Species Recognition Of Wild Edible Mushrooms In Yunnan Markets

In the world wild mushroom trade market,China’s Yunnan Province is famous for its rich resources of wild edible fungi.In this study,3,585 wild edible fungi samples from 35 cities and counties in Yunnan Province were collected and sorted by the project team from 2010 to 2019.Using ITS as the molecular marker gene for identification and classification of edible fungi,the number of wild edible fungi in Yunnan was widely counted and the phylogenetic identification of wild edible fungi was carried out.Great diversity of wide（hidden）species and 21 new records were found.The feasibility of using ITS sequence as the identification marker of wild mushroom species was also discussed.Multigene phylogeny and morphological analyses were conducted on the genus Ramaria and Auricularia,new species were described.The use of barcode technology can provide a solid foundation for the description of new species,and the identification of their origin and authenticity,finally provide scientific guidance for market management,and can also provide strategies for the effective protection and utilization of these sustainable resources.The results of the study are as following:1.A total of 3585 specimens of wild edible fungi were collected and sorted,covering 35 counties in 13 prefectures of Yunnan Province.According to the morphological characteristics of the specimens and the experience of local people,all the specimens were roughly identified as about 41 species by their local names.With ITS being used as a molecular marker for identification and classification of wild edible fungi in Yunnan,CTAB method is used to extract genomic DNA from 3585 samples,3381 available genomic DNA were successfully extracted.From the total 2198 samples that we successfully obtained their ITS sequences,we found a total of 107 species matched to existing species（representing 62.73% of the total ITS sequences）when the within-species ITS sequence similarity was set at >97% based on Blast match against NCBI database.For samples the failed to classify to species level based on 97%sequence identity,we progressively used other threshold values 95%-97%,90%-95%and lower than 90% to detect additional species.The relaxed criteria allowed us to identify additional 17（6.18% of the ITS se-quences）,33（21.97% of the ITS sequences）and 25（1.14% of the ITS sequences）known species that respectively matched to our samples.Thus,using the very loose criteria,we found at least 182 species with binomial names matching our samples.Together,these 182 species belonged to 50 different genera,28 families,9 orders,3 classes and 1 phyla,with 37.27% of the species and the known sequence of similarity is lower than 97%,they may be potentially new（cryptic）species,new genera or even new families.2.We calculate respectively in big classes within phylum Basidiomycotina:Agaricomycetes the range of genetic distances within intra and interspecies,between genera and families combined with online download data.Our comparisons showed that the greatest intraspecific K2 P distance within Agaricomycetes was 0.012.In comparison,interspecific divergence within individual genera ranged from 0 to 0.33 within Agarico-mycetes.The intra-and inter-species genetic divergence greatly overlapped each other in both classes and the same occurs at the intergenus and interfamily levels.Combined with the high PCR amplification rate and sequencing success rate,and the amount of data in the online database,it was suggested that ITS could be used as the DNA barcode gene for the primary identification of most wild edible fungi in Yunnan.The specific species identification depends on the phylogenetic and multi-gene fragments combined with the identification method of morphological characteristics.3.With the method of phylogenetic species identification to compare the sample size and classification background data of Boletus and allies,Cantharellus cibarius species complex,Lactarius,Lyophyllum,Russula virescens Ally,Termitomyces clypeatus species complex and the Thelephora ganbajun species complex.A total of110 phylogenetic species were identified,with a total 20 additional provisional species in the selected 1536 ITS sequences.4.A total of fifteen new species of Ramaria were identified by combining with RBP2,ITS,TEF1 and ATP6 fragments phylogeny and morphological characters from analysis of 23 Ramaria species.And six new species of Auricularia were identified by morphological description and phylogenetic analysis 8 Auricularia species by combining RBP2,ITS and LSU sequence,which greatly enriched the biodiversity of these groups of fungi.