Species Diversity Of Soil Dematiaceous Hyphomycetes From Yangtse River And Yellow River Source Area Of China

Soil fungi are those which have some parts or whole life cycle either in or directly associated with the soil environment. They play an important role in the soil ecosystem. The species diversity of fungi are the most abundant in soil, of which dematiaceous Hyphomycetes account for more than one half. The Yangtse River and Yellow River source area lie in the hinterland of the Qinghai-Tibetan Plateau. The natural environment is extremely harsh and the mailly show in two aspects: one is high altitude, low temperature and drought, the other is low vegetation and eco-efficiency. Its special geographical conditions breeds an extremely an fragile state and low conversion cold-arid ecosystems. Special ecological environment in these area gives a special species diversity of soil fungi. This study is a part of a whole set of diversity survey of dematiaceous Hyphomycetes in soil of China. It was aimed at investigating the species diversity and distribution of soil dematiaceous hyphomycetes in Yangtse River and Yellow River source area to enrich the biological diversity of China and lay a useful foundation for the development and utilization of fungi resources.The classification of mitosporic fungi have been maily based on morphology and lack of molecular information supporting. The morphological classification of mitosporic fungi are according to the cinidiomata type (hyphal, synnematal, sporodachial, pycnidial, etc.), conidial groups (amerosporae, didymosporae, phragmosporae, dictyosporae, scolicosporae, helicosporae and staurosporae) and formation pattern of conidia. Based on the 32 genera in the paper, phylogenetic relationships were studied to provide molecular support to taxonomic criteria at genus level of mitosporic fungi. Molecular systematics on represented species of Doratomyces, Trichurus and Graphium were also carried out.A total 206 soil samples were collected from the Yangtse River and Yellow River source area, including grassland, desert, gobi, alpine meadow, forest, wetland, farmland,wasteland eight ecosystem types from 2006 to 2007. From these samples, 700 fungi isolates of dematiaceous Hyphomycetes were isolated by using the soil plate and dilution plates methods. From them, 92 species in 35 genera were identified. Among them, 9 species in 3 genera are Stilbellales, 8 species in 2 genera belong to Tuberculariales and 74 species in 30 genera are Hyphomycetales, of which 17 are new species, 8 species are new records to China and 1 new combination. All genera and species are fully described and illustrated morphologically. Distribution sites and habitat are also cited. Dried and living cultures of all the fungi studied have been deposited in the Herbarium of Shandong Agricultural University: Plant Pathology (HSAUP).1 Species diversity of dematiaceous HyphomycetesNew species: Doratomyces globosae H.Q. Pan & T.Y. Zhang, Myrothecium advena var. terrum H.Q. Pan & T.Y. Zhang, Cirrenalia qinghaiensis H.Q. Pan & T.Y. Zhang, Monodictys macrosporum H.Q. Pan & T.Y. Zhang, Monodictys huangheensis H.Q. Pan & T.Y. Zhang, Oidiodendron tshawytschae H.Q. Pan & T.Y. Zhang, Pleurophragmium nanus H.Q. Pan & T.Y. Zhang, Rhinocladiella variabile H.Q. Pan & T.Y. Zhang, Scolecobasidium chinensis H.Q. Pan & T.Y. Zhang, Scolecobasidium laevis H.Q. Pan & T.Y. Zhang, Scolecobasidium longisporum H.Q. Pan & T.Y. Zhang, Scopulariopsis humicola H.Q. Pan & T.Y. Zhang, Scopulariopsis penicillin H.Q. Pan & T.Y. Zhang, Scopulariopsis verruculosum H.Q. Pan & T.Y. Zhang, Stachybotrys longisporum H.Q. Pan & T.Y. Zhang, Trichocladium microsporium H.Q. Pan & T.Y. Zhang, Veronaea gobica H.Q. Pan & T.Y. Zhang, Veronaea latispora H.Q. Pan & T.Y. Zhang.New records to China: Acroconidiella eschscholtziae(Harkn.) M.B. Ellis, Botrytis byssoidea Walker, Gliomastix inflate (C.H. Dickinson) W. Gams, Scolecobasidium dendroides Pirozynski & Hodges, Scopulariopsis hibernica A. Mangan, Veronaea coprophila (Subram. & Lodha) M.B. Ellis, Wardomyces anomalus Brooks & Hansf., Wardomyces humicola Hennebert & G.L. Barron.There are also 7 undeterminate species: Acremonium sp., Cephaliophora sp., Cladosporium sp., Scopulariopsis sp.1,Scopulariopsis sp.2, Verticillium sp., Wardomyces sp..The other 57 species have been known to China.Richness, Berger-Parker dominance index, Shannon-Wiener diversity index, evenness, niche breadth and niche overlap were used to analyze the community structure and eco-niche of soil dematiaceous hyphomycetes in different ecosystem-types.The results show that the amount and species distribution of soil dematiaceous hyphomycetes had an obvious variability in different ecosystem-types, and that the dominant genus species varied in the eight ecosystem-types studied. Species diversity was closely related to vegetation. The species diversity of soil dematiaceous hyphomycetes in forest and grassland was much higher than those in gobi and desert. The species of soil dematiaceous hyphomycetes in Yangtse River and Yellow River source area were less compared with these area in low altitude. There were rich of soil dematiaceous hyphomycetes with smaller conidia and lack of helminthosporioid fungi.2. The experience and improvement on taxonomic theory and methodsAssociated with identification work undertaken, the researcher paid great attention to the basal taxonomic theory at generic and species ranks of dematiaceous hyphomycetes and the improvement the fungal isolation and culture methods.(ⅰ) Taxonomic criteria at genus levelThe patterns of conidia formation and conidiophores (conidiogenous cells) proliferation are the main criteria used to separat diferent genera in dematiaceous Hyphomycetes. The conidiogensis patterns of almost all fungal genera isolated from soil in this study are holoblastic or enteroblastic. But the proliferation patterns of conidiophores (conidiogenous cells) are various, for example sympodial, phialidic, annellidic etc.. The morphological characters of conidiogenous cells and conidiophores, patterns of conidia germination and the order of septum (distoseptum) ontogeny are very important in differentiate those genera whose patterns of conidia formation and conidiophores proliferation are the same. The characters, color and growth traits of colonies are useful assistant characteristics to differentiate some genera while absence or presence of setae is also helpful to differentiate a few genera.(ⅱ) Taxonomic criteria at species levelThe morphological characters of conidia, especially the shape, size, color, septum and ornamentation etc. are important characters in identificating species. Aiming to reducing the influence of environment, all species in one genus should be cultured at the same or being close to the same conditions. The conidia shape and size of some species in some genera are very easily influenced by environment factors, it should be considered that the culture conditions used by the origional author(s) should be followed as closely as possible. The characters of conidiophores and conidia are also very useful in identificating species in some genera. Characters of setae may be used to separat species in a few genera, such as Trichurus Clements & Shear and Myrothecium Tode. Molecular methods are also used in identificate the species with less differences between each other in one genus.(ⅲ) The improvements of isolateting and culture methodsThe mainly used culture media are PDA, PCA, CMA and TWA+W in isolating and culturing the soil dematiaceous Hyphomycetes. The suitable conditions for soil dematiaceous Hyphomycetes is cultured for about 1-2 weeks under 20-25℃with 12h /12h alternate light and dark. According to the different altitude and temperature in Yangtse River and Yellow River source area, the author reduced the culture temperature to 18℃and added the soil extract into the medium. Mechanical damnification and stimulation with lower temperature etc. were also helpful in stimulating the fungus sporulation.3 Analysis of classification at genus level baced on rDNA-ITS sequencePhylogenetic relationships among 32 genera involved in this paper were studied based on 5.8S rDNA-ITS sequences by NJ (Neighboring-joining). The resultes showed the conidiogensis pattern'blastic'could be easily reflected in the phylogenetic tree while the the conidiogensis pattern'thallic'could not be reflected. The conidiogensis patterns'holoblastic'and'enteroblastic'were distincted in the phylogenetic tree. Also two major distinct clades were found within the genera whose conidiogensis patterns were enteroblastic-phialidic and enteroblastic-tredic. The similarity of 5.8S rDNA-ITS sequences between several genera with different conidiogensis patterns was much higher because of the different evolutionary rate between them.Taxonomic criteria at genus level, such as holoblastis, which used in morphology, could be well reflected in molecular systematics. Multi-gene sequences analysis could be used to reveal the conidiogensis patterns'arthric'and other patterns of'holoblastic'.4 Molecular systematics on represented species of Doratomyces, Trichurus and GraphiumIn this study, phylogenetic analysis of 5.8S rDNA-ITS and mt-SSU sequence data sets by NJ (Neighboring-joining)with a broad taxon sampling of Doratomyces, Trichurus and Graphium were conducted for inferring evolutionary relationships between the three genera. The results of analysis with 5.8S rDNA-ITS and analysis with SSU-rDNA were basically the same. The genus Graphium could be divided with Doratomyces and Trichurus on the phylogenetic tree. And the genus Doratomyces and Trichurus could not be divided from each other. The similarity of 5.8S rDNA-ITS sequences and SSU-rDNA sequences between all the species in Doratomyces and Trichurus were between 97% and 100%. These two genes were highly conservative in Doratomyces and Trichurus and the species with different morphology couldn't be distinguished by analysis of 5.8S rDNA-ITS and SSU-rDNA sequences.Based on 5.8S rDNA-ITS sequences analysis, three distinct clades were found within the genus Graphium, of which Graphium dubautiae divided with other species. Four clades were found on the phylogenetic tree based on SSU-rDNA sequences. The similarity between the species were 100% and the similarity between the four clades were only between 63% and 94%. There was a little difference between the results of 5.8S rDNA-ITS and SSU-rDNA sequences analysis. The morphological results were not supported by the molecular data. More gene regions should be used with multi-gene molecular phylogeny to inferring evolutionary relationships between the three genera. Because 5.8S rDNA-ITS and mt-SSU sequence were highly conserved in the three genera, not enough variability was present to distinguish genera and species clearly, which implied that 5.8S rDNA-ITS and mt-SSU sequence were not enough when used them to study the lower taxa such as at genus and species leval. Multi-gene genealogy were suggested to ues in testing the correct taxonomic status of these species.