Introduction

Phellinus igniarius has both medicinal properties and is edible1. It grows on the trunk of mulberry trees, and its fruiting body is yellowish brown, giving it common names like mulberry ear and mulberry minister. China, South Korea, Japan, and other east Asian nations account for the majority of distribution2. Phellinus igniarius is a general term for a class of fungi that belongs to Basidiomycota, Agaricomycetes, Hymenochaetales and Hymenochaetaceae.

Phellinus igniarius is a medically important fungi and has been used as a traditional medicine. The first use of Phellinus igniarius was reported in the oldest Chinese medicinal book, Shennong’s Compendium of Materia Medica, which reported the use of Phellinus igniarius for the treatment of various disease conditions, such as stomach pain and amenorrhea; the use of this fungus is also correlated with prolonged life, detoxication, and improved digestion after long-term use3,4,5. In the past few years, several studies have examined the phylogenetic relationships within Phellinus igniarius6,7,8. Currently, the genus is approved for thirteen species.

As eukaryotic cells’ normal cellular metabolism depends on energy, mitochondria are essential organelles for generating energy in the form of adenosine triphosphate (ATP) via the electron-transport chain and oxidative phosphorylation system (OXPHOS)9. The established research discovered the existence of genetic material in mitochondria for the first time10. Since then, scientists have discovered that mitochondria contain all the components necessary for protein replication, transcription, and translation, including DNA polymerase, RNA polymerase, tRNA, and rRNA, demonstrating the existence of a largely independent genetic transcription system in mitochondria. Regarding the mitochondrial genome, there are three genetic traits: Typical matrilineal inheritance also includes a conservative coding area, a control region that evolves quickly, and a high mutation rate. In other words, mitochondrial DNA (mtDNA) plays a significant role in the study of molecular evolution.

In order to deepen our understanding of the Phellinus igniarius species in terms of its mitochondrial genome, we conducted complete mitochondrial genome sequencing and performed bioinformatic analysis to explore various aspects such as tRNA structure, mitochondrial genome configuration, nucleotide diversity within complete mitochondrial genomes, identification of interspersed repetitive sequences, and phylogenetic analyses. The findings from this study will provide valuable insights into the phylogenetic relationship of Phellinus igniarius and contribute to a better understanding of this species11.

Results

Statistics of whole genome sequencing results

Table 1 Sequencing data statistics of Phellinus igniarius.
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Table 2 Statistics of base shift analysis.
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Table 3 Overview of mitochondrial genome of Phellinus igniarius.
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Table 4 Codon usage of coding genes in Phellinus igniarius.
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Table 5 Statistical analysis of simple repeat sequences.
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Table 6 KaKs analysis of mitochondrial genome coding gene of Phellinus igniarius.
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Table 7 The positively selected genes in the mitochondrial genome of Phellinus igniarius.
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The table displayed the sequencing data statistics following the sample’s complete genome sequencing. A total of 7,134,012,300 raw read pairs with a GC content of 46.04, a Q20 of 97.29, and a Q30 of 92.57 were produced (Table 1). The average coverage of assembled mitochondrial genomes was 4380.7078. This sequencing’s genomic insert size is typically 250–350 bp, and the randomness of the sequencing is good (Fig. 1).

Fig. 1
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Distribution of inserted fragments.

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Fig. 2
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Mitochondrial genome circular map. Gene map shows 86 annotated genes with different functional groups. The forward coding gene is located outside the circle while the reverse coding gene inside. The inner gray circle represents GC content.

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Fig. 3
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Secondary structure prediction of tRNAs.

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Fig. 4
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Use of relative synonymous codons in the genome of Phellinus igniarius.

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Fig. 5
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Comparative analysis of mitochondrial structure.

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Fig. 6
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Mitochondrial sequence homology analysis. The rectangular blocks in the figure represent the similarity between genomes, and the lines between the rectangular blocks represent a collinear relationship. The short squares represent the gene positions of each genome. Among them, white represents CDs, green represents tRNA, and red represents rRNA.

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Fig. 7
figure 7

Maximum likelihood (ML) phylogenetic tree based on 20 Phellinus igniarius homologous sequence species. Numbers beside nodes indicate bookstrap support values.

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Gene characteristics of mitochondrial genome

Phellinus igniarius ’s mitochondrial genome is a 172,449 bp (Genbank accession number: ON990064.1) double-stranded circular molecule that contains 51 PCGs, 33 tRNA genes, and 2 rRNA genes. The GC content percentage in the mitochondrial genome is 25.31%, indicating a significant bias towards AT (AT = 74.69%). As depicted in Table 2, A, T, G, and C make up 37.12%, 37.57%, 13.24%, and 12.07% of the genome’s bases, respectively. As illustrated in Table 2, the A+T content of the entire mitochondrial genome, PCGs, tRNAs, and rRNAs in Phellinus igniarius was remarkably similar, at 74.69%, 73.33%, 61.76%, and 66.97%, respectively. GC and AT base composition skews in Table 2 could be calculated as (G-C)/(G + C) and (A-T)/(A + T)12 .

86 genes, including 51 PCGs, 33 tRNA genes, and 2 rRNA genes (rrnL and rrnS), were identified in the mitochondrial genome of Phellinus igniarius. The annotated genes’ physical location and functional classification are depicted in Fig. 2. The Table 3 contains eight categories for the 51 different proteins (rsp19 has two copies): laglidadg endonuclease (23 genes), giy endonuclease (6 genes), DNA polymerase (7 genes), cytochrome c oxidase (3 genes), NADH dehydrogenase (7 genes), panthenol cytochrome c reductase (1 gene), ATP synthase (3 genes), ribosomal protein (SSU; 1 gene). Almost all protein coding genes use ATG as the start codon, mainly TAA and TAG as the stop codon. The coding direction of most genes is forward coding, only a few are reverse coding: trnD, trnG, trnK, trnR, trnS2, dpo-1, dpo-3, dpo-4, dpo-5, dpo-7 and lagli-23. In this genome, the arrangement of genes is relatively loose, forming multiple spacers, and the largest interval between trnD and lagli-23 is 10,965 bp. It is also found that a gene overlap: lagli-18/lagli-19, overlapping genes play an important role in the regulation of genes expression13.

PCGs, transfer and ribosomal RNAs

The mitochondrial genome of the Phellinus igniarius has 33 tRNA genes. The length of the mitochondrial tRNA genes is 2419 bp, and the percentages of A + T and G + C are 61.76% and 38.24%, respectively. Each tRNA is roughly 67–79 bp in size. Two rRNA molecules were 4632 base pairs long and had a 66.97% A+T composition. The majority of the tRNAs in the Phellinus igniarius exhibit conventional secondary clover structures, such as the amino acid acceptor stem (AAS), dihydrouridine stem and loop (DSL), anticodon stem and loop (ASL), and thymidine stem and loop (TSL) (Fig. 3). There are insertions, deletions and mutations in the tRNA gene. Except trnS2 which lacks dihydrouracil (DHU) arm, the secondary structure of all tRNAs shows a typical clover structure. And in the majority of the tRNA secondary structures in the mitochondrial genome of the Phellinus igniarius, G-U matching has also been discovered.

In organisms, amino acids are usually encoded by multiple codons, however, it does not evenly use all available codons. So, the inequality of synonymous codon use is Relative Synonymous Codon Usage (RSCU). The formula for calculating RSCU value is the number of codons that encode the amino acid / the total number of codons that encode the amino acid.

ATG is usually always used as the start codon in the mitochondrial genome of the sample, whereas TAA and TAG are typically used as the stop codons. The findings of the RSCU calculation revealed that GTA (Met) and TTA (Leu) were the most commonly used amino acid coding codons (Fig. 4). The relative codon usage frequencies of 51 PCGs are shown in Table 4.

Interspersed repetitive sequences analysis

Interspersed repetitive sequences are a type of repetitive sequences that is different from tandem repeats, and they are distributed in a dispersed manner throughout the genome. As showed in Table 5, there were a large number of scattered repeat sequences in the mitochondrial genome, including 376 forward repeat sequences, 31 reverse repeat sequences, 133 palindromic repeat sequences and 22 complementary repeat sequences. In the interspersed repetitive sequences of Phellinus igniarius, the proportion of forward and palindromic repeats is relatively high. Most of the repeat sequences are between 30 and 38 bp in length.

Comparative analysis of mitochondrial genome

Based on the close phylogenetic relationships between these species, chosing Phellinotus piptadeniae (MW255595.1), Ganoderma lucidum (NC021750.1), Sanghuangporus sanghuang (NC039931.1), Porodaedalea pini (NC044675.1), Coniferiporia sulphurascens (NC044678.1) and Phellinus lamaoensis (NC044677.1) for comparison analysis in order to better understand the gene content and structure of this species. The findings demonstrated that the entire mitochondrial genome’s gene composition varied significantly. They all have genes that code for the cytochrome coxidase complex of adenosine triphosphate synthase and the cytochrome b subunit, two parts of the oxidative phosphorylation pathway. It is quite traditional. The comparison results show that the homology of this sample with Sanghuangporus sanghuang (NC039931.1) is close to 55%, and that with other species is close to 40%. The difference mainly occurs in the coding region (CDS region) (Fig. 5). The results showed that there were significant differences in the gene content of the whole mitochondrial genome.

Compared with Phellinotus piptadeniae (137,790 bp), Ganoderma lucidum (60,635 bp), Sanghuangporus sanghuang (112,060 bp), Porodaedalea pini (144,970 bp), Coniferiporia sulphurascens (58,959 bp), Phellinus lamaoensis (45,604 bp), the length of the mitochondrial genome in Phellinus igniarius (172,449 bp) is the largest, which may be related to the content of repetitive sequences and introns in the genome. Meantime, we compared the GC content of these seven species. There is also a certain difference in the GC content among these seven species, with the maximum difference reaching 11.34%. The GC content of these seven different species ranges between 23.21% (Sanghuangporus sanghuang) and 34.55% (Phellinus lamaoensis) , with the GC contents of the other species being as follows: Phellinotus piptadeniae (24.10%), Phellinus igniarius (25.31%), Ganoderma lucidum (26.67%), Porodaedalea pini (28.26%), Coniferiporia sulphurascens (33.17%). The GC content is relatively low (23.21–34.55%), which is consistent with the characteristically high AT content found in fungal mitochondria.

We compared gene sequences of Phellinotus piptadeniae and Phellinus igniarius through synteny analysis and found a high degree of sequence similarity, along with evidence of gene rearrangement. The 51 coding genes of Phellinus igniarius mitochondrial genome were compared with other mitochondrial genome sequences for KaKs analysis. We used mafft v7.310 (https://mafft.cbrc.jp/alignment/software/) to conduct gene sequence alignment and applied KaKs_Calculator v2.0 (https://sourceforge.net/projects/kakscalculator2/) to calculate the Ka/Ks ratio of genes. Ka/Ks ratio can be used to explore the evolution of genes in response to environmental pressures14. The results showed that most of the genes were purified and selected (Ka/Ks < 1), a few genes were positively selected (Ka/Ks > 1). Among them, giy-3, giy-6, rps3, lagli-3, lagli-5, lagli-11 and lagli-17 genes contained positive selection sites (Tables 6 and 7).

Sequence homology analysis

Using the Mauve software for genome alignment (with default parameters), the visualization of the alignment results was shown in the Fig. 6. The mitochondrial genomes of Hymenochaetaceae species contained several gene rearrangements, according to comparative genomic analyses. As presented in Fig. 6, the overall mitochondrial genome may have shrunk as a result of the loss of some homologous clusters throughout the course of evolutionary time. The placement of homologous clusters indicates that Ganoderma species have significant homology. Aside from that, gene rearrangements are regularly observed in other Hymenochaetaceae species.

Phylogenetic analysis

The entire mtDNA sequences of 20 homologous sequence species were chosed for phylogenetic analysis and created a phylogenetic tree using the maximum likelihood approach in order to monitor the evolutionary position of the examined Phellinus igniarius samples. It showed different branches, including 3 orders, including Polyporales, Hymenochaetales and Agaricales. There are 9 different families, including Poronaceae, Lateral Auriculae, Coruscaceae, Reticulodaceae, Ganodermaceae, Echinococcaceae, Dermachaceae, Tricholomidae and Gallinaceae. In addition, studies have shown that Inonotus obliquus has a close phylogenetic relationship with Phellinus igniarius15. Phellinus igniarius sample is homologous with Inonotus obliquus and has a strong nodule support rate (bootstrap = 100) (Fig. 7).

Methods

Sample preparation and DNA extraction of Phellinus igniarius

Samples were collected from Aiwa Village in Mountain Tai (36.25N/117.16E) . Voucher specimens (ZJ22006) had been identificated by Professor Yanyou Su and been deposited in Specimens Laboratory, School of Life Sciences, Shandong First Medical University. Samples were kept at a temperature of − 4 °C until they were used for DNA extraction. After washing the samples in sterile water, silica beads were used to extract the DNA16. 60 µL of diluted DNA were used. DNA was stored in a refrigerator at temperatures of − 70 °C.

Genome sequencing and de novo assembly of mitochondrial genome of Phellinus igniarius

Using a PCR-amplified library, Illumina NovaSeq raw sequences were acquired. Under the environment of Genome Information System (GeIS; http://geis.infoboss.co.kr/)17, raw sequence were filtered by fastp (version 0.20.0, https://github.com/Open Gene/fastp) and subjected to de novo assembly process done by SPAdes v3.10.1(http://cab.spbu.ru/software/spades/) in order to obtain the complete mitochondrial genome sequences18. Using SSPACE v2.0 (https://www.baseclear.com/services/bioinformatics/basetools/sspace-standard/) software, connect contig sequences to get scaffolds. Filling gap sequences as well as circular test were conducted with Gap filler v2.1.1, until we get the complete pseudo genome sequence. After that, the sequencing sequence was compared to pseudo genome for genome correction; according to the structure of mitochondria, the corrected pseudo genome was rearranged in coordinates to obtain a complete mitochondrial circular genome sequence.

Mitochondrial genome annotation

Based on sequence alignment with the mitochondrial genomes of sibling species, Mitos2 (http://mitos2.bioinf.uni-leipzig.de) was used to annotate the mitochondrial genome. The annotation results are manually rectified and drawn into the final tRNA secondary structure when the necessary settings (E-value Exponent = 5, Maximum Overlap = 100, ncRNA overlap = 100) are established.

Identification of interspersed repetitive sequences on Phellinus igniarius mitochondrial genome

Using Vmatch software to identify repetitive sequences, the minimum length=20 bp and hamming distance = 3 are set to analyze the mitochondrial interspersed repetitive sequences of Phellinus igniarius, the identification takes four forms: forward, palindromic, reverse, complement.

Genetic and phylogenetic analysis

The sequences were collected and added to the GenBank database. By comparing BLASTN with reference sequences in the GenBank database, genetic similarity was ascertained. Using Clustal, the sequences obtained for this investigation and those downloaded from the GenBank database were manually corrected. DNA Sp v.5.10.01 was utilized to perform a sliding window analysis. The analytical mode was decided upon as being DNA polymorphism. The window length was set to 600 bp, and the step size was 200 bp to gather data on haplotype diversity (hd), nucleotide diversity (PI), and other relevant topics19. The PROTGAMMAJTT model with bootstrap = 1000 was used to create the phylogenetic tree in Raxml v8.2.10 (https://cme.h-its.org/exelixis/software.html).

Discussion

The mitochondrial genome of Hymenochaetales ranges in size from 60,635 to 156,348 base pairs. According to some research, the elements that affect how the mitochondrial genome varies in size within and between fungal species include introns, mitochondrial plasmids, gene spacers, repetitive sequences, mobile genetic factors, hypothetical genes and others20,21,22.

Inonotus obliquus belongs to the family Hymenochaetales, Inonotus. Inonotus obliquus has a complete mitochondrial genome of 119,110 bp23. It is composed of 90 genes, including 58 protein coding genes, 2 rRNAs (RNL and RNS) and 30 tRNAs (covering all 20 amino acids). The 58 protein coding genes encode 14 conserved mitochondrial proteins, including 3 cytochrome oxidase (cox1-3), cytochrome b (cob), 7 nad dehydrogenase (nad1-6 and nad4l) and 3 adenosine triphosphate synthetases (atp6, atp8 and atp9). The sample’s mitochondrial genome is 172,449 bp long overall. It contains a total of 86 genes, 51 of which encode proteins, along with 2 rRNAs and 33 tRNAs. Three cytochrome oxidases (cox1-3), cytochrome b (cob), seven nad dehydrogenases (nad1-6 and nad4l), and three adenosine triphosphate synthetases (atp6, atp8 and atp9) are among the 51 protein-coding genes. The AT nucleotides in the Phellinus igniarius skewed slightly, and the content was 74.69%, which is significantly higher than that of other Phellinus igniarius species like Inonotus obliquus. Strong base composition skews can result from asymmetrical mutational pressures caused by inversions of the origin of replication in mitochondrial genomes24 . AT skew was -0.006, indicating the occurrence of A less than T, and revealed a negative AT skew value and a positive GC skew value, which were biased toward G and T bases. Regrettably, we have not clarified the mechanism underlying this phenomenon. Nevertheless, preliminary research revealed that the GC skew’s value was linked to replication orientation rather than gene direction, while the value of the AT skew could vary across gene direction, replication, and codon positions25. Codon usage bias has influence on the level of mRNA and expression of foreign genes26,27. 46 PCGs of the 51 PCGs utilize the ATN as the starting codon, with the exception of lagli-4, lagli-16, lagli-22 starting with TTA and nad3, cox1 utilize TTG. And aside from several genes stop with TAG, the majority of PCGs stop with TAA.

Phellinus igniarius and Inonotus obliquus possess a comparable mitochondrial structure and number. The total number of tRNA genes that code for all 20 common amino acids can range from 25 to 29 in the mitochondrial genome of porous bacteria. Each of the amino acids arginine, methionine, and serine found in the mitotic body of porous bacteria is all encoded by at least two tRNAs. However, all amino acids other than methionine in the whole mitochondrial genome of this Phellinus igniarius sample are encoded by a minimum of two tRNAs. Secondary structure analyses revealed that all of the tRNAs in Phellinus igniarius’s mitochondrial genome were folded into a traditional clover shape. The size of the tRNAs ranged from 67 to 79 bp. We discovered that the big outer frontal arms of trnS, trnL, and trnY were the cause of this length discrepancy. However, little research has been done on tRNA mutations in large fungi. The implications of tRNA mutations on the mitochondrial genome and how these changes impact fungal growth and development require further research.

An examination of the sample’s phylogenetic relationships revealed that Phellinus igniarius, Sanghuangporus vaninii, and Inonotus obliquus belong to different genera, Sanghuangporus and Inonotus. This sample is categorized as an Inonotus, as is Inonotus obliquus. All of them have a strong nodule support rate (bootstrap = 98, bootstrap = 100). In summary, this fungus has a close association with Inonotus obliquus.

Mitochondrial genome analysis has been made extensive use in phylogeny,population genetics and evolution theory28,29. Due to its challenging morphological differentiation and limited morphological characteristics30, it is unable to distinguish subspecies or closely related species based solely on morphological traits. Therefore, mitochondrial whole genome sequencing is a helpful method for accurate species identification31.

Current scholarly investigations pertaining to Phellinus igniarius are predominantly concentrated on elucidating the therapeutic mechanisms underlying its constituent components. Relevent genetic inquiries are primarily directed towards the exploration of specific genetic elements within this species. The literatures regarding the mitochondrial genome of Phellinus igniarius are relatively underreported. Phellinus igniarius, which has geographical uniqueness and distinctive characteristics from Mount Tai, as our research sample and conducted a comprehensive analysis of the mitochondrial genome of Phellinus igniarius using classic sequencing and analysis methods.

Through genomic research, the genetic diversity of Phellinus igniarius populations can be assessed, which is of great significance for species conservation, breeding, and the sustainable use of resources.

This study presents, for the first time, the mitochondrial genome sequence of Phellinus igniarius, contributing valuable insights into its phylogeny. Additionally, it expands the database of mitochondrial genomes and serves as a reference for future investigations into the mitochondrial genomes of other species within the Phellinus. The findings can serve as a benchmark, allowing researchers to more effectively analyze the variations and adaptability of Phellinus igniarius across different regions. The data contributes to elucidating its evolutionary relationships with other fungi, especially closely related species, providing molecular evidence for the systematic classification of fungi.

Utilizing genomic methodologies to investigate the interactions between Phellinus igniarius and organisms, including both parasitic and symbiotic relationships, enhances our understanding of its ecological role.

The results of phylogenetic tree analysis of Phellinus igniarius mitochondrial genome further supported the phylogenetic relationship of nuclear genome data construction, indicating that fungal mitochondrial genome research can not only address the issue of fungal classification, but also offer crucial auxiliary evidence for the study of mitochondrial evolution and fungal phylogeny.

The subsequent studies can further augment the quantity of samples from the aspects of morphology, transcriptome and genetics, so as to furnish more efficacious information for the establishment of its ultimate categorization status and the research of the function of gene expression products.

Limitations

We selected six additional species for genetic comparison analysis, with low species diversity. Currently, there are few reports on conspecific species, and the number of species available for comparative analysis in this study is limited, resulting in a relatively small scale and limited scope of comparison. Therefore, we will continue to monitor the progress of research on congeneric species, and in subsequent studies, we should increase the number of species for comparison to further deepen the depth of comparative research.

This study has, for the first time, completed the sequencing and analysis of the complete mitochondrial genome of species Phellinus igniarius growing in Mount Tai, establishing a data foundation for future genomic studies and comparisons within the same genus. In future research, it is essential to expand the study samples to achieve more comprehensive results in the areas of population genetics and developmental evolution. This expansion will enable a deeper understanding of genetic diversity, population structure, and the evolutionary processes that shape species over time.