Studies On Phylogeny And Mitogenomes Of The Genus Tolypocladium

The species of the genus Tolypocladium are widely distributed in the world,with rich host/substrate diversity,can adapt to a large range of altitude/temperature and produce a variety of important metabolites,and has high theoretical research value and practical application value.From "only morphology-based" to "combining morphological characteristics with molecular data",during the process of changes of identification method in Tolypocladium species,there may be some species with wrong taxonomic treatment,due to the lack of molecular data of some species.The mitochondrial genomes of fungi contain important evolutionary information and can be used as a good molecular marker in phylogenetic research.There are few studies on mitochondrial genome of Tolypocladium species,which hinders the understanding of this important fungal group.In this study,through the collection,collation and comparison of morphological characteristics and molecular biological information of Tolypocladium species,combined with the results of observation and sequencing of research materials based on morphological methods and molecular systematics methods,the taxonomic identification and phylogenetic analysis of Tolypocladium species were carried out,and the phylogenetic framework of Tolypocladium was constructed,taxonomic status of one new species was determined,4 new strains of known species were identified,and one species with wrong classification was revised.Two strains of Tolypocladium isolated from the same naturally occurring Ophiocordyceps sinensis(Berk.)G.H.Sung,J.M.Sung,Hywel-Jones & Spatafora were identified as a new species of T.reniformisporum H.Yu,Y.Wang,Y.F.Liu & Y.B.Wang sp.nov.,ex-type living culture YFCC 1805002,and a known species of Tolypocladium,T.cylindrosporum W.Gams,strain YFCC 1805001.Cordyceps cicadae S.Z.Shing is a new taxonomic unit of Tolypocladium,which is revised as T.cicadae(S.Z.Shing)H.Yu,Y.F.Liu,W.B.Zeng & Y.B.Wang,comb.nov.,and it is also identified that T.dujiaolongae Y.P.Cao& C.R.Li should be regarded as the synonym of T.cicadae.A Tolypocladium strain YFCC 881 B isolated from the soil of Baima Snow Mountain was identified as T.inflatum W.Gams and YFCC 8579 from Zixi Mountain was identified as T.ophioglossoides(J.F.Gmel.)C.A.Quandt,Kepler & Spatafora.Tolypocladium sp.Salcha MEA-2,which was reported to produce a new natural product “maximiscin”,was identified as T.inflatum.The growth temperature test of T.reniformisporum YFCC 1805002 and T.cylindrosporum YFCC 1805001 showed that they were cold-adapted fungi,the optimum growth temperature was 18-22 ℃,and the minimum growth temperature was below 5 ℃.The complete mitochondrial genomes of 6 species(13 strains)of Tolypocladium were assembled,annotated and analyzed by high-throughput sequencing and raw reads of the whole genome of Tolypocladium collected in Gen Bank.And the mitochondrial genome sequences of six Tolypocladium taxa collected in Gen Bank were re-annotated and analyzed.The complete mitochondrial genomes of T.reniformisporum(isolates YFCC 1805002),T.cicadae(isolates YFCC 8003),T.cylindrosporum(isolates YFCC 1805001),T.inflatum(isolates YFCC 881B),T.ophioglossoides(isolates YFCC 8579),and T.geodes(isolates Sup5 PDA-1)were described in detail from the basic characteristics,codon usage of protein coding gene,t RNAs and its secondary structure,r RNAs,gene overlap and interval.The mitochondrial genomes of six Tolypocladium species were analyzed and compared with variation among their whole genomes.The mitochondrial genomes of different strains of the same species were compared and analyzed in six T.cicadae strains,six T.inflatum strains,three T.ophioglossoides strains and two T.cylindrosporum strains,which revealed the genetic variation of mitochondrial genomes among different individuals within the species.The results showed that the shape,gene composition/order of mitochondrial genomes of different species of Tolypocladium and different strains of the same species were highly conserved,abundant variations are mainly caused by the presence/deletion or movement of introns,the differences in the number and length of ORFs,and the differences in intergenic regions.The Ka/Ks values of 15 core protein-coding genes were all less than 1,indicating that these genes were all selected by purification.An intron ⅠA containing rps3 gene in rrn L was found in the mitochondrial genomes of 6 Tolypocladium species(all 19 strains)and even Ophiocordyceps species,it was the only common intron,while introns in cob,cox1,cox2,cox3,nad1 and nad6 were newly obtained.All introns belong to group Ⅰ,which have four types: ⅠA,ⅠB,ⅠC1 and ⅠD.There is a base overlap between nad4 L and nad5,and nad2 and nad3 are closely linked,indicating that the functions of these genes are highly correlated.In both mitochondrial and nuclear genomes,intergenic regions have greater variability than gene-coding regions.The variation among mitochondrial genomes was lower than that among nuclear genomes.The mitochondrial genome characteristics of T.cicadae are highly similar to those of T.inflatum,but very different from that of Isaria cicadae,the obvious differences in mitochondrial genomes confirmed that T.cicadae and I.cicadae are not sexual anamorphs of each other,but completely different species.The SNP variation among the mitochondrial genomes of T.cicadae strains from different provinces in China was obvious.With the increase of geographical distance,the SNP increased significantly.The SNP/Indel variations among the mitochondrial genomes of six T.inflatum strains(isolated from soil)from six different countries was low.High SNP/Indel variations were detected in the mitochondrial genomes of 3 strains(isolated from teleomorphs)of T.ophioglossoides and 2 strains(isolated from different substrates)of T.cylindrosporum from different countries.Based on atp6-rrn S intergenic sequence data sets and nad3-atp9 intergenic sequence data sets of mitochondrial genomes,a Tolypocladium strain Sup5 PDA-1,which can produce a new natural product Gichigamin A,was identified as T.geodes W.Gams.Phylogenetic analysis was carried out based on 14 core PCGs data sets,atp6-rrn S intergenic sequence data sets and nad3-atp9 intergenic sequence data sets of mitochondrial genomes,and the results were consistent with those of phylogenetic analysis based on 5-gene or ITS.The reliability of mitochondrial gene as a good molecular marker was verified.Compared with phylogenetic analysis based on single intergenic region sequence data set,phylogenetic analysis based on 14 core PCGs combination data sets can better distinguish strains.This study increased the data of morphology and molecular biology of Tolypocladium species,which will help to lay a solid foundation for taxonomic and phylogenetic studies of Tolypocladium and its related species,and enhanced the understanding of the evolutionary characteristics of the mitogenomes of Tolypocladium species,contributed to a better understanding of the origin and evolution of Tolypocladium species,has great innovative significance and scientific value.