Phylogeography of Francisella tularensis from Tibet, China: Evidence for an asian origin and radiation of holarctica-type Tularemia

Abstract

The geographical origin and radiation of holarctica-type tularemia, which has spread across the northern hemisphere, is open to scientific debate. Here, through phylogenetics, we show that five Tibetan Francisella tularensis isolates subsp. holarctica cluster between basal-positioned Japanese isolates and all other subspecies strains in the world, providing evidence for a previously unknown intermediate lineage next to the Japanese isolates. Importantly, identification of this new intermediate lineage complements current knowledge of tularemia epidemiology, supporting a geographical origin and radiation of the subsp. holarctica in Asia. In addition, thirteen Tibetan isolates belonging to a clade previously found only in North America and Scandinavia, further increases the diversity of holarctica strains in Asia. In summary, this study provides evidence for an Asian origin and radiation of holarctica-type tularemia.

Introduction

Tularemia, a disease caused by the bacterium Francisella tularensis (subsp. tularensis, type A; subsp. holarctica, type B), is a public health issue across the northern hemisphere (Rotz et al., 2002). Additionally, concerns regarding the potential use of this bacterium in bioterror or biowarfare have been raised (Dennis et al., 2001, Rotz et al., 2002, CDC, 2005). Type A is the most virulent subspecies and is found almost exclusively in North America, appearing only once in central Europe (Gurycova, 1998). Type B is less virulent in humans, but has spread widely across the northern hemisphere. F. tularensis also includes two low virulence subspecies, novicida and mediaasiatica (Parola and Raoult, 2001, Jackson et al., 2012). These four subspecies are closely related and share most biochemical characteristics but differ in their genetic diversity. F. tularensis subsp. tularensis has two distinct subpopulations (A.I and A.II) (Farlow et al., 2005), as determined by a variety of molecular typing methods (CDC, 2013). F. tularensis subsp. holarctica is divided into five genotypic clades (B.I-B.V) as defined by multi-locus variable-number tandem repeat analysis (MLVA). The five clades are further subdivided into ten subclades as determined by real-time-PCR based canonical single nucleotide polymorphism (canSNP) genotyping (Johansson et al., 2004, Vogler et al., 2009). The holarctica subspecies MLVA clades correlate with the subclades determined by canSNP. Genetic diversity in novicida and mediaasiatica subspecies is largely unknown.

Previous genetic studies have provided insight into the origin, evolution and distribution of the four subspecies. Their highly similar genetic content and unidirectional genomic deletion events suggests they evolved from a common ancestor by vertical descent (Svensson et al., 2005). A genome-wide SNP phylogeny revealed close relationships between the four subspecies (Vogler et al., 2009). In this phylogeny, the basal lineage of the four subspecies is novicida, followed by a tularensis lineage associated with mediaasiatica, while the holarctica lineage stands alone. Although confined to North America, but exhibiting different geographical distributions, the two subpopulations of the tularensis subsp. likely evolved from a common ancestor in North America. The holarctica subsp. is thought to have recently emerged through a genetic bottleneck and spread to its current geographic locations, thus explaining the low genetic diversity of F. tularensis (Vogler et al., 2009). The geographic origin of the holarctica subsp. in Asia is suggested by the basal positioning of the holarctica strains that are found only in Japan (Vogler et al., 2009). However, those Japanese strains have diverged from the clades associated with the holarctica radiation. Thus, a North American origin for the holarctica radiation with multiple dispersal events between North America and Eurasia was hypothesized (Vogler et al., 2009). Consequently, the geographic origin and transmission history of holarctica subspecies is controversial.

Here, we report the characterization and differentiation of 18 Tibetan F. tularensis strains by using recently developed high-resolution genetic typing methods. Six of these strains were partially analyzed using the canSNP method (Wang et al., 2014). We found that F. tularensis strains from Tibet are grouped into two separate clades −a clade genetically linking the basal-positioned Japanese isolates and all other subspecies strains in the world and a second clade linked to strains previously found only in North America and Scandinavia.