Comparative analysis of the complete mitochondrial genomes of four cordyceps fungi

Abstract Cordyceps is a large group of entomogenous, medicinally important fungi. In this study, we sequenced, assembled, and annotated the entire mitochondrial genome of Ophiocordyceps xuefengensis, in addition to comparing it against other three complete cordyceps mitogenomes that were previously published. Comparative analysis indicated that the four complete mitogenomes are all composed of circular DNA molecules, although their sizes significantly differ due to high variability in intron and intergenic region sizes in the Ophiocordyceps sinensis and O. xuefengensis mitogenomes. All mitogenomes contain 14 conserved genes and two ribosomal RNA genes, but varying numbers of tRNA introns. The Ka/Ks ratios for all 14 PCGs and rps3 were all less than 1, indicating that these genes have been subject to purifying selection. Phylogenetic analysis was conducted using concatenated amino acid and nucleotide sequences of the 14 PCGs and rps3 using two different methods (Maximum Likelihood and Bayesian analysis), revealing highly supported relationships between O. xuefengensis and other Ophiocordyceps species, in addition to a close relationship with O. sinensis. Further, the analyses indicated that cox1 and rps3 play important roles in population differentiation. These mitogenomes will allow further study of the population genetics, taxonomy, and evolutionary biology of medicinally important cordyceps species.

'Cordyceps' comprises four genera including Cordyceps, Ophiocordyceps, Metacordyceps, and Elaphocordyceps. The fungi have been used in traditional Chinese medicine, as dietary supplements, or as tonic edibles with broad pharmacological properties including antibacterial, antiviral, and antitumor activities, in addition to the ability to enhance human immunity (Yue et al., 2013;Zhong et al., 2019). Although Cordyceps have considerable economic and medicinal significance, wild cordyceps resources are on the verge of exhaustion, leading to many investigators seeking alternatives. Among these, O. xuefengensis is a newly identified cordyceps resource that we previously discovered on Xuefeng mountain in Hunan province of China (Wen et al., 2013). The novel taxa has been widely used as an ethnopharmacological invigorant by the Yao group for a long time . Further, we have successfully cultivated this cordyceps fungi and also characterized some of its chemical metabolites . Cordyceps and Ophiocordyceps are the two most widely used groups of cordyceps fungi. In particular, O. sinensis is popularly used to treat many diseases in China. Consequently, the cultivation, novel metabolite compounds, and pharmacology of cordyceps fungi have been extensively researched, although further molecular phylogenetic and species identification studies are critically needed.
In this study, the complete mitogenome from O. xuefengensis was sequenced, assembled, and compared to other three complete cordyceps mitogenomes that were previously reported. This study thereby represents the first comparative analysis of the genomic structure, base composition, substitution, and evolutionary rates among four cordyceps species, in addition to the comprehensive molecular phylogenetic analysis of cordyceps in the Hypocreales order. This study aimed to expand our knowledge of the diversity of mitochondrial genomes and phylogenetic relationships of cordyceps.

| Fungal materials and DNA extraction
Ophiocordyceps xuefengensis strain HCMA001 was used in this study for mitogenomic analysis. In vitro mycelial fermentation in liquid culture was conducted in Erlenmeyer flasks with shaking at 150 rpm in an incubator at 25°C for 10 days. Mycelia were collected by centrifugation and quickly frozen in liquid nitrogen. Total genomic DNA was then extracted using an improved extraction method (Chen et al., 2011). Genomic DNA was quantified using an TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High quality DNA samples (OD 260/280 = 1.8-2.0, >6 μg) were used to construct fragment libraries for genomic sequencing.

| Mitochondrial DNA sequencing and genome assembly
About 8 μg of purified DNA was sequenced using a combination of third-generation sequencing technologies including PacBio RS and Illumina sequencing platforms. The Illumina data were used to evaluate the complexity of the genome and correct the PacBio long reads.
The raw paired-end reads were trimmed, and quality controlled using Trimmomatic with parameters including SLIDINGWINDOW: 4:15 MINLEN: 75 (version 0.36). Clean data obtained from the above quality control procedures were then used for further analyses.

| Repetitive element analysis
Tandem repeats (>10 bp in length) in the four mitogenomes were detected using the Tandem Repeats Finder program (Benson, 1999), with default parameters. In addition, repeat sequences were identified using the REPuter program (Kurtz et al., 2001) to identify forward (direct), reverse, complemented, and palindromic (reverse complemented) repeats across the four mitogenomes, using a minimum repeat size of 30 and a hamming distance set to 3.

| Phylogenetic analysis of Hypocrealean species
To determine the phylogenetic relationships of the cordyceps species  (Ronquist et al., 2012). Other analyses and evaluation methods were conducted based on previously described protocols (Qiang et al., 2018).

| Data availability statement
The newly sequenced mitogenome of O. xuefengensis strain HCMA001 was submitted to GenBank under the accession number SAMN16236787.

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Across all 14 PCGs and rps3 genes that were evaluated, rps3 has the greatest K2P genetic distance among the mitogenomes, followed by cox1. atp8 has the least genetic distance among the genes investigated, indicating a high degree of conservation. Ka analysis indicated that rps3 and cox1 have relatively high nonsynonymous substitution rates. Further, the Ks of nad1 is the highest across all four mitogenomes, while that of atp8 is the lowest. The Ka/Ks values for all 14 PCGs and rps3 are <1, suggesting that these genes are subject to purifying selection ( Figure 6).

| Gene rearrangements and phylogenetic analysis
The relative positions of the mitochondrial genes (including rnl, rns, rps3, the 14 core PCGs, and the tRNAs) are highly conserved in the four cordyceps mitogenomes. The O. xuefengensis mitogenome only lacks trnI, consistent with that of O. sinensis (Figure 7). Genome synteny analysis also identified several instances of gene rearrangement in the mitogenomes of the four cordyceps. All four cordyceps mitogenomes could be divided into two homologous regions (Figure 8) xuefengensis and O. sinensis was highly supported (Figure 9).

| DISCUSS ION
Cordyceps are fungal species used in traditional medicine that are represented by the well-known fungal resource, O. sinensis. However, natural populations of O. sinensis have been overharvested to the extent that it is an endangered species. It is consequently necessary to explore alternative cordyceps resources, because artificial cultivation of O. sinensis is difficult to achieve .
Ophiocordyceps xuefengensis is a new potential cordyceps resource that was found on Xuefeng mountain in Hunan. Investigation of its artificial cultivation and biometabolite biosynthesis yielded some positive results . Several previous studies have investigated the phylogenetic relationships of cordyceps, although detailed phylogenetic relationships for the species remain poorly understood, especially since increasing numbers of related species have been reported (Ghikas et al., 2010;Li et al., 2015;. It is difficult to accurately classify fungal species based on limited morphological characters and overlapping morphological features . Consequently, mitogenomes have been widely used in the phylogenetic analysis of eukaryotes due to many advantages, including uniparental inheritance, rapid evolutionary rates, and the presence of several molecular markers (Boore, 1999). Here, we report a new mitogenome sequence for O. an adaptation to their host genome that may be due to their decreased mobility (Novikova & Belfort, 2017), or alternatively, to their potential important role in stabilizing the gene that hosts the intron (Genetics, 2003;Korovesi et al., 2018). Other introns may be acquired late in the divergence of taxa, either through HGT events or through active transposition (Mardanov et al., 2014;Wu et al., 2015).
The transposition of some introns to other genomic regions with less sequence similarity can occur more frequently under stress-induced conditions (Coros et al., 2009;Robbins et al., 2011).  et al., 2014). Moreover, the accumulation of repeat sequences could also be highly related to gene recombination and gene loss F I G U R E 8 Collinearity analysis of four cordyceps mitogenomes, generated with Mauve 2.4.0. Two homologous regions covered by two different colored blocks were detected across the four mitochondrial genomes overall. The boundaries of colored blocks usually indicate the breakpoints of genome rearrangement, unless sequence has been gained or lost in the breakpoint region. The crossing "X" pattern of lines, which happen to occur in the vicinity of the predicted rearrangements in these organisms F I G U R E 9 Molecular phylogeny of 20 fungal species based on Bayesian inference (BI) and Maximum likelihood (ML) analysis of 14 PCGs and rps3 genes. Support values are Bayesian posterior probabilities (before slash) and bootstrap (BS) values (after slash). Species and NCBI accession numbers for genomes used in the phylogenetic analyses are provided in Table 1 ( Zou, Jakovlić, et al., 2017). A variety of models have been proposed to investigate these mitochondrial gene rearrangements (Xia et al., 2016 These observations collectively indicate that selection upon the genomes by environmental factors is relatively weak among the four cordyceps fungi evaluated here, resulting in highly conserved phylogenetic relationships. Thus, we could also infer that differentiation of the cordyceps genomes remains in early stages, consistent with the molecular phylogenetic analyses. One reasonable explanation for these contradictory results is that the larger pro-

| CON CLUS IONS
This study extends the known mitogenomes of cordyceps fungi and identified variation in PCGs, tRNA genes, and rRNA genes among medicinal cordyceps species, including O. xuefengensis.
Comparative mitochondrial genome analysis of four cordyceps fungi was conducted, revealing that their mitochondrial genomes are conserved and that differentiation of their mitogenomes is still in the early stages. Nevertheless, results indicated that the cox1 and rps3 genes likely play important roles in population differentiation among the taxa. Mitogenome analyses, like those reported here, will allow further study of the population genetics, taxonomic relationships, and evolutionary biology of medicinally important cordyceps species.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.