Gene trees showing nucleotide sequence clusters of 68 Borrelia burgdorferi isolates at 1 chromosomal locus (panel A: rrs-rrlA spacer, or intergenic spacer [IGS]) and 3 plasmid loci (panels B, C, and D: ospC on cp26, dbpA on lp54, and BBD14 on lp17, respectively). Trees were inferred based on nucleotide sequence alignments and were rooted by using the Ri5, SV1, or both, sequences as outgroups. The DNADIST and neighbor-joining programs of the PHYLIP package (33) were used for distance calculation and the APE software package (34) was used for tree plotting. Isolates were grouped as clonal groups (A through U), which are named by their typical ospC alleles. Five isolates (Bol26, VS219, MI409, MI415, and MI418) showing atypical allelic associations with ospC alleles, likely caused by recombination, were labeled in orange. Red, European isolates; blue, northeastern US isolates; green, midwestern US isolates. Scale bars indicate number of nucleotide substitutions per site.

Species phylogeny based on concatenated sequences at housekeeping loci. Seventeen isolates include 1 Borrelia garinii strain (PBi), 1 B. afzelii strain (PKo), 1 B. bissettii strain (DN127), 2 strains of an unnamed genomic species (SV1 and Ri5), and 12 B. burgdorferi sensu stricto isolates. These strains were selected for reconstructing interspecies phylogeny (hence species samples), as well as for resolving the clade containing clonal groups A and B (A, B1, and B2 are represented by 2 isolates). Sequences at 6 chromosomal housekeeping loci (gap, alr, glpA, xylB, ackA, and tgt) were obtained for each strain, with B31 and PBi sequences from published genomes (29,36), N40, JD1, PKo, and DN127 sequences from draft genomes (S. Casjens et al. pers. comm.). Sequences of the remaining strains were obtained by direct sequencing. The total length of concatenated alignment is 7,509 nt. A) Consensus of maximum likelihood trees obtained by using DNAML of the PHYLIP package (33). Branch support values are based on 100 bootstrapped replicates of the original alignment. B) Enlarged view of B. burgdorferi sensu stricto subtree. Tips were colored by geographic origin of the isolate (blue, North America; red, Europe) and were labeled with ospC major-group allele type. Scale bars indicate number of nucleotide substitutions per site.