The research team selected five species—T. borealicremeolina, T. crassimultiformis, T. lavendulaceus, T. sinopurpurascens, and T. sinoscaurus—and sequenced their entire mitogenomes using Illumina short-read technology, supplemented by PacBio continuous long reads for one species. The resulting assemblies revealed that all five mitogenomes are circular molecules, but their sizes vary dramatically, from 35,902 base pairs in T. sinopurpurascens to 106,732 base pairs in T. crassimultiformis. GC contents ranged from 24.35% to 29.15%, with AT content consistently higher across all five species.

Despite this striking size variation, the researchers found a remarkably conserved core gene repertoire. Each mitogenome contains exactly 15 core protein-coding genes—atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3—along with two rRNA genes (rns and rnl) and 25 to 26 tRNA genes covering all 20 standard amino acids. The core gene order is also identical across the five species, suggesting that mitochondrial gene rearrangements have not occurred within this genus, even though gene order differs markedly among related genera.

The study’s comparative analysis showed that the pronounced differences in total mitogenome size are not driven by variation in the core coding regions themselves. Instead, the combined lengths of the 15 core protein-coding genes ranged only from 14,487 to 14,826 base pairs among the five species, indicating that the size variation must come from regions outside the core genes. The researchers turned their attention to introns and repeat sequences as the primary sources of this structural diversity.

Intron dynamics emerged as a key factor. Across 12 related mitogenomes examined, the team annotated 147 introns in total, with 57 of them—38.78%—residing within the cox1 gene alone. Pearson correlation analysis revealed a significant positive correlation between total intron abundance and mitogenome size (R = 0.8465, P = 0.0005). Among the five Thaxterogaster species, T. crassimultiformis and T. borealicremeolina contained 17 and 16 introns respectively, and their mitogenomes were markedly larger than those of the other three species, which harbored no introns at all. Further classification of cox1 introns into positional classes showed that the two larger-genome species shared several homologous insertion sites while each also harbored species-specific variants.

Repeat sequences added another layer of variation. The researchers identified 192 simple sequence repeats, 94 tandem repeats, and 184 pairs of interspersed repeats across the five mitogenomes. T. borealicremeolina possessed the highest SSR count at 87, while T. sinopurpurascens had only 9. Mononucleotide repeats dominated the SSR repertoire in all species. The numbers of different repeat types varied markedly among species, indicating differential repeat accumulation within the mitogenomes of this genus.

The study also predicted putative C-to-U RNA editing sites using a convolutional neural network approach, recovering 318 high-confidence candidate sites across 14 of the 15 core protein-coding genes. The gene-wise distribution differed significantly from a cytosine-proportional expectation, with cob significantly enriched and cox3 significantly depleted. Of the 318 candidate sites, 287 were predicted to result in nonsynonymous changes, and cross-species mapping identified 138 homologous site groups, of which 77 were shared by at least two species—a number significantly greater than expected by chance, suggesting conservation of editing sites across lineages.

To evaluate the phylogenetic utility of mitochondrial core genes, the researchers reconstructed a tree based on the concatenated amino acid sequences of the 15 core protein-coding genes from 51 species. Both maximum likelihood and Bayesian inference produced largely congruent topologies. The five Thaxterogaster species formed a well-supported distinct clade within Cortinariaceae, positioned adjacent to related lineages of Hymenogastraceae and Strophariaceae. Within the Thaxterogaster clade, T. crassimultiformis and T. borealicremeolina formed a fully supported sister pair, while T. sinoscaurus and T. sinopurpurascens constituted another sister subclade, with T. lavendulaceus occupying the basal position.

The mitogenome-based tree showed a high degree of congruence with previous multigene and nuclear genomic phylogenies, with no obvious conflicts at the family or genus level. Notably, the mitochondrial tree yielded higher nodal support values than previous analyses, further demonstrating the utility of mitochondrial core protein-coding genes in resolving relationships within Thaxterogaster and closely related lineages. However, the researchers caution that because mitochondrial genes are inherited as a single linkage group, the high support values should not be interpreted as evidence from multiple independent loci.

Collectively, these findings enhance understanding of mitogenomic structural and evolutionary features in Thaxterogaster and provide a valuable reference for future studies into mitogenome evolution in Cortinariaceae and allied taxa. The researchers note that the limited taxonomic sampling in this study cannot fully represent the mitogenomic diversity of the genus, and they call for broader species sampling and combined nuclear-mitogenome datasets in future work to improve resolution of intergeneric relationships within Cortinariaceae.

Subject of Research: Comparative mitogenomics and phylogenetics of five Thaxterogaster mushroom species

Article Title: Assembly and comparative analysis of the complete mitochondrial genomes of five Thaxterogaster species ( Cortinariaceae , Basidiomycota ): Insights into mitochondrial genome evolution and intrageneric phylogenetic relationships

Article References: Ou, L.-Y., Xie, M.-L., Li, Y., & Li, C.-T. (2026). Assembly and comparative analysis of the complete mitochondrial genomes of five Thaxterogaster species (Cortinariaceae, Basidiomycota): Insights into mitochondrial genome evolution and intrageneric phylogenetic relationships. MycoKeys, 140, 217-239. https://doi.org/10.3897/mycokeys.140.205520

Image Credits: AI Generated

DOI: 10.3897/mycokeys.140.205520

Keywords: Thaxterogaster, mitochondrial genome, Cortinariaceae, phylogenetics, Basidiomycota, introns, Assembly, comparative, analysis, complete, mitochondrial, genomes