Phylogenetic Analysis Reveals a Cryptic Species Blastomyces gilchristii, sp. nov. within the Human Pathogenic Fungus Blastomyces dermatitidis Elizabeth M. Brown 1,2,3 , Lisa R. McTaggart 1 *, Sean X. Zhang 4 , Donald E. Low 1,2,3 , David A. Stevens 5 , Susan E. Richardson 1,3,6 1 Public Health Laboratories Toronto, Public Health Ontario, Toronto, Ontario, Canada, 2 Department of Microbiology, Mount Sinai Hospital, Toronto, Ontario, Canada, 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, 4 Department of Pathology, Division of Medical Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 5 Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, California, United States of America, 6 Division of Microbiology, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada Abstract Background: Analysis of the population genetic structure of microbial species is of fundamental importance to many scientific disciplines because it can identify cryptic species, reveal reproductive mode, and elucidate processes that contribute to pathogen evolution. Here, we examined the population genetic structure and geographic differentiation of the sexual, dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. Methodology/Principal Findings: Criteria for Genealogical Concordance Phylogenetic Species Recognition (GCPSR) applied to seven nuclear loci (arf6, chs2, drk1, fads, pyrF, tub1, and its-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2, drk1, pyrF, and its-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenated chs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy with all isolates falling into one of two evolutionarily independent lineages. Phylogenetic species were genetically distinct with interspecific divergence 4-fold greater than intraspecific divergence and a high Fst value (0.772, P,0.001) indicative of restricted gene flow between PS1 and PS2. Whereas panmixia expected of a single freely recombining population was not observed, recombination was detected when PS1 and PS2 were assessed separately, suggesting reproductive isolation. Random mating among PS1 isolates, which were distributed across North America, was only detected after partitioning isolates into six geographic regions. The PS2 population, found predominantly in the hyper-endemic regions of northwestern Ontario, Wisconsin, and Minnesota, contained a substantial clonal component with random mating detected only among unique genotypes in the population. Conclusions/Significance: These analyses provide evidence for a genetically divergent clade within Blastomyces dermatitidis, which we use to describe a novel species, Blastomyces gilchristii sp. nov. In addition, we discuss the value of population genetic and phylogenetic analyses as a foundation for disease surveillance, understanding pathogen evolution, and discerning phenotypic differences between phylogenetic species. Citation: Brown EM, McTaggart LR, Zhang SX, Low DE, Stevens DA, et al. (2013) Phylogenetic Analysis Reveals a Cryptic Species Blastomyces gilchristii, sp. nov. within the Human Pathogenic Fungus Blastomyces dermatitidis. PLoS ONE 8(3): e59237. doi:10.1371/journal.pone.0059237 Editor: Anastasia P. Litvintseva, Duke University Medical Center, United States of America Received May 22, 2012; Accepted February 14, 2013; Published March 22, 2013 Copyright: ß 2013 Brown et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by Public Health Ontario (http://www.oahpp.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lisa.mctaggart@oahpp.ca Introduction Microorganisms are the most abundant life forms on earth in terms of protoplasmic biomass [1,2] and second only to insects in estimated species diversity [3]. Yet, only an estimated 1–10% of all microbial species have been described [3]. Among fungi, only ,74,000 species are known even though biodiversity species estimates range from 500,000 to 9.9 million [4]. The differenti- ation of fungal species has historically relied on morphologic, phenotypic, and reproductive characterization that satisfies a biological species concept [5,6]. However, the advent of DNA sequencing and advancement of phylogenetic analysis has provided a powerful way to study the differentiation of fungal species at a more fundamental level by examining evolutionary processes such as population-level gene flow and genetic isolation that lead to the evolution of differential phenotypic characteristics. More recently, fungal species have been defined according to the phylogenetic species concept through multilocus sequence analysis of populations of individuals, rather than by tedious pair-wise mating tests or infrequent observation of reproductive structures that fulfill a biological species definition [7,8]. Genealogical PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e59237