Discussion

SMRT sequencing，which was recently developed by Pacific Biosciences，can achieve extraordinarily long reads(up to 40 kb)with no GC bias，so that it is suitable for sequencing the genomes with low GC and high repeat content.Moreover，SMRT sequencing enables direct genome-wide detection of diverse base modifications by monitoring the kinetic variations of single bases.Thus，SMRT sequencing is convenient for sequencing the mt genome of O.sinensis and revealing the base modification pattern of the mt genome.It has long been debated whether modified bases exist in mt genomes.

Previously，O.sinensis has been called Cordycepssinensis or Cordyceps sp.due to its morphology.However，the colony characteristics of O.sinensis cultures are significant different from other Cordyceps spp.Most Cordyceps species produce bright color and fleshy stromata，while the stromata of O.sinensis are often darkly pigmented，tough，fibrous to pliant，and have aperithecial apices，which are the characteristics of Ophiocordyceps(Sung et al.，2007).Based on these diagnostic characters and several loci sequence analyses(nrSSU，nrLSU，tef1，rpb1，rpb2，tub，and atp6)，C.sinensis was classified into Ophiocordycipitaceae and renamed“O.sinensis”in 2007(Sung et al.，2007).Our phylogenetic tree(Fig.2)based on the 14 PCGs confirms the phylogenetic position of O.sinensis in Ophiocordycipitaceae.

The mt genome intron number dynamics in the compared fungi(from 1 to 54)are most likely caused by the gain or loss of introns(Haugen et al.，2005；Cuenca et al.，2016).The introns are probably acquired from ancestors or gained through horizontal gene transfer(Haugen et al.，2005；Lang et al.，2007).HE domains in group I introns help to splice the transcribed intronic RNA，and are then removed from the transcribed pre-mRNA resulting in a contiguous RNA transcript in the process of gene transcription(Taylor and Stoddard，2012).However，not all group I introns possess auto-catalytic splicing activity，especially for the mini-introns(e.g.rnl_I5 and nad6_I1)，which lack the typical features(Schafer et al.，1991；Lang et al.，2007).It is proposed that the loss of intron splicing activity may be compensated by some other assistant proteins(Kreike et al.，1987；Lambowitz and Perlman，1990；Lang et al.，2007).If the numerous HE motifs in the O.sinensis mtDNA are active，the HE domains might regulate the transcription of their target genes or modify their target genes，as seen in bacterial viruses and the animal-pathogenic Cryptococcus spp.(Ma et al.，2009；Stoddard，2011).In addition，the existence of HEs within fungal mtDNA genes could promote intron mobility，genetic diversity and adaptive responses for mt genomes，when the allelic recombination events may be impossible or rare in mtDNA due to maternal inheritance(Barr et al.，2005；Basse，2010).

Within the fungi，rps3 is extremely diverse in location and organization：some are lost，some are free-standing，some are incorporated into group I intron，and others have been invaded by HEs(Sethuraman et al.，2009).Among the Ascomycete fungi，group I intron-encoded rps3 seems to have a rather complex evolutionary history(Sethuraman et al.，2009).The phylogeny of rps3 genes inferred in this study displays an evolutionary pattern that is different from that based on 14 conserved PCGs in Hypocrealean fungi，similar to the report of Lin et al.(2015).The rps3 in A.fuci was a free standing gene and was grouped with the intron encoded rps3，implying that the rps3 gene in A.fuci was somehow relocated and the rnl intron was lost.rps3 is prone to insertion and deletion，is a refuge for HE genes，and is subject to recombination，making it problematic to resolve taxonomic relationships using rps3(Sethuraman et al.，2009).(https://www.daowen.com)

To evaluate the balance between purifying selection，neutral evolution and positive selection acting on rps3，the d N/dS ratio，an indicator of evolutionary pressure on a gene，was examined in Hypocreales fungi.A d N/dS value＜1.0 in Hypocreales indicates that rps3 has evolved under functional constraints.However，the significance of LRT showed a clear signal of positive selection in rps3.In addition，the d N/dS values that were＞1 in Hypocreales in relation to the outgroup Penicillium nordicum and the 36 explored positive selection sites suggest that rps3 is under positive selection in Hypocreales fungi，which is in accordance with the results of Lin et al.(2015).rps3 is involved in DNA repair and potentially has endonuclease activities in Schizosaccharomyces pombe and nuclear versions of rps3 in human and Drosophila melanogaster(Neu et al.，1998；Lyamouri et al.，2002；Jang et al.，2004).Except for these model organisms，no functional studies confirm that mtDNA-encoded rps3 genes are actually functional(Kim et al.，2009).Further studies are needed to determine if the rps3 genes in the mt genomic introns produce functional proteins and how these intron-encoded rps3 genes are expressed.

Base modifications are indispensable parts of comprehending biological processes such as host-pathogen interactions，DNA damage and DNA repair(Trygve，2010).Among base modifications，DNA methylation has been one of the most studied modifications.It has emerged as a significant phenotypic determinant for disease susceptibility and pathogenesis in eukaryotes，and involved in the Restriction Modification system in prokaryotes(Bernstein et al.，2010；Ershova et al.，2015).However，whether mtDNA can be the site of epigenetic modifications has long been mired in controversy because the mt genome is multi-copy，lacks canonical CpGislands and histones(D'Aquila et al.，2017).SMRT sequencing is a more innovative and sensitive technology by comparing to the first and second generation DNA sequencing.Its advent makes the discovery of mitochondrial epigenetics，including various DNA modifications，more convenient.In this study，we revealed a mitogenome wide epigenetic DNA modification pattern of O.sinensis by SMRT sequencing，resolving the issue of whether or not epigenetic modifications exist in the mt genome of fungi.

Among the DNA modifications in the mt genome of O.sinensis，several 6mA and 4mC methylations have been identified，which are always found in prokaryotes and have the function of protecting against restriction enzymes，regulating virulence and controlling DNA replication，repair and expression(Ratel et al.，2006).Bisulfite sequencing has enabled genome-wide surveys of 5mC methylation(Masser et al.，2016)，a wellestablished epigenomic mark in eukaryotes，but the historic absence of tools for studying 6mA and 4mC modifications has precluded more comprehensive studies of 6mA and 4mC methylation.Recently，Mondo et al.(2017)firstly analyzed 6mA in fungi by SMRT，and revealed its role as a gene-expression-associated epigenomic mark.In this study，SMRT sequencing revealed that not only 6mA but also 4mC were presented in the fungal mt genome.Nucleotide modifications are one of the most evolutionarily conserved properties of RNA，and the sites of modification are under strong selective pressure(Li and Mason，2014).Mitochondria are largely thought to originate from endosymbioticα-proteobacteria species(Gray，2012).Thus，the 6mA methylations，which are often found inα-proteobacteria，in the fungal mitogenome might be remnants of the methylation system fromα-proteobacteria.However，the function of these epigenetic modifications in the mt genome of O.sinensis remains unknown.

Mitochondria are crucial for responses to hypobaria，hypothermia，and hypoxia due to their central role in energy production and consumption(Chitra and Boopathy，2013；Chandel and Schumacker，2000).Cold temperature and low oxygen pressure are the two most remarkable characters of high-altitude environments，where O.sinensis is endemic(Li et al.，2011；Yu et al.，2011).Current associations between the mt genome and high-altitude adaption is focused on the mtDNA content and polymorphisms of mt genes，including NADH dehydrogenases，cytochrome c oxidases，ATP synthases and cytochrome b(Luo et al.，2013).However，no studies had been conducted on mt epigenetics.In this study，we found only three methylation affecting coding regions(in nad2，nad4L and nad5)，while the rest is spread between intergenic regions and introns.ComplexⅠin the mitochondria，including NAD1-6，NAD4L，are involved in collecting electrons from various donors and passing them to coenzyme Q，which then passes the electrons to ComplexesⅢ(including COB)andⅣ(including COX1-3)，and finally to the final electron acceptor O2 to complete the electron transport chain.At the end of this chain，ComplexⅤ(including ATP6，ATP8，and ATP9)catalyzes the reaction between ADP and inorganic phosphorus to form ATP(Saraste，1999).The DNA methylations in eukaryotes are closely related with DNA transcription and translation(Volpe，2005).Therefore，we supposed that the methylations across the whole O.sinensis mt genome，covering protein-coding regions，introns，and intergenic regions，might be closely related to the expression of the electron transport subunits(nad1—nad2，nad4L，nad6 in ComplexⅠ，cob in ComplexⅢ，cox1—cox2 ComplexⅣ，and atp6 in ComplexⅤ)，and thereby modulate the enzymatic activity of OXPHOS and the mt respiratory rate，and then change the ability to capture O2 and produce energy.Moreover，methylation of mtDNA is supposed to play an important role in mitochondrial biogenesis by affecting the binding affinity of transcription factor A to mtDNA，impacting the relative activity of promoters(van der Wijst and Rots，2015).As the mentioned above，the methylation of mtDNA in O.sinensis might be a genetic feature for adaptation to the cold and low PO2 environment at high altitudes(Volpe，2005；Luo et al.，2013).