The Application Of DNA Barcoding Technology In Species Identification Of Clitopilus,Clitocella And Clitopilopsis

DNA barcoding technology is widely used in the classification and identification of species.The fungal species in the genus Clitopilus have important edible and medicinal values,but some species have not been well recognized due to the lack of detailed studies on morphological characters and molecular phylogeny in the genus.Therefore,in this study,DNA barcoding technology was applied to Clitopilus and its allied genera,Clitocella and Clitopilopsis,to obtain the most suitable DNA barcoding fragments.Then we used the suitable fragments to accurately identify and distinguish the species of each genus.We tried to analyze the diversity of fungal species and the affinities of these three genera in China.The main conclusions were as follows:1.DNA barcoding study of Clitopilus,Clitocella and ClitopilopsisIn this study,five gene fragments,viz the internal transcribed spacer region(ITS),large subunit ribosomal RNA gene(LSU),the translation elongation factor 1-? gene(tef1),RNA polymerase II second largest subunit(rpb2)& ATPase subunit 6(atp6)and three combined gene fragments(ITS-LSU-rpb2,ITS-tef1-rpb2 & LSU-tef1-rpb2-atp6)were selected for evaluation.Based on primer universality,sequencing quality,barcoding gap,and accuracy of genus or species identification,the study indicated that ITS,LSU and atp6 should not be used as suitable DNA barcodes for these three genera.Further analysis indicated that in intraspecific variation,rpb2 > ITS > tef1 > atp6 > LSU,and in interspecific variation,tef1 > rpb2 > ITS > atp6 > LSU.In the analysis of the three joint fragments,the intra-and interspecies variation were consistent,all of which were LSUtef1-rpb2-atp6 > ITS-tef1-rpb2 > ITS-LSU-rpb2.But in terms of identification accuracy in genus and interspecies level,only the LSU-tef1-rpb2-atp6 could accurately identify all genera and species.Comprehensive evaluation shows that rpb2 in single fragments was the most suitable as the DNA barcoding for Clitopilus and its allies.Among combined gene fragments,LSU-tef1-rpb2-atp6 was the most suitable as the DNA barcoding for Clitopilus and its allies.2.Application of DNA barcoding technology in the taxonomy of Clitopilus,Clitocella and ClitopilopsisThe phylogenetic trees of Clitopilus,Clitocella and Clitopilopsis were constructed using the optimal DNA barcoding in the above combined gene fragments,and the species diversity of this group was analyzed in combination with morphological taxonomy.The results showed that:(1)Clitopilus could be divided into three sections,viz sect.Clitopilus,sect.Scyphoides and sect.Crispi,the last one is a new section erected in this study;(2)Clitocella and Clitopilopsis were two genera new to China and seven new species were described;(3)In total,there were 17 species in China,including ten in Clitopilus,two in Clitocella,one in Clitopilopsis and four insufficiently known species.