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 fungusBlastomyces 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, andits-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2,drk1, pyrF, andits-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenatedchs2-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 withinBlastomyces 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.

B. dermatitidis Isolates and Culture Growth Conditions

Seventy-eight geographically and temporally diverseB. dermatitidis strains isolated from human, canine, and environmental sources were studied (Table S1). Region of isolation represents the site of patient diagnosis, patient residence, or the site of environmental isolation. Strains were selected to represent all traditional endemic regions, in addition to diverse geographic localities within Ontario, Canada. For analysis purposes, the isolates were divided into six regional analysis groups to represent geographic concentrations of human cases of blastomycosis separated by ecological barriers or physical distance: (1) northwestern Ontario (21 isolates from Kenora, Ontario and the area within a 400 km radius); (2) central and southern Ontario (12 isolates); (3) Alberta and Saskatchewan (5 isolates); (4) Wisconsin, Minnesota (20 isolates); (5) areas of southeastern and central United States (South Carolina, Georgia, Louisiana, Mississippi, Kentucky, and Illinois (17 isolates)) and (6) southern Africa (South Africa, Rwanda, and Zimbabwe (3 isolates)). Ontario isolates were partitioned from those of Wisconsin and Minnesota to reflect the physical barrier of the Great Lakes separating these two geographic areas. Likewise, isolates from Ontario were split into two analysis groups to better reflect two distinct ecological zones: the Canadian shield of northwestern Ontario and the mixed wood plains of central and southern Ontario (http://www.mnr.gov.on.ca/en/Business/Biodiversity/2ColumnSubPage/STEL02_166891.html, accessed 2011 Nov 12).

Isolates were grown on potato dextrose agar (BD, Mississauga, ON) at 25°C for 3–7 days. Strains were then sub-cultured onto Formula D agar[36] and incubated at 37°C for 1–3 weeks until sufficient yeast growth was present for extraction of DNA.

DNA Extraction, PCR Amplification, and Nucleotide Sequence Determination

Genomic DNA was extracted from yeast cells using the MasterPure yeast DNA purification kit (Epicentre Biotechnologies, Madison, WI), according to the manufacturers protocol with the following modifications. Approximately 10–15 acid-washed glass beads (Sigma-Aldrich, Oakville, ON) were added to each tube of lysis buffer inoculum. Samples were subsequently vortexed for 2 minutes using the Disruptor Genie (Scientific Industries Inc., Bohemia, New York) and incubated at 65°C for 45 minutes. Extracted DNA was suspended in 160 µl of TE buffer (Epicentre Biotechnologies).

Regions from seven nuclear genes were selected for multi locus sequence typing (MLST): (1) chitin synthase (chs2)[37], (2) histidine kinase (drk1)[38], (3) fatty acid desaturase (fads), (4) orotidine 5′-phosphate decarboxylase (pyrF), (5) alpha tubulin (tub1)[39], (6) ADP ribosylation factor 6 (arf6), and (7) internal transcribed spacer 2 (its2)[39]. Exons, introns, 5′ and 3′ untranslated regions were utilized to maximize nucleotide sequence variation among isolates.

PCR amplification of the target regions was performed using the Platinum Pfx DNA Polymerase kit (Life Technologies, Carlsbad, CA). Each PCR reaction contained 1.0 U of Platinum Pfx DNA Polymerase, 1× amplification buffer, 1000 µM MgSO₄, 300 µM deoxynucleotide triphosphate mix (Life Technologies), 0.2 µM forward primer, 0.2 µM reverse primer (seeTable 1), and 2 µl of genomic DNA in a 50 µl reaction. Reactions were denatured at 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 55–57°C for 30 seconds (seeTable 1), and 68°C for 1 minute, followed by a final extension of 68°C for 5 minutes. PCR products were diluted 1∶20 in distilled deionized H₂O in preparation for traditional Sanger sequencing.

Bi-directional cycle sequencing reactions were performed using PCR primers and the BigDye ® Terminator v3.1 Cycle Sequence Kit (Life Technologies). Sequencing reactions contained 1 µl of BigDye ® Terminator v3.1, 1× of sequencing buffer, 0.5 µM of PCR primer, and 2 µl of diluted template DNA in a 20 µl reaction. Reactions were cycled according to the manufacturer’s instructions and the products were subsequently sequenced by capillary electrophoresis utilizing the 3730xl DNA Analyzer (Life Technologies).

Sequence, Polymorphism, and Population Genetic Analysis

DNA sequences were trimmed, aligned, and primer sequences were removed using Bionumerics v.6.1 (Applied Maths, Sint-Martens-Latem, Belgium). Nucleotide polymorphisms were visually identified from sequence alignments of each gene region. The ratio of non-synonymous to synonymous amino acid changes (d_N/d_S) was calculated for each complete gene sequence to ensure that locus segments selected were under neutral selective pressure[40]. DnaSP v.5 was used to estimate the average pairwise divergence between isolates (pi) and the average pairwise divergence between populations (Dxy)[41]. The extent of gene flow between populations was inferred from the fixation index (Fst) calculated using Multilocus v.1.3b[42]. The signficance of the Fst value was assessed by comparing the original observed data set to the distribution of Fst values for 1000 randomized data sets in which alleles had been artificially re-sampled at each locus using Multilocus v.1.3.b[42].

Phylogenetic Analysis

In Bionumerics v.6.1 (Applied Maths), majority consensus maximum parsimony trees were constructed for each gene region and the 3573 bp concatenated sequence of all nuclear sequences in the following orderchs2-drk1-fads-pyrF-tub1-arf6-its2, with midpoint rooting applied to the concatenated gene phylogeny. Similarly, Bayesian analyses were performed using MrBayes v3.1[43] for each gene region and the concatenatedchs2-drk1-fads-pyrF-tub1-arf6-its2 sequences. For each analysis, parameters and prior were modified to reflect the evolutionary model that best represented the data according to jModeltest[44] (JC forarf6,fads, andtub1, F81 forchs2 andpyrF, and K80 fordrk1 andits2). The concatenated data set was partitioned to reflect each gene region with all parameters, priors and evolutionary rates estimated independently for each locus. Four Markov chains were run independently in two separate analyses for 1 000 000 generations, with samples taken every 100 generations. All analyses were performed twice to ensure that they were not trapped at local maxima. For each replicate, posterior probability values and the overall tree topology were compared to ensure analyses converged on a similar phylogeny.

To assess the robustness of clades identified by phylogenetic analyses, maximum parsimony bootstrap (MPB) values were calculated with 1000 randomizations in Bionumerics v.6.1 (Applied Maths) and Bayesian posterior probabilities (BPP) values were estimating using MrBayes v3.1[43]. Consistency indices (CI) for majority consensus maximum parsimony trees were calculated using Bionumerics v6.1 (Applied Maths).

Recombination and Linkage Disequilibrium Analysis

To assess mixis as an implicit measure of recombination, the degree of association among loci was quantified using the Index of Association (I_A)[45],[46] and r_d values calculated with Multilocus v.1.3b[42]. Values were calculated for all isolates, for isolates within each clade identified in the phylogenetic analysis, and for isolates within each geographic regional analysis group. Based on these groupings, values were calculated for both the full data set and when the data set was reduced to unique genotypes (i.e. corrected for clonal genotypes)[46]. Complete panmixia expected in a fully recombining population will generate values near 0, while I_A and r_d values that are significantly greater than 0 signify deviation from panmixia, that is linkage disequilibrium[46]. To assess the significance of I_A and r_d, the values for the observed data were compared to the distribution of I_A or r_d values calculated for 1000 randomized data sets that had been artificially recombined by re-sampling alleles at each locus without replacement with single nucleotide polymorphisms (SNP) at each locus shuffled together as a single linkage group[11]. The null hypothesis of panmixia was rejected if the I_A or r_d values of the observed data set were significantly larger than the distribution of I_A and r_d values for the randomized data set[11].

The Parsimony Tree Permutation Length Test (PTPLT) was performed to detect random mating in the phylogenetic data[11]. Phylogenetic trees were constructed based on multilocus genotypes in PAUP using parsimony, with loci treated as loci and alleles as character states[42]. Statistical significance was determined by comparing the actual length of the observed tree to the distribution of tree lengths of 1000 data sets that were artificially recombined as described above. Values were calculated for all isolates in the population, in addition to isolates within each clade identified in the phylogenetic analysis, and isolates within each geographic region. As above, this test was performed for both the full data set and when each dataset was reduced to unique genotypes[46].

To explicitly detect recombination in the sequence data, phylogenetic network analysis using split decomposition[47] was performed with SplitsTree v.4[48], using the pairwise genetic distances of the concatenatedchs2-drk1-fads-pyrF-tub1-arf6-its2 sequences estimated in Bionumerics (Applied Maths). Bootstrapping values for the split decomposition network was calculated with 1000 replicates. The Phi test for recombination[49] was applied to the entire population and each phylogenetic species in SplitsTree v.4.

Finally, the simple four-gametes test was performed within each locus and between loci for each phylogenetic species using DnaSP v.5[41]. Data were considered to have failed the simple four-gametes test if the minimum number of recombination events (R_M) needed to explain the alleles was ≥1.[50].

Mating Type Locus Determination

The mating type of each isolate was determined by amplifying the MAT loci as described by Meece et al.[35]. PCR products were analyzed by gel electrophoresis on a 1.5% agarose gel with 0.5 mg/L ethidium bromide. An isolate was considered positive for a specific mating type if a band was present for one locus and absent for the other mating type locus[35]. Mating type strains ATCC 18188 (+, containing the α box allele) and ATCC 18187 (-, containing the HMG box allele) available from the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands) and the American Type Culture Collection (Manassas, VA) were used to confirm the expected presence/absence of the a mating type locus. Mating type frequencies were assessed against the null hypothesis of a 1∶1 ratio using the chi square test with a significance level ofP<0.05.

Geographic Mapping

The distribution of isolates from Ontario and Wisconsin pertaining to eachB. dermatitidis phylogenetic species was mapped using ArcGIS software (ESRI, Toronto, ON) and maps imported from the U.S. Geological Survey (http://data.geocomm.com/readme/usgs/atlas/html/statesmt.html, accessed 2011 Apr 20) and CanMap Route Logistics 2009v.4.

Nomenclature

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide print copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefixhttp://www.mycobank.org/MycoTaxo.aspx?Link=T&Rec=MB803330. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Polymorphism Analysis

Nucleotide sequences for the 7 loci analyzed in this study have been deposited in the Genbank database under the following accession numbers:chs2 (JN561872–JN561949),drk1 (JN561950–JN562027),fads (JN562028–JN562105),pyrF (JN562184–JN562261),tub1 (JN562262–JN562339), arf6 (JN561794–JN561871), andits2 (JN562106–JN562183). Nucleotide sequence alignments are available inFigure S1.

A total of 46 nucleotide polymorphisms were identified in the 3573 bp of sequence analyzed, yielding an overall genetic diversity of 1.29%. The characteristics of each locus are summarized inTable 1. The number of polymorphic sites identified at each locus ranged from 3 infads, its2 andarf6 to 13 indrk1. With the exception of thetub1 locus, no insertions or deletions were detected in any of the sequenced fragments. A 5 bp insertion was detected in thetub1 locus of 2 of the isolates. The insertion was viewed to have arisen from a single evolutionary event and counted as a single polymorphism. The ratio of nonsynonymous to synonymous amino acid substitutions (d_N/d_S) was below 1 for all genes, suggesting that all gene loci were under stabilizing selection[40]. Among the 78 isolates studied, 36 unique multilocus sequence types (ST) were identified. Of the 10 environmental isolates examined in our study, 7 were genetically identical to patient isolates and 3 had genetically unique sequence types.

Identification of Phylogenetic Species withinB. dermatitidis

Single gene phylogenies for each of the 7 loci were examined by constructing majority consensus maximum parsimony trees. Tree topologies derived from maximum parsimony and Bayesian analyses were very similar or identical for all loci with the exception oftub1, therefore only maximum parsimony trees for each locus are displayed with MPB and BPP values indicated on tree branches (Figure 1). The branching topology of thetub1 maximum parsimony tree differed slightly from that predicted based on Bayesian analyses. For this locus, BPP values are displayed only on branches of the maximum parsimony tree that were also present in Bayesian analysis.Figure 2 displays thechs2-drk1-fads-pyrF-tub1-arf6-its2 concatenated maximum parsimony tree with MPB and BPP values displayed on the branches.

Two distinct monophyletic clades were identified by the phylogenetic analysis. To determine whether the clades represented independent evolutionary lineages and whether these lineages could be defined as phylogenetic species, we applied the criteria previously outlined by Dettman et al.[5] for phylogenetic species recognition by genealogical concordance and non-discordance (GCPSR). Briefly, a clade was recognized as an independent evolutionary lineage if it satisfied either of the following criteria: (1) Genealogical concordance: the clade was observed in the majority of single locus genealogies; or (2) Genealogical non-discordance: the clade was strongly supported by at least one genealogy with high MPB (≥70%) and BPP (≥0.95) values and was not contradicted by any other single locus genealogy with the same level of support[5]. Independent evolutionary lineages were defined as phylogenetic species if the concatenatedchs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy revealed: (1) Genetic Differentiation: the phylogenetic species was distinct and well differentiated from other species; and (2) Exhaustive subdivision: all individuals in the population were placed within a phylogenetic species[5].

Four of the seven single-locus genealogies (chs2,drk1, pyrF, andits-2) separated clades 1 and 2 as independent evolutionary lineages, thereby satisfying criterion 1 of GCPSR (Figure 1a, b, d, g). The genealogies ofchs2 anddrk1 also strongly supported the separation of clades 1 and 2, with MPB ≥70% and BPP≥0.95 (Figure 1a, b). Therefore clades 1 and 2 represent independent monophyletic evolutionary lineages as recognized by genealogical concordance of single-locus genealogies[5].

To determine whether these two independent evolutionary lineages represent phylogenetic species, a majority consensus maximum parsimony tree for the concatenated sequence for all nuclear loci was examined (Figure 2). All isolates were partitioned into one of the two genetically distinct clades. The clades were well differentiated (MPB = 100%, BPP = 1.0 separating clade 1 from clade 2), with 12 fixed nucleotide differences separating clades 1 and 2. Based on the GCPSR criteria proposed by Dettman et al.[5], the currently defined speciesB. dermatitidis contains two phylogenetic species: Phylogenetic Species 1 (PS1) (clade 1) and Phylogenetic Species 2 (PS2) (clade 2) (Figures 1 and2).

A substantial amount of internal phylogenetic structure was observed within PS1, which contained an internal clade. While the separation of this clade from PS1 was well supported by thedrk1 locus genealogy (Figure 1b, MPB = 89%, BPP = 1.0) and in the combined concatenated analyses (Figure 2, MPB = 70%, BPP = 1.0), this lineage was not supported as a monophyletic clade in any locus examined and therefore was not recognized as a phylogenetic species using the criteria applied by Dettman et al.[5].

The average pairwise divergence between phylogenetic species, 5.45×10⁻³ (σ = 5.40×10⁻⁴), was approximately 4-fold greater than the intraspecific pairwise divergence of either PS1 or PS2 (1.76×10⁻³ (σ = 1.20×10⁻⁴) and 0.69×10⁻³ (σ = 0.50×10⁻⁴) respectively).

To estimate gene flow between the phylogenetic species, Fst, which can range from 0 (no differentiation between populations due to unrestricted gene flow) to 1 (complete isolation due to the absence of gene flow) was calculated[20]. A statistically significant Fst value (0.77235,P<0.001) suggests restricted gene flow and genetic isolation between the two phylogenetic species.

Assessment of Genetic Recombination and Linkage Disequilibrium

The extent of linkage disequilibrium and recombination between and within each phylogenetic species was assessed using (i) the Index of Association (I_A) and r_d values; (ii) the Parsimony Tree Permutation Length Test (PTPLT); (iii) the Phi test and split decomposition analysis; and (iv) the four-gametes test and minimum number of recombination events (R_M).

Panmixia as implicit evidence of recombination was assessed for all isolates, and separately within PS1 and PS2, by calculating I_A and r_d values both with and without correction for clone genotypes[46]. To account for recombination within but not between either phylogenetic species or distant geographic regions, the data were partitioned prior to randomization such that loci were shuffled within but not between the specified groups[11],[42] (Table 2). Panmixia was not detected among the 78B. dermatitidis isolates when treated as a single population (P<0.001) or partitioned to mimic recombination within but not between either the two phylogenetic species (P<0.001) or the six geographic regions (P<0.001) (Table 2). Likewise, reducing the three datasets to unique genotypes also failed to detect panmixia (P<0.001) (Table 2). However, when the I_A and r_d values were calculated separately for each phylogenetic species, evidence for linkage disequilibrium disappeared for PS1 when the data were partitioned into six geographic regions, thereby implying recombination among isolates of the same geographic region (P = 0.121) (Table 2). Panmixia was not detected among the 39 isolates of PS2 either with or without partitioning into 4 geographic regions (P<0.001). After reducing the dataset to 7 unique genotypes by removing clones[46] the I_A and r_d values (Table 2) declined but remained significant (P = 0.020) signifying a deviation from panmixia. These findings indicate that a considerable portion of the association between alleles was contained within the population structure separating PS1 from PS2.

The PTPLT was used to assess the extent of random mating among isolates by comparing the length of the observed parsimony tree to the lengths of trees constructed from 1000 artificial recombinations of the data(Figure 3). In a clonal population, loci would be expected to have the same evolutionary topology and therefore the observed tree length should be significantly shorter than the distribution of tree lengths for the artificially recombined datasets[11]. Conversely, in a population undergoing random mating, different genealogies are expected to have varying topologies, and the observed tree length would be expected to be within the distribution of tree length randomizations[11]. The parsimony tree length of the observed data set was significantly shorter (P<0.001) than the distribution of tree lengths from 1000 permutations of the data randomly recombined across the entire population both with and without reduction of the dataset to unique genotypes (Figure 3a). Likewise, partitioning the data such that loci were randomized within but not between each of the two phylogenetic species or 6 geographic regions both with and without reduction to unique genotypes also generated distributions of tree lengths that were significantly shorter than the observed tree (P<0.001) (Figure 3b, c). PTPLT of all isolates of PS1 and PS2 analyzed separately also failed to detect random mating (P<0.001) (Figure 3d, e). However, partitioning by geographic region and reducing the dataset to 30 unique genotypes (i.e. clone correction[46]) rendered the observed tree length of PS1 isolates statistically undifferentiated from those of the artificially recombining population (Figure 3d) (P = 0.073). While PTPLT partitioned by geographic regions failed to detect random mating among the PS2 isolates, amalgamating the data from 39 isolates into 7 unique genotypes generated a distribution of tree lengths that were not statistically different from the observed tree (P = 0.092) (Figure 3e).

The Phi test for recombination identified statistically significant evidence for recombination within PS1 (P<0.01). The Phi test did not identify statistically significant evidence for recombination within PS2 (P = 0.3069), however the split decomposition analysis revealed reticulate networks suggesting the possibility of recombination in PS2 independent of the PS1 population (Figure 4). Low genetic diversity, as was observed in PS2, is known to reduce the statistical power of the Phi test[51].

Finally, the simple four-gametes test was applied to explicitly assess recombination within PS1 and PS2. Although intralocus recombination was only observed within PS1, interlocus recombination between pairs of loci was observed in both PS1 and PS2, suggesting recombination is occurring within each phylogenetic species (Table 3).

In summary, the four-gametes test, PTPLT and split decomposition analysis detected random mating and recombination within PS2 when analyses were reduced to unique genotypes. Recombination, random mating, and linkage equilibrium was also detected within PS1 by the four-gametes test, the Phi test, the clone-corrected PTPLT and the I_A and r_d tests, but for the later two tests only when isolates were partitioned into 6 geographic regions, indicating that sexual reproduction among this group, which is broadly distributed across North America, may be limited by geographic distance. In contrast, none of these tests detected evidence of recombination in the PS1–PS2 combined dataset. Collectively, these analyses demonstrate thatB. dermatitidis is not a single freely recombining species. Rather, recombination was only detected within each of the two phylogenetic species.

Distribution of Mating Type Genes

Both mating types were found within each phylogenetic species (Table S1), however the distribution of mating types was significantly different from the expected 1∶1 ratio (PS1P = 0.0112; PS2P = 0.0374), with each species containing more HMG box alleles. PS1 contained 25 HMG box positive isolates and 10 α box positive isolates, whereas PS2 contained 26 HMG box positive isolates and 13 α box positive isolates. Interestingly 8 of the 9 environmental isolates examined contained the HMG box group allele. Five of the PS2 ST’s in our study contained both mating type alleles.

Phylogeography ofB. dermatitidis

PS1 encompassed 39 isolates, from southeastern and central United States (17 isolates), southern and central Ontario (9 isolates), Alberta and Saskatchewan (5 isolates), Wisconsin and Minnesota (3 isolates), northwestern Ontario (4 isolates), and southern Africa (1 isolate). PS2 consisted of 39 isolates from northwestern Ontario (17 isolates), Wisconsin and Minnesota (17 isolates), 3 isolates from southern and central Ontario, and 2 additional isolates of southern African origin (Figure 2).

The geographic distribution of each phylogenetic species was geospatially mapped across Ontario and Wisconsin to illustrate species distribution across the most thoroughly sampled geographic region (Figure 5). PS2 was primarily restricted to northwestern Ontario, Wisconsin, and Minnesota (34 isolates) whereas isolates belonging to PS1 were found in these areas in addition to central and southern Ontario.

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