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\begin{document}

\title{Comparative analysis of the complete mitochondrial genomes of four
cordyceps fungi}



\author[1]{can zhong}%
\author[1]{jian jin}%
\author[1]{rongrong zhou}%
\author[1]{hao liu }%
\author[1]{jing xie}%
\author[1]{dan wan}%
\author[2]{shengen xiao}%
\author[1]{shuihan zhang}%
\affil[1]{Hunan Academy of Chinese Medicine}%
\affil[2]{Hunan Agricultural University}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Cordyceps is a large group of entomogenous, medicinally important fungi.
In this study, we sequenced, assembled, and annotated the entire
mitochondrial genome of O. xuefengensis, in addition to comparing it
against three other 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 O. 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.%
\end{abstract}%



\sloppy


Article

Comparative analysis of the complete mitochondrial genomes of four
cordyceps fungi

\textbf{Can Zhong \textsuperscript{1,2}, Jian Jin \textsuperscript{2},
Rongrong Zhou \textsuperscript{3}, Hao Liu \textsuperscript{2}, Jing
Xie\textsuperscript{2}, Dan Wan\textsuperscript{2}, Shengen
Xiao\textsuperscript{1}*, and Shuihan Zhang \textsuperscript{1,2}*}

\textsuperscript{1} Horticulture and Landscape College, Hunan
Agricultural University, Changsha 410128, P. R.
China,\textsuperscript{2} Institute of Chinese Materia Medica, Hunan
Academy of Chinese Medicine, Changsha 410013, P. R.
China,\textsuperscript{3} Changchun University of Chinese Medicine,
Changchun 130117, P. R. China.\textbf{*} Correspondence:
2395265030@qq.com (S. X.),\emph{zhangshuihan0220@126.com}(S.Z.)
Received: date; Accepted: date; Published: date

\textbf{Abstract:} Cordyceps is a large group of entomogenous,
medicinally important fungi. In this study, we sequenced, assembled, and
annotated the entire mitochondrial genome of \emph{O. xuefengensis} , in
addition to comparing it against three other 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 \emph{O.
sinensis} and \emph{O. xuefengensis} mitogenomes\emph{.} 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\emph{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
\emph{rps3} using two different methods (Maximum Likelihood and Bayesian
analysis), revealing highly supported relationships between\emph{O.
xuefengensis} and other \emph{Ophiocordyceps} species, in addition to a
close relationship with \emph{O. sinensis} . Further, the analyses
indicated that \emph{cox1} and \emph{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.

\textbf{Keywords:} cordyceps, mitochondrial genome, phylogenetic
analysis, introns

1. Introduction

Mitochondrial genomes are powerful tools that can be used in
phylogenetic and evolutionary studies {[}1-4{]}, owing to their small
size, high copy numbers, conserved orthologous genes, rare instances of
recombination, and high evolutionary rates {[}5{]}. The rapid
development of new genome sequencing technologies in recent years has
led to the production of more than 180 fungal mitogenomes. Fungal
mitochondria range in length from approximately 11 kbp
(\emph{Hanseniaspora uvarum} ) to 272.2 kbp (\emph{Morchella importuna}
), depending on the species, but usually contain 14 conserved genes
{[}6{]}. In addition, mitogenome characteristics, including genome size,
genome structure, gene content, gene arrangement, tRNA number, intron
number, codon usage, and repeat content, can provide useful information
to understand the origin, evolution, and systematics of eukaryotes
{[}7-9{]}.

''Cordyceps'' comprises four genera including \emph{Cordyceps}
,\emph{Ophiocordyceps} , \emph{Metacordyceps} , and
\emph{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 {[}10, 11{]}. Although\emph{Cordyceps} have considerable
economic and medicinal significance, wild cordyceps resources are on the
verge of exhaustion, leading to many investigators seeking alternatives.
Among these,\emph{O. xuefengensis} is a newly identified cordyceps
resource that we previously discovered on Xuefeng mountain in the Hunan
province of China {[}12{]}. The novel taxa has been widely used as an
ethnopharmacological invigorant by the Yao group for a long time
{[}13{]}. Further, we have successfully cultivated this cordyceps fungi
and also characterized some of its chemical metabolites {[}14{]}.
\emph{Cordyceps} and\emph{Ophiocordyceps} are the two most widely used
groups of cordyceps fungi. In particular, \emph{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 the present study, the complete mitogenome from \emph{O.
xuefengensis} was sequenced, assembled, and compared to three other
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. The present
study aimed to expand our knowledge of the diversity of mitochondrial
genomes and phylogenetic relationships of cordyceps.

2. Materials and Methods

\emph{2.1 Fungal materials and DNA extraction}

\emph{O. xuefengensis} strain HCMA001 was used in this study for
mitogenomic analysis. \emph{In vitro}mycelial fermentation in liquid
culture was conducted in Erlenmeyer flasks with shaking at 150 rpm in an
incubator at 25\selectlanguage{ngerman}°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 {[}40{]}. Genomic DNA was
quantified using an TBS-380 fluorometer (Turner BioSystems Inc.,
Sunnyvale, CA). High quality DNA samples
(OD\textsubscript{260/280}=1.8\textasciitilde{}2.0, \textgreater{} 6 ug)
were used to construct fragment libraries for genomic sequencing.

\emph{22 Mitochondrial DNA sequencing and genome assembly}

About 8 \selectlanguage{greek}μ\selectlanguage{english}g of purified DNA was sequenced using a combination of
third-generation sequencing technologies including PacBio RS and
Illumina sequencing platforms. The Illumina data was 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\emph{http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic}).
Clean data obtained from the above quality control procedures were then
used for further analyses.

\emph{2.3 Annotation and comparative analysis of the O. xuefengensis
mitogenome}

Ab initio prediction was used to generate gene models for the \emph{O.
xuefengensis} mitogenome. Gene models were identified using MFannot
(https://megasun.bch.umontreal.ca/cgi-bin/dev\_mfa/mfannotInterface.pl).
Gene models were then compared against the non-redundant (NR) NCBI
database using BLASTp, in addition to comparison against the SwissProt,
KEGG, and COG databases to establish functional annotation, also using
BLASTp. tRNAs were identified using the tRNAscan-SE program (v1.23,
http://lowelab.ucsc.edu/tRNAscan-SE) and rRNAs were identified using the
RNAmmer program (v1.2,\emph{http://www.cbs.dtu.dk/services/RNAmmer/}).
Circular maps of the four mitochondrial genomes were drawn using OGDRAW
(\emph{https://chlorobox.mpimp-golm.mpg.de/OGDraw.htmlonline}) {[}41{]}.

\emph{2.4 Sequence analysis}

Nucleotide compositional skew was calculated according to the formulae:
AT-skew = (A-T)/(A+T) and GC-skew = (G-C)/(G+C) {[}42{]}. The Sequence
Manipulation Suite program package
(http://www.bioinformatics.org/sms2/codon\_usage.html) was used to
analyze codon usage based on genetic code ``4'' {[}43{]}. Genetic
distances between each pair of the 14 core PCGs (\emph{atp6, atp8, atp9,
cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5,}
and\emph{nad6} ) were calculated with MEGA X {[}44{]}, using the
Kimura-2-parameter (K2P) substitution model. DnaSP v6 {[}45{]} was used
to calculate the nonsynonymous (Ka) and synonymous (Ks) substitution
rates for the 14 core PCGs among the four mitogenomes. Genomic synteny
among the four mitogenomes was analyzed using the Mauve v2.4.0 program
{[}46{]}.

\emph{2.5 Repetitive element analysis}

Tandem repeats (\textgreater{} 10 bp in length) in the four mitogenomes
were detected using the Tandem Repeats Finder program {[}47{]}, with
default parameters. In addition, repeat sequences were identified using
the REPuter program {[}48{]} 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.

\emph{2.6 Phylogenetic analysis of Hypocrealean species}

To determine the phylogenetic relationships of the cordyceps species
among the Hypocreales order, 19 mitogenomes were downloaded from the
NCBI database and subjected to Maximum Likelihood (ML) and Bayesian
phylogenetic analysis using the aforementioned 14 PCGs and \emph{rps3} .
\emph{Rhizopogon salebrosus} and \emph{Rhizopogon vinicolor} were used
as outgroups for the analyses (Table S1). Single mitochondrial genes
were first aligned using MAFFT v7.037 {[}49{]} and these alignments were
then concatenated using SequenceMatrix v1.7.8
(\emph{http://www.softpedia.com/get/Science-CAD/Sequence-Matrix.shtml})
{[}50{]}. The ML tree was then calculated for the combined gene set
using RAxML v8.0.0 {[}51{]}, while Bayesian inference (BI) analysis was
performed with MrBayes v3.2.6 {[}52{]}. Other analyses and evaluation
methods were conducted based on previously described protocols {[}9{]}.

\emph{2.7 Data availability statement}

The newly sequenced mitogenome of \emph{O. xuefengensis} strain HCMA001
was submitted to GenBank under the accession number SAMN16236787.

3. Results

3.1. Characterization of the four cordyceps mitogenomes

Comparative analysis indicated that the complete mitogenomes of the four
cordyceps all comprised circular DNA molecules. The mitogenome length
of\emph{O. xuefengensis} (78,744 bp) was much smaller than that
of\emph{O. sinensis} (157,559 bp), but longer than those of
\emph{Cordyceps militaris} (33,277 bp) and\emph{C. brongniartii} (33,926
bp). All four mitogenomes encoded 14 core PCGs involved in energy
metabolism, \emph{rps3} involved in translation, and 2 rRNAs. The PCG
lengths varied, but the protein coding regions themselves were
conserved. The tRNA numbers of the four mitogenomes ranged from 25
(\emph{C. brongniartii} ) to 27 (\emph{O. sinensis} ). The tRNAs
comprised those for all 20 amino acids and ranged in size from 71 to 86
nucleotides (Table S1). The nucleotide composition of the \emph{O.
xuefengensis} mitogenome was identical to that of \emph{O. sinensis} ,
but not to \emph{C. militaris} and \emph{C. brongniartii} . The \emph{O.
sinensis} mitogenome had the highest GC content (30.20\%), followed by
those of \emph{O. xuefengensis}(29.94\%), \emph{C. brongniartii}
(27.34\%), and \emph{C. militaris}(26.79\%). The high frequency of A and
T usage in codons contributes to the high AT content of the four
mitochondrial genomes, with AT content reaching 70.06\% in \emph{O.
xuefengensis} , 69.80\% in \emph{O. sinensis} , 73.21\% in \emph{C.
militaris} , and 72.66\% in \emph{C. brongniartii} . Both GC skew and AT
skew were positive for all four mitogenomes (Table 1).

The \emph{O. sinensis} mitogenome exhibited an unusually enlarged size
(157,559 bp). The compositions of regions were thus analyzed for the
mitogenomes, indicating that the protein coding regions among the four
mitogenomes are basically identical. A significant difference was
observed when comparing intronic and intergenic regions, wherein the
total number of intronic nucleotides of the \emph{O. sinensis}
mitogenome was much higher than for those of the other cordyceps, and
the total number of intergenic nucleotides of the\emph{O. xuefengensis}
mitogenome is the highest among the four mitogenomes (Fig 2).

The number of introns also significantly differed among the four
mitogenomes. Fifty-four introns were present in the mitogenome
of\emph{O. sinensis} , 18 in that of \emph{O. xuefengensis} , 8 in that
of\emph{C. militaris,} and 6 in that of \emph{C. brongniartii} (Table.
1). Further, the host genes of introns also differ among the four
cordyceps. Introns are distributed in the \emph{cob} (no. of introns:
1),\emph{cox1} (1), \emph{cox2} (1), \emph{cox3} (1), and \emph{rnl} (4)
genes of the \emph{C. militaris} mitogenome, and in the \emph{cob}
(1),\emph{cox1} (2), \emph{nad1} (1), and \emph{rnl} (2) genes of
the\emph{C. brongniartii} mitogenome. In \emph{O. xuefengensis} , the
introns are distributed in \emph{cob} (2), \emph{cox1} (3),
\emph{cox2}(1), \emph{cox3} (3), \emph{nad1} (2), \emph{nad2} (2),
\emph{nad5} (1),\emph{rnl} (3), and \emph{rns} (1) genes, while in the
\emph{O. sinensis} mitogenome, the introns are distributed in the
\emph{cob} (6), \emph{cox1} (14),\emph{cox2} (6), \emph{cox3} (2),
\emph{nad1} (3), \emph{nad2} (2),\emph{nad4L} (1), \emph{nad5} (5),
\emph{nad6} (1), \emph{atp6} (2),\emph{atp9} (1), \emph{rnl} (8), and
\emph{rns} (1) genes (Table S2). The most common intron type among the
mitogenomes are group IB introns. Introns also harbor 0--2 homing
endonuclease genes, including the LAGLIDADG homing endonuclease and the
GIY-YIG endonuclease.

Table 1. Characteristics of cordyceps mitogenomes.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Characteristic & \emph{Ophiocordyceps xuefengensis} &
\emph{Ophiocordyceps sinensis} & \emph{Cordyceps militaris} &
\emph{Cordyceps brongniartii}\tabularnewline
\midrule
\endhead
Size (bp) & 78,744 & 157,559 & 33,277 & 33,926\tabularnewline
A (\%) & 36.08 & 36.70 & 36.98 & 36.51\tabularnewline
T (\%) & 33.98 & 33.10 & 36.23 & 36.15\tabularnewline
G (\%) & 16.33 & 15.70 & 15.20 & 15.13\tabularnewline
C (\%) & 13.61 & 13.70 & 11.59 & 12.21\tabularnewline
Rate of GC (\%) & 29.94 & 30.20 & 26.79 & 27.34\tabularnewline
Rate of AT (\%) & 70.06 & 69.80 & 73.21 & 72.66\tabularnewline
AT-skew & 0.03 & 0.05 & 0.01 & 0.005\tabularnewline
GC-skew & 0.09 & 0.09 & 0.14 & 0.11\tabularnewline
PCGs (n) & 14 & 14 & 14 & 14\tabularnewline
Introns (n) & 18 & 54 & 8 & 6\tabularnewline
Intronic ORFs (n) & 16 & 45 & 8 & 5\tabularnewline
GIY-YIG (n) & 4 & 10 & 4 & 4\tabularnewline
LAGLIDADG (n) & 9 & 27 & 3 & 1\tabularnewline
rRNAs (n) & 1 & 1 & 1 & 1\tabularnewline
nRNAl (n) & 1 & 1 & 1 & 1\tabularnewline
tRNAs (n) & 26 & 27 & 26 & 25\tabularnewline
PCG regions (bp) & 34,994 & 104,257 & 17,735 & 18,329\tabularnewline
Intronic regions (bp)\textsuperscript{a} & 21,146 & 107,288 & 9,812 &
8,452\tabularnewline
Exon regions (bp)\textsuperscript{b} & 13,848 & 13,287 & 12,978 &
13,098\tabularnewline
tRNA genes (bp) & 1,934 & 2,010 & 1,924 & 1,867\tabularnewline
rRNA genes (bp)\textsuperscript{c} & 7,225 & 7,144 & 4,666 &
4,801\tabularnewline
Intergenic regions (bp) & 34,591 & 27,831 & 3,797 & 5,708\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}: Introns in PCGs and rRNA; \textsuperscript{b}:
Exons in PCGs; \textsuperscript{c}: \emph{rnl} and \emph{rns} including
exons.

3.2. Protein-coding genes and codon usage among the four mitogenomes

Most PCGs and \emph{rps3} are initiated with the ATG codon,
whereas\emph{cox1} and \emph{nad6} in the \emph{O. xuefengensis}
mitogenome are initiated with the TTG codon. Further, \emph{cox3} in the
\emph{O. sinensis} mitogenome and \emph{atp9} in both \emph{O.
xuefengensis} and\emph{O. sinensis} mitogenomes are initiated with the
GTG codon. In addition, most PCGs and \emph{rps3} are terminated with
the TAA codon, but \emph{cox1} , \emph{rps3,} and \emph{cox3} in the
\emph{O. sinensis}mitogenome, and \emph{cox1} and \emph{cob} of the
\emph{O. xuefengensis}mitogenome are terminated with the TAG codon
(Table S3). Comparative analysis indicated that all the PCGs and
\emph{rps3} encode 20 amino acids, with similar amino acid compositions
among the cordyceps mitogenomes. The most overused amino acid is Leu
(ranging in prevalence from 628 to 652), whereas the least used amino
acid is Cys (32 -- 48 residues). The usage of Asn, Lys, Asp, and Glu in
the \emph{O. xuefengensis}mitogenome is much higher than in those of the
other three cordyceps (Fig. 3, Table S4). The relative synonymous codon
usage (RSCU) of the 14 PCGs and \emph{rps3} in the four cordyceps
mitogenomes was also analyzed. A total of 4,616, 4,429, 4,326 and 4,366
codons were observed among the PCGs and \emph{rps3} of the \emph{O.
xuefengensis, O. sinensis, C. militaris,} and \emph{C. brongniartii}
mitogenomes, respectively. The most used codon among all four
mitogenomes is UUA, but the most frequently used codon is AGA in the
\emph{O. xuefengensis} and\emph{O. sinensis} mitogenomes (Fig. 4, Table
S5).

3.3. Variation, genetic distance, and evolutionary rates of common genes

The lengths and/or GC contents of the 14 core PCGs and rps3 are not all
consistent across the four mitogenomes, with the exception of atp8,
atp9, and nad4L PCGs. In particular, the length of nad5 in O.
xuefengensis is about 1,000 bp longer than in the other three cordyceps
mitogenomes. Further, the GC content of all 14 PCGs and rps3 differ,
indicating that the core PCGs and rps3 have been modified within
different cordyceps. Across all PCGs in the mitogenomes, GC content is
highest in atp9 within all four cordyceps mitogenomes, and lowest in
atp8 within the O. xuefengnensis and O. sinensis mitogenomes, but lowest
in rps3 within the C. militaris and C. brongniartii mitogenomes. AT skew
is negative in most PCGs except rps3, while GC skew is present in most
PCGs except atp8. In particular, the AT skew of atp6 in the C. militaris
mitogenome and nad6 in the O. xuefengnensis mitogenome are negative,
while the GC skew of only rps3 in the C. brongniartii mitogenome is
negative (Fig. 5).

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 \textless{} 1, suggesting that these genes are subject to
purifying selection (Fig. 6, Table S6).

3.4. Repetitive elements

A total of 18 repeat sequences were identified in the mitogenome of O.
xuefengensis, 250 in that of O. sinensis, 6 in that of C. militaris and
23 in that of C. brongniartii (Table S7). The length of repeat sequences
ranges from 38 bp to 372 bp, with pairwise nuleotide similarities
ranging from 74.73\% to 100\%. The largest repeat region was observed in
the O. sinensis mitogenome, within ORF322 and ORF308. The largest repeat
region in the O. xuefengensis mitogenome is 220 bp long and located in
the intronic region of cox2 and ORF301, while the largest repeat region
in the C. militaris mitogenome is 259 bp long and located in the
intronic region of rnl. Lastly, the largest repeat region in the C.
brongniartii mitogenome is 191 bp long and located between cox1 and
trnR.

Seven tandem repeats were detected in both the O. xuefengensis and C.
brongniartii mitogenomes, 43 in the O. sinensis mitogenome, and 5 in the
C. militaris mitogenome (Table S8). The longest tandem sequence is found
in the O. sinensis mitogenome, comprises 123 bp, and is located in the
in the ORF138 coding region. Of all tandem repeats in the four
mitogenomes, most exist in one or two copies. REPuter identified forward
(F) and palindromic (P) repeats in the mitogenome of each cordyceps
(Table S9), including 69 F and 34 P repeates in the mitogenome of O.
xuefengensis that account for 10.16\% of the total mitogenome, along
with 16 F and 1 P repeats in the C. militaris mitogenome that account
for 4.95\% of its total mitogenome. In addition, 38 F and 19 P repeats
were identified in the C. brongniartii mitogenome that account for
14.54\% of its total mitogenome, and a total of 754 F, 538 P, 31 C, and
60 R repeats were identified in the O. sinensis mitogenome that account
for 65.55\% of its total mitogenome.

3.5. 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
cordycep mitogenomes. The O. xuefengensis mitogenome only lacks trnI,
consistent with that of O. sinensis (Fig. 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, with the sizes and relative
positions of these homologous regions substantially differing among the
four species. Mitogenomic rearrangements were not observed among the
four species (Fig. 8).

A complete mitogenome was produced for O. xuefengensis in this study and
used with 19 other complete mitogenome sequences from six families for
phylogenetic reconstruction to further investigate the phylogenetic
position of O. xuefengensis within the Hypocreales order. Species and
NCBI accession numbers used in these phylogenetic analyses are listed in
Supplementary Table S10. Bayesian inference (BI) based on mitochondrial
gene datasets using the GTR + I + G nucleotide substitution model
yielded an identical and well-supported topology in which all major
clades are well supported. The 20 fungal species comprised six major
clusters, corresponding to the major groups of the orders Hypocreales
and Agaricomycetes. The four cordyceps species were divided into two
groups, wherein the close relationship between O. xuefengensis and O.
sinensis was highly supported (Fig. 9).

4. Discussion

Cordyceps are fungal species used in traditional medicine that are
represented by the well-known fungal resource, \emph{O.
sinensis.}However, natural populations of \emph{O. sinensis} have been
over harvested to the extent that it is an endangered species. It is
consequently necessary to explore alternative cordyceps resources,
because artificial cultivation of \emph{O. sinensis} is difficult to
achieve {[}15{]}. \emph{O. 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 {[}14{]}. 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 {[}16-18{]}. It is difficult to accurately classify fungal
species based on limited morphological characters and overlapping
morphological features {[}19{]}. 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 {[}20{]}. Here, we report
a new mitogenome sequence for \emph{O. xuefengensis} that could provide
new insights into the evolutionary histories of cordyceps fungal
mitogenomes and the taxonomic relationships among cordyceps.

Mitogenome sizes significantly differ among fungi, ranging from
approximately 11.09 kbp (\emph{Hanseniaspora uvarum} ) {[}21{]} to 272.2
kbp (\emph{Morchella importuna} ) {[}22{]} in length. The variation in
size of mitogenomes among different fungal species primarily results
from the presence and extent of intergenic regions and introns {[}23{]}.
The Ascomycota cordyceps fungus \emph{O. sinensis} contained a
mitogenome with introns accounting for 68.09\% of the whole mitogenome,
while introns in the mitogenomes of\emph{O. xuefengensis,C. militaris,}
and \emph{C. brongniartii} only accounted for 26.85\%, 29.49\% and
24.91\% of their mitogenomes, respectively. Further, intergenic regions
within the\emph{O. xuefengensis} mitogenome accounted for 43.93\% of the
whole mitogenome, but only 11.41\%-17.66\% of the mitogenomes of the
other three cordyceps species. Thus, considerable variation in the size
of introns and intergenic regions are the major reasons underlying the
significant differences in the mitogenome sizes of\emph{O. sinensis} and
\emph{O. xuefengensis} . In contrast, \emph{C. militaris} and \emph{C.
brongniartii} exhibited similarly sized mitogenomes, due to similar
numbers and sizes of intergenic regions and introns.

However, the large difference in introns did not affect the
taxonomically close relationship between \emph{O. sinensis} and \emph{O.
xuefengensis.} Maximum Likelihood and Bayesian phylogenetic analyses
were constructed using 14 PCGs and \emph{rps3} from 20 fungi to reveal
the taxonomic relationships of these fungi. The phylogenetic analysis
highly supported the close relationship between \emph{O.
xuefengensis}and \emph{O. sinensis} . This apparent domestication of
ancestral introns may indicate an adaptation to their host genome that
may be due to their decreased mobility {[}24{]}, or alternatively, to
their potential important role in stabilizing the gene that hosts the
intron {[}25, 26{]}. Other introns may be acquired late in the
divergence of taxa, either through HGT events or through active
transposition {[}27, 28{]}. The transposition of some introns to other
genomic regions with less sequence similarity can occur more frequently
under stress-induced conditions {[}29, 30{]}. A recent study
demonstrated that the number and Pcl of introns are highly variable
between two \emph{Rhizopogon} species {[}31{]}. Further, the introns in
nuclear genes of \emph{S. cerevisiae}play crucial roles in the survival
of the organism under starvation conditions {[}32{]}. Consequently, the
abundance of introns in the mitochondrial genome of \emph{O. sinensis}
relative to other cordyceps species may be a consequence of the
particular environments they inhabit.

The accumulation of repeated evolutionary events in fungal mitogenomes
could lead to over-dispersal of repeat sequences and the introduction of
new genes through HGT, thereby contributing to dynamic changes in genome
structure and gene order {[}33{]}. Moreover, the accumulation of repeat
sequences could also be highly related to gene recombination and gene
loss {[}34{]}. A variety of models have been proposed to investigate
these mitochondrial gene rearrangements {[}35{]}. Here, the mitogenome
of \emph{O. sinensis} harbored a larger proportion of repeat sequences
than the mitogenomes of \emph{O. xuefengensis} , \emph{C. militaris}
or\emph{C. brongniartii} . Interestingly, collinearity analysis revealed
that the mitogenomic gene order in these four fungi was not highly
variable. In addition, positive selection of core genes was not observed
by Ka/Ks analysis. This differs from observations in plants, wherein
mitochondrial gene order is highly variable due to high rates of
recombination {[}33{]}. 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 remain in early stages,
consistent with the molecular phylogenetic analyses. One reasonable
explanation for these contradictory results is that the larger
proportion of repeat sequences in the genome of \emph{O. sinensis}exists
in intronic regions, but not in intergenic regions, leading to little
influence on their evolution.

Mitochondrial genomes are obtained from a common ancestor and have been
widely used in population genetics and evolutionary studies {[}36,
37{]}. Different barcode genes, including ITS, LSU, RPB2, and EF1\selectlanguage{greek}α, \selectlanguage{english}have
been used for phylogenetic analysis {[}9, 38{]}. However, the large
number of available molecular markers in mitogenomes and their
independent evolutionary histories make them attractive tools for
reconstructing phylogenetic relationships {[}39{]}. Here, we
reconstructed a well-resolved phylogeny based on the combined alignment
of 14 PCGs and the \emph{rps3} gene that separated 20 fungal species
into major clades. In addition, K2P and Ka/Ks analysis indicated
that\emph{cox1} and \emph{rps3} divergence play important roles in
population differentiation. Given that reliable genetic molecular
markers are critical for further understanding the phylogenetics and
classification of species, these two genes could be further evaluated as
barcodes for fungal species identification.

5. Conclusions

This study extends the known mitogenomes of cordyceps fungi and
identified variation in PCGs, tRNA genes, and rRNA genes among medicinal
cordyceps species, including \emph{O. xuefengensis} . Comparative
mitochondrial genome analysis of four cordyceps fungi were 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 \emph{cox1} and
\emph{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.

\textbf{Supplementary Materials:} The following materials are available
online. Table S1: General tRNA features in the four cordyceps
mitogenomes. Table S2: The number of introns and their host genes. Table
S3: Complete mitochondrial genome characteristics for the four cordyceps
analyzed here. Table S4: Amino acid composition of mitogenomes among the
four cordyceps species. Table S5: The RSCU of 14 PCGs and \emph{rps3} in
the four cordyceps mitochondrial genomes. Table S6: Genetic analysis of
14 core PCGs and \emph{rps3} among the four cordyceps genomes. Table S7:
Local BLAST analysis of the four cordyceps mitogenomes against
themselves. Table S8: Tandem repeats detected in the mitogenomes of
cordyceps using the Tandem Repeats Finder program. Table S9:
Distribution of repeat loci in the four cordyceps mitogenomes identified
by REPuter. Table S10: The GenBank accessions for genomes used in the
phylogenetic analysis.

\textbf{Author Contributions:} Conceptualization: C.Z., S.X., and. S.Z.
Methodology: J.J., C.Z., R.Z., and H.L. Software analysis: C.Z., J.J.,
and J.X. Investigation: R.Z. Resources: C.Z. and H.L. Data curation:
C.Z., J.J., and J.X. Writing (original draft preparation): C.Z. Writing
(review and editing): S.Z., J.J., and S.X. Visualization: J.X. Project
administration: D.W. Funding acquisition: S.Z. All authors have read and
agreed to the published version of the manuscript.

\textbf{Funding:} This research was funded by the National Natural
Science Foundation of China (No. 81673585), the Natural Science
Foundation of Hunan Province (2018JJ3309, 2020JJ5330), the Program of
Survey and Monitoring of Chinese Medicines for National Drugs
({[}2017{]} 66) and the Key Project at the Central Government Level for
the Ability Establishment of Sustainable Use for Valuable Chinese
Medicine Resources (2060302).

\textbf{Conflicts of Interest:} The authors declare no conflicts of
interest.

\textbf{Acknowledgment}

We thank LetPub (\emph{www.letpub.com}) for its linguistic assistance
and scientific consultation during the preparation of this manuscript.

Fig. 1. Circular maps of the mitochondrial genomes of four cordyceps
species. Genes are represented by different colored blocks. Colored
blocks outside each ring indicate that the genes are on the direct
strand, while colored blocks within the ring indicate that the genes are
located on the reverse strand.

Fig. 2. The abundances of protein-coding, intronic, intergenic, and RNA
gene regions (rRNAs and tRNAs) of the entire mitochondrial genomes from
four cordyceps species.

Fig. 3. Codon usage in the mitochondrial genomes of the four cordyceps
species. Codon numbers are plotted on the y-axis for A, Ophiocordyceps
xuefengensis; B, Ophiocordyceps sinensis; C, Cordyceps militaris; D,
Cordyceps brongniartii.

Fig. 4. RSCU values for 14 PCGs and \emph{rps3} in the mitochondrial
genomes of four cordyceps.

Fig. 5. Variation in the length and base composition of each of the 14
PCGs and rps3 among four cordyceps mitogenomes. A, PCG length variation;
B, GC content across PCGs; C, AT skew; D, GC skew.

Fig. 6. Genetic analysis of 14 PCGs and \emph{rps3} across four
cordyceps mitogenomes. K2P, the overall mean Kimura-2-Parameter
distance; Ka, the mean number of nonsynonymous substitutions per
nonsynonymous site; Ks, the mean number of synonymous substitutions per
synonymous site.

Fig. 7. Gene order comparison among four cordyceps mitogenomes.

Fig. 8. Colinearity analysis of four cordyceps mitogenomes, generated
with Mauve 2.4.0. Two homologous regions were detected across the four
mitochondrial genomes overall.

Fig. 9. Molecular phylogeny of 20 fungal species based on Bayesian
inference (BI) and Maximum likelihood (ML) analysis of 14 PCGs
and\emph{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 Supplementary Table S10.

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