Fungal mitochondrial genomes evolve faster than nuclear DNA in Cordyceps militaris

The prized medicinal fungus Cordyceps militaris, long valued for bioactive compounds such as cordycepin and now cultivated commercially as a novel food in China, has yielded some of its deepest evolutionary secrets. A new study published in IMA Fungus presents the first large-scale, integrated mitochondrial and nuclear genomic analysis of this entomopathogenic species, assembling complete mitogenomes from 118 geographically diverse samples and comparing them against 90 nuclear genomes. The findings reveal a mitochondrial genome that is structurally conserved yet remarkably dynamic, evolving at an accelerated rate and engaging in ongoing DNA transfer to the nucleus.

The research team, drawing samples from major culture collection centers and academic institutions across nine countries including Canada, the United States, Denmark, the United Kingdom, Germany, Russia, China, Japan, and South Korea, taxonomically validated every specimen using a multilocus approach targeting six nuclear regions. Sequencing was performed on an Illumina NovaSeq 6000 platform, and mitogenomes were assembled with two independent programs, NOVOPlasty and GetOrganelle, with gaps resolved through targeted PCR amplification and Sanger sequencing. This rigorous workflow ensured complete circular mitogenome assemblies for all 118 samples.

The resulting mitogenomes ranged in size from 26,527 base pairs in a Russian strain to 40,362 base pairs in a Chinese strain, with a mean length of 31,315 base pairs and tightly constrained GC contents between 26.12 and 27.24 percent. Despite this size variation, all samples carried a conserved suite of 14 protein-coding genes essential for oxidative phosphorylation, along with two rRNA genes and a nearly complete set of tRNA genes distributed across three primary clusters. Intriguingly, 5 percent of samples lacked the trnG_1(acc) gene while retaining its counterpart trnG_2(ucc), a finding confirmed by targeted PCR validation that hints at functional diversity in mitochondrial protein synthesis.

Comparative analysis identified 71 distinct mitochondrial haplotypes, or mitotypes, across the 118 samples. The distribution was markedly skewed: the two most prevalent mitotypes alone accounted for 33 percent of all samples, while 60 mitotypes were each represented by a single specimen. Notably, wild fruiting bodies from two locations in Liaoning Province, China, each displayed high mitotype diversity, whereas commercial fruiting bodies contained only two mitotypes, demonstrating severe genetic bottlenecks during artificial breeding and mass cultivation. Haplotype network analysis revealed no discernible association between mitotype clustering and geographic origin, suggesting extensive gene flow and an absence of reproductive isolation across the sampled range.

Structural comparisons using Mauve revealed nine core locally collinear blocks consistently preserved across all samples, underscoring the remarkable evolutionary stability of mitogenome organization. Yet the dominant force shaping mitogenome size variation emerged as intron dynamics rather than intergenic spacer accumulation. Introns exhibited substantial variability, ranging from 3,020 to 14,327 base pairs per mitogenome, and showed strong positive correlations with total mitogenome size, with correlation coefficients between 0.948 and 0.969. In contrast, intergenic regions showed only a negligible biological impact on mitogenome expansion.

A total of 18 distinct intron insertion sites were identified across eight protein-coding and rRNA genes, all belonging to the group I family and classified into six subtypes. Each intron harbored one or two intronic open reading frames encoding homing endonucleases of the LAGLIDADG or GIY-YIG families, ribosomal protein S3, or hypothetical proteins. Ancestral state reconstruction uncovered complex evolutionary trajectories, with loss events significantly outnumbering gain events across all loci at an approximately 3:2 ratio. Online BLAST searches provided compelling evidence that horizontal gene transfer, particularly among closely related Hypocreales taxa including species of Cordyceps, Beauveria, and Epichloe, has played a significant role in intron acquisition and diversification.

The study also detected evidence of mitochondrial-to-nuclear DNA transfer, producing nuclear mitochondrial DNA segments known as NUMTs. Comparative BLASTN analysis revealed considerable interstrain variability, with the number of insertions per strain ranging from one to 34 and cumulative fragment lengths reaching over 5.5 kilobases. Of particular interest, one strain harbored a NUMT exceeding 1,300 base pairs, and its authenticity was confirmed by PCR validation together with significantly lower sequencing depth than the mitogenome, effectively excluding sequencing artifacts or contamination. This provides direct evidence of ongoing mitochondrial-to-nuclear DNA transfer in C. militaris.

Perhaps the most striking finding concerns evolutionary rate. Genome-wide SNP analysis across 90 strains identified 182,967 nuclear SNPs, representing 0.57 percent of the genomic sites analyzed, whereas the mitogenome exhibited an overall SNP frequency of 1.58 percent. This means the mitochondrial genome accumulated mutations approximately 2.8-fold faster than the nuclear genome, a result that contrasts sharply with a previous locus-based study reporting moderately faster nuclear variation. The disparity underscores how evolutionary rate estimates can differ substantially depending on whether limited loci or whole genomes are analyzed.

Phylogenomic reconstructions based on mitochondrial and nuclear genomes yielded largely congruent topologies, yet subtle discordances were statistically significant, with both Shimodaira-Hasegawa and Kishino-Hasegawa tests returning P values below 0.0001. This mitochondrial-nuclear discordance may result from past hybridization, incomplete lineage sorting, or differential selective pressures acting on the two genomic compartments. The absence of any association between phylogenetic clustering and geographic origin suggests that spatial isolation has played a limited role in shaping evolution, consistent with patterns reported in other fungi such as Flammulina filiformis and Corynespora cassiicola.

Collectively, these findings establish a foundational map of intraspecific organellar diversity in one of the most economically important medicinal fungi. They demonstrate that although mitochondrial architecture is structurally conserved, its evolution is remarkably dynamic, driven by frequent intron turnover, recurrent mitochondrial DNA transfer to the nucleus, and a significantly accelerated rate of variation compared with the nuclear genome. The work provides an important foundation for future investigations into fungal genome evolution and organelle-nuclear genome interactions, and highlights the critical role of mitogenomes in understanding coevolution between the two genomic compartments.

Subject of Research: Evolutionary insights into Cordyceps militaris from mitogenome dynamics and genome-wide variation

Article Title: Evolutionary insights into Cordyceps militaris from mitogenome dynamics and genome-wide variation

Article References: Ren, L.-Y., Zhang, S., & Zhang, Y.-J. (2026). Evolutionary insights into Cordyceps militaris from mitogenome dynamics and genome-wide variation. IMA Fungus, 17, Article e195879. https://doi.org/10.3897/imafungus.17.195879

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.195879

Keywords: Cordyceps militaris, mitogenome, evolutionary biology, fungal genomics, NUMTs, horizontal gene transfer, phylogenomics, IMA Fungus, Evolutionary, insights, Cordyceps, militaris