Mitochondrial genome assembly and phylogenetics
Short-read shotgun sequencing produced a minimum 150-fold read coverage per sample that was used to reconstruct the mitochondrial genomes from six Nannizziopsis and one Paranannizziopsis reference strains. Variability in mitochondrial DNA length between species ranged from 24.5 to 30.8 Kb (TABLE 1). The overall GC content was fairly consistent among species ranging between 23.4 to 24.5%, consistent with the AT-rich composition of fungal mitochondrial genomes. The N. barbatae mtDNA is gene-dense and contains the same gene order reported for other Onygenaceae (FIGURE 2) (de Melo Teixeira, Lang, Matute, Stajich, & Barker, 2021).
A total of 3,780 positions were aligned across the 13 PCGs annotated for each of the genomes used in this study (FIGURE 3). Topology of theNannizziopsis branches in the phylogenetic tree resemble results from previous studies (Peterson et al., 2020; Sigler et al., 2013) showing congruence between the approach used in this study and the conserved nucleotide targets of the ITS and 28S ribosomal regions. Only two out of the 3,757 aligned positions observed between the assemblies of N. barbatae were different suggesting a high level of conservation in PCGs between these strains. Likewise, only two out of the 3,760 aligned amino acids between N. vriesii andN. dermatiditis were found to be different. Given the high degree of sequence conservation between these two pairwise comparisons, whole mtDNA alignments were performed to determine the degree of similarity at the nucleotide level. Whole mtDNA comparisons between N. barbataestrains USC001 and UAHM 11185 resulted in 1,675 differences being observed among 27,757 aligned nucleotide positions (93.97% identical). However, the alignment between N. vriesii and N. dermatiditis resulted in only 154 differences out of 24,572 aligned nucleotide positions (99.37% identical). TheN. barbatae strain USC001 assembly is slightly larger than the UAHM 11185 strain owing to the inclusion of two introns, one in the cox1 gene and another in the large ribosomal subunit RNA gene (rnl ) gene of approximately 1 Kb each in size. The cox1 intron was found to contain an LAGLI-DADG endonuclease motif and the rnl intron an GIY-YIG endonuclease motif. The smaller assembly size for N. vriesii when compared with N. dermatiditis is due to the former missing an approximately 1 Kb intron in the nad1 gene, explaining the absence of PCR amplification with the primers from this assay. This intron also contains an GIY-YIG endonuclease motif.