The bacterium Treponema paraluisleporidarum causes syphilis in Lagomorphs. In a set of 1,095 samples from four species – European brown hare (Lepus europaeus), mountain hare (Lepus timidus), Corsican hare (Lepus corsicanus) and European rabbit (Oryctolagus cuniculus) – we genotyped the strains that infect wild lagomorphs. Samples originate from Sweden, the Netherlands, the United Kingdom, Germany, the Czech Republic and Italy. The phylogenetic analyses of two informative gene targets (tp0488 and tp0548) showed high genetic diversity among the lagomorph-infecting treponemes. More specifically, we found a high number of nucleotide variants and various short repeat units in the tp0548 locus which have not been described for human syphilis and primate yaws causing Treponema pallidum. While the functional aspect of these short repeat units remains subject to ongoing investigations, it likely enables the pathogen to better survive in its lagomorph host. Our data did not support any geographic clustering which is equally reflected in the host population genetics as shown by mitochondrial genome data corresponding to the sampled lagomorph populations. This is unexpected and in contrast with what has been shown for nonhuman primate infection with T. pallidum. In the future, the combination of multi-locus sequence typing and WGS from modern and ancient samples from a wide geographic range and multiple lagomorph species will contribute to a better understanding of the epidemiology and evolutionary path of lagomorph-infecting treponemes. In conclusion, our current study demonstrates a high genetic variation of the syphilis-causing pathogen in a higher number of positively PCR-tested European lagomorphs (n=496/1095).

1. Microsatellite genotyping is an important genetic method for a number of research questions in biology. Given that the traditional fragment length analysis using polyacrylamide gel or capillary electrophoresis has several drawbacks, microsatellite genotyping-by-sequencing (GBS) has arisen as a promising alternative. Although GBS mitigates many of the problems of fragment length analysis, issues with allelic dropout and null alleles often remain due to mismatches in primer binding sites and unnecessarily long PCR products. This is also true for GBS in catarrhine primates where cross-species amplification of loci (often human derived) is common. 2. We therefore redesigned primers for 45 microsatellite loci based on 17 available catarrhine reference genomes. Next, we tested them in singleplex and different multiplex settings in a panel of species representing all major lineages of Catarrhini and further validated them in wild Guinea baboons (Papio papio) using faecal samples. 3. The final panel of 42 microsatellite loci can efficiently be amplified with primers distributed into three amplification pools. 4. With our microsatellite panel, we provide a tool to universally genotype catarrhine primates via GBS from different sample sources in a cost- and time-efficient way, with higher resolution, and comparability among laboratories and species.

AbstractIn pair-living mammals, genetic monogamy is extremely rare. One possible reason is that in socially monogamous animals, mate choice can be severely constrained, generating a risk of inbreeding or incompatibility between partners. To escape these constraints and minimize inbreeding, individuals might engage in extra-pair copulations. Alternatively, inbreeding can be avoided by natal dispersal. However, little is known about the interactions between mating system, mate choice and dispersal in pair-living mammals. Here we show that coppery titi monkeys (Plecturocebus cupreus), are mostly genetically monogamous, as parentage analyses indicated no cases of extra-pair paternity. We did not find evidence for relatedness- or heterozygosity-based mate choice. Despite the lack of evidence for active inbreeding avoidance via mate choice, mating partners were on average not related. We further found that dispersal was opportunistic, with both sexes dispersing over varying distances. Our findings indicate that even opportunistic dispersal, as long as it is not constrained, can generate sufficient genetic diversity to prevent inbreeding. This, in turn, can render both active inbreeding avoidance via mate choice and extra-pair copulations unnecessary, thus helping to maintain genetic monogamy.Key wordsGenetic monogamy, mate choice, dispersal, titi monkeys, Plecturocebus