Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutans species complex (AVSC). Their identification is clinically relevant because of antifungal drug resistance and refractory infections. Species boundaries in the AVSC are unresolved since most species have uniform morphology and produce interspecific hybrids in vitro. Clinical and environmental strains from six continents (n = 110) were characterized by DNA se- quencing of four to six loci. Biological compatibilities were tested within and between major phylogenetic clades, and ascospore morphology was characterised. Species delimitation methods based on the multispecies coalescent model (MSC) supported recognition of ten species including one new species. Four species are confirmed op- portunistic pathogens; A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infections, while A. felis followed by A. udagawae and A. wyomingensis are agents of feline sino-orbital aspergillosis. Recently described human-pathogenic species A. parafelis and A. pseudofelis are synonymized with A. felis and an epitype is designated for A. udagawae. Intraspecific mating assay showed that only minor part of the heterothallic species can readily generate sexual morph in vitro. Interspecific mating assay revealed that five different species combinations were biologically compatible. Hybrid ascospores had atypical surface ornamentation and significantly different dimensions compared to parental species. This suggests that species limits in the AVSC are maintained by both pre- and post-zygotic barriers and these species display a great potential for rapid adapta- tion and modulation of its virulence. This study highlights that a sufficient number of strains representing genetic diversity within a species is essential for meaningful species boundaries delimitation in cryptic species complexes. MSC-based delimitation methods are robust and suitable tools for evaluation of boundaries between these species.
Alignment - section Fumigati; concatenated data from benA, CaM, act and RPB2 loci, 76 taxa
Combined dataset of benA, CaM, act and RPB2 sequences for 76 taxa belonging to section Fumigati. Aspergillus clavatus NRRL 1 (sect. Clavati) represents an outgroup. BenA: positions 1-534; CaM: positions 535-1231; act: positions 1232-1662; RPB2: positions 1663-2661.
Fumigati benA caM actin RPB2 76 taxa.fas
Phylogeny of Aspergillus sect. Fumigati inferred from combined data: benA, CaM, act and RPB2
Phylogenetic relationships of the section Fumigati members inferred from Bayesian analysis of the combined, 4-gene data set of β-tubulin (benA), calmodulin (CaM), actin (act) and RNA polymerase II second largest subunit (RPB2) genes. Bayesian posterior probability (PP) and Maximum likelihood bootstrap support (BS) are appended to nodes; only PP ≥ 95% and BS ≥ 70% and are shown; lower supports are indicated with a hyphen, whereas asterisks indicate full support (1.00 PP or 100% BS); ex-type strains are designated by a superscript T; species names in quotes are considered synonyms. The tree is rooted with Aspergillus clavatus NRRL 1. The reproductive mode of each species is designated by icons before the species name (see legend).
Aspergillus section Fumigati phylogeny.pdf
Combined alignment: benA, CaM, act and RPB2 loci; Aspergillus viridinutans complex
Combined dataset of benA, CaM, act and RPB2 sequences for 110 taxa belongmembers of Aspergillus viridinutans complex. Aspergillus lentulus NRRL 35552 represents an outgroup. BenA: positions 1-475; CaM: positions 476-1172; act: positions 1173-1516; RPB2: positions 1517-2483.
BenA CaM actin RPB2 viridinutans complex Dryad.fas
Phylogeny of Aspergillus viridinutans species complex phylogeny inferred from combined data: BenA, CaM, act, RPB2
Phylogenetic relationships of the section Aspergillus viridinutans species complex members inferred from Bayesian analysis of the combined, 4-gene data set of β-tubulin (benA), calmodulin (CaM), actin (act) and RNA polymerase II second largest subunit (RPB2) genes. Bayesian posterior probability (PP) and Maximum likelihood bootstrap support (BS) are appended to nodes; only PP ≥ 90% and BS ≥ 70% and are shown; lower supports are indicated with a hyphen, whereas asterisks indicate full support (1.00 PP or 100% BS); ex-type strains are designated by a superscript T; species names in quotes are considered synonyms. The tree is rooted with Aspergillus lentulus NRRL 35552. The geographic origin, and reproductive mode with MAT idiomorph (if known) is designated by icons before the isolate number while substrate of origin is designated by icons after isolate number (see legend).
Aspergillus viridinutans complex phylogeny.pdf
Alignment - Aspergillus viridinutans complex, A. felis clade: concatenated data of act, benA, CaM, mcm7, RPB2 and tsr1
Combined dataset of act, benA, CaM, mcm7, RPB2 and tsr1 sequences for 33 taxa belonging to Aspergillus viridinutans complex - A. felis clade only. act: positions 1-329; benA: positions 330-803; CaM: positions 804-1484; mcm7: positions 1485-2107; RPB2: positions 2108-3074; tsr1: positions 3075-3835.
actin benA CaM mcm7 RPB2 tsr1 Dryad.fas
Species delimitation in Aspergillus felis clade based on six genetic loci
Schematic representation of results of species delimitation methods in Aspergillus felis clade based on six genetic loci. The results of multilocus method (STACEY) are compared to results of single-locus methods (mPTP, bGMYC, GMYC). The results of STACEY are shown as tree branches with different colours, while the results of single-locus methods are depicted with coloured bars highlighting congruence across methods. The displayed tree is derived from IQtree analysis based on a concatenated dataset and is used solely for the comprehensive presentation of the results from different methods. The species validation analysis results (BP&P) are appended to nodes and shown in gray bordered boxes; the values represent posterior probabilities calculated in three scenarios having different prior distributions of parameters θ (ancestral population size) and τ0 (root age). The top value represents the results of analysis with large ancestral population sizes and deep divergence: θ ~ G (1, 10) and τ0 ~ G (1, 10), the middle value ); large ancestral populations sizes and shallow divergences among species: θ ~ G (1, 10) and τ0 ~ G (2, 2000) and the bottom value small ancestral population sizes and shallow divergences among species: θ ~ G (2, 2000) and τ0 ~ G (2, 2000).
