Taxonomy of Aspergillus series Versicolores: species reduction and lessons learned about intraspecific variability

Abstract

Aspergillus series Versicolores members occur in a wide range of environments and substrates such as indoor environments, food, clinical materials, soil, caves, marine or hypersaline ecosystems. The taxonomy of the series has undergone numerous re-arrangements including a drastic reduction in the number of species and subsequent recovery to 17 species in the last decade. The identification to species level is however problematic or impossible in some isolates even using DNA sequencing or MALDI-TOF mass spectrometry indicating a problem in the definition of species boundaries. To revise the species limits, we assembled a large dataset of 518 strains. From these, a total of 213 strains were selected for the final analysis according to their calmodulin (CaM) genotype, substrate and geography. This set was used for phylogenetic analysis based on five loci (benA, CaM, RPB2, Mcm7, Tsr1). Apart from the classical phylogenetic methods, we used multispecies coalescence (MSC) model-based methods, including one multilocus method (STACEY) and five single-locus methods (GMYC, bGMYC, PTP, bPTP, ABGD). Almost all species delimitation methods suggested a broad species concept with only four species consistently supported. We also demonstrated that the currently applied concept of species is not sustainable as there are incongruences between single-gene phylogenies resulting in different species identifications when using different gene regions. Morphological and physiological data showed overall lack of good, taxonomically informative characters, which could be used for identification of such a large number of existing species. The characters expressed either low variability across species or significant intraspecific variability exceeding interspecific variability. Based on the above-mentioned results, we reduce series Versicolores to four species, namely A. versicolor, A. creber, A. sydowii and A. subversicolor, and the remaining species are synonymized with either A. versicolor or A. creber. The revised descriptions of the four accepted species are provided. They can all be identified by any of the five genes used in this study. Despite the large reduction in species number, identification based on phenotypic characters remains challenging, because the variation in phenotypic characters is high and overlapping among species, especially between A. versicolor and A. creber. Similar to the 17 narrowly defined species, the four broadly defined species do not have a specific ecology and are distributed worldwide. We expect that the application of comparable methodology with extensive sampling could lead to a similar reduction in the number of cryptic species in other extensively studied Aspergillus species complexes and other fungal genera.

Phylogenomics reveals extensive misidentification of fungal strains from the genusAspergillus

“…These findings suggest that multiple strains from multiple species may be misidentified. Furthermore, ATCC 9577 and CBS 583.65 are reportedly synonymous strain names (Sklenář et al, 2022); the fact that there are two different genome sequences available is suggestive of contamination or erroneous metadata for at least one of these strains. These findings suggest that the entire clade may benefit from taxonomic revision and closer scrutiny of strain identity.…”

“…These findings suggest that the entire clade may benefit from taxonomic revision and closer scrutiny of strain identity. To this point, a recent analysis of a five-gene, 213-taxon dataset proposed species in the series Versicolores be reduced from 17 species to four, citing intraspecific variation as a driver of over splitting of species boundaries by taxonomists (Sklenář et al, 2022). Evolutionary relationships under this new analysis differ slightly from our genome-scale phylogeny (Fig S2).…”

Reducing the number of accepted species inAspergillusseriesNigri

“…The species reduction proposed in this study is in line with some other studies using MSC model-based methods, genomic data or more complex approaches for species delimitation in fungi. For example, species number was significantly reduced in the plant pathogens from the Diaporthe eres complex ( Hilário et al 2021 ) and Alternaria section Alternaria ( Woudenberg et al 2015 ), the edible mushroom Flammulina ( Wang et al 2018 ), the lichen-forming fungus Bryoria ( Boluda et al 2019 ), the indoor fungi from Aspergillus series Versicolores ( Sklenář et al 2022 ) and the opportunistic human and animal pathogens from Aspergillus series Viridinutantes ( Hubka et al 2018 ). Numerous examples can also be found outside the Fungi kingdom ( Li et al 2019 , Feng et al 2021 , Parker et al 2021 ).…”

A monograph of Aspergillus section Candidi

“…In general, the studies of intraspecific variability in Aspergillus are relatively rare and limited to a few extensively studied species or species complexes ( Peronne et al 2006 , Drott et al 2021 , Lofgren et al 2021 , Bian et al . 2022 , Sklenář et al . 2022 ).…”