Determining Gene Order Patterns in the Suillus and Boletales through Comparative Analysis of Their Mitogenomes

Suillus is one of the most important genera of ectomycorrhizal fungi. As a model for studying host specificity, its molecular fragments and nuclear genome have been analyzed. However, its mitochondrial genome has not yet been reported. In this study, we assembled five mitogenomes of Suillus and analyzed and compared their basic characteristics. Owing to the large number of introns as well as intergenic regions, the mitogenomic lengths of species of Suillus were greater than those of other species of Boletales. We identified two main patterns of gene order arrangement in the members of the order Boletales. The Ka/Ks values of 15 protein-coding genes were <1 for the mitochondrial genes of 39 Boletales species, indicating their conserved evolution. Phylogenetic trees, reconstructed using the mitogenomes, indicated that the genus Suillus was monophyletic. Phylogenetic results based on the internal transcribed spacer region and mitogenome were used to confirm the distribution of Suillus placidus in China. The results showed that the mitogenome was superior in distinguishing species compared with a single molecular fragment. This is the first study to investigate the mitogenome of Suillus, enriching the mitogenome information and providing basic data for the phylogeny, resource conservation, and genetic diversity of this genus.


Introduction
The genus Suillus, belonging to the family Suillaceae, forms a notable symbiotic relationship with the family Pinaceae.A review suggested that Suillus species are one of the ecological drivers of global pine invasions [1] owing to their five main ecological traits: their long-distance dispersal capacity, establishment of positive biotic interactions with mammals, capacity to generate a resistant spore bank, rapid colonization of roots, and long-distance exploration type.Therefore, based on its host specificity patterns and wide distribution, Suillus has been widely used to investigate the diversity of ectomycorrhizal fungi and conservation of Pinaceae.In addition, suillin, the ethyl acetate extract of the mushroom S. placidus, has been found to be an effective therapeutic agent for liver cancer [2].Some species of Suillus are also widely consumed as food items in China [3].
Most species of the genus Suillus have a cylindrical, central stipe.The pileus surface may be sticky and slippery when wet, and the cap is usually circular [4].Suillus was originally classified as a genus belonging to the family Boletaceae, but after further study, Suillus was assigned to the family Suillaceae, which was mainly established on the basis of chemical data [5].The genus Suillus reportedly comprises three parts: Paragyrodon, Boletinus, and Suillus [6].However, Paragyrodon is currently recognized as belonging to the family Paxillaceae.Researchers have reported a range of Suillus species from Asia [3,7,8].Due to the subjective nature of morphological identification, researchers have also investigated and identified Suillus through methods such as chemical systematics and molecular phylogeny [9,10].Molecular fragment-based phylogenetic studies of Suillus first appeared in 1996 [11] and were later supplemented by Nguyen et al. [12].However, none of these previous studies have focused on a phylogenetic analysis of the mitochondrial genome (mitogenome).
As an independent genetic system outside the nucleus, the mitogenome is characterized by rapid evolution, lack of recombination, high copy number, and maternal inheritance [13].As a result, it can be used as a new molecular marker and can provide more phylogenetic information than the nuclear gene.Mitogenome sequencing is more accessible and affordable than whole-genome sequencing.With the development of next-generation sequencing (NGS) technology, mitogenomes have been widely employed in the fields of molecular phylogenetics, population genetics, molecular diagnostics, and the geosystematics of fungi [14,15].Mitogenomes have been used to probe the phylogenetic relationships of the species belonging to the order Boletales, and some comparative analyses of the basic structure and evolution of these mitogenomes have been conducted [16][17][18][19][20][21][22][23].According to the Macrofungal Taxonomic System and Economic Mushrooms (nmdc.cn),there are approximately 107 species of Suillus in the world.Nevertheless, the complete mitogenomes of Suillus species have not yet been reported.This has greatly hindered the development of molecular systematics research in this genus.
In this study, five Suillus species collected from Guizhou and Yunnan Provinces, China, were sequenced: S. bovinus, S. huapi, S. placidus, S. sibiricus, and Suillus sp.This study had three main objectives.First, to describe the mitogenomic composition of the abovementioned Suillus species.Second, to compare the basic composition, evolutionary rates, and conserved sequences of these five mitogenomes.Third, to reconstruct the phylogenetic relationships of Boletales using the complete mitogenomes, in order to explore the evolutionary relationships between members of the genus Suillus and other Boletales species.This study provides a theoretical basis for the comprehensive understanding of the structural characteristics and evolutionary diversity of the Suillaceae mitogenome, as well as laying a foundation for the elucidation of the biological function and genome evolution of the Suillaceae.
dehydrogenase subunits (nad1, nad2, nad3, nad4, nad4l, nad5, and nad6), and 1 cytochrome b (cob) and 2 rRNAs.All 4 mitogenomes contained 25 transfer RNAs (tRNAs), which encoded the 20 standard amino acids, except for the mitogenome of S. huapi, which contained 26 tRNAs.This was due to the presence of an extra copy of trnL2 (TAA).Dpo, which is present in the mitogenomes of Boletaceae, was not detected in the mitogenomes of Suillus.

PCGs of the Five Suillus Mitogenomes
Among all PCGs, cox1 showed the highest length in all five Suillus mitogenomes, with an average length of 10,902 bp. S. bovinus contained the longest cox1, while S. sibiricus contained the shortest cox1.Atp8 was the shortest PCG in all five Suillus mitogenomes, with an average length of 176 bp. S. sibiricus, S. placidus, and S. bovinus contained the longest atp8, whereas S. huapi and Suillus sp.contained the shortest atp8.The start codons for PCGs in the five mitogenomes included all start codons in the genetic code table 4, except for GTG.Similarly, the termination codons of PCGs in the 5 mitogenomes contained all stop codons in the genetic code table 4, except for TGA.Gene duplication was a frequent occurrence in the mitogenomes of Suillus.Duplication of atp8 occurred in the mitogenomes of S. huapi and Suillus sp.; duplication of nad6 and nad4l occurred in the

PCGs of the Five Suillus Mitogenomes
Among all PCGs, cox1 showed the highest length in all five Suillus mitogenomes, with an average length of 10,902 bp. S. bovinus contained the longest cox1, while S. sibiricus contained the shortest cox1.Atp8 was the shortest PCG in all five Suillus mitogenomes, with an average length of 176 bp. S. sibiricus, S. placidus, and S. bovinus contained the longest atp8, whereas S. huapi and Suillus sp.contained the shortest atp8.The start codons for PCGs in the five mitogenomes included all start codons in the genetic code table 4, except for GTG.Similarly, the termination codons of PCGs in the 5 mitogenomes contained all stop codons in the genetic code table 4, except for TGA.Gene duplication was a frequent occurrence in the mitogenomes of Suillus.Duplication of atp8 occurred in the mitogenomes of S. huapi and Suillus sp.; duplication of nad6 and nad4l occurred in the mitogenome of S. bovinus; and duplication of nad4 and nad2 occurred in the mitogenome of S. sibiricus.Cytochrome c oxidase showed no duplication in the mitogenomes of Suillus.

Gene Rearrangement in the Five Suillus Mitogenomes
After gene synteny analysis of the five Suillus mitogenomes, two homologous regions (A and B) were identified (Figure 2).The order of arrangement of regions A and B was the same between S. huapi and Suillus sp. and between S. bovinus, S. sibiricus, and S. placidus.The size of region B in the mitogenome of S. sibiricus was significantly smaller than that in the remaining four mitogenomes.This may be due to the fact that the mitogenome of S. sibiricus was also the smallest among the five species.Overall, the results of synteny analysis indicated that Suillus evolved conservatively.However, currently, there is insufficient mitogenomic data from Suillus, highlighting the need for further research.

Gene Rearrangement in the Five Suillus Mitogenomes
After gene synteny analysis of the five Suillus mitogenomes, two homologous regions (A and B) were identified (Figure 2).The order of arrangement of regions A and B was the same between S. huapi and Suillus sp. and between S. bovinus, S. sibiricus, and S. placidus.The size of region B in the mitogenome of S. sibiricus was significantly smaller than that in the remaining four mitogenomes.This may be due to the fact that the mitogenome of S. sibiricus was also the smallest among the five species.Overall, the results of synteny analysis indicated that Suillus evolved conservatively.However, currently, there is insufficient mitogenomic data from Suillus, highlighting the need for further research.The gene order of the five Suillus mitogenomes was more complex than the results of the synteny analysis suggested (Figure 3).This was probably caused by the duplication and insertion of genes.Four conserved gene clusters were detected in five Suillus mitogenomes: rrnl-cox2, nad2-nad3, rrns-nad1-cob-cox3, and nad5-nad4.Rrnl and cox2 were in conserved gene order despite non-overlapping regions and long spacer regions.One to four bases overlapped between nad2 and nad3.The number of introns in the mitogenomes of Suillus was significantly high.These introns were predominantly found in cox1, cob, cox3, and nad5.Furthermore, one intron was identified in nad6 of Suillus sp.The gene order of the five Suillus mitogenomes was more complex than the results of the synteny analysis suggested (Figure 3).This was probably caused by the duplication and insertion of genes.Four conserved gene clusters were detected in five Suillus mitogenomes: rrnl-cox2, nad2-nad3, rrns-nad1-cob-cox3, and nad5-nad4.Rrnl and cox2 were in conserved gene order despite non-overlapping regions and long spacer regions.One to four bases overlapped between nad2 and nad3.The number of introns in the mitogenomes of Suillus was significantly high.These introns were predominantly found in cox1, cob, cox3, and nad5.Furthermore, one intron was identified in nad6 of Suillus sp.

Phylogenetic Relationships of Boletales
The application of two methods (maximum likelihood, ML, and Bayesian inference, BI) on the 5 datasets (PCG, PCG12, PCGRNA, PCG12RNA, and AA) resulted in nearly identical outcomes.Boletales can be divided into 7 main branches: Boletaceae, Paxillaceae, Boletinellaceae, Sclerodermataceae, Suillaceae, Rhizopogonaceae, and Gomphidiaceae (Figures 4 and S1-S9).The monophyly of each family was supported, which justified the existing classification system.Suillus were obviously monophyletic, suggesting that they evolved from a common ancestor and were more closely related to each other than to any other species.Throughout the course of evolution, Suillus and Rhizopogon share a close phylogenetic relationship.Pisolithus was once considered to belong to Suillus but has since been moved out as a separate genus, which was confirmed by our study.Despite their morphological similarities, Suillus is more distantly related to the remaining Boletes species than Pisolithus.The 3 families, Suillaceae, Rhizopogonaceae, and Gomphidiaceae, belonging to the suborder Suillineae, also showed close affinities in these phylogenetic trees.

Gene Order Analysis of Boletales
To further investigate the associations between gene order and phylogenetic relationships in Boletales, a comparison of mitogenomic gene order was performed for 39 species from the Boletales, covering 20 genera from 7 families.During the evolution of the Boletales, a large number of gene rearrangement events occurred (Figure 5).However, these changes in the location and direction of genes were non-random; there were certain patterns to be found.The orientation of rps3 was reversed in all but six species, which were in a rather confusing order in Boletaceae.In contrast, in all other families of the Boletales, rps3 was positively orientated.In the Boletales, dpo was only detected in the family Boletaceae, and its position and orientation were relatively random.With the exception of gene order disruption due to the insertion of duplicate genes, the gene order of most species followed a specific pattern (cox1-atp6-cox2-rps3-nad6-atp9-atp8-nad2-nad3-nad1-cob-cox3-nad4l-nad5-nad4) in Suillaceae, Rhizopogonaceae, and Gomphidiaceae.The positions of atp9-atp8 and nad2-nad3 were switched in Paxillaceae and Sclerodermataceae.In contrast, after a certain amount of gene rearrangement as well as phylogenetic evolution, the gene order tended to develop in a pattern (cox1-atp6-cox2-atp9-nad3-nad2-nad6-rps3-nad4-nad5-nad4l-cox3-cob-nad1-atp8) in the Boletaceae.

Gene Order Analysis of Boletales
To further investigate the associations between gene order and phylogenetic relationships in Boletales, a comparison of mitogenomic gene order was performed for 39 species from the Boletales, covering 20 genera from 7 families.During the evolution of the Boletales, a large number of gene rearrangement events occurred (Figure 5).However, these changes in the location and direction of genes were non-random; there were certain patterns to be found.The orientation of rps3 was reversed in all but six species, which were in a rather confusing order in Boletaceae.In contrast, in all other families of the Boletales, rps3 was positively orientated.In the Boletales, dpo was only detected in the family Boletaceae, and its position and orientation were relatively random.With the exception of gene order disruption due to the insertion of duplicate genes, the gene order of most species followed a specific pattern (cox1-atp6-cox2-rps3-nad6-atp9-atp8-nad2-nad3-nad1-cob-cox3-nad4l-nad5-nad4) in Suillaceae, Rhizopogonaceae, and Gomphidiaceae.The positions of atp9-atp8 and nad2-nad3 were switched in Paxillaceae and Sclerodermataceae.In contrast, after a certain amount of gene rearrangement as well as phylogenetic evolution, the gene order tended

Evolutionary Rates of Boletales
To further explore the evolutionary dynamics of Boletales, PCGs were analyzed for selection pressure at the amino acid level.Due to the skewedness of the data, the median was used to represent the distribution (Figure 6).The nonsynonymous substitution (Ka) rate of rps3 was significantly higher than that of the other PCGs.Meanwhile, the Ka rate of nad4l was the lowest among the 15 PCGs.In general, the Ka values of all 15 genes were found to be less than 0.3.Among these 15 genes, nad2 had the highest synonymous substitution (Ks) rate, while atp9 had the lowest.The Ks values of these 15 PCGs were all less than 0.8.The values of Ka/Ks in the 15 PCGs in Boletales were all less than one, and this purifying selection at the amino acid level played a dominant role in each species.Nad4l and cox1 had significantly lower Ka/Ks values, suggesting that these genes had undergone stronger evolutionary selection pressure.Among the 15 PCGs, rps3 had significantly higher values of Ka/Ks.

Evolutionary Rates of Boletales
To further explore the evolutionary dynamics of Boletales, PCGs were analyzed for selection pressure at the amino acid level.Due to the skewedness of the data, the median was used to represent the distribution (Figure 6).The nonsynonymous substitution (Ka) rate of rps3 was significantly higher than that of the other PCGs.Meanwhile, the Ka rate of nad4l was the lowest among the 15 PCGs.In general, the Ka values of all 15 genes were found to be less than 0.3.Among these 15 genes, nad2 had the highest synonymous substitution (Ks) rate, while atp9 had the lowest.The Ks values of these 15 PCGs were all less than 0.8.The values of Ka/Ks in the 15 PCGs in Boletales were all less than one, and this purifying selection at the amino acid level played a dominant role in each species.Nad4l and cox1 had significantly lower Ka/Ks values, suggesting that these genes had undergone stronger evolutionary selection pressure.Among the 15 PCGs, rps3 had significantly higher values of Ka/Ks.

Evolutionary Rates of Boletales
To further explore the evolutionary dynamics of Boletales, PCGs were analyzed for selection pressure at the amino acid level.Due to the skewedness of the data, the median was used to represent the distribution (Figure 6).The nonsynonymous substitution (Ka) rate of rps3 was significantly higher than that of the other PCGs.Meanwhile, the Ka rate of nad4l was the lowest among the 15 PCGs.In general, the Ka values of all 15 genes were found to be less than 0.3.Among these 15 genes, nad2 had the highest synonymous substitution (Ks) rate, while atp9 had the lowest.The Ks values of these 15 PCGs were all less than 0.8.The values of Ka/Ks in the 15 PCGs in Boletales were all less than one, and this purifying selection at the amino acid level played a dominant role in each species.Nad4l and cox1 had significantly lower Ka/Ks values, suggesting that these genes had undergone stronger evolutionary selection pressure.Among the 15 PCGs, rps3 had significantly higher values of Ka/Ks.

Discussion
Although fungal mitogenomes are intermediate in size between those of animals and plants, they exhibit the greatest variation in size [24].The size of the fungal mitogenome changes due to several factors, including the accumulation of repetitive sequences, expansion and contraction of introns, and the length of intergenic regions [25,26].This is true in Boletales, where the presence of introns makes Suillus and Rhizopogon the two genera with the longest mitogenomes.Interestingly, both Suillus and Rhizopogon are one of the ecological drivers of global pine invasions [1].And they are always one of the first fungi to appear on young pine plantations.Perhaps this genomic difference has led to differences in their behavior during the global pine invasions.The length of the intergenic regions is an important factor in the variation in mitogenome length of both Boletaceae and Suillaceae.The variation in mitogenome size within Boletaceae is largely attributed to the accumulation of repetitive sequences.This phenomenon, however, is not observed in the other six families of Boletales.
Although mitochondrial genes are generally conserved owing to their role in cellular metabolism, the order of fungal mitochondrial genes exhibits great variability, which may be attributed to recombination [27].The mitogenomes of fungi exhibit distinct variations in gene order, both between species and within the same species [28], which was confirmed by the results of this study.The mitogenomic gene order in Boletales species varies considerably between genera and between species of the same genus.Gene duplication causes the highly disorganized gene order in the mitogenomes of Suillus species.In these mitogenomes, genes undergo random insertion into a new position after a duplication event, leading to a change in their order.This study broadly categorized the mitogenome gene order of Boletales species into two patterns, consistent with previous research [20].Intra-molecular recombination of the mitogenome of the common ancestor of the Boletaceae may account for the difference between these two patterns [29].As a former member of the Agaricales, the PCGs in the mitogenomes of Boletales species exhibited an evolutionary rate that was approximately equivalent to that observed in the mitogenomes of Agaricales species [30].The Ka/Ks ratios of the 15 PCGs in the mitogenomes of the two orders were less than one, indicating that these genes are still undergoing phylogenetic change.
Previous studies have raised questions regarding the distribution of Suillus placidus in China [31].We concluded that the species previously misidentified as S. placidus is actually a well-supported independent lineage, known as S. huapi.The two species have distinct physical appearance and ecological characteristics.In S. huapi, the color of the pileus changes to brown with growth, whereas in S. placidus, it remains white throughout the life cycle.Although all species of Suillus form symbiotic relationships with Pinus species, the specific species differ.This study provides evidence of the existence of S. placidus in China.During our study of the mitogenomes, we observed a clear distinction between S. placidus and S. huapi.To verify this, ITS fragments and the mitogenome were used to distinguish between S. placidus and S. huapi.The ITS was used to reconstruct the phylogenetic tree using the same methods and parameters used in the study by Xue et al. (Figures 7 and S10).The molecular fragment reconstruction results indicated a clear division of the ITS from the two species into two branches: species S. placidus from China exhibited close affinities with species from Asia and Europe.There also appeared to be a clustering of species from the Americas and Australia, while most of the ITS of S. huapi belonged to Chinese species.Phylogenetic trees inferred from mitogenomes also indicated the presence of S. placidus in China.Taken together, the molecular fragments contain less information and are slightly less effective than the mitogenome in distinguishing between easily confused species.

Sample Collection, DNA Isolation, and Sequencing
The five Suillus specimens were collected from Guizhou and Yunnan province, China (Table S1).After examination and description, the dried specimens were deposited at Guizhou Medical University.They were first identified by their macroscopic characteristics, such as the pileus, stipe, and hymenophore, and their microscopic characteristics, such as the size of the spores.Then, their identities were confirmed by molecular analysis.DNA samples were extracted from entities of the five specimens using Fungi Genomic DNA Extraction Kits (Solarbio, Beijing, China) and Fungal DNA Kits (Omega, GA, USA), according to the manufacturers' instructions.The DNA samples were stored at −20 °C until sequencing.The complete mitogenomes of these species were obtained via NGS (Illumina HiSeq 4000 and 6 Gb raw data; Berry Genomic, Beijing, China).

Assembly, Genome Annotation, and Sequence Analysis
The obtained sequences were assembled using Geneious (Available online: https://www.geneious.com,accessed on 5 February-1 March 2024) and compared with homologous sequences of Aureoboletus raphanaceus (NC079662) and Pulveroboletus ravenelii (NC061666) [16].They were then identified through BLAST searches in the NCBI database.The mitogenomes of five Suillus species were annotated using MITOS based on the Galaxy platform [32] and genetic code table 4 (mold protozoan mitochondrial).The

Sample Collection, DNA Isolation, and Sequencing
The five Suillus specimens were collected from Guizhou and Yunnan province, China (Table S1).After examination and description, the dried specimens were deposited at Guizhou Medical University.They were first identified by their macroscopic characteristics, such as the pileus, stipe, and hymenophore, and their microscopic characteristics, such as the size of the spores.Then, their identities were confirmed by molecular analysis.DNA samples were extracted from entities of the five specimens using Fungi Genomic DNA Extraction Kits (Solarbio, Beijing, China) and Fungal DNA Kits (Omega, GA, USA), according to the manufacturers' instructions.The DNA samples were stored at −20 • C until sequencing.The complete mitogenomes of these species were obtained via NGS (Illumina HiSeq 4000 and 6 Gb raw data; Berry Genomic, Beijing, China).

Assembly, Genome Annotation, and Sequence Analysis
The obtained sequences were assembled using Geneious (Available online: https://www.geneious.com,accessed on 5 February-1 March 2024) and compared with homologous sequences of Aureoboletus raphanaceus (NC079662) and Pulveroboletus ravenelii (NC061666) [16].They were then identified through BLAST searches in the NCBI database.The mitogenomes of five Suillus species were annotated using MITOS based on the Galaxy platform [32] and genetic code table 4 (mold protozoan mitochondrial).The locations of the large ribosomal subunit (rrnl) and small ribosomal subunit (rrns) were ascertained via comparison with homologous mitogenomes.The locations of 15 protein-coding genes (PCGs) were confirmed using ORF Finder in Geneious.Finally, all genes with reference were compared to verify their validity.Five circular mitogenomic maps were constructed using OGDraw version 1.3.1 [33].The strand asymmetry was calculated using the following formulas: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [34].
Phylogenetic trees were reconstructed using ML and BI analyses.ML analysis was conducted in IQ-TREE version 1.6.12 with 1000 bootstrap replicates to estimate node support using the ultrafast bootstrap approach [43].BI trees were constructed using MrBayes v.3.2.7 [44] with the default settings to simulate four independent runs for 1 million generations, with sampling every 1000 generations.After the average standard deviation of split frequencies decreased to <0.01, the initial 25% of the samples were discarded as burn-in, and the remaining trees were used to generate a consensus tree and to calculate the posterior probabilities.Finally, the phylogenetic trees were visualized using FigTree version 1.4.4 (Available online: tree.bio.ed.ac.uk/software/Figtree/, accessed on 7 May 2024) and enhanced using Adobe Illustrator CC version 22.1.
To determine whether S. placidus and S. huapi were the same species and explore the differences in phylogenetic relationships obtained using mitogenomes and molecular fragments, we chose ITS data, a molecular fragment with the most data, for these two species to reconstruct the phylogenetic relationship.All sequences were aligned using the MAFFT algorithm in PhyloSuite [45].Poorly aligned sites were removed using Gblocks 0.91b., and MrBayes v.3.2.7 was used to construct BI trees.ML analysis was conducted via IQ-TREE.Visualization of phylogenetic trees was performed using FigTree and Adobe Illustrator CC version 22.1.

Conclusions
Suillus is a crucial ectomycorrhizal fungus as well as a valuable edible and medicinal mushroom.Owing to their similar morphology, researchers have conducted studies to differentiate the species of Suillus [46,47].However, there is limited information available on molecular fragments, which hinders the efforts to differentiate species.Mitochondrial genomes are commonly used to differentiate species and phylogeny because of their high copy rate and easy accessibility [48,49].Studies have reported mitogenomes for many species of Boletales, but not for any species of Suillus.In this study, we collected, sequenced, assembled, and annotated the mitogenomes of five Suillus species.These five mitogenomes were analyzed comparatively and phylogenetically with those of other species of Boletales.All five mitogenomes were probed for at least 15 PCGs, 25 tRNAs, and 2 ribosomal RNAs.This study revealed that these mitogenomes had common duplications of the ATP synthase subunit and NADH dehydrogenase subunit.The results of gene synteny and gene order analyses were slightly different between the mitogenomes of Suillus species.The former suggested that species of Suillus were more conserved, while the latter indicated that Suillus species were more disordered due to the insertion of duplicate genes.Phylogenetic analysis validated the existing taxonomic system of Boletales species at the family level, with support for the monophyly of the seven families.There were two main patterns of gene order in Boletales, and the insertion of duplicated genes and dpo may disrupt the gene order.Analysis of 15 PCGs of Boletales for selection pressure showed that these genes were subject to purifying selection.This study revealed that the mitogenome provides insights into the evolutionary relationships of Suillus and Boletales, aids in constructing accurate phylogenetic trees, and serves as a molecular marker for species identification.Further research is warranted to better understand the mitogenomes of Suillus species, including their evolution patterns and mechanisms, as well as the relationship between mitogenomes and fungal functions and adaptations.

Figure 1 .
Figure 1.Circular maps of 5 mitogenomes of Suillus.Each gene is represented by the corresponding color block.Color blocks outside each ring indicate that the genes are on the direct strand, and color blocks within the ring indicate that the genes are located on the reverse strand.

Figure 1 .
Figure 1.Circular maps of 5 mitogenomes of Suillus.Each gene is represented by the corresponding color block.Color blocks outside each ring indicate that the genes are on the direct strand, and color blocks within the ring indicate that the genes are located on the reverse strand.

Figure 2 .
Figure 2. Synteny analysis of the 5 Suillus mitogenomes.The symbols * indicate that the posterior probability of this node is 1.Homologous regions between Suillus species are color-coded and connected by lines.

Figure 2 .
Figure 2. Synteny analysis of the 5 Suillus mitogenomes.The symbols * indicate that the posterior probability of this node is 1.Homologous regions between Suillus species are color-coded and connected by lines.

13 Figure 3 .
Figure 3. Gene order analysis of Suillus.The symbols * indicate that this gene contains introns.Each colored block represents a distinct gene type.15 PCGs and 2 rRNAs were utilized for analysis.

Figure 3 .
Figure 3. Gene order analysis of Suillus.The symbols * indicate that this gene contains introns.Each colored block represents a distinct gene type.15 PCGs and 2 rRNAs were utilized for analysis.

Figure 4 .
Figure 4. Phylogenetic relationship of Boletales using MrBayes.The symbols * indicate that the posterior probability of this node is 1.Five newly sequenced mitogenomes of Suillus highlighted in lavender.

Figure 4 .
Figure 4. Phylogenetic relationship of Boletales using MrBayes.The symbols * indicate that the posterior probability of this node is 1.Five newly sequenced mitogenomes of Suillus highlighted in lavender.

Figure 5 .
Figure 5. Gene order analysis of Boletales.The symbols * indicate that the posterior probability of this node is 1.Five newly sequenced mitogenomes of Suillus were highlighted in lavender.Each colored block represents a distinct gene type.16 PCGs were utilized for analysis.

Figure 6 .
Figure 6.The rate of gene evolution in species of the Boletales.Ka, the number of nonsynonymous substitutions per nonsynonymous site; Ks, the number of synonymous substitutions per synonymous site.

Figure 5 .
Figure 5. Gene order analysis of Boletales.The symbols * indicate that the posterior probability of this node is 1.Five newly sequenced mitogenomes of Suillus were highlighted in lavender.Each colored block represents a distinct gene type.16 PCGs were utilized for analysis.

13 Figure 5 .
Figure 5. Gene order analysis of Boletales.The symbols * indicate that the posterior probability of this node is 1.Five newly sequenced mitogenomes of Suillus were highlighted in lavender.Each colored block represents a distinct gene type.16 PCGs were utilized for analysis.

Figure 6 .
Figure 6.The rate of gene evolution in species of the Boletales.Ka, the number of nonsynonymous substitutions per nonsynonymous site; Ks, the number of synonymous substitutions per synonymous site.

Figure 6 .
Figure 6.The rate of gene evolution in species of the Boletales.Ka, the number of nonsynonymous substitutions per nonsynonymous site; Ks, the number of synonymous substitutions per synonymous site.

Figure 7 .
Figure 7. Phylogenetic relationship of Suillus placidus and Suillus huapi inferred from an ITS dataset using Bayesian inference.Our newly sequenced molecular fragments were highlighted in orange.

Figure 7 .
Figure 7. Phylogenetic relationship of Suillus placidus and Suillus huapi inferred from an ITS dataset using Bayesian inference.Our newly sequenced molecular fragments were highlighted in orange.

Table 1 .
Basic composition of 5 new mitogenomes of Suillus.

Table 1 .
Basic composition of 5 new mitogenomes of Suillus.