Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (Bbsl) complex. Borrelia spirochetes circulate in obligatory transmission cycles between tick vectors and different vertebrate hosts. To successfully complete this complex transmission cycle, Bbsl encode for an arsenal of proteins including the PFam54 protein family with known, or proposed, influences to reservoir host and/or vector adaptation. Even so, only fragmentary information is available regarding the naturally occurring level of variation in the PFam54 gene array and its impact on Borrelia pathogenesis. Utilizing whole genome data from isolates (n=141) originated from the three major LB-causing Borrelia species across Eurasia (B. afzelii, B. bavariensis, and B. garinii), we aimed to characterize the diversity of the PFam54 gene array in these isolates to facilitate understanding the evolution of PFam54 orthologs on an intra- and interspecies level. We found an extraordinarily high level of variation in the PFam54 gene array with 39 PFam54 paralogs belonging to 23 orthologous groups including five novel paralogs. Even so, the gene array appears to have remained fairly stable over the evolutionary history of these Borrelia species. Interestingly, genes outside Clade IV previously associated with host or, proposed, vector adaptation more frequently displayed signatures of diversifying selection. Taken together, our findings support the idea that non-Clade IV orthologs could play a larger role in host and/or vector adaptation than previously thought.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host shifts can lead to geographic expansion and the emergence of new diseases. Three etiological agents of human Lyme borreliosis (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus) but how and in which order they colonized each continent remains unknown. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we show that all three Borrelia genospecies evolved from an Asian origin, suggesting that successful expansion into Europe resulted through invading a novel vector. The pattern of gene flow between continents is different between genospecies and most likely conditioned by reservoir host association and their dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion through vector shifts, but potentially differ in their capacity as emergent pathogens.