Complete mitochondrial genome revealed different taxonomic assignment of the fungus Pleurocordyceps sinensis (Hypocreales, Ascomycota)

Abstract

The complete mitochondrial (mt) genome of Pleurocordyceps sinensis, a fungus originally isolated from Ophiocordyceps sinensis was sequenced, and assembled as a single circular DNA of 31,841 bp. The mt genome encoded 15 conserved proteins (rps3, cox1, cox2, cox3, cob, atp6, atp8, atp9, nad1, nad2, nad3, nad4, nad4L, nad5 and nad6), 2 rRNA (rnl and rns) and 25 tRNA, as well as 10 additional non-conserved open reading frames. Comparative analyses showed that mt genomes within the order Hypocreales encoded same number and synteny of conserved protein coding genes despite an obvious size variation among this group of fungi. Phylogenetic analyses using 14 conserved protein sequences revealed that this fungus may not belong to the current designated family Ophiocordycipitaceae but is more closely related to the species of Clavicipitaceae. The mt genome presented herein would aid to clarify the phylogenetic position of species of Polycephalomyces s. l. and also gave valuable information on reconstructing the evolutionary history of clavicipitaceous fungi.

Introduction

Pleurocordyceps sinensis (Q.T. Chen & et al.) Y.J. Yao & et al. was first isolated from the sclerotium of Ophiocordyceps sinensis collected in Kangding, Sichuan, China in June 1980, and was described as a new species named Paecilomyces sinensis Q.T. Chen & et al. (Chen et al. 1984). The species gained broad scientific attention since 1980s in China though only one authentic strain was isolated (termed CN80-2). Large numbers of pharmacological studies have been carried out using this strain. The species was reported to have various pharmacological activities such as anti-implantation (Lin et al. 1988), anti-inflammatory (Li et al. 1983), anti-oxidant (Liu et al. 1987; Liu et al. 1989; Liu et al. 1991), anti-tumor (Wu et al. 1986; Huang et al. 1988), fertility regulation (Li and Lin 1991), immune modulation (Zheng et al. 1983; Lin et al. 1987; Ge et al. 1989; Zhang et al. 1998), and treating conditions of coronary arteriosclerotic heart disease (You et al. 1986) and immunological liver injury (Zeng et al. 2000; Cheng et al. 2005). Because of the similarities in the pharmacological effects and chemical components compared with O. sinensis, Pleurocordyceps sinensis has once been recognized as the possible anamorph of the later. This viewpoint was widely accepted and cited when summarizing the anamorph of O. sinensis (Liu 1990; Fang 1991; Liang 1991). The idea of using P. sinensis as a substitute of O. sinensis was thus proposed (Li et al. 1983; Zeng et al. 2000; Cheng et al. 2005). However, several independent researches based on molecular evidences rejected the anamorph-teleomorph relationship between P. sinensis and O. sinensis (Zhao et al. 1999; Li et al. 2000; Chen et al. 2001; Jiang and Yao 2002; 2003). Wang et al. (2012) placed the species in Polycephalomyces based on morphological and molecular analysis and designated Polycephalomyces group (Polycephalomyces s. l.) as a new clade of clavicipitaceous fungi and stated that this new clade is distinct from the known families of Hypocreales. Thus, Pleurocordyceps sinensis is based on Paecilomyces sinensis with Polycephalomyces sinenses as another synonym; they all refer to the same species.

The genus Polycephalomyces was erected by Kobayasi (1941) with P. formosus as the type. Only three species, i.e., P. paludosus, P. cylindrosporus and P. tomentosus, were described in the last century (Mains 1948; Samson et al. 1981; Seifert 1986). Until recently, after the recombination of Paecilomyces sinensis (Wang et al. 2012) and several species of Cordyceps s. l. into this genus (Kepler et al. 2013), more and more new species were discovered and described, especially from China and Southeast Asia (Wang et al. 2015a; Wang et al. 2015b; Crous et al. 2017; Xiao et al. 2018; Yang et al. 2020). A total of 24 species name are currently recorded by the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp), among which 4 were segregated from the genus to Perennicordyceps (Matočec et al. 2014). Although the ‘Polycephalomyces clade’ (Polycephalomyces sensu lato) may represent a family level clade (Kepler et al. 2013; Wang et al. 2021), thus, more evidence is required to resolve this issue. Recently, Wang et al. (2021) proposed a new genus Pleurocordyceps for one of the subclade within the ‘Polycephalomyces clade’ based on morphological and molecular analyses. Ten species were included in the new genus including P. sinensis. The taxonomic position of species of Polycephalomyces based on the type P. formosus was not resolved (Kepler et al. 2013), though species of this group were tentatively placed in Ophiocordycipitaceae in Index Fungorum.

Mitochondria play various essential roles in eukaryotic cells, including respiratory metabolism, energy production, calcium homeostasis and also involved in cell death and aging (Basse 2010). Mitochondrial (mt) genomes usually have a rapid rate of evolution compared with nuclear genomes, and thus considered as powerful tools in evolutionary biology (Chris et al. 1994; Berbee and Taylor 2001). A previous study revealed that the gene contents and synteny of mt genomes of hypocrealean species were largely conserved, but in the meantime, the genome sizes expanded greatly in species such as Ophiocordyceps sinensis (Li et al. 2015). Complete mt genomes have been reported for a number of species of the three clavicipitaceous fungal families, i.e., Cordycipitaceae (Kouvelis et al. 2004; Sung 2015; Fan et al. 2019; Zhang et al. 2021), Ophiocordycipitaceae (Li et al. 2015; Zhang et al. 2016; Zhang and Zhang 2020; Abuduaini et al. 2021) and Clavicipitaceae (Winter et al. 2018; Sun et al. 2021), while not reported for species of the ‘Polycephalomyces clade’ so far.

In this study, the complete mt genome of the type strain (CN 80 − 2) of the species P. sinensis was sequenced, and genome characteristics were compared with other hypocrealean species. Phylogenetic analyses were also performed using 14 conserved protein sequences to resolve the taxonomic position of P. sinensis and to clarify the phylogenetic relationship between species of the ‘Polycephalomyces clade’ and other clavicipitaceous fungal groups.

Materials And Methods

Fungal isolation and cultivation

The ex-type strain (CN80-2) of Pleurocordyceps sinensis used in this study was isolated from a sclerotium of O. sinensis collected in Kangding, Sichuan, China in June 1980 (Chen et al. 1984). Stock strain was maintained at 4°C on Potato Dextrose Agar (PDA) slants. Seed cultures were grown in 250-ml Erlenmeyer flasks, containing 50 ml liquid potato-dextrose medium, shaking 100 rpm at 25°C for 10 d. Mycelia were harvested, washed with distilled water using vacuum filtration to remove extracellular polysaccharides, frozen with liquid nitrogen, and then vacuum freeze dried using a freeze dryer. Dried mycelia were then sent to the genome sequencing company.

DNA extraction and genome sequencing

Genomic DNA was extracted using TIANamp Yeast DNA Kit (TIANGEN Biotech Co., Ltd., Beijing, China) according to the manufacturer's instruction. The amount and quality of total DNA was visualized by 1% agarose gel electrophoresis and quantified with a Qubit2.0^® Fluorometer (Life Technologies, New York, USA). A 20 K library was prepared from sheared genomic DNA (containing both mitochondrial and nuclear sequences) using a 20-Kb template library preparation workflow. Twelve SMRT cells were sequenced on PacBio RS II sequencing platform (Pacific Biosciences, Menlo Park, CA) with P6 polymerase and C4 sequencing chemistry at Tianjin Biochip Corporation (Tianjin, China).

Mitochondrial genome assembly and annotation

Mitochondrial genome of the strain CN80-2 was assembled and annotated following a procedural described in Li et al. (2015). The adapter sequences, reads with length < 50 bp or average quality < 0.75 (defined as low-quality) were filtered before assembling. The mt sequences were extracted from filtered reads, matching each read against the fungal mitochondrial genome database (https://www.ncbi.nlm.nih.gov/genome/browse#!/organelles/), preassembled and corrected using BLASR (Chaisson and Tesler 2012). Corrected reads were retained and then re-assembled with the Celera Assembler program (Myers et al. 2000) and assembly was further refined with Quiver (Chin et al. 2013). A circular double stranded DNA was finally obtained and proceeded to an online annotation tool MFannot using the Mold, Protozoan, and Coelenterate Mitochondrial Code (Beck and Lang 2010). The annotated mt genome was submitted to GenBank under the accession number OK017430. The annotated mt genetic map was generated with Circos software (Krzywinski et al. 2009) and modified by Adobe Illustrator^® CS5 (Version 15.0.0, Adobe^®, San Jose, CA).

Phylogenetic analyses and comparative genomics

All the 82 complete mt genomes available from GenBank (accessed on 28 March, 2021) within the order Hypocreales were downloaded and used for phylogenetic and comparative genomic analysis. Three mt genomes from two species of Colletotrichum lindemuthianum (NC_023540) and Verticillium dahlia (NC_008248 and CM019738) which belongs to the order Glomerellales were used as outgroups (Table S1). Phylogenetic tree was constructed using 14 conserved protein-coding genes including cox1, cox2, cox3, cob, atp6, atp8, atp9, nad1, nad2, nad3, nad4, nad5, nad4L and nad6. Protein sequences were aligned using BioEdit version 7.0.9.0 (Hall 1999) and manually refined. Phylogenetic analyses using maximum likelihood (ML) were performed with the LG substitution matrix and default parameters using RAxML v.7.2.659 (Stamatakis 2006). Bootstrap values were calculated with 1000 re-sampling iterations using an approximate likelihood ratio test. Ophiocordyceps camponoti-floridani EC05 (CM022976) was only used for genome comparison while not included in phylogenetic analyses as it was probably not well assembled, and an un-classified mt genome (NC_049089) was also excluded due to the large numbers of possible sequencing errors or assembly mistakes. A number of mt genomes were found to be incorrectly or incompletely annotated. For example, the rps3 gene was usually not predicted certain species. Those genomes were re-checked with missing genes replenished and wrongly predicted genes manually corrected.

The contents and synteny of the 15 conserved protein coding genes of mt genomes were compared within the order Hypocreales and related outgroup species.

Results

Genome sequencing and assembly

A total of 23,757 reads (171,039,810 bp) were identified as mitochondrial among 601,168 reads (5,005,308,071 bp) of the raw sequencing output for the whole genome of Pleurocordyceps sinensis. The lengths of the putative mitochondrial reads ranged from 276 bp to 45,245 bp with an average length of 7,200 bp, reaching a coverage depth of 5,371× over the mt genome of the species. The mitochondrial reads were passed through the program BLASR and assembled with Celera Assembler program and Quiver, resulting in a circular DNA of 31,841 bp (Fig. 1).

Conserved protein genes and non-conserved open reading frames (ncORFs)

The mt genome of Pleurocordyceps sinensis had a low GC content of 25.46% and encoded 15 protein genes conserved within the order Hypocreales (Li et al. 2015) including seven subunits of the electron transport complex I (nad1, nad2, nad3, nad4, nad4L, nad5 and nad6), cytochrome b (cob), three subunits of complex IV (cox1, cox2 and cox3), three F0 subunits of the ATP-synthase complex (atp6, atp8 and atp9) and the rps3 gene which encodes 40S ribosomal protein S3 (Table 1, Fig. 1). In addition to those genes, 10 ncORFs (7,194 bp totally in length) were also predicted, among which two (ncORF3 and ncORF9) were found to encode homing endonucleases with motif patterns GIY-YIG and LAGLIDADG, respectively (Table 1).

All conserved protein coding genes and ncORFs were found on the positive strand and oriented clockwise except for ncORF1 and ncORF2 which were on the negative strand and anticlockwise oriented. As found in Pleurotus ostreatus (Wang et al. 2008), Rhizoctonia solani (Losada et al. 2014) and Ophiocodyceps sinensis (Li et al. 2015), the nad2/nad3 genes were joined and nad4L/nad5 genes were fused, i.e., the initial codon of the nad3 gene (ATG) followed the terminal codon of the nad2 gene (TAA); the terminal codon of nad4L (TAA) uses the same nucleotide A with the initial codon (ATG) of nad5 (Fig. 1, Table 1). Other protein coding genes and ncORFs were separated by either long or short intergenic regions (Fig. 1).

All the 15 protein-coding genes and 10 predicted ncORFs employed the standard fungal mitochondrial start codon ATG, except cox1 and ncORF10, which were initiated by ATA. In addition, 24 of those genes used TAA as the stop codon except ncORF8 which used TAG (Table 1).

Noncoding RNAs

In addition to the 15 protein-coding genes, a large and a small ribosomal RNA (rnl and rns) and 25 tRNA genes corresponding to 20 amino acids were also identified (Table 1). The tRNA genes ranged in size from 71 to 87 bp. A majority of amino acids were coded by only one tRNA gene; however, Serine (Ser), Arginine (Arg), Methionine (Met) and Leucine (Leu) had 2, 2, 3 and 2 tRNA genes, respectively (Table 1). All noncoding RNAs (tRNA, rRNA) were found on the positive strand and oriented clockwise.

Intronic and intergenic regions

Exons of protein-coding genes, rRNA and tRNA genes had a total length of 20,873 bp accounting for 65.55% of the mt genome. Ten ncORFs (7,194 bp) accounted for 22.59% of the mt genome. Two group I introns were predicted, including one further classified into subgroup IA (1,643 bp) in rnl and one classified into subgroup IB in cox1 (1,036 bp) respectively, making up 8.5% of the entire mito-genome. The intergenic sequences had a total length of 1,070 bp covering 3.4% of the genome.

Gene component and synteny

Although different numbers of ncORFs (hypothetical proteins) would be predicted for hypocrealean fungi, the content and synteny of 15 protein coding genes (rps3, cox1, cox2, cox3, cob, atp6, atp8, atp9, nad1, nad2, nad3, nad4, nad5, nad4L and nad6) remained largely conserved in this order, except in a few species. As predicted, location of the cox2 gene was shifted in three species of Acremonium chrysogenum, A. fuci and Clonostachys rose compared to other hypocreales (Table S1). An additional copy of rps3 and atp9 gene was found in Beauveria malawiensis and Fusarium solani IISc-1 (CM023198), respectively. In F. oxysporum UASWS AC1 (KR952337), an extra copy was found for nad1 and nad4, while in F. oxysporum f. sp. matthiolae (CM019668), the location of the two genes were found to be reversed. The mt genome of F. oxysporum f. sp. fragariae GL1381 (CM029251) was found to lose cox3 and nad6 genes and possess an extra reversed copy of genes of cob, cox1, nad1, nad4, atp8 and atp6. An extreme case was found in Sarocladium implicatum in which three genes (cob, cox3 and nad6) were lost and the nad4 gene shifted its location from the nad1-atp8 junction to a position between rps3 and nad2 (Table S1).

Phylogenetic analyses

Eighty-one complete mt genome representing 63 distinct species from the order Hypocreales were included in phylogenetic analyses. After excluding the ambiguous aligned regions, a total of 4345 amino acid sequences of 14 conserved proteins were retained and used. All species of Hypocreales formed a well-supported clade (BP = 100%) in ML analysis. Within the clade, four family level subclades were recognized with very strong supports (BP = 100%), i.e. Nectriaceae, Bionectriaceae, Hypocreaceae and Clavicipitaceae (Fig. 2). Species in the family Ophiocordycipitaceae were clustered into two subclades, one subclade that consists of four Tolypocladium species showed a sister group relationship with the Clavicipitaceae clade with low bootstrap support (BP = 75%), the other highly supported (BP = 100%) subclade comprised of four Hirsutella species (H. minnesotensis, H. rhossiliensis, H. thompsonii and H. vermicola) and Ophiocordyceps sinensis (Fig. 2). It is interesting to find that Pleurocordyceps sinensis was clustered with the Clavicipitaceae clade with 100% bootstrap support, while not grouped with either two subclades of Ophiocordycipitaceae.

Discussion

The 31,841 bp complete mt genome from the ex-type strain CN 80 − 2 of the species Pleurocordyceps sinensis described here is the first report of the complete mt genome of the new genus Pleurocordyceps and the proposed ‘Polycephalomyces clade’ (Wang et al. 2012; Kepler et al. 2013; Wang et al. 2021). The mt genome sizes varied greatly in hypocrealean fungi, from 22,376 bp of Sarocladium implicatum (Yao et al. 2016) to 272,497 bp for Ophiocordyceps camponoti-floridani (Will et al. 2020). The mt genome of P. sinensis is rather compact compared with other Hypocreales species, especially Ophiocordyceps sinensis, from which P. sinensis was isolated. The genome sizes of the two sequenced isolates of O. sinensis were 157,510 bp (KP835313) and 157,539 bp (NC_034659), respectively, almost five times larger than P. sinensis. Despite the size variation of mt genome, the gene contents and synteny (gene order) are largely conserved within the order Hypocreales, generally encoding 15 known proteins and 2 rRNAs (rnl and rns) (Li et al. 2015). The observed genome size variation in hypocrealean fungi was probably not associated with taxonomic classification since notable variation was also observed within the genus Fusarium (30,629 bp to 110,525 bp) or even within species F. oxysporum (34,477 bp to 52,424 bp) (Table S1), but largely due to the presence of various introns and the lengths of intergenic regions (Burger et al. 2003).

Intronic ncORFs, particularly those encoding mobile elements of reverse transcriptases (RTs) and homing endonucleases (HEs) were considered as the main drive of mt genome expansion (Li et al., 2015). Zhang et al. (2015) compared mt genomes of three isolates of Cordyceps militaris, and found that larger genomes usually contained more introns and more intronic HEs genes. In this study, only two introns (subtype IA of 1643 bp in rnl and subtype IB of 1036 bp in cox1) and one intronic nrORFs (ncORF10) was recognized, comparing to the 52 introns and 49 intronic nrORFs observed in O. sinensis (Li et al. 2015), those differences between the two species could interpret the compact small mt genome size of P. sinensis. Of the two types of mobile elements, RTs were usually encoded in group II introns whereas HEs genes were almostly found in group I (Lang et al. 2007). As in O. sinensis, all 32 HEs genes (21 LAGLIDADG and 11 GIY-YIG endonuclease) were located in group I introns with the 10 RTs genes included in group II (Li et al. 2015). Megarioti and Kouvelis (2020) recently proposed an “aenaon” model for the evolution of HEs genes and their host introns, thus, free-standing introns and HEs genes were the ancestral form and could invade intron-free coding genes together; HEs genes and their host introns coevolved through recombination, transposition, and horizontal gene transfer. The two HEs genes found in P. sinensis, i.e., one GIY-YIG (ncORF3) and one LAGLIDADG endonuclease (ncORF9), were located in intergenic regions (free-standing or sole mobile in other words) of tRNA genes (Table 1) rather than invaded into intronic regions of coding genes (intron homing), which was also different from O. sinensis. It indicates that the species was earlier diverged than O. sinensis according to the “aenaon” model. Considering that the species was isolated from O. sinensis and might be a fungal parasite of the later, moreover, several other species in Polycephalomyces s. l. have often been found associated with entomopathogenic Cordyceps s. l. (Kobayasi 1941), it is reasonable to hypothesis that species of Polycephalomyces s. l. gained hyperparasitic ability to entomophagous fungi during the evolutionary process rather than a host driven speciation.

Fungal mt genomes displayed remarkable variation between and within the major fungal phyla in terms of gene order, genome size, composition of intergenic regions, and presence of repeats, introns and associated ncORFs (Aguileta et al. 2014). Although the genome size, composition of intergenic regions, and presence of repeats, introns and associated ncORFs may vary, the gene content and synteny remained highly conserved within the Hypocreales. A few exceptional cases observed in this study (listed in Table S1) were probably due to the wrong assembly which should be re-checked, or probably due to the undetermined taxonomic status (incertae sedis). It would be interesting to know the mt genome evolutional process, i.e., gene gain and loss events happened during the evolutionary history of different major fungal groups.

GC contents of mt genomes ranged from 25.27% in Hirsutella vermicola (Zhang et al. 2017) to 30.20% in O. sinensis (Li et al. 2015) within hypocrealean fungi, rather converged compared with other ascomycetic groups. In Saccharomycetales (Saccharomycetes) for example, GC contents varied from 8.39% in Saccharomycodes ludwigii to 52.67% in Candida subhashii (https://www.ncbi.nlm.nih.gov/genome/browse#!/organelles/). Pleurocordyceps sinensis is obviously on the low side (25.46%) among hypocrealean species, little has been done to clarify those GC contents discrepancies among different species and fungal groups.

Most protein coding genes and ncORFs used standard mitochondrial initial and terminal codons (ATG and TAA respectively) in P. sinensis, except ncORF8, cox1 and ncORF10. ncORF10 and cox1 were initiated by ATA and ncORF8 was terminated by TAG. It is noteworthy that cox1 is usually found to use nonstandard start codons such as TCG, ACC, CGA, CTA, CCG and AAA in insect mt genomes (Fenn et al. 2007; Wei et al. 2010), and ATA has been recorded to be used in organisms like Pseudocohnilembus persalinus (Gao et al. 2018), Wellcomia siamensis (Park et al., 2011) and Calanus sinicus (Wang et al. 2011). Although most hypocrealean species used ATG as the initial codon of cox1 gene, exceptional cases were also reported in Hirsutella rhossiliensis (NC_030164) and Calonectria ilicicola (Gai et al. 2020), in which TTG were used.

The relationship between Ophiocordyceps sinensis and Pleurocordyceps sinensis has been debated, P. sinensis has once been considered as the possible anamorph of O. sinensis (Li et al. 1983; Zeng et al. 2000; Cheng et al. 2005). Until the wide application of DNA sequence into taxonomic and phylogenetic studies, this incorrect view point has been clarified (Zhao et al. 1999; Li et al. 2000; Chen et al. 2001; Jiang and Yao 2002; 2003). Recent studies based on multi-gene phylogeny and morphological characteristics comparison also rejected the anamorph-teleomorph relationship between the two species. The mt genome released in this study provided additional evidence that P. sinensis is not the anamorph of O. sinensis but represent another fungal.

In ML phylogenetic analyses applying 14 conserved protein sequences, P. sinensis was clustered with Clavicipitaceae species with very strong supports (BP = 100%) (Fig. 2). Species within the ‘Polycephalomyces clade’ were thus considered as more closely related to Clavicipitaceae rather than Ophiocordycipitaceae. Although mitochondrial DNA provided valuable information, the taxonomic assignment of this group of fungi could not be resolved in this study and better taxon sampling especially for the ‘Polycephalomyces clade’ would be needed. It is also noteworthy that the family Ophiocordycipitaceae was paraphyletic which contradicts previous studies applying multi-gene phylogeny (Sung et al. 2007a). Although the paraphyly was not well supported (Fig. 2), it should be clarified whether the possible contradiction was due to the incongruence of phylogenies revealed by different molecular markers since mitochondrial DNA may tell different evolutionary stories than nuclear genes (Burger et al. 2003), or just caused by the insufficient sampling. Sung et al. (2007b) conducted multi-gene phylogenetic analyses of clavicipitaceous fungi and compared performance of seven loci including: the nuclear ribosomal small and large subunit DNA (nrSSU and nrLSU), ß-tubulin, elongation factor 1α (EF-1α), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), and one mitochondrial protein coding gene ATP Synthase subunit 6 (mtATP6), and found that seven genes gave incongruent topologies in higher level relationships from each other and also from the combined dataset. It also showed that the only mt fragment (mtATP6) used in the study possessed localized incongruence and simultaneously provided increased level of support for certain nodes. Phylogenetic incongruence revealed by different markers, especially those from mitochondrial and nuclear fragments respectively, has been frequently reported and compared in different organisms (e.g. Kimball et al. 2021; Mikula et al. 2021; Zhang et al. 2021). It is still remains unclear that whether the nuclear genome sequences (fragments or whole genome data) or the mitogenome sequences (fragments or complete data) could provide better resolution of fungal phylogeny or could those genome level data perform better than simply using the nuclear ribosomal genes alone (nrSSU and nrLSU).

As in the case of clavicipitaceous fungi, almost all the later publications on taxonomy and phylogenetic studies accepted the backbone phylogeny created by Sung et al. (2007a,b), and continued to use the five gene dataset which excluded the ß-tubulin and mtATP6 loci (e.g. Kepler et al. 2013; Xiao et al. 2018; Wang et al. 2021). While the above two studies (Sung et al. 2007a,b) failed to include species that assigned to Polycephalomyces s. l. group. Kepler et al. (2013) then included several species of this group and found those species represented a clade distinct from other clavicipitoid genera or even families, and considered them as incertae sedis of Hypocreales as DNA data that been used were unable to infer deeper relationships in Hypocreales. Further studies are needed to reconstruct a reliable phylogenetic relationship of clavicipitaceous fungi, especially the assignment of species of Polycephalomyces s. l.. An increasing number of whole mt genomes have recently been sequenced and released for hypocrealean species, and even more being proceeded, since the gene content in this group of fungi is largely conserved and the conserved protein coding genes almost possess the same exon length, transcribed amino sequences could be easily aligned and phylogenetically informative sites are plentiful, thus the whole mt genome data would provide valuable information. While, publically released data should be carefully treated since they could probably include assembly and annotation errors as observed in Ophiocordyceps camponoti-floridani EC05 (CM022976) and Ophiocordycipitaceae sp. (NC_049089), correct annotation and characterization are always necessary (Kortsinoglou et al. 2019; Megarioti et al. 2020).

Conclusions

The phylogenetic position of species of Polycephalomyces s. l. has not been fully resolved due to the insufficiency of molecular markers that been used. This study sequenced and assembled the complete mt genome of Pleurocordyceps sinensis, a fungus originally isolated from Ophiocordyceps sinensis. Comparative genomics showed that the gene number and synteny of conserved protein coding genes remained conserved in the order Hypocreales despite an obvious size variation among this group of fungi. Phylogenetic analyses using 14 conserved protein sequences demonstrated that this fungus may not belong to the current designated family Ophiocordycipitaceae but is more closely related to the species of Clavicipitaceae. It indicated that mitogenomic phylogeny could probably clarify the phylogenetic position of species of Polycephalomyces s. l. and could also gave valuable information on mitochondrial evolution of clavicipitaceous fungi.

Abbreviations

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The complete sequence and annotation of the mt genome of Polycephalomyces sinensis has been submitted to GenBank under the accession number OK017430.

Competing interests

The authors declare that they have no competing interests.

Funding

This work is supported by the National Science Foundation of China (31400018, 31170017, 31700009, 32170001) and the Natural Science Fund for Colleges and Universities in Jiangsu Province (17KJB350003).

Author contributions

YL and YJY designed the experiments. YL, YHW, KW and XCZ conducted the experiments. YL, JL, KW, RLW and XCZ analysed the data. YL, JL, RLW and YJY wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to thank Sheng-Rong Xiao for his generous donation of the ex-type strain CN80-2.

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