Aspergillus nidulans has long-been used as a model organism to gain insights into the genetic basis of asexual and sexual developmental processes both in other members of the genus Aspergillus, and filamentous fungi in general. Paradigms have been established concerning the regulatory mechanisms of conidial development. However, recent studies have shown considerable genome divergence in the fungal kingdom, questioning the general applicability of findings from Aspergillus, and certain longstanding evolutionary theories have been questioned. The phylogenetic distribution of key regulatory elements of asexual reproduction in A. nidulans was investigated in a broad taxonomic range of fungi. This revealed that some proteins were well conserved in the Pezizomycotina (e.g. AbaA, FlbA, FluG, NsdD, MedA, and some velvet proteins), suggesting similar developmental roles. However, other elements (e.g. BrlA) had a more restricted distribution solely in the Eurotiomycetes, and it appears that the genetic control of sporulation seems to be more complex in the aspergilli than in some other taxonomic groups of the Pezizomycotina. The evolution of the velvet protein family is discussed based on the history of expansion and contraction events in the early divergent fungi. Heterologous expression of the A. nidulans abaA gene in Monascus ruber failed to induce development of complete conidiophores as seen in the aspergilli, but did result in increased conidial production. The absence of many components of the asexual developmental pathway from members of the Saccharomycotina supports the hypothesis that differences in the complexity of their spore formation is due in part to the increased diversity of the sporulation machinery evident in the Pezizomycotina. Investigations were also made into the evolution of sex and sexuality in the aspergilli. MAT loci were identified from the heterothallic Aspergillus (Emericella) heterothallicus and Aspergillus (Neosartorya) fennelliae and the homothallic Aspergillus pseudoglaucus (=Eurotium repens). A consistent architecture of the MAT locus was seen in these and other heterothallic aspergilli whereas much variation was seen in the arrangement of MAT loci in homothallic aspergilli. This suggested that it is most likely that the common ancestor of the aspergilli exhibited a heterothallic breeding system. Finally, the supposed prevalence of asexuality in the aspergilli was examined. Investigations were made using A. clavatus as a representative ‘asexual’ species. It was possible to induce a sexual cycle in A. clavatus given the correct MAT1-1 and MAT1-2 partners and environmental conditions, with recombination confirmed utilising molecular markers. This indicated that sexual reproduction might be possible in many supposedly asexual aspergilli and beyond, providing general insights into the nature of asexuality in fungi.

Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.

A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits.

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Introduction A. fumigatus is a saprotrophic fungus that is involved in environmental carbon and nitrogen recycling is a ubiquitous filamentous fungus in the environment. It is abundant in soils and decomposing organic materials. It is essential that A. fumigatus survive and flourishes in nutrient limited environments, 1 and the most widespread airborne human pathogen, accountable for approximately 90% of all invasive aspergillosis (IA) cases. 2 Spores in A. fumigatus can be produced by two different forms of cellular reproduction, asexual reproduction (produce conidia) or sexual reproduction (produce ascospore). Sexual reproduction involves the production of eight ascospores normally produced in each ascus. These asci form within an ascocarp with a closed structure called cleistothecium. The species has a heterothallic breeding system, isolates of complementary mating types are necessary for sex to take place. 3 Sexual reproduction may bring about increased genetic variation, due to recombination of genes from the parents that can occur by crossing over, gene conversion, or independent segregation of centromeres, permitting a species to react to a change in the environment and evolve. 4 Germination consequently forms a first and critical stage Background and objective: Aspergillus fumigatus is the most common airborne fungal pathogen of humans. It is an opportunistic human pathogen in immunocompromised hosts. Following the recent discovery of the sexual reproductive cycle ascospores of this fungus were available and their germination was studied in the present study. This study aimed to study factors influencing germination of ascospores of A. fumigatus. Methods: A total of 12 environmental A. fumigatus were chosen in both mating type, examined for sexual reproduction by crossing complementary mating type, following removing of cleistothecia, preparation of ascospore suspensions in 0.05% Tween 20, then heat treated to kill any remaining conidia. Results: Ascospore germination of greater than 75% occurred at 28 °C on a wide variety of mycological media, 0.5% (ACM) was chosen for use in subsequent studies. Ascospores did not require a heat treatment prior to germination; however, heat treatment of ascospores at 75 °C for 1 hour previous to incubation enhanced germination to 89% and also served to inactivate contaminating conidia. Ascospores also exhibited a psychrotolerance following exposure at-80 °C for 96 hours. Inoculum concentration did not have a significant effect on germination rates between the range of 0.125×10 6 to 4×10 6 ascospores ml-1 (P >0.05). Conclusion: This study demonstrated that ascospore germination of greater than 75% occurred at 28 °C on a wide variety of mycological media. Ascospores of A. fumigatus are thermotolerant also exhibited a psychrotolerance whilst at the same time retaining viable sexual ascospores, to assess percentage germination a drop of Lactophenol Cotton Blue was added.