Studies On Molecular Phylogeny,Biogeographvy And Mitochondrial Genome Of Orbiliaceae

Nematode-trapping hyphomycetes belong to a modern lineage of the broad predatory fungi.Those fungi prey on nematodes by means of specialized trapping structures,such as adhesive traps and constricting rings.There is a great interest in using these fungi as biological control agents against parasitic nematodes and as model organisms to study adaptive evolution.However,there have been significant confusions in the systematics and taxonomy of this fungi group.Furthermore,due to lack of thorough studies on the sample all over the world,the distribution,dispersal and biogeography of nematode-trapping fungi have been poorly discussed.With the samples gathered from a broad survey for predatory fungi and related taxa,as well as sequence downloaded from NCBI database,molecular phylogeny,biogeographical and mitochondria analyses were conducted using multiple nucleotide and amino acid sequences of multiple loci.The main results are as following:1.Molecular phylogeny and biogeography of nematode-trapping fungiWe conducted a comprehensive,multi-gene phylogenetic analysis of the nematode-trapping hyphomycetes and related taxa,and investigated the potential genes which can be used as DNA barcodes.A total of 860 sequences from 390 strains representing 70 nematode-trapping hyphomycetes and related taxa were included.These sequences came from six marker loci(ITS,RPB2,nr SSU,mt LSU,?-tubulin,and TEF-1?).Our analyses identified that the combined ITS + RPB2 sequence is the most promising DNA barcode for this group of fungi and the barcode gap of 4.2% should be used as threshold for species identification.Based on the phylogeny and barcoding criterion,we redefined 59 strains,and proposed 3 new species among our analyses.Our combined ITS+RPB2 phylogeny strongly supports the placement of non-predatory taxa separately from the predatory taxa with 100% bpp.Then predatory fungi gained the trapping structure in the subsequent evolutionary process.Our analyses revealed a definitive pattern of evolutionary development for Orbilia and their trapping structures.Molecular clock calibration based on two fossil records revealed that Orbilia diverged from saprophytism about 311 Mya,shortly after the occurrence of the Devonian extinction event about 365 Mya.Predatory fungi origined from about 117 Mya,after the Triassic–Jurassic extinction event about 201.4 Mya.Predatory fungi(fungi with constricting rings)and passive carnivorism(fungi with adhesivetraps)diverged from each other around 98 Mya,the major adhesive traps evolved around 77–87 Mya.But none structure possess geographic specificity.Historical distribution reconstructions based on Bayesian Binary MCMC analysis strongly supported that these fungi originated during the Eocene in the East Asia,followed by dispersal to and subsequent speciation in other parts of Asia,Europe,and Americas from the Oligocene through the early Miocene.2.Mitochondrial genome analyses of nematode-trapping fungiBased on the download mitochondrial sequences,we completed the gap of Arthrobotrys oligospora(160613bp)and Dactylellina haptotyla(146101bp)mitochondrial genome by PCR,and was annotated automatically using the MFannot tool and RNAweasel based on the old mitochondrial genetic code 4 and successfully predict 14 protein-encoding genes ATP6,ATP8,ATP9,COX1,COX2,COX3,COB,NAD1,NAD2,NAD3,NAD4,NAD4 L,NAD5,NAD6,2 ribosomal RNAs(r RNAs),transfer RNAs(t RNAs)and intron types,respectively.The predictions were individually verified by blastx searches.In the result,A.oligospora mitochondrial genome contains 24 t RNAs and 33 introns while Da.haptotyla has 26 t RNAs and 27 introns.COX1 has most introns(12 and 9,respectively)in both two mitochondrial genomes.And ORFs were found in most introns and coded two types of homing endonuclease.At the same time,we download all ascomycetes mitochondrial genome which had been published on literature or available in NCBI database.Although 14 protein-coding genes phylogenetic analyses of ascomycetes by Bayesian inference,our mitochondrial phylogenetic result of Orbilia placement is consistent with nuclear phylogeny,mitochondrial genes evolve faster than nuclear gene.