Diversity of Francisella tularensis Subsp. holarctica Lineages, China

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.
T ularemia is a disease caused by distinct subspecies and phylogenetic groups within the bacterial species Francisella tularensis (1,2). These groups exhibit distinct phylogeographic patterns; F. tularensis subsp. tularensis (type A) is restricted to North America, whereas F. tularensis subsp. holarctica (type B) is found throughout many parts of the Northern Hemisphere (3) and has been reported recently in Tasmania (4). Both subspecies exhibit highly clonal population structures, as determined by phylogenetic analysis using data from multilocus variable number tandem repeat analysis, single-nucleotide polymorphisms (SNPs), and indels (5)(6)(7). The wide geographic distribution and low diversity of F. tularensis subsp. holarctica isolates have been used to argue that this clade is recently emerged and highly fit (3), but the geographic origin of its emergence has not been determined.
F. tularensis subsp. holarctica has been further subdivided by whole-genome sequencing and canonical SNP (canSNP) genotyping into multiple clades (7) (Figure 1). The most basal clade consists of strains assigned to the biovar japonica; this biovar had previously only been reported from Japan (8), but a recent report suggests that it may be found in Turkey (9). The next derived clade (B.2/3) has been described only from 2 isolates from California, USA (7). Isolates from these 2 most basal clades are rare, and apparently geographically restricted, but still provide insights into the origin of F. tularensis subsp. holarctica. The global expansion of the more derived clades is extensive, and closely related isolates are common and widely distributed. The source for emergence of the main type B has been proposed for either North America or Scandinavia, on the basis of the presence of the OSU18 clade isolates in both locations (6,7). However, a sampling bias toward both of these geographic regions has left F. tularensis subsp. holarctica diversity in much of the rest of the world poorly understood. We analyzed 10 isolates of F. tularensis subsp. holarctica from China (10) to determine their placement within the current global phylogeographic framework of this pathogen.

The Study
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, http://wwwnc. cdc.gov/EID/article/20/7/13-0931-T1.htm). 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, 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).
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 as- 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 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 7, July 2014 Previous studies (6,7,11) defined the major lineages of this subspecies on the bases of whole-genome sequences and single-nucleotide polymorphism analysis. The other F. tularensis subspecies and closely-related Francisella species are shown at the root. 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.

Conclusions
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.  The global distribution of different clades (indicated by colored stars, circles, and circle sections) and their phylogenetic relationships (tree) are shown as described (6,7,11). Stars indicate sequenced reference strains. The phylogenetic positions of the 10 isolates from China (boxes on tree) and their sites of isolation (circles within China) are indicated. The exact lineage of strain 410105 (black circle) was not determined.