延边朝鲜族自治州敦化市寒葱岭红叶谷进行大型真菌资源调查的过程中发现了2个物种。【目的】确定这2个物种的分类地位。【方法】采集大型真菌标本,对其形态进行详细观察和描述,并提取DNA、测定rDNA ITS序列,基于最大似然法和贝叶斯法构建分子系统发育树。【结果】鉴定结果表明,这2个蘑菇目物种分别是径边刺毛鬼伞(Tulosesus callinus)和拉氏红菇(Russula lakhanpalii),在形态上与原始描述高度吻合,系统发育分析也验证了形态学的鉴定结果。【结论】拉氏红菇(R. lakhanpalii)系中国首次报道,径边刺毛鬼伞(T. callinus)为吉林省新记录种。     

Molecular Systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses

This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the Sordariales. Multiple gene sequences (partial 28 S rDNA, ITS/5.8 S rDNA and partial β-tubulin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to infer phylogenies. Phylogenetic analyses show that currently recognised Zopfiella species are polyphyletic. Based on sequence analyses and morphology, it appears that Zopfiella should be restricted to species having ascospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies. Our analyses show that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In addition, there are several strongly supported clades, characterised by species possessing divergent morphological characters. It is difficult to predict which characters are phylogenetically informative for delimiting these clades.     

A new species of Melampsora on Populus yunnanensis from China

Melampsora nujiangensis, a rust fungus found on Populus yunnanensis in China, is described as a new species. Light and scanning electron microscopy with herbarium specimens of the rust fungus show that the shape of its urediniospores differs from that of other known species of Melampsora, and its urediniospore walls are thinner than the other species. Furthermore, in phylogenetic trees based on the DNA sequences (28S and ITS) the rust fungus is phylogenetically separated by high bootstrap values. These results indicate that the fungus is an isolated species among the genus Melampsora. Contribution no. 202 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.     

Two new species of <Emphasis Type="Italic">Trichoderma</Emphasis> from Yunnan,China

Two new species of the fungal genus Trichoderma, Trichoderma compactum and Trichoderma yunnanense, isolated from rhizosphere of tobacco in Yunnan Province, China are described based on morphological characters and phylogenetic analyses of nucleotide sequences. Our DNA sequences included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1 and ITS2), and partial sequences of the translation elongation factor 1-alpha (tef1) and a fragment of the gene coding for endochitinase 42 (ech42). The analyses show that T. compactum belongs to the Harzianum clade, and T. yunnanense belongs to the Hamatum clade.     

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.     

Isolation of basidiomycetous anamorphic yeast-like fungus <Emphasis Type="Italic">Meira argovae</Emphasis> found on Japanese bamboo

A basidiomycetous anamorphic yeast-like fungus, isolated from new bamboo shoots collected in Japan, was assigned to Meira argovae by comparison of conidial morphology, physiological characteristics, rDNA sequences, and DNA-DNA relatedness with the ex-type strains of Meira species. This is the first record of the finding of M. argovae from other than mite cadavers and in regions other than Israel. Phylogenetic analysis based on the D1-D2 domain demonstrated that Meira species and teleomorphic Dicellomyces species, which include a bamboo leaf parasite, D. gloeosporus, formed sister clades.     

Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?

Genetic, reproductive and morphological variation were studied in 193 global strains of the marine diatom species Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle to assess potential intraspecific variation and biogeographic distribution patterns. Genetic differentiation between allo- and sympatric strains was investigated using the ITS1–5.8S–ITS2 rDNA region. Three ITS clades were found. Clones of opposite mating type were sexually compatible within clades I or II, and viable F1 hybrid offspring were produced in crosses between them. The molecular differences between these clades were correlated with slight but consistent morphological differences. At present, nothing can be said about morphology and mating behavior for clade III clones because only ITS data were available. The three ITS clades showed different geographic distributions. Clade II was restricted to the NE Pacific, whereas clones belonging to clade III originated from geographically widely separated areas (Vietnam, China and Mexico). ITS clade I was recovered in all locations studied: the North Sea (Belgium, The Netherlands, France), the eastern and western N Atlantic (Spain, Canada), the NW and S Pacific (Japan, New Zealand) and the NE Pacific (Washington State). Clade I thus appears to be globally distributed in temperate coastal areas and provides the first strong evidence to date for the global distribution of a biologically, genetically and morphologically defined diatom species.     

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus <Emphasis Type="Italic">Phormidium</Emphasis> (Oscillatoriales) from different habitats and geographical sites

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.     

Morphological and ITS1, 5.8S,and Partial ITS2 Ribosomal DNA Sequence Distinctions Between Two Species <Emphasis Type="Italic">Playtygyra</Emphasis> (Cnidaria: Scleractinia) from Hong Kong

Two sympatric species of Platygyra have been identified from Hong Kong waters: i.e., P. sinensis and P. pini. The former has been further subdivided into 4 morphotypes based on colony growth form as follows: classic, encrusting, hillocky, and long-valley. Taxonomic confusion raised by overlapping morphological variations and frequent sympatric occurrences, however, has posed problems in relation to Platygyra ecology and population dynamics. This study attempted to differentiate Platygyra pini and morphotypes of P. sinensis by both morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence analysis. Morphological data based on 9 skeletal characters were subjected to multivariate analysis. No clear groupings were obtained using a multidimensional scaling plot. Most parsimony analysis was conducted using either the rDNA data set including ITS1, 5.8S, and partial ITS2 or the ITS1 region only. Maximum parsimony (MP) and neighbor-joining (NJ) trees obtained from both data sets, clustered samples of P. sinensis and P. pini into 2 clades. The interspecific Kimura 2-parameter sequence divergence value (k2) obtained by the former rDNA data set was 14.275 ± 0.507%, which is greater than the intraspecific values (1.239 ± 1.147% for P. sinensis and 0.469 ± 0.364% for P. pini), indicating that this marker of ITS1, 5.8S, and ITS2 contains substantially high levels of inherent diversity and is useful in resolving the problematic taxonomy of Platygyra.     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

中国菊科一新种——香紫菀北大核心CSCD

该文描述了在中国福建北部山区茫荡山发现的菊科(Compositae)紫菀属(Aster L.)一新种——香紫菀(A.artemisiaodorum X.X.Su,S.P.Chen&L.Ma sp.nov.)。基于2个核基因(ITS、ETS)和1个叶绿体基因(trn L-F)片段构建的系统发育树显示,香紫菀具有独立性,与白舌紫菀[A.baccharoides(Benth.)Steetz]互为姐妹类群,但该种形态与白舌紫菀相差较大,其全部叶两面无毛、总苞筒状、头状花序1.0~1.8 cm、舌状花5~8朵以及全株具芳香气味等特征。形态特征分析与系统发育分析均支持该新种成立。模式标本现存于福建农林大学标本馆。