Chromosome assembly and phylogeny reconstruction
Illumina reads were first trimmed for Illumina MiSeq adapter sequences
using Trimmomatic v. 0.38 (Bolger, Lohse, & Usadel, 2014) before being
assembled using SPAdes v. 3.13.0 (Bankevich et al., 2012), which has
been shown to be the best option for de novo assemblies ofBorrelia genomes (Becker et al., 2020). Pacific Bioscience
sequences were obtained for three B. bavariensis isolates (PBi,
A104S, and NT24) (Becker et al., 2020) and three B. gariniiisolates (PHeI, PBr, and NT31; see Suppl. Met.). Additionally, threeB. afzelii chromosomes were downloaded from GenBank for use as
references and inclusion in all analyses: PKo (CP009058.1), K78
(CP002933.1), and ACA-1 (NZ_ABCU00000000.2). SPAdes contigs were then
mapped to reference chromosomes using NUCmer v. 3.23 from the package
MUMmer (Delcher, Phillippy, Carlton, & Salzberg, 2002; Kurtz et al.,
2004). Final chromosomes were produced according to the mapping protocol
outlined in Becker et al. (2020) (see Suppl. Met.). Three additionalB. bavariensis chromosomes were downloaded from GenBank and used
in further analyses: SZ (CP007564.1), BgVir (CP003151.1), and NWJW1
(CP003866.1).
Final assembled chromosomes were aligned using MAFFT v. 7.407 (Katoh,
Misawa, Kuma, & Miyata, 2002; Katoh & Standley, 2013). Recombination
is known to be low on the Borrelia chromosome (Gatzmann et al.,
2015) but as recombinant regions could bias the phylogenetic signal, we
searched for areas of the chromosome violating the four-gamete condition
(Richard R Hudson & Kaplan, 1985) (as described in Gatzmann et al.
(2015); see Suppl. Met.). Regions with strong violation of the
four-gamete condition were assumed to be recombinant and were removed
from the final alignments (final alignment length: 936908bp). Phylogeny
reconstruction was done in MrBayes v. 3.2.6 (Huelsenbeck & Ronquist,
2001; Ronquist et al., 2012) with ploidy set to haploid and a GTR
(Tavaré, 1986) substitution model with gamma distributed rate variation.
Three independent runs were launched and ran for 5 million generations
at which point convergence of parameters was checked with Tracer v.
1.7.1 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018). Consensus trees
were built using the sumt command from MrBayes using a respective
burn-in of 25%. Convergence to a single topology in all three
independent runs was checked manually in FigTree v. 1.4.4
(http://tree.bio.ed.ac.uk/software/figtree/) which was also used to plot
the tree shown in Figure 2. Trees were midpoint rooted on the longest
branch, which corresponded to the well-established delineation betweenB. afzelii and the monophyletic group containing B.
bavariensis and B. garinii (Becker et al., 2016; Gabriele
Margos, Vollmer, Ogden, & Fish, 2011).