Treponema paraluisleporidarum multi-locus sequence typing
We obtained positive PCR results from 405 samples from the tp0488gene (Germany: n=343/938;, Sweden: n=4/4; England: n=12/25; Italy: n=14/81; the Netherlands: n=18/32; Czech Republic: n=14/15) and 439 sequences from the tp0548 gene (Germany: n=380/938;, Sweden: n=4/4; England: n=13/25; Italy: n=14/81; the Netherlands: n=13/32; Czech Republic: n=15/15; Table S1). In 57 samples we could only amplify thetp0488 gene and in 91 samples we were only able to amplify thetp0548 gene target. A total of 349 sequences (tp0488 = 195 and tp0548 = 154) had to be excluded from analysis due to low sequence quality or high background noise due to superimposed sequences. This resulted in 212, 296 and 194 sequences for tp0488 ,tp0548 and the concatenated gene target sequences, respectively, including the reference sequence from the TP eC strain Cuniculi A (CP002103.1; locus tags TPCCA_RS02365 and TPCCA_RS02685 ) and T. pallidum ssp. endemicum strain Iraq B (CP032303.1; locus tags TENDIB_0488 and TENDIB_0548). We generated a maximum-likelihood tree based on the concatenated sequences of thetp0488 and tp0548 genes and added the geographic origin as attributes, with samples grouped into Northern (Schleswig Holstein and northern part of Lower Saxony), Central and Western (southern part of Lower Saxony, Hesse and North Rhine-Westphalia) and Southern Germany (Bavaria and Baden-Wuerttemberg) as well as the Netherlands, Italy, Czech Republic and the United Kingdom. Overall, there is no clustering of the samples according to their geographic origin (Figure 2). While bootstrap support for nodes in the phylogenetic tree is generally low, there are some significantly supported distinctive features that are noteworthy. The tree exhibits an initial split into two clades of which one contains sequences obtained from EBHs sampled in Baden-Wuerttemberg and Bavaria (Southern Germany) and one EBH from North Rhine-Westphalia (Western Germany). In addition, the clade contains the TPe C reference strain Cuniculi A and three strains of mountain hares from Sweden. A sample from the fourth Swedish mountain hare (V1313_03_L1) is found in the second clade and clusters together with all other EBH samples as well as a strain that was found in a pet rabbit in Hesse (Central Germany). The latter is identical to a strain obtained from a EBH from Lower Saxony, approximately 140 km from the pet rabbit sampling location. Within both main clusters, a number of statistically supported subclades of geographically related samples e.g., from the Czech Republic or Northern Germany, were found.
In our analysis we identified positively selected sites (codons) in each of the target genes including tp0488 (n = 29) and tp0548(n = 54) (Table S1). Those sites were removed from the alignments and only non-positively selected parsimony-informative sites as well as singletons were used for network constructions. The minimum spanning network that resulted from non-positively selected single nucleotide variants within the tp0548 locus (Figure S1) does not change the overall topology of the maximum-likelihood tree shown in Figure 2 and equally lacks the overall geographic clustering of the samples. Maximum-likelihood trees for individual loci can be found in the Supplemental Material (Figure S2 and S3).
********************
Add Figure 2 about here
********************