The F. tularensis subsp. holarctica isolates we analyzed were collected over a long period but have been preserved by lyophilization and have been verified every 5 years since they were isolated (Table 1, Appendix). We assigned these isolates into previously defined (6,7) phylogenetic clades and conducted a phylogeographic analysis by using a panel of 12 canSNPs specific for F. tularensis subspecies or clades within F. tularensis subsp. holarctica (Table 2); these canSNPs were obtained from previous reports (6,7). The canSNP analysis was PCR based and performed as described (7). Table 1, Appendix, lists the derived or ancestral allele status for these isolates and for 13 control isolates. These data facilitated the assignment of the 10 F. tularensis subsp. holarctica isolates to major phylogenetic subgroups previously identified within this subspecies (6,7).

Figure 2

Figure 2. Phylogeography of Francisella tularensis (F.t.) subspholarctica. The global distribution of different clades (indicated by colored stars, circles, and circle sections) and their phylogenetic relationships (tree) are shown as described (

The isolates were of wide phylogenetic diversity for isolates from a single country. The 10 isolates we analyzed were assigned to 4 distinct phylogenetic clades: 3 were assigned to the basal japonica clade (B.16), 3 to the OSU18 clade (B.4), 3 to the FSC200 clade (B.20), and 1 to clade B.6 (Figure 2; Table 1, Appendix). Two of these clades are very basal (B.16 and B.4; Figure 1), whereas the other 2 are relatively derived (B.6 and B.20). Regardless, these results demonstrate the presence of multiple distinct F. tularensis subsp. holarctica lineages in China. Within China, isolates from the Tibetan plateau in the areas bordering Nepal, Bhutan, India, and central Asia were particularly diverse; all 7 strains assigned to clades B.4, B.6, and B.16 were from this region.

The substantial diversity of F. tularensis subsp. holarctica from the Tibetan region provides evidence for an Asian ancestral focus of this subspecies. With the exception of the rare B.2/3 California group, all major basal lineages were represented in this small sample from this region. The center of diversity rationale would suggest that F. tularensis subsp. holarctica diversified in Asia and then spread outward to the rest of the world. The presence of representatives of the basal japonica and OSU18 lineages further argues for ancestral populations in this region. In contrast, the derived and terminal position of the B.20 (FSC200) isolates in northern China suggests that this lineage was introduced to this region from other regions—perhaps Europe, in which B.20 is found (6)—after ancestral strains dispersed to other regions from Asia and diversified in these new locations. The analyses used in this current study show that the B.4 (OSU18) isolates from China are indistinguishable from B.4 isolates from North America or Europe and could represent an ancestral population or a reintroduction after global dissemination.

Although strong evidence shows that F. tularensis subsp. holarctica is a highly fit and recently emerged clone (3), we know little about the basis for its great fitness. It is possible that certain, as yet unidentified, adaptive features developed that led to an increase in its fitness. Alternatively, a stochastic event may have led to the emergence and subsequently circumpolar expansion of this subspecies. However, our understanding of the ecology of F. tularensis subsp. holarctica is severely limited, so the dispersal mechanisms that led to its wide geographic distribution have yet to be identified.

Wide diversity in F. tularensis subsp. holarctica strains, including basal lineages, has been observed in China and underscores a lack of phylogeographic knowledge of this subspecies. Previous arguments (1) about the emergence of this highly fit subspecies have been based on highly biased sampling of strains in North America, Europe, and Japan. Our data suggest a broader distribution in Asia of the japonica clade (B.16) in particular. The OSU18 clade (B.4) also appears to have a broader distribution in Asia than has been observed from both North America and Europe. These clades are thought to be basal to the highly fit clonal expansion on these continents. Sampling of additional regions in Asia and characterization of those isolates would greatly advance the literature on the phylogeography of F. tularensis subsp. holarctica.