The Diversity Of Endophytic Fungi In Aquatic Plants By High-throughput Sequencing And Phylogenetic Analysis Of Two Genera Of Aquatic Hyphomycetes

Endophytic fungi of aquatic plants refer to a group of microorganisms that live in aquatic plant tissues and do not cause the host plants to show obvious symptoms of infection.Aquatic hyphomycetes widely distributed,and mainly distributed on the rotten leaves and branches in freshwater environments.In addition,a small part of them belongs to endophytes of plants.At present,there are few studies on endophytic aquatic hyphomycetes in aquatic plants.In this study,we use second-generation and third-generation sequencing technologies to mainly reveal the diversity of endophytic fungi of aquatic plants collected from aquatic environments of partial high altitude areas in Yunnan Province,Sichuan Province and Tibet Autonomous Region.Second-generation sequencing targets the ITS1 locus,while third-generation sequencing targets the full length of ITS and partial 28 S.Combined with morphological and molecular phylogenetic studies,we have conducted further research on two genera of aquatic hyphomycetes which are Mycocentrospora and Filosporella respectively.The main findings are as follows:1.The second-generation sequencing results based on ITS1 about the diversity of endophytic fungi in Yunnan and Sichuan Province were show that most of them belong to 6 phylums(Ascomycota ? Basidiomycota ? Chytridiomycota ? Zygomycota ?Glomeromycota and Rozellomycota),20 classes,49 orders,90 families and 193 genera,and the remaining 396 OTUs can not be identified as any phylums.Based on the ITS1,Neighbor Joining analysis was performed on 311 OTUs only annotated as fungal kingdom.We further identified that these OTUs were identified as Ascomycota(22%)?Basidiomycota(29%)? Chytridiomycota(29%)?Mucoromycota(11%)respectively and 29 OTUs could not find a clearly matched sequence in NCBI and UNITE(9%).2.The third-generation sequencing results based on ITS and partial 28 S about the diversity of endophytic fungi in Tibet Autonomous Region were show that most of them belong to 5 phylums(Ascomycota?Basidiomycota?Chytridiomycota?Zygomycota and Rozellomycota),8 classes,11 orders,7 families and 12 genera,and the remaining 93 OTUs can only be identified to the genera.Based on the partly 28 S,Neighbor Joining analysis was performed on OTUs(73.1%)only annotated as Ascomycota and OTUs(12.9%)annotated as Basidiomycota respectively.Except for three of them have short sequences and cannot be aligned,the remaining 65 Ascomycota OTUs combined with 160 reference sequences for phylogenetic tree analysis can be further identified in known families(53.8%),known orders(18.5%),possibly new families(1.5%),possibly new classes(1.5%),and possibly new classes or divisions(24.6%)respectively.Except for one OTU sequence is short and cannot be aligned,the remaining 11 Basidiomycota OTUs combined with 112 reference sequences for phylogenetic tree analysis can be further identified in known orders(72.7%)and possibly new classes(27.3%)respectively.3.Combined with the four loci of 18 S,28S,TEF and RPB2,Bayesian and Neighbor Joining analysis were performed on 11 strains of the genus Mycocentrospora.Combining morphological comparisons,we identified four new species: Mycocentrospora daochengensis,Mycocentrospora litangensis,Mycocentrospora isotiana,Mycocentrospora hydrillanus?4.Combined the five loci of ITS,18 S,28S,TEF and RPB2,Bayesian and Neighbor Joining analysis were performed on 17 strains of the genus Filosporella,and we determine the phylogenetic position of Filosporella for the first time.Combining morphological comparisons,we identified nine new species: Filosporella kasensis,Filosporella myriophyllanus,Filosporella hippuris,Filosporella myriophyllana,Filosporella litangensis,Filosporella bungeiana,Filosporella wasensis,Filosporella yunnansis?...