e Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

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