Phylogenomics reveals extensive misidentification of fungal strains from the genus Aspergillus

Abstract

Modern taxonomic classification is often based on phylogenetic analyses of a few molecular markers, although single-gene studies are still common. Here, we leverage genome-scale molecular phylogenetics (phylogenomics) of species and populations to reconstruct evolutionary relationships in a dense data set of 710 fungal genomes from the biomedically and technologically important genus Aspergillus. To do so, we generated a novel set of 1,362 high-quality molecular markers specific for Aspergillus and provided profile Hidden Markov Models for each, facilitating their use by others. Examining the resulting phylogeny helped resolve ongoing taxonomic controversies, identified new ones, and revealed extensive strain misidentification (7.59% of strains were previously misidentified), underscoring the importance of population-level sampling in species classification. These findings were corroborated using the current standard, taxonomically informative loci. These findings suggest that phylogenomics of species and populations can facilitate accurate taxonomic classifications and reconstructions of the Tree of Life.IMPORTANCEIdentification of fungal species relies on the use of molecular markers. Advances in genomic technologies have made it possible to sequence the genome of any fungal strain, making it possible to use genomic data for the accurate assignment of strains to fungal species (and for the discovery of new ones). We examined the usefulness and current limitations of genomic data using a large data set of 710 publicly available genomes from multiple strains and species of the biomedically, agriculturally, and industrially important genus Aspergillus. Our evolutionary genomic analyses revealed that nearly 8% of publicly available Aspergillus genomes are misidentified. Our work highlights the usefulness of genomic data for fungal systematic biology and suggests that systematic genome sequencing of multiple strains, including reference strains (e.g., type strains), of fungal species will be required to reduce misidentification errors in public databases.

Keywords: Aspergillaceae; Penicillium; genomics; pathogen; pathogenicity; plant pathogen; taxogenomics; taxonomy; virulence.

Fig 1

Phylogenomic tree of 725 genomes based on analysis of 1,362 genes (6,378,237 nucleotide sites). The evolutionary history of 710 Aspergillus species and 15 outgroup genomes was reconstructed from a 1,362-gene matrix. The phylogeny is depicted without branch lengths (a) and with branch lengths, representing substitutions per site (b). Colors represent different sections—taxonomic ranks above species and below genus. Note, Versicolores is a series within the section Nidulantes.

Fig 2

Concatenation- (left) and coalescence-based (right) phylogenies of taxonomic sections in the genus Aspergillus are highly congruent. The evolutionary relationships among sections are depicted. Species-level concatenation- and coalescence-based phylogenies differed at two bipartitions (represented by a red dot). Branch lengths and triangle sizes have no meaning. Note, Versicolores is a series within the section Nidulantes.

Fig 3

Phylogenomics underscores known taxonomic uncertainties and reveals new ones. (a) A. neoellipticus NRRL 5109 is an A. fumigatus strain or the clade formed by the 16 A. fumigatus strains and A. neoellipticus NRRL 5109 is, in fact, A. neoellipticus, a species sister to A. fumigatus. (b) Strain misidentification occurs between Aspergillus pseudoterreus and Aspergillus terreus and (c) Aspergillus pseudonomiae and Aspergillus nomiae. (d) Strains identified as Aspergillus welwitschiae are A. niger. (e) Aspergillus strain ATCC 9577 is misidentified as Aspergillus versicolor but is Aspergillus sydowii. (f) Strains of A. oryzae, A. flavus, Aspergillus parasiticus, and Aspergillus minisclerotigenes appear to have polyphyletic origins, a result that is likely due to extensive strain misidentification [e.g., see also Houbraken et al. (22), detailing misidentification of five strains of A. minisclerotigenes as A. flavus (22)]. Topologies presented were inferred using the concatenation approach. See Fig. S3 at https://doi.org/10.6084/m9.figshare.21382131 for topologies inferred using coalescence. Different colors represent different species. Isolates with no known species are depicted in black. Triangles represent collapsed linages with multiple isolates. The number of isolates in each collapsed lineage is shown next to the species name in parentheses. See Table 1 for the revised taxonomy.

Fig 4

A species-level phylogeny of the genus Aspergillus. A genome-scale view of the Aspergillus phylogeny and the identification of a new sister lineage, clade A, to a clade of the rest of Aspergillus species whose genomes have been sequenced may help understand the early evolution of this biomedically and technologically relevant lineage. Inset represents the same phylogeny with branch lengths representing substitutions per site. Note, Versicolores is a series within the section Nidulantes.