Phylogeography of Francisella tularensis: global expansion of a highly fit clone

Abstract

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

FIG. 1.

Summary flow chart of three SNP analyses used. The methods for the three principal SNP analyses (whole-genome SNP analysis, MIP SNP analysis, and canSNP analysis) are presented.

FIG. 2.

Whole-genome SNP phylogeny of 13 F. tularensis strains. Maximum parsimony was used to construct this phylogeny from 29,774 SNPs discovered among the whole-genome sequences of these 13 strains. Red branches indicate the branches along which SNPs were discovered for the MIP SNP analysis, as only eight whole-genome sequences were available for SNP discovery when the MIP SNPs were selected. This analysis was highly robust due to the large number of characters and the very high consistency index of >0.99.

FIG. 3.

MIP SNP phylogeny indicating canSNPs, clades, and subclades. Stars indicate terminal subclades defined by one of the eight F. tularensis genomes used for MIP SNP discovery. Circles represent collapsed branch points along the lineages that contain subgroups of isolates. These subclades are named for the two flanking canSNPs. canSNP names and positions are indicated in red. Branch A.II.Br.006 has a branch length of 1 SNP but is presented as a longer, dashed line to avoid obscuring other parts of the tree.

FIG. 4.

Distribution of F. tularensis subsp. tularensis subclades throughout North America. The F. tularensis subsp. tularensis portion of the canSNP phylogeny from Fig. 3 is presented along with a map indicating the frequencies and geographic distribution of F. tularensis subsp. tularensis subclades across North America. The number of isolates (N) and number of MLVA genotypes (G) within each subclade are indicated. Subclades are color coded to facilitate mapping. For the purposes of mapping and determining isolate and MLVA genotype totals, strains belonging in strain-specific subclades (A.II.Br.ATCC 6223 and A.II.Br.WY96-3418) are included within the subclade immediately basal to their actual subclade. Colors within the mapped pie charts correspond to the subclade color designations. For explanation of stars and circles, see the legend to Fig. 3.

FIG. 5.

Worldwide distribution of F. tularensis subsp. holarctica subclades. The F. tularensis subsp. holarctica portion of the canSNP phylogeny from Fig. 3 is presented along with a map indicating the frequencies and geographic distribution of F. tularensis subsp. holarctica subclades throughout the world. The number of isolates (N) and number of distinct MLVA genotypes (G) within each subclade are indicated. Subclades are color coded to facilitate mapping. For the purposes of mapping and determining isolate and MLVA genotype totals, the strain belonging in the strain-specific subclade B.Br.LVS is included within the subclade immediately basal to its actual subclade, B.Br.013/014. Colors within the mapped pie charts correspond to the subclade color designations. Gray regions indicate the known distribution of F. tularensis, by country (19). For explanation of stars and circles, see the legend to Fig. 3.