Molecular identification of the mycorrhizal fungi of the epiparasitic plant Monotropastrum humile var. glaberrimum (Ericaceae)

Achlorophyllous monotropoid plants (Monotropoideae, Ericaceae) are epiparasites that obtain all of their carbon from their host plants via connections with mycorrhizal fungi. The mycorrhizal fungi of the epiparasitic monotropoid Monotropastrum humile var. glaberrima were identified based on mitochondrial, large ribosomal DNA sequences, and were compared with those of another variety, M. humile var. humile. The fungi that inhabit M. humile var. glaberrimum belong to the Thelephoraceae, whereas that of M. humile var. humile is a member of the Russulaceae. Two explanations are possible for this phenomenon: a misunderstanding of the taxonomic position of M. humile var. glaberrimum, or a change in the fungal partner within the Monotropastrum.     

秦岭植物分布3新记录属

报道了秦岭植物分布3新记录属:鹿蹄草科(Pyrolaceae)的沙晶兰属(Monotropastrum Andres)、列当科(Orobanchaceae)的藨寄生属(Gleadovia Gamble et Prain)和爵床科(Acanthaceae)的十万错属(Asystasia Blume)。相应的新记录种或变种为无毛沙晶兰[M.humile(D.Don)H.Hara var.glaberrimum H.Hara]、藨寄生(G.ruborum Gamble et Prain)和白接骨[A.neesiana(Wallich)Nees]。     

Plagiochila cucullifolia var. anomala var. nov. from Ecuador, with notes on discordant molecular and morphological variation in Plagiochila

Plagiochila cucullifolia Jack & Steph. var. anomala J. Heinrichs & Gradst. var. nov. is described and illustrated. The new variety is known from a single locality in southern Ecuador and differs from P. cucullifolia var. cucullifolia by the flat, not saccate leaves, somewhat smaller plant size and weaker leaf dentation. According to phylogenetic analyses of 35 nrDNA ITS1 and ITS2 sequences of Plagiochila, the two varieties of P. cucullifolia form a monophyletic lineage and are placed in a well supported clade together with five other species of Plagiochila sect. Hylacoetes: P. dimorpha Lindenb. & Gottsche var. ecuadorica (Inoue) J. Heinrichs, P. flabelliflora Steph., P. patriciae J. Heinrichs & H. Anton, P. macrostachya Lindenb. and P. turgida Herzog. Within Plagiochila, nrITS sequence variation is not concordant with morphological diversification. ITS sequences of Plagiochila cucullifolia s.str. and of P. dimorpha var. ecuadorica differ in only 23 aligned positions whereas two sequences of P. subplana Lindenb. differ in 86 aligned positions. Morphologically, P. cucullifolia s.str. and P. dimorpha var. ecuadorica differ in more than 20 characters and previously these two taxa were placed in separate genera. P. subplana phenotypes show considerable variation in leaf shape and dentation but the extremes are linked by numerous intermediates.     

Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan

We explored the diversity of mycorrhizal fungi associated with Monotropastrum humile in the central part of Japan's main island. We collected 103 M. humile individuals from 12 sites with various forest types. We analyzed the DNA sequences of the internal transcribed spacer region from fungal and plant nuclear ribosomal DNAs to assess the genetic diversity of the fungi associated with M. humile roots and to position the plant with respect to known Monotropoideae groups, respectively. The plants formed a monophyletic clade with other members of M. humile but were separated from M. humile var. glaberrimum and other monotropes (97% bootstrap support). Of the 50 fungal phylotypes, 49 had best matches with the Russulales, and the other had highest similarity with the Thelephoraceae. Our phylogenetic analysis suggests that M. humile roots have a highly specialized association with fungal partners in the Russulaceae. Moreover, a few fungal phylotypes from the M. humile roots had positions neighboring those from Monotropa uniflora roots. These results indicated that the genetic diversity of mycorrhizal fungi of M. humile was highly specific to the Russulaceae, but with high diversity within that family, and that the fungi associated with M. humile differ from those associated with M. uniflora.     

Molecular variation of Spiranthes sinensis (Orchidaceae) in Japan, with special reference to systematic treatment of seasonally differentiated groups and a dwarf form, f. gracilis, from Yakushima Island

Molecular variations of Spiranthes sinensis Ames var. australis (R.Br.) H. Hara et Kitam. ex Kitam. in Japan were examined to evaluate the validity of the seasonally differentiated groups and a dwarf form of the species, which is endemic to Yakushima Island, Japan. Sequence differences in the plastid trnL-F locus clearly distinguished Japanese S. sinensis var. australis from S. sinensis var. sinensis collected from Ryukyu. In contrast, the trnL-F sequence of S. sinensis var. australis from Sabah, Malaysia, clearly differed from that of Japanese S. sinensis var. australis, suggesting genetic heterogeneity of Spiranthes sinensis var. australis in Asia. Moreover, a molecular analysis based on the sequences of nuclear ITS1 regions indicated that there are two major groups of S. sinensis var. australis in Japan, with a geographic distribution boundary on Kyushu Island. However, the trnL-F and ITS1 sequences did not support the genetic differentiation of the seasonally differentiated groups or the dwarf form from the other Japanese individuals. Based on these molecular data, the systematic treatment of physiological and morphological variations in the Japanese population of S. sinensis. var. australis is discussed.     

Morphological and molecular phylogenetic analysis of <Emphasis Type="Italic">Melampsora</Emphasis> species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M. rostrupii was found from either morphological characteristics or sequence analysis.Contribution no. 185 Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

The phylogenetic relationships of Cynoglottis (Boraginaceae- Boragineae) inferred from ITS, 5.8S and trnL sequences

A molecular phylogenetic analysis of Cynoglottis was performed to evaluate previous hypotheses based on non-molecular evidence concerning the position of this genus within Boraginaceae tribe Boragineae. ITS-5.8S and trnL_UAA sequences from the nuclear and chloroplast non-coding genomes were obtained for four Cynoglottis taxa and selected members of the related genera Anchusa, Anchusella, Gastrocotyle, Brunnera and Pentaglottis. Cynoglottis is monophyletic, but neither trnL nor ITS support a close relationship with Brunnera, unlike previously supposed on morphological grounds. Brunnera is, instead, related to the southwestern European monotypic genus Pentaglottis, with which it forms a basal clade. ITS-5.8S sequences show that Anchusa thessala, a southeastern European annual species of Anchusa subg. Buglossellum, is sister to Cynoglottis and that the two taxa form a clade which also includes the Balkan endemic Gastrocotyle macedonica. Species of Anchusa subg. Anchusa form a separate lineage with high bootstrap support, suggesting that this heterogeneous genus is paraphyletic with respect to Cynoglottis. ITS sequences also discriminate between the Balkan-Apenninic diploid C. barrelieri and the Anatolian tetraploid C. chetikiana, albeit with low support. The molecular results are discussed in the light of karyological, morphological and chorological aspects.This work has been supported by M.I.U.R. 40% 2003 and the University of Firenze.     

Phylogeny of Bonatea (Orchidaceae: Habenariinae) based on molecular and morphological data

The genus Bonatea is widely distributed throughout southern and eastern Africa. Considerable debate surrounds the generic status of Bonatea, but there have been neither previous studies of evolutionary relationships among Bonatea species, nor any tests of the monophyly of the genus in relation to its close relative Habenaria. We investigated phylogenetic relationships between Bonatea and selected Habenaria species using morphology, as well as sequences of the chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. A fully resolved cladogram was obtained using morphological data, but neither the ITS, matK, nor combined data sets yielded well-resolved and well-supported phylogenetic structure for Bonatea. There is poor congruence between ITS and matK data for interspecific relationships in Bonatea, whilst the morphological results are largely congruent with the ITS analysis. Relative to Habenaria, there is little sequence variation between Bonatea species, which could indicate a recent and rapid radiation of Bonatea. Although the sampled Bonatea species form a distinct clade, more extensive sampling of Habenaria would be required to establish unambiguously whether or not Bonatea is monophyletic.     

Flowering time of two saprophytic plants,Monotropa uniflora L. andMonotropastrum humile (D. Don) Hara in Japan

The flowering time of two Japanese saprophytic species,Monotropastrum humile andMonotropa uniflora, was examined. The dependence of flowering time on assumed mean temperature is found to be different between the two species.Monotropa uniflora showed no relationship between assumed mean temperature and its flowering time, whileMonotropastrum humile showed a tight correlation.     

西畴重楼生物学特征补述及其系统位置

西畴重楼(Paris cronquistii var.xichouensis)为云南省特有物种,野外调查发现了广西(田林、那坡)的省级新分布,针对该新分布居群,观测并描述西畴重楼植物形态学特征,记录物候学特征,野外观测其地理分布特征,综上对其生物学特性进行补述,并结合前人基于细胞核ITS、叶绿体psbA-trnH和trnL-trnF等序列的分子系统学研究结果探讨其系统关系。结果表明:西畴重楼叶背面无紫色斑块,花瓣较宽而显著区别于凌云重楼(P.cromquistii var.cromquistii),但西畴重楼部分植株沿叶脉有紫色斑纹,表明与凌云重楼存在形态过渡,二者雄蕊数量均以3轮为主,而且变化范围一致;西畴重楼的物候期与同域分布的凌云重楼相近,同南重楼(P.vietnamensis)差异显著;西畴重楼多条序列的分子系统学研究显示西畴重楼和凌云重楼始终聚为一支,证实二者高度的亲缘关系。该研究结果完善了西畴重楼的生物学特性描述,首次报道了西畴重楼与凌云重楼形态特征存在过渡类型,物候学特征较一致,地理分布中心重叠,分子系统学位置相邻,仍支持李恒系统将西畴重楼作为凌云重楼的一变种。     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

     

A phylogenetic analysis of Pisum based on morphological characters, and allozyme and RAPD markers

Cladistic analyses of 17 wild and cultivated pea taxa were performed using morphological characters, and allozyme and RAPD (random amplified polymorphic DNA) markers. Both branch-and-bound and bootstrap searches produced cladograms that confirmed the close relationships among the wild species and cultivars of Pisum proposed by a variety of systematic studies. Intraspecific rankings were supported for northern P. humile, southern P. humile, P. elatius and P. sativum, which together comprise a single-species complex. P. fulvum, while clearly the most divergent of the pea taxa, could also be assigned to the same species complex without violating the hierarchial logic of the cladogram. Its inclusion or exclusion depends on whether the level of interfertility it displays with other pea taxa or its overall morphological and chromosomal distinction are emphasized. As suggested by previous studies, northern P. humile was the most likely sister taxon to cultivated P. sativum; although, rigorous phylogenetic evaluation revealed a close genealogical affinity among P. elatius, northern P. humile and P. sativum. Despite their limited number, the 16 morphological characters and allozyme markers used precisely organized the pea taxa into established taxonomic groupings, perhaps in part reflecting the role morphology has played historically in pea classification. The RAPD data also generally supported these same groupings and provided additional information regarding the relationships among the taxa. Given that RAPDs are relatively quick and easy to use, are refractory to many environmental influences, can be generated in large numbers, and can complement traditional characters that may be limited in availability, they provide a valuable new resource for phylogenetic studies.     

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.     

南美蟛蜞菊霜霉病病原鉴定及系统发育分析

[目的]为从天敌病原生物方面探索外来入侵植物南美蟛蜞菊的生物防治,对新发现的南美蟛蜞菊霜霉病进行病原鉴定和系统发育分析。[方法]在广东省广州市对南美蟛蜞菊霜霉病的发生及危害情况进行调查,并通过病害症状识别、病原显微形态记录与比较、病原菌及其近似种ITS序列结构比较、LSU序列和ITS序列系统发育分析,对南美蟛蜞菊霜霉病病原进行鉴定和系统发育分析。[结果]南美蟛蜞菊霜霉病在广州零星发生,但该病害在华南农业大学校园内发生较严重,发病率达50%~70%,病情指数为30~35。经鉴定,其病原菌为南美蟛蜞菊单轴霉,是国内一新记录种。基于病原菌LSU序列和ITS序列的系统发育分析显示,侵染菊科植物的单轴霉属菌种聚在一个分枝,亲缘关系密切,与侵染其他不同科寄主植物的单轴霉亲缘关系较远。ITS序列结构比较显示,寄生于菊科向日葵族植物的单轴霉属菌种的ITS2区包含多个重复序列,不同菌种间的ITS2区重复序列相似度不同,说明侵染菊科向日葵簇植物的单轴霉属菌物可细分成多个种,而不是只有向日葵单轴霉。[结论]广州发生的南美蟛蜞菊霜霉病是该寄主上首次正式报道的病害,鉴定的病原菌也是国内一新记录种;寄生在菊科植物上的单轴霉属种类不尽相同,但亲缘关系紧密。     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.     

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.     

Molecular taxonomy and ecology of Pseudallescheria, Petriella and Scedosporium prolificans (Microascaceae) containing opportunistic agents on humans

The main purpose of the present paper is to establish the connection between phylogenetic and morphological data and ecological features of strains of Pseudallescheria, Petriella, and Scedosporium. For the phylogenetic analysis sequences of the ITS region and the large subunit (partial sequences) of the rDNA were used. Cultural characteristics were observed on MEA 2 % and Weitzman-Silva Hutner Agar. Results showed, that three major groups could be differentiated, corresponding to Pseudallescheria, Petriella and S. prolificans. Among Petriella species only Pe. setifera is reasonably delimited. Pe. musispora was found to be synonymous with Pe. setifera. S. prolificans proved to be a homogenous species on the basis of ITS-sequences. Morphologically, Pseudallescheria and Petriella are distinguished by ostiolate vs non-ostiolate ascomata, a bipartition reflected also in ITS sequence data. We hypothesise a secondary loss of the ostiole of Pseudallescheria due to its ecological preferences. Infraspecific grouping within the highly variable species P. boydii is consistent for at least one clade in the ITS tree. The evolution of lineages with increased virulence within P. boydii is discussed.     

A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.