Figure 1. Origins of the independent lineages of H99.

Since the initial publication, the isolate has lost virulence following laboratory passage (possibly multiple independent times) and was subsequently passaged through the rabbit model of infection to increase virulence and distributed to many labs. All variants were derived from the original sequenced H99 isolate (H99O), and the major strain variants of this study have been termed H99W, H99E, and H99S. The origins of this strain series are as follows. During laboratory passage by repeated growth on YPD rich medium, the H99W/H99ED isolates arose from the H99O original stock (frozen in 1994). H99W and H99ED are distinguished from the parental strain by reduced melanin production, impaired mating, and attenuated virulence. This isolate or a closely related derivate of H99O was sent to the Lodge laboratory (Washington University, St Louis, USA) (H99E), and was subsequently distributed to the Madhani laboratory (University of California, San Francisco, USA) (H99CMO18, hereafter named H99C). Thus, isolates H99W and H99ED (Duke University), H99E (Washington University), and H99C (UCSF) are all closely related to one another. Additionally, John Perfect (Duke University Medical Center, USA) derived the H99S isolate via passage of a mixed H99 frozen stock through the well-validated rabbit model of central nervous system (CNS) infection. The pedigree was constructed based on SNPs and indels identified from sequence analysis. Specific mutations separating independent strains are annotated.