The Resources Of Hgrophorus In Southwestern China

Objective:In order to find more resources of Hygrophorus in China,and to establish a natural classification framework of the genus,collections mainly from southwestern China are investigated by overall macro-microscopic and molecular phylogeny studies.Methods:Molecular studies include DNA extraction,amplification,and sequencing,based on molecular phylogenetic analyses of two genes(ITS,nrLSU).Microchemical reactions studies are conducted using seven chemical reagents(C6H5OH=10%(weight/volume)phenol,EtOH=95%(volume/volume)ethanol,FeCl3=10%(w/v)ferric chloride,FeSO4=10%(w/v)ferrous sulfate,KOH=5%(w/v)potassium hydroxide,Melzer's=Melzer's reagent,NH4OH=10%(w/v)ammonium hydroxide).Ultrastructure studies are to scan and photograph basidiospores and mycelial hyphae.Taxonomy includes macro-morphological descriptions,micro-morphological characters,and combined with geographical information and ecological habitat.Results:Twenty eight new phylogenetic species are recognized among the studied collections,which belong to H.sect.Chrysodontes?H.sect.Olivaceoumbrini?H.sect.Pudorini?H.sect.Aurei?and H.sect.Hygrophorus.Hygrophorus robustus has not been found in the studied collections.In addition,the molecular phylogenetic and morphology studies have been carried on species of H.russula complex in H.sect.Pudorini.There are at least 11 new phylogenetic species,and five of them are described as new species:H.parvirussula H.Y.Huang&L.P.Tang,H.crispulus H.Y.Huang&L.P.Tang,sp.nov.,H.orientalis H.Y.Huang&L.P.Tang,sp.nov.,H.tunuis H.Y.Huang&L.P.Tang,sp.nov.,H.yunnanensis H.Y.Huang&L.P.Tang,sp.nov.A key to the morphologically recognizable taxa of H.russula complex is presented.The diagnostic features of H.russula complex can be summarized as following:basidiomata solitary to sparsely scattered on soil;pileus surface dry,slimy when wet,pale flesh pink to reddish purple;lamellae adnate to slightly decurrent;stipe central to slightly eccentric,surface pale white to flesh pink;basidiospores ellipsoid,yellowish in Melzer's reagent,hyaline;basidia clavate;cystidia scattered,narrowly clavate,spathulate,subcylindric,flexuous,lamellar trama divergent;pileipellis an ixotrichoderm,consisting of narrow hyphae in gluten;clamp connections present in all tissues.In addition,the six species vary in the distribution area,basidiomata size,basidia length,and basidiospores size.The microchemical result shows that it makes little sense to distinguish the species of Hygrophorus.Conclusions:In this study,none is confirmed that 47 known species from Europe and North America distributed in China;H.sect.Chrysodontes,H.sect.Hygrophorus,and H.subsect.Aurei are identified monophyletic;H.sect.Olivaceoumbrini and H.sect.Pudorini need further study.Among four DNA markers,ITS is the best gene in species identification bescause of high amplifation rate and discrimination.By contrast,nrLSU is more difficult in PCR amplification,but it is better to discriminate some sibling species.tefl-a has lower PCR amplification and sequencing success.Due to multiple bands,it needs to design new primers of rpb2.