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. 2026 Mar 11;27:390. doi: 10.1186/s12864-026-12739-2

Complete mitochondrial genome of Laetiporus ailaoshanensis (Laetiporaceae, Basidiomycota): features, evolution, and phylogeny

Jin-Xin Ma 1, Hai-Jiao Li 2, Hao Wang 1, Lu-Xin Tang 1, Can Jin 1, Jing Si 1,✉, Bao-Kai Cui 1,✉
PMCID: PMC13088608  PMID: 41814158

Abstract

Background

Laetiporus ailaoshanensis is a fungus within the Laetiporaceae family that causes brown rot in wood through its ability to degrade lignocellulose. However, the mitochondrial genome (mitogenome) of members of the genus Laetiporus has not been extensively investigated.

Results

After complete sequencing and assembly, we annotated and characterized the mitogenome of L. ailaoshanensis and selected Polyporales species for comparative analyses, including gene composition and number, AT/GC content, and skew, codon usage, genetic distance, nonsynonymous and synonymous substitution rates (Ka and Ks), intron dynamics, and phylogenetics. The mitogenome of L. ailaoshanensis was found to contain 117,203 bp and 15 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 25 transfer RNA (tRNA) genes, and 12 introns. Phylogenetic analysis based on mitogenomes further confirmed its phylogenetic relationships within Polyporales. Two Laetiporus species were grouped together with Wolfiporia cocos on the same branch and had a high level of support.

Conclusions

This study represents the first report on the mitogenome of L. ailaoshanensis, filling an important gap in the mitogenome database of the Laetiporaceae and providing a cornerstone for comparative genomics within this clade. Additionally, in phylogenetic analysis, the highly supported topological structure clearly placed Laetiporus and Wolfiporia on the same branch, which laid a solid foundation for further clarification of taxonomic classification.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12739-2.

Keywords: Polyporales, Wood-decaying fungi, Mitochondrial genetics, Comparative genomics, Phylogenetics

Introduction

Wood-decaying fungi are an important part of forest ecosystems. They can degrade dead branches, fallen leaves, and rotten wood, therefore participating in the material cycle, energy flow, and metabolic balance of the whole ecosystem [1, 2]. The genus Laetiporus comprises an important group of wood-decaying fungi that can cause cubical brown rot in hardwoods and conifers. Its members are distributed throughout the world, from boreal to tropical zones. Some Laetiporus species are forest pathogens, whereas others have medicinal properties [3, 4]. Laetiporus ailaoshanensis, first discovered in Ailaoshan of Yunnan Province, China [5], causes brown rot in wood, and is a member of the Laetiporaceae family in the phylum Basidiomycota [6]. Its related species, Laetiporus sulphureus, has been extensively studied. L. sulphureus is edible and possesses anticancer medicinal properties, thus presents significant utilization value [7–9]. However, despite being a closely related species, relatively little is known about L. ailaoshanensis. In 2014, Song et al. reported the morphological and ecological characteristics of L. ailaoshanensis, which is distributed in the subtropical areas of southwestern China [4]. This species is characterized by an orange-yellow to reddish orange pileal surface and cream to buff pores when fresh, an azonate to indistinctly zonate pileus, and ovoid to ellipsoid basidiospores (5.0 − 6.2 × 4.0–5.0 μm). As a brown rot fungus, L. ailaoshanensis can break down lignocellulose, accelerating the decomposition process of diseased and decayed wood. However, current knowledge about this fungus is limited to its ecological distribution and taxonomic aspects. Therefore, this study aimed to deepen our understanding of this fungus at the level of mitogenomes.

Mitochondria are essential organelles in eukaryotic cells and play an important role in respiratory metabolism and energy cycle generation [10–12]. Mitochondria have characteristics such as high copy number, abundant molecular markers, rapid evolutionary rates, and involvement in numerous cellular processes [13–18]. The mitochondrial genome (mitogenome) presents a potentially viable avenue to investigate the genetic and evolutionary relationships among eukaryotes and to identify subtle differences between species [19]. However, compared with plants and animals, fungal mitogenomes are less studied. Current database resources indicate that fungal mitogenomes contain a set of conserved protein-coding genes (PCGs) that play an important role in maintaining homeostasis and cellular energy supply [20, 21].

In this study, the mitogenome of L. ailaoshanensis was assembled, annotated, and characterized. Subsequently, the phylogenetic relationships between L. ailaoshanensis and its relatives were analyzed and a comparative analysis of the mitogenomes of selected closely related species was performed, highlighting their differences and similarities in structural, gene content, and gene order. Despite the lack of research on L. ailaoshanensis, it has potential ecological and economic value. Studies on the mitogenome of L. ailaoshanensis will improve our understanding of the taxonomy and evolutionary biology of this genus. Additionally, our findings will contribute to the fungal mitochondrial database and provide important reference data for future research.

Materials and methods

Fungal isolates, DNA extraction, and genome sequencing

The fungal strain analyzed was isolated from the fruiting bodies of L. ailaoshanensis, which were collected from a decaying angiosperm tree in the Daxueshan National Nature Reserve, located in Yongde County, Lincang, Yunnan Province, China (Fig. 1). The genomic DNA from the mycelium of this individual species was extracted and used for subsequent sequencing analysis. The specimen is preserved at the Herbarium of the School of Ecology and Nature Conservation, Beijing Forestry University, Beijing, China. Mycelial cultures were grown and isolated on potato dextrose agar medium (composed of 200 g/L potato, 20 g/L glucose, 20 g/L agar, and 1 L of distilled water). After incubation at 28 ℃ for 10 to 14 days, the resulting mycelia were harvested directly or stored at 4 ℃ for short-term preservation. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method [22]. Whole-genome sequencing was conducted by GrandOmics (https://www.grandomics.com/, Wuhan, China) employing both the PacBio Sequel and MGISEQ-2000 platforms (Nextomics Biosciences Co., Ltd., Wuhan, China), following the manufacturer’s protocols. A CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing, China) was used to obtain DNA from mycelial cultures. The PCR procedure for internal transcribed spacer (ITS) was as follows: initial denaturation at 95 ℃ for 3 min, followed by 34 cycles at 94 ℃ for 40 s, annealing at 54 ℃ for 45 s and extension at 72 ℃ for 1 min, and a final extension of 72 ℃ for 10 min. The PCR products were purified and sequenced at the Beijing Genomics Institute, China, using the same primers.

Fig. 1.

Fig. 1

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Fruiting body of Laetiporus ailaoshanensis, collected from a decaying angiosperm tree in the Daxueshan National Nature Reserve, located in Yongde County, Lincang, Yunnan Province, China. Photograph by Hai-Jiao Li

Assembly and annotation of the mitogenome

High-quality reads obtained after filtering were assembled into the mitogenome using GetOrganelle version 1.7.7.0 [23]. The sequencing depth of the assembled mitogenome was analyzed using the Bowtie2 version 2.5.2 (Fig. S1) [24]. The circular structure of the assembled mitogenome was confirmed using Bandage (Fig. S2) [25]. Initial annotation was performed in Geneious Prime (https://www.geneious.com), followed by manual refinement. Ribosomal RNA (rRNA) genes and PCGs were annotated based on alignment with the mitogenomes of phylogenetically related fungal species. Transfer RNA (tRNA) genes were identified employing tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) [26]. Mitochondrial repeat elements were categorized into three types for further analysis: simple sequence repeats (SSRs), which were detected with MISA version 2.1 [27, 28]; tandem repeats, identified using TRF [29]; and interspersed repeats, determined using REPuter [30]. Circos version 0.69.9 [31] was employed to visualize these repeat elements. The mitogenome map of L. ailaoshanensis was generated using the Proksee web tool [32]. The final assembled mitogenome sequence was submitted to GenBank under accession number PV364146.

Codon usage and genetic evolution analysis

PCG sequences extracted from the mitogenomes of two Laetiporus species (PhyloSuite) were analyzed for codon usage bias with CodonW [33] and the resulting data were processed and visualized in R version 4.3.1. To evaluate base composition asymmetry in Polyporales PCGs, AT-skew and GC-skew were calculated using the formulas AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C), respectively. GC content, gene length variation, and skewness values were also represented graphically in R. For genetic evolution analysis, species phylogenetically close to L. ailaoshanensis were selected. The mitogenomes of Polyporales species were aligned using Muscle [34], and the substitution rates of nonsynonymous (Ka) and synonymous (Ks) were computed with KaKs_Calculator version 2.0 (https://sourceforge.net/projects/kakscalculator2/) using the MLWL method [35]. Generally, a Ka/Ks ratio significantly ≤ 1 indicates purifying selection, where deleterious nonsynonymous mutations are removed by natural selection. A value not significantly different from 1 is consistent with neutral evolution, where mutations are fixed randomly without selective constraint. A value significantly ≥ 1 provides evidence for positive selection, where advantageous amino acid changes are driven by natural selection [36, 37]. Genetic distances based on the Kimura-2-parameter (K2P) model were estimated using MEGA version 11 [38].

Comparative mitochondrial genomics and intron dynamics

Comparative analyses of mitogenome size and intron composition among Polyporales species are detailed in Supplementary Information 1. The arrangement of PCGs and rRNA genes within the mitogenomes of Polyporales was compared. Genomic collinearity and structural rearrangements between homologous regions of L. ailaoshanensis and L. sulphureus were examined using Mauve version 2.4.0 [39]. BLAST [40] analyses of related species were performed, and syntenic relationships were visualized using the TBtools-Ⅱ software suite [41].

The relationship between mitogenome size and intron count was evaluated using the Pearson’s correlation coefficient across 46 Polyporales species. Intron classification within cox1 genes of 12 closely related species was carried out based on position classes (Pcls) following the criteria described by Férandon et al. [42]. The cox1 gene sequences were aligned with those of Ganoderma calidophilum using Clustal W [43, 44], with each Pcl consisting of introns occupying the same relative positions and exhibiting high sequence similarity. The distribution of homing endonuclease genes (HEGs) of the LAGLIDADG (LAG) and GIY-YIG (GIY) families in the mitogenome of L. ailaoshanensis was identified using EMBOSS and HMMER [45, 46].

Phylogenetic analysis

The ITS sequences in the Laetiporus species were aligned using muscle algorithm in MEGA11 prior to phylogenetic analysis [38]. Subsequently, a maximum likelihood (ML) bootstrap analysis was performed using PhyloSuite to reveal the phylogenetics of Laetiporus species [47]. The ITS sequence of L. ailaoshanensis was compared with other six species in the same genus. At the same time, Trametes duplexa and Jorgewrightia fusiformis were selected as the outgroup to construct the evolutionary tree (Fig. S3).

Fungal mitogenome data were retrieved from the National Center for Biotechnology Information (NCBI) database, focusing on species within the order Polyporales. Details of the 46 selected species are provided in Table S1. PCGs were extracted and aligned using MAFFT version 7.471 [48, 49]. The alignments were refined with MACSE version 2 [50] and trimmed using Gblocks [51]. The resulting gene alignments were concatenated, and PartitionFinder version 2.1.1 [52] was used to determine the best partition schemes and evolutionary models. Phylogenetic relationships were constructed using MrBayes version 3.2 and IQ-TREE, employing Bayesian inference (BI) and ML methods [53, 54]. All analyses were performed on the PhyloSuite platform [47], and the resulting phylogenetic trees were annotated and visualized using the iTOL web tool (https://itol.embl.de/) [55].

Results

Composition and characteristics of the mitogenome of L. ailaoshanensis

The complete mitogenome of L. ailaoshanensis is a closed circular DNA molecule of 117,203 bp and has a GC content of 36.0%. It comprises two rRNA genes, 15 PCGs, and 25 tRNA genes (Fig. 2, Table S2). All typical fungal mitochondrial PCGs were identified, including three ATP synthase subunits (atp9, atp8, atp6), one cytochrome c reductase (cob, complex Ⅲ), three cytochrome c oxidase (cox3, cox2, cox1, complex IV), seven NADH dehydrogenase subunits (nad6, nad5, nad4L, nad4, nad3, nad2, nad1, complex Ⅰ), and one non-core gene, rps3, encoding ribosomal protein S3. The total length of the 15 PCGs was 22,358 bp, comprising 19.08% of the mitogenome. Individual PCG lengths varied significantly, from 156 bp (atp8) to 5,352 bp (cox1). The two rRNA genes, rnl and rns, measured 4,434 bp and 2,038 bp, respectively, and encoded the small and large mitochondrial ribosomal subunits. The 25 identified tRNA genes included trnA, trnC, trnD, trnE, trnF, trnG, trnH, trnI, trnK, trnL; three trnM genes, trnN, trnP, trnQ; two trnR genes; two trnS genes; two trnT genes; trnV, trnW, and trnY. The mitogenome was AT-rich, with both AT and GC skews showing negative values (Table S1). A total of 12 introns, ranging from 384 to 1,762 bp, were identified within nad2, cob, nad5, cox1, and cox2. Intergenic regions spanned from 0 to 9,021 bp, with the longest located between atp6 and cox3. Notably, nad2 and nad3 were positioned adjacent to each other, consistent with the mitogenomic organization in many related species.

Fig. 2.

Fig. 2

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Circular map of the mitogenome of Laetiporus ailaoshanensis, which contains 15 PCGs, 25 tRNA genes, two rRNA genes, and 12 introns. The distinct gene types are color-coded. The outer ring shows the positive strand. GC content and GC skew are presented from outer to inner rings

The 25 tRNAs contained in the mitogenome of L. ailaoshanensis folded into classic clover structures (Fig. S4). The 25 tRNA genes ranged in length from 71 to 86 bp and encoded 20 standard amino acids. These genes in the mitogenome of L. ailaoshanensis were scattered and distributed throughout the loop. Owing to their sizable extra arms, the tRNA encoded by the trnS and trnL genes had the largest and second largest volumes of all detected tRNA. A total of 43 base mismatches were found in the G − U genes. The mitogenome of L. ailaoshanensis contained three methionine-encoding trnMs with identical anticodons, as well as two trnRs, two trnSs, and two trnTs encoding arginine, serine, and threonine, respectively. In addition, the tRNAs of L. sulphureus and L. ailaoshanensis were compared and only one tRNA differed, namely trnT, highlighted in blue in Fig. S4.

Among the 15 PCGs, three (nad1, nad6, rps3) had ATA as the start codon, whereas the reminder had ATG. Meanwhile, the TAA stop codon was predominant, with only one gene (nad2) having TAG as the stop codon. Codon usage analysis in the mitogenomes of L. ailaoshanensis and L. sulphureus revealed TTT (phenylalanine), TTA (leucine), AAA (lysine), AAT (asparagine), ATT (isoleucine), and TAT (tyrosine) as the most frequently used codons (Fig. 3A, Table S3). The frequent use of A and T in codons may explain the high AT content in both genomes. Additionally, amino acid usages were almost identical between the two species; however, L. ailaoshanensis showed significantly higher threonine and methionine usage than L. sulphureus, whereas L. sulphureus exhibited a significantly higher frequency of isoleucine and leucine usage than L. ailaoshanensis (Fig. 3B).

Fig. 3.

Fig. 3

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Comparative usage of codon and amino acids in the mitogenomes of Laetiporus ailaoshanensis and L. sulphureus. A Codon usage; (B) Usage of amino acids

Repeat sequence analysis revealed four SSRs, consisting of three dinucleotides and one hexanucleotide SSR (Tables S4 and S5). Eight tandem repeats were also identified, ranging from 7 to 21 bp in length and exhibiting a minimum similarity of 77% (Table S6). A total of 49 interspersed repeat pairs were detected, including 32 forward, 13 palindromic, and four reverse repeats (Table S7). These interspersed repeats spanned 3,626 bp in total, accounting for 3.09% of the mitogenome. The longest palindromic and forward repeats were 65 bp and 118 bp, respectively. The highest density of interspersed repeats occurred within the cox3 gene and the intergenic region between atp6 and cox3. The full distribution of all these repeats is illustrated in Fig. S5.

Genetic distance, evolutionary rates, and gene variation

A comparison of GC content of each gene among eight Polyporales fungi revealed that the GC content of several genes−atp6, atp9, cob, cox1, nad3, nad5, nad6, rps3, and rns − was higher in L. ailaoshanensis and L. sulphureus than in other fungi (Fig. 4). This may account for the high overall GC content in the mitogenomes of these species. Moreover, among the species depicted in Fig. 4, the GC content in multiple genes (atp6, atp9, cob, cox1, cox3, nad3, nad4L, nad5, nad6, rps3, and rns) of L. ailaoshanensis was higher than that of the other species (Table S8). This was also consistent with the high overall GC content in the mitogenome of L. ailaoshanensis. Two genes (atp6 and atp8) of L. ailaoshanensis were the same length as their counterparts in L. sulphureus and Wolfiporia cocos (Table S8). The AT and GC skews were found to differ among the eight species. However, the AT and GC skews of L. ailaoshanensis were similar to those of L. sulphureus.

Fig. 4.

Fig. 4

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Variations in individual mitochondrial genes across Polyporales species. A Gene length; (B) GC content; (C) AT skew; (D) GC skew

We selected 12 PCGs present in the eight species of Polyporales fungi for evolutionary analysis, that is, to determine the genetic distances of K2P and substitution rates. The results showed that the cox3 gene has the maximum average genetic distance of K2P, followed by cob and nad6, indicating that these genes have diverged considerably throughout evolution (Fig. 5). The nad1 gene showed the lowest average genetic distance of K2P, followed by atp8, indicating that these genes are highly evolutionarily conserved. Regarding substitution rates, atp8 exhibited relatively small Ka and Ks values, with cox3 displaying the highest Ka and nad4 the highest Ks. The Ka/Ks ratios for these PCGs were all less than 1 but not equal, indicating that they all underwent different degrees of purifying selection. The atp9 gene demonstrated the highest average Ka/Ks value (0.14) and nad1 the lowest (0.03).

Fig. 5.

Fig. 5

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Genetic characteristics of conserved mitochondrial PCGs in eight Polyporales fungi. Ka, the mean number of nonsynonymous substitutions per nonsynonymous site; Ks, the mean number of synonymous substitutions per synonymous site; K2P, genetic distance of Kimura-2-parameter

Gene rearrangement and collinearity analysis

The rRNA genes and PCGs arrangements were compared among the mitogenomes of the 12 Polyporales fungi (Fig. 6A). The gene order for all species assessed in this study is listed in Table S1. The results showed that the order of the genetic sequence varies even among species within the same genus. Two gene pairs, nad4L/nad5 and nad2/nad3, were found in L. ailaoshanensis; however, no inversion was found in either pair. In Grifola frondosa, Pappia fissilis, and Fomitopsis palustris, these two genes appeared in the opposite order. The positions of the three genes (nad1, trnI, and cob) in the mitogenomes of most Polyporales fungi were relatively stable and always appeared in the order of nad1, trnI, and cob. Our findings also indicated that mitogenomes have undergone extensive gene rearrangements. Collinearity analysis identified 33 homologous regions in the mitogenomes of both L. ailaoshanensis and L. sulphureus (Fig. 6B). An evaluation of the arrangement or distribution of homologous regions between L. ailaoshanensis and L. sulphureus suggested that gene rearrangements occurred in both mitogenomes. Similarly, differences in size between homologous regions in different mitogenomes indicated that gene contraction or expansion occurred in the two mitogenomes. Furthermore, the collinearity among the mitogenomes of 12 Polyporales species showed extensive gene rearrangement, contraction, and expansion (Fig. S6).

Fig. 6.

Fig. 6

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A Comparison of gene order among 12 Polyporales mitogenomes, beginning with cox1. B Collinearity analysis between Laetiporus ailaoshanensis and L. sulphureus. Homologous regions are denoted by identically colored blocks and connecting lines

Intron dynamics of cox1 genes

The distribution of introns in mitogenome genes is very uneven, with a clear prevalence in the cox1 gene, which serves as the main intron host. A total of 261 introns were detected in the mitogenomes of 46 Polyporales species, of which 123 were located in the cox1 gene, accounting for 47.12% of the total number of introns (Table S1). Pearson’s correlation analysis further revealed that a high correlation between intron number and mitogenome size (R = 0.87, P = 8.1e-15) (Fig. 7A). The introns of the cox1 genes of 12 Polyporales species were categorized into 44 Pcls based on the cox1 gene of Ganoderma calidophilum, with introns within each Pcl considered to be homologous (Fig. 7B). The large number of Pcls was indicative of the high abundance of intron types. Of the 123 introns identified in the 12 Polyporales fungi, one was unknown, two belonged to group Ⅱ, and the rest belonged to group Ⅰ. Of the 44 intron types, P1305 was the most common type found in eight Polyporales species, followed by P383, P706, and P1107. In addition, multiple introns were identified only once in the 12 species. As shown in Fig. 7B, the location and type of intron varied even among related species belonging to the same branch. We additionally investigated the distribution of LAG and GIY families. Only three LAGs were found in L. ailaoshanensis, one located in the cox3 intron and the other two in the nad2 intron. GIYs was not found in L. ailaoshanensis.

Fig. 7.

Fig. 7

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A Pearson’s correlation between intron number and mitogenome sizes in 46 Polyporales fungi. B Classification of introns with Pcl in cox1 genes across 12 species, using Ganoderma calidophilum as a reference. Phylogenetic relationships were inferred via BI and ML analyses of concatenated mitochondrial genes. Species abbreviations: Fdick, Fomitopsis dickinsii; Fpalu, Fomitopsis palustris; Fpini, Fomitopsis pinicola; Gfron, Grifola frondosa; Laila, Laetiporus ailaoshanensis; Lsulp, L. sulphureus; Pfiss, Pappia fissilis; Pradi, Phlebia radiata; Rmicr, Rigidoporus microporus; Scris, Sparassis crispa; Tcamp, Taiwanofungus camphoratus; Wcoco, Wolfiporia cocos. The accession numbers are listed in Table S1

Comparative and phylogenetic analyses of mitogenomes

Due to a lack of information related to genera within the family Laetiporaceae in the NCBI database, in addition to L. sulphureus of the same genus, ten Polyporales species were selected for a comparative analysis of mitogenomes. Among the mitogenomes of these 12 species of fungi, the largest was 3.61 times the size of the smallest. The average genome size was 114,668 bp and the GC content ranged from 22.6% to 36.3% (Table S1). Three species−Grifola frondosa, Fomitopsis palustris, and Phlebia radiata−show a positive AT skew, whereas the rest display a negative AT skew. Meanwhile, L. ailaoshanensis, L. sulphureus, and Grifola frondosa had a negative GC skew, and the remaining species had a positive GC skew. Except for Grifola frondosa, which encodes no rRNA genes, the remaining mitogenomes all contained two genes encoding rRNAs. In the mitogenomes of the 12 species, the tRNA gene number varied from 25 to 28, whereas the intron number ranged from 3 to 52.

A phylogenetic tree comprising 46 fungal species within Polyporales was constructed using concatenated sequences of conserved PCGs, employing both BI and ML methods (Fig. 8). The species names, NCBI accession numbers, and mitogenome characteristics included in the phylogenetic analysis are presented in Table S1. Among the 43 branching nodes, 30 displayed bootstrap values that exceeded 90, with 27 nodes showing bootstrap values of 100. Conversely, 40 nodes had Bayesian posterior probability (BPP) values above 0.90, including 38 nodes with BPP values of 1.00. L. ailaoshanensis, L. sulphureus, and Wolfiporia cocos were placed in the same clade, which was relatively consistent with the results of the genome analysis and was indicative of a close relationship among the three species.

Fig. 8.

Fig. 8

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Phylogenetic tree of 46 Polyporales species based on conserved mitochondrial PCGs, reconstructed using BI and ML methods. BPP and ML bootstrap values are shown next to branches. The asterisks denote complete support (BPP = 1, bootstrap = 100). GenBank accession numbers follow species names

Discussion

Mitogenome composition and introns

In this study, the mitogenome of L. ailaoshanensis was sequenced, assembled, and analyzed. Further, L. ailaoshanensis was found to have a complete set of PCGs, which are primarily involved in energy metabolism and regulation, as well as two rRNA and 25 tRNA genes. One of the PCGs, nad4L, plays an important role in maintaining intracellular homeostasis and regulating cellular responses to environmental stimuli [56]. All available and valid mitogenomes of Polyporales fungi were selected for comparative and phylogenetic analyses with reference to the study of Liu et al. [57]. Among the mitogenomes of the 46 Polyporales species included in this study, the largest was 4.85 times the size of the smallest. The GC content of the mitogenomes ranged from 22.6% to 36.3%. Most mitogenomes harbored two rRNA genes, whereas Grifola frondosa had none. Additionally, the number of tRNAs varied from 22 to 29, whereas intron numbers, which showed the greatest variation, ranged from 0 to 52. From these data, the differences in size and structure among different species of Polyporales are significant. L. ailaoshanensis and L. sulphureus had the most similar GC content, with a mitogenome size difference of 16,092 bp. The mitogenomes from these two species exhibited identical PCG composition, numbers of rRNA and tRNA genes (2 and 25, respectively), and total number of introns (12); however, they differed in the number of introns in the cox1 gene. Moreover, the mitogenomes of these two Laetiporaceae species shared many similarities with that of Wolfiporia cocos, including GC content and the size of the mitogenomes.

However, the number and types of introns differed markedly between the two Laetiporaceae species and Wolfiporia cocos, suggesting that intron variation underlies the observed difference in mitogenome size. Introns act as mobile genetic elements in the mitogenomes of eukaryotes, and dynamic changes in their location, number, and type can significantly affect the mitogenome [58–60]. Thus, introns are widely recognized as the main contributors to mitogenome amplification. Consistent with this, the number of introns was highly correlated with the size of the mitogenomes of Polyporales species. Mitogenomes of animals have been shown to typically lack introns, mitogenomes of plants predominantly contain group II introns, and those of fungi primarily contain group I introns [60, 61]. Using Ganoderma calidophilum as a reference, the introns of the cox1 genes of 12 Polyporales fungi were divided into different Pcls according to their insertion position in the protein-coding region and most of the introns belonged to group I, which was in line with the previous research. Introns with the same Pcls were categorized as homologous [42]. P824 and P934 intron types were found in the mitogenome of L. ailaoshanensis but not in that of the closely related L. sulphureus, whereas the opposite was observed for P547, P821, P894, and P941. However, the findings that are parts of these introns present in relatively distant relatives may indicate the occurrence of intron transfer events in these fungal species. Moreover, multiple introns were identified in just one species. This variation in introns across species is suggestive of dynamic variability in intron distribution and frequent intron gain and loss in these fungal species [62, 63]. Introns often encode homing endonucleases, enabling their insertion into specific locations in the mitogenome [64–66]. This characteristic suggests that they have potential as gene editing tools, an area that merits further investigation. In addition, the distribution of HEGs in the mitogenome was also identified. In the mitogenome of L. ailaoshanensis, only LAGs were identified and GIYs were not found. In the same species L. sulphureus, the presence of GIYs was also not detected. This might be an important characteristic of Laetiporus. Further research will require additional mitogenome data from Laetiporus to conduct a more in-depth study. HEGs were located mainly in the introns of the cox, cob, nad, and rRNA genes, and played an important role in regulating the diversity of fungal mitogenomes, and can also alter the organization and size of the mitogenome [67, 68]. Moreover, some HEGs may also possess additional biological functions. The presence of HEGs significantly promotes the diversity and evolution of fungal mitogenomes [69]. Therefore, the significance of HEG has also enhanced the need for in-depth research on introns.

Gene rearrangements and repeats

The gene arrangements in the mitogenomes of 46 species belonging to Polyporales were compared. Wide variations in gene arrangements were observed among the studied species, with differences apparent even within the same genus. These included gene relocations, position exchanges, and other rearrangements, indicating that mitochondrial genes are highly variable. The arrangement of mitochondrial genes is closely related to the genetic evolution and phylogeny of fungal species [15, 17, 70]. This study further revealed that even the core genes of the mitogenomes of closely related species still exhibit differences. Such differences may play a crucial role in adaptive evolution of different ecological communities [71]. The mechanisms underlying mitochondrial gene rearrangements in fungi remain unclear but appear to be more complex than those of animals [72, 73]. One contributing factor is the greater abundance of repetitive sequences in fungal mitochondria, which not only leads to changes in mitogenome size but also drives dynamic changes in gene structure through homologous recombination and gene rearrangement [74]. In the mitogenome of L. ailaoshanensis, three types of repeats were detected, namely, SSRs, tandem repeats, and interspersed repeats. In addition to their rich genetic information content, repeats also participate in the regulation of gene expression as part of gene regulatory networks, interacting with other signaling molecules or homologous expression elements [75]. Some repeats can specifically bind to certain proteins, thus facilitating the assembly of higher structures [76]. However, at the same time, repeats also significantly increase the probability of homologous recombination errors, thereby leading to a high frequency of structural rearrangement phenomena. Therefore, repeats not only play a significant role in promoting evolution and generating diversity, but also may be a factor that leads to mitogenome rearrangements. The study of repetitions is a crucial link in understanding and recognizing the evolution of mitogenomes [77, 78].

Codon usage and gene characteristics

Among the PCGs, three had ATA as the start codon and one had TAG as the stop codon. The remainder had typical start (ATG) and stop (TAA) codons. We further analyzed codon and amino acid usage, which also influences gene expression levels and protein structure [79]. Codon usage can affect the translation elongation rate and the efficiency of mRNA translation. Furthermore, the frequency of codon usage is correlated with the corresponding tRNA level and copy number [80–83]. The size and base composition of tRNAs can affect the efficiency of protein synthesis [84, 85]. Studies on genome-wide expression profiling have also revealed a positive correlation between codon usage and mRNA levels in both prokaryotic and eukaryotic organisms [79, 86–89]. Thus, codon usage and preference may also play an important role in mitogenomes, highlighting the need for further research into codon usage in fungal mitogenomes.

A more comprehensive analysis of PCGs was performed in the mitogenomes of different Polyporales species, including an investigation of their length, GC content, AT/GC skew, Ka/Ks, and genetic distance of K2P. The GC and AT skews of PCGs differed among the different species, which highlights their genetic differentiation and distinct environmental selection pressures [90]. The PCGs of the Polyporales species also differed in length, base composition, and evolution rates; however, our analysis indicated that all PCGs are under purifying selection. The finding that all PCGs have evolved under purifying selection (Ka/Ks < 1) is consistent with their essential functions within the mitogenome. This conservatism indicates that the amino acid sequences of these genes are restricted. Genes involved in core functions are particularly subject to strong structural constraints, as even minor alterations may disrupt energy production processes and have fatal effects on the organism. This result highlights the stability and functional significance of these genes in the evolution of the mitogenome of L. ailaoshanensis.

Phylogenetic relationships based on mitochondrial genes

Unlike the nuclear genome, the mitogenome undergoes rapid evolution, offering distinct advantages for studies on fungal population genetics and phylogenetic analysis [91–94]. In this study, a phylogenetic tree of 46 Polyporales fungi was constructed based on mitochondrial gene sets. The tree contained all available and valid Polyporales data retrieved from NCBI. Our analysis showed that L. ailaoshanensis is closely related to species in the genus Wolfiporia, which is consistent with results based on the DNA sequence analysis [57]. Among the three species forming a branch, the bootstrap values and BPP support rates were 100 and 1, respectively. In this highly supported phylogenetic context, we simultaneously noticed that all three species belonged to the brown rot type of wood-decaying fungi. This common and crucial ecological functional characteristic provides strong biological support for their close phylogenetic relationship, indicating that the brown rot habit might be a conserved feature of this lineage. By maintaining the core degradation ability of the brown rot mechanism, the reproductive structures and host preferences of the three species have undergone adaptive evolution. Laetiporus typically produces large, annual fruiting bodies that grow on living trees or fallen logs [4]. In contrast, Wolfiporia cocos produces underground mycelial bodies and is often associated with pine trees [95]. This difference may indicate adaptive evolution for different survival strategies and reproductive methods. The mitochondrial gene dataset has been proven to be a reliable molecular marker, particularly of mitochondrial-specific genes (atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), and has great potential for application in the evaluation of the phylogenetic relationships of fungi [96–99]. A better understanding of the phylogenetic relationships among the Polyporales species necessitates extensive sequencing and research on the mitogenomes of its constituent fungal species.

Conclusions

In the present study, the mitogenome of L. ailaoshanensis was assembled and annotated for the first time. The size of the mitogenome of L. ailaoshanensis was 117,203 bp, which contains two rRNA genes, 15 PCGs, 25 tRNA genes, and 12 introns. In addition, the mitogenome of L. ailaoshanensis was assessed for gene composition and structural features, codon usage, tRNA structure, GC content, gene length and AT/GC skew, gene rearrangements, collinearity, genetic distance, evolutionary rates and variation, intron dynamics, and phylogenetics. Particularly, in the phylogenetic analysis, Wolfiporia cocos and two Laetiporus species were grouped together in a branch with a high support rate. These three fungi are all brown rot fungi, which may indicate that this brown rot characteristic is a conserved feature on this branch. The findings of this study, which represents a preliminary exploration of the mitogenome of L. ailaoshanensis, enhances our understanding of the genetics and evolution of L. ailaoshanensis. The data gathered here also expand gene data relative to fungal mitogenomes, therefore providing an effective reference for further research on fungal evolution and genetic development.

Supplementary Information

12864_2026_12739_MOESM1_ESM.xlsx (57.4KB, xlsx)

Supplementary Material 1: Table S1. Species information and GenBank accession numbers used in phylogenetic analysis. Table S2. Details of annotation and characterization of the mitogenome of Laetiporus ailaoshanensis. Table S3. Relative synonymous codon usage of individual amino acid pairs of codons in the mitogenome of two Laetiporus species. Table S4. Simple sequence repeats (SSRs) identified in the mitogenome of Laetiporus ailaoshanensis. Table S5. Summary of SSR characteristics in the mitogenome of Laetiporus ailaoshanensis. Table S6. Tandem repeats in the mitogenome of Laetiporus ailaoshanensis. Table S7. Interspersed repeats in the mitogenome of Laetiporus ailaoshanensis. Table S8. Variations in individual mitochondrial genes across Polyporales species. (A) Gene length; (B) GC content; (C) AT skew; (D) GC skew.

12864_2026_12739_MOESM2_ESM.docx (11MB, docx)

Supplementary Material 2: Fig. S1. Sequencing depth and coverage map of the mitogenome of Laetiporus ailaoshanensis. Fig. S2. Circular diagram of the mitogenome of Laetiporus ailaoshanensis. Fig.S3. Phylogeny of Laetiporus constructed by maximum parsimony analysis based on internal transcribed spacer sequences. Fig. S4. Predicted secondary structures of tRNAs in the mitogenome of Laetiporus ailaoshanensis, arranged in genomic order starting from trnK. tRNA, which differs from those of L. sulphureus, are highlighted in blue. Fig. S5. Distribution of repeats in the mitogenome of Laetiporus ailaoshanensis. The outermost circle represents the mitogenome. Moving inward, the locations of SSRs and tandem repeats are displayed sequentially. The innermost lines represent interspersed repeats. Fig. S6. Collinearity analysis among the mitogenomes of 12 Polyporales fungi. Bars represent the individual mitogenomes; ribbons denote homologous sequences shared between adjacent species.

Acknowledgements

We thank the Editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript.

Authors’ contributions

J.X.M: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing-original draft, Writing-review & editing. H.J.L: Formal analysis, Methodology, Resources, Software, Visualization, Writing-original draft. H.W: Formal analysis, Methodology, Writing-original draft. L.X.T: Formal analysis, Methodology, Writing-original draft. C.J: Formal analysis, Methodology, Software. J.S: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Writing-original draft, Writing-review & editing. B.K.C: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing-review & editing.

Funding

This work was funded by the National Natural Science Foundation of China (32270016 and 32470002), the Fundamental Research Funds for the Central Universities (QNTD202509), the National Natural Science Foundation of China (32070016 and 32325001), and the Beijing Nova Program (20230484322).

Data availability

The complete mitogenome of Laetiporus ailaoshanensis has been deposited in the GenBank database under the accession number PV364146. The phylogenetic files can be viewed via 10.5061/dryad.xsj3tx9v4. All data generated or analyzed during this study are included in this article [and its Additional files].

Declarations

Ethics approval and consent to participate

All materials used in this study comply with international and national legal standards. The collected species material does not pose a threat to other species, and the collection of the species is recognized by the relevant authorities.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jing Si, Email: jingsi1788@126.com.

Bao-Kai Cui, Email: cuibaokai@bjfu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12864_2026_12739_MOESM1_ESM.xlsx (57.4KB, xlsx)

Supplementary Material 1: Table S1. Species information and GenBank accession numbers used in phylogenetic analysis. Table S2. Details of annotation and characterization of the mitogenome of Laetiporus ailaoshanensis. Table S3. Relative synonymous codon usage of individual amino acid pairs of codons in the mitogenome of two Laetiporus species. Table S4. Simple sequence repeats (SSRs) identified in the mitogenome of Laetiporus ailaoshanensis. Table S5. Summary of SSR characteristics in the mitogenome of Laetiporus ailaoshanensis. Table S6. Tandem repeats in the mitogenome of Laetiporus ailaoshanensis. Table S7. Interspersed repeats in the mitogenome of Laetiporus ailaoshanensis. Table S8. Variations in individual mitochondrial genes across Polyporales species. (A) Gene length; (B) GC content; (C) AT skew; (D) GC skew.

12864_2026_12739_MOESM2_ESM.docx (11MB, docx)

Supplementary Material 2: Fig. S1. Sequencing depth and coverage map of the mitogenome of Laetiporus ailaoshanensis. Fig. S2. Circular diagram of the mitogenome of Laetiporus ailaoshanensis. Fig.S3. Phylogeny of Laetiporus constructed by maximum parsimony analysis based on internal transcribed spacer sequences. Fig. S4. Predicted secondary structures of tRNAs in the mitogenome of Laetiporus ailaoshanensis, arranged in genomic order starting from trnK. tRNA, which differs from those of L. sulphureus, are highlighted in blue. Fig. S5. Distribution of repeats in the mitogenome of Laetiporus ailaoshanensis. The outermost circle represents the mitogenome. Moving inward, the locations of SSRs and tandem repeats are displayed sequentially. The innermost lines represent interspersed repeats. Fig. S6. Collinearity analysis among the mitogenomes of 12 Polyporales fungi. Bars represent the individual mitogenomes; ribbons denote homologous sequences shared between adjacent species.

Data Availability Statement

The complete mitogenome of Laetiporus ailaoshanensis has been deposited in the GenBank database under the accession number PV364146. The phylogenetic files can be viewed via 10.5061/dryad.xsj3tx9v4. All data generated or analyzed during this study are included in this article [and its Additional files].

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