Insights into Species Evolution and Dispersal Northwest : Whole-Population Genome Analysis Provides in North American Pacific Cryptococcus gattii 2014

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. IMPORTANCE Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic. Received 15 June 2014 Accepted 16 June 2014 Published 15 July 2014 Citation Engelthaler DM, Hicks ND, Gillece JD, Roe CC, Schupp JM, Driebe EM, Gilgado F, Carriconde F, Trilles L, Firacative C, Ngamskulrungroj P, Castañeda E, Lazera MDS, Melhem MSC, Pérez-Bercoff Å, Huttley G, Sorrell TC, Voelz K, May RC, Fisher MC, Thompson GR, III, Lockhart SR, Keim P, Meyer W. 2014. Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal. mBio 5(4):e01464-14. doi:10.1128/mBio.01464-14. Editor Arturo Casadevall, Albert Einstein College of Medicine Copyright © 2014 Engelthaler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Paul Keim (population genetics and genomics questions), paul.keim@nau.edu, or Wieland Meyer (mycology and molecular epidemiology questions), wieland.meyer@sydney.edu.au. This article is a direct contribution from a member of the American Academy of Microbiology. RESEARCH ARTICLE July/August 2014 Volume 5 Issue 4 e01464-14 ® mbio.asm.org 1 m b.asm .rg on O cber 5, 2014 P ubished by m b.asm .rg D ow nladed fom Cryptococcus gattii, an environmental basidiomycetous yeast and the primary cause of cryptococcosis in immunocompetent patients, has been previously primarily associated with central nervous system disease in tropical and subtropical regions of the world (1). This distribution and disease phenotype are in contrast with those of another causative agent of cryptococcosis, Cryptococcus neoformans, which is primarily an opportunistic pathogen of immunosuppressed patients and is distributed worldwide. In addition, C. neoformans is classically found in bird excreta and decaying wood from a wide variety of trees (2–4), whereas C. gattii is primarily found growing in tree bark, tree detritus, and decaying wood. C. gattii has been closely associated with a number of trees, notably Eucalyptus species, which were thought to be the dominant environmental source allowing for transportation of C. gattii out of Australia where it is endemic to locales, such as California, Greece, and elsewhere (5, 6). It has since been shown to be endemic in South America where it is found in close association with a large number of tropical trees (2, 7–9). C. gattii is currently classified into four distinct populations, referred to previously as “major molecular types”: VGI (AFLP4), VGII (AFLP6), VGIII (AFLP5), and VGIV (AFLP7) (10), based on multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP), and other subgenomic genotyping methodologies. Each of these populations or molecular types can be divided further into distinct lineages or subtypes, as defined by AFLP profiling or MLST sequence typing, which identify individual clonal lineages within each molecular type. In the late 1990s, infections caused by C. gattii molecular type VGII were reported on Vancouver Island in British Columbia, Canada (11–13); until this time, reports of C. gattii infection were uncommon in North America (1). Two clonal subtypes within the VGII molecular type known as VGIIa (AFLP6A) and VGIIb (AFLP6B) were identified as the cause of the outbreak, which subsequently spread to the Canadian mainland (14) and other regions of the Pacific Northwest (PNW) (15, 16). Within a decade, an additional novel VGII subtype, VGIIc, appeared in the PNW, primarily in the states of Oregon and Washington (16). Given the genomic similarity of isolates within each subtype and finding only the mating type locus, all three subtypes were thought to be distinct clones (17). While all three PNW subtypes of C. gattii display a shared novel biome preference (presence in a temperate versus tropical climate), only VGIIb maintains the classical clinical features of neurologic dominance (18). The VGIIa and VGIIc subtypes, on the other hand, share distinct novel phenotypes (dominantly pulmonary disease and increased virulence, both in vitro and in vivo) (18). Given their geographic proximity, time frame of emergence, and genotypic and phenotypic similarity, it was proposed that VGIIa and VGIIc originated relatively recently from a common ancestor (15, 19). Several studies were conducted to better understand the genotypic and phylogenetic nature of the C. gattii strains associated with these outbreaks (11, 13, 15, 17). VGIIa and VGIIb strains have now been found in other non-North American locales, and genotyping studies have provided evidence that these strains emerged independently from a South American source (9). The VGIIc subtype has not been identified elsewhere thus far. A number of studies have described in vitro and in vivo clinical phenotypic variances (i.e., virulence in animal model, clinical presentation, etc.) between the emergent North American strains (15, 18, 20). In animal models of infection, VGIIa and VGIIc strains have been shown to be more virulent than VGIIb and other C. gattii molecular types (9, 21). It is also clear that the virulence profile within subtype VGIIa does not remain consistent globally with isolates of lesser virulence being described (13, 21; C. Firacative and W. Meyer, unpublished data). Niche adaptation and phenotypic changes in pathogenic fungi are known to happen through a series of evolutionary mechanisms, including gene duplication, sexual recombination, base mutation, and to some degree, horizontal gene transfer (22). The potential genetic causes for the phenotypic and niche differences in the PNW C. gattii strains are not well understood, although potential contributing factors have been deduced from targeted expression analyses (20, 23). Whole-genome analyses provide a more comprehensive view of phylogenetic relationships and genomic evolution. Here we apply population-level wholegenome approach with the following aims: (i) to more thoroughly understand the population genomics of the C. gattii VGII molecular type; (ii) to explore the emergence of its subtypes in North America; and (iii) to identify possible genetic causes for its changing phenotype.