The complete mitochondrial genome of enigmatic mycoparasitic fungus Squamanita imbachii

Abstract The complete mitochondrial genome of Squamanita imbachii I. Saar, is unveiled in this research for the first time. It covers 76,643 base pairs (bp) and exhibits a guanine-cytosine (GC) content of 23%. The genome includes 14 conserved protein-coding genes, 1 DNA polymerase gene, 2 ribosomal RNA gene (RNS and RNL), 25 transfer RNA (tRNA) genes and 18 open reading frames (ORFs). Phylogenetic analysis, utilizing a mitochondrial gene dataset from 15 taxa across seven families within the Agaricales order, was conducted employing the maximum-likelihood (ML) approach. This analysis identified a close phylogenetic relationship between S. imbachii and Floccularia luteovirens (Alb. & Schwein.) Pouzar 1957, positioning both within the Squamanitaceae family.


Introduction
Squamanita is a genus in the family Squamanitaceae (Vizzini et al. 2019;Kalichman et al. 2020;Liu et al. 2021), which may represent one of the most unique basidiomycetes fungi in the world as nearly all the species are parasitic on other living fungi.Saar et al. (2022) split Squamanita into two monophyletic groups, viz.Squamanita and Dissoderma (A.H.Sm. & Singer) Singer 1973.Fourteen and nine species are accepted in Squamanita and Dissoderma worldwide, respectively (Species Fungorum 2024).

Genomic DNA extraction and sequencing
The CTAB method (Doyle and Doyle 1987) was utilized to extract the entire genomic DNA from the desiccated S. imbachii specimen.Standard protocols were followed for wholegenome sequencing on an Illumina sequencing platform (HiSeq PE150).Paired-end libraries with 150 bp were created, resulting in approximately 6 GB of raw data.

Assembly and annotation
The assembly process was carried out employing GetOrganelle v. 1.7.5 with default settings (Jin et al. 2020).depth and coverage was calculated following the procedure outlined in Ni et al. (2023).
Squamanita imbachii and F. luteovirens exhibited a close relationship in the phylogenetic tree and both of them belonged to Squamanitaceae (Figure 3).

Discussion and conclusion
In this study, we conducted sequencing and characterization of the first mitochondrial genome from the rare genus Squamanita.Phylogenetic analysis revealed that S. imbachii and F. luteovirens are affiliated with Squamanitaceae based on mitochondrial protein-coding gene, a finding consistent with Liu et al. (2021) that was based on multiple nuclear genes.Notably, our current phylogenetic tree (Figure 3) reveals that the family Squamanitaceae does not cluster with Nidulariaceae.Given the maternally inherited nature of the mitochondrial genome, evolutionary histories infering based on mitochondrial genomes often differ from those based on biparentally inherited nuclear genomes.Further sequencing and the generation of additional mitochondrial genomes from a broader range of representative taxa within Suborder Pluteineae would enhance our understanding of the evolutionary relationships among these remarkable mycoparasitic basidiomycetes fungi.
Initial annotation was automated using the MFannot tool(Beck and Lang 2010), relying on genetic code 4 (Mold, Protozoan, and Coelenterate Mitochondrial genetic code).Verification of rRNA and tRNA genes was performed using RNAweasel(Beck and Lang 2010) and tRNAscan-SE 2.0 (Lowe and Chan 2016), respectively.To ensure accurate annotation, the intron and exon boundaries of Protein-Coding Genes (PCGs) were individually validated by aligning gene sequences with homologous alleles sequences (obtained via Extract Regions in Geneious Prime) from previously annotated mitogenomes of Squamanitaceae using the MAFFT program in Geneious Prime 2020.0.3 (BioMatters, Ltd., Auckland, New Zealand).OGDraw v. 1.2 (Lohse et al. 2013) was employed to generate a physical circular map of S. imbachii.Sequencing

Figure 2 .
Figure 2. Circular map of the mitochondrial genomes of S. imbachii.Genes are represented by different colored blocks.Colored blocks outside each ring indicate that the genes are on the direct strand, while colored blocks within the ring indicate that the genes are located on the reverse strand.The inner grayscale bar graph shows the GC content of the mitochondrial sequences.The circle inside the GC content graph marks the 50% threshold.Label intron-containing genes with � .