DOI: 10.5061/dryad.7m0cfxpth
title: Pathogen-mediated selection favours the maintenance of innate immunity gene polymorphism in a widespread wild ungulate

description: Toll-like Receptors (TLR) play a central role in recognition and host
 frontline defence against a wide range of pathogens. A number of recent
 studies have shown that TLR genes (Tlrs) often exhibit large polymorphism
 in natural populations. Yet, there is little knowledge on how this
 polymorphism is maintained and how it influences disease susceptibility in
 the wild. In previous work, we showed that some Tlrs exhibit similarly
 high levels of genetic diversity as genes of the Major Histocompatibility
 Complex (MHC), and signatures of contemporary balancing selection in roe
 deer (Capreolus capreolus), the most abundant cervid species in Europe.
 Here, we investigated the evolutionary mechanisms by which
 pathogen-mediated selection could shape this innate immunity genetic
 diversity by examining the relationships between Tlr (Tlr2, Tlr4 and Tlr5)
 genotypes (heterozygosity status and presence of specific alleles) and
 infections with Toxoplasma and Chlamydia, two widespread intracellular
 pathogens known to cause reproductive failure in ungulates. We showed that
 Toxoplasma and Chlamydia exposures vary significantly across year and
 landscape structure with few co-infection events detected, and that the
 two pathogens exert antagonistic selection on Tlr2 polymorphism. By
 contrast, we found limited support for Tlr heterozygote advantage. Our
 study confirmed the importance of looking beyond Mhc genes in wildlife
 immunogenetic studies. It also emphasized the necessity to consider
 multiple pathogen challenges and their spatiotemporal variation to improve
 our understanding of vertebrate defence evolution against pathogens.
description: - 433 annual captures corresponding to 328 different individuals
 (190 females and 138 males, 164 juveniles and 164 adults, 1 to 5 captures
 per individual) - The <i>Tlr</i>
 (<i>Tlr2</i>, <i>Tlr4</i> and
 <i>Tlr5</i>) genes of 157 roe deer from the area (VCG) had
 been genotyped in a previous immunogenetic study (Quéméré <i>et
 al.</i>, 2015 Molecular Ecology). We
 completed this dataset by genotyping 171 new individuals using exactly the
 same procedure. DNA was extracted from alcohol-preserved tissues using the
 DNeasy Blood and Tissue kit (QIAGEN). <i>Tlr</i> genes were
 genotyped using a two-step approach: a pilot study on 32 individuals was
 first performed to identify polymorphic sites (SNPs) by screening almost
 the entire coding region of the three <i>Tlr</i> genes (82% in
 average) including the leucine-rich extracellular region of receptors
 involved in antigen-binding (between 2081 and 2436 bps sequenced per
 gene). A total of 38 SNPs (6-16 SNPs per locus) were uncovered at this
 step. Details about primer sequences, SNP positions and codon changes are
 provided in Quéméré et al. (2015). Then we applied the exact test of
 linkage disequilibrium (LD) implemented in ARLEQUIN 3.5
 (Excoffier & Lischer, 2010). Based on LD scores and
 <i>P</i>‐values (after Bonferonni correction), we identified
 linkage disequilibrium (LD) groups. For each group, we selected one SNP
 (primarily targeting non-synonymous sites) that was genotyped for all
 individuals using the KASPar allele-specific genotyping system provided by
 KBiosciences (Hoddesdon, UK). A total of 13 SNPs (out of 38) were typed
 including 5, 3 and 5 SNPs for <i>Tlr2</i>,
 <i>Tlr4</i> and <i>Tlr5</i> respectively. Lastly,
 haplotypes were reconstructed from the phased SNPs using the procedure
 implemented in DNASP v5 (Librado & Rozas, 2009). All sequences
 have been submitted to NCBI Genbank (Accession nos. are in Table S2 fot
 the ms, Supporting information). 
 - The serological status of roe deer for
 <i>Toxoplasma gondii</i> and/or <i>Chlamydia abortus
 </i>was analysed using classical enzyme-linked immunosorbent assays
 (ELISA) with specific commercial kits (Sevila <i>et al.</i>,
 2014). The specificity and sensitivity of these kits were respectively
 97.4 and 99.4 % for <i>T. gondii</i> and 92.2 and 89 % for
 <i>C. abortus</i>. Although the kits were developed for
 domestic ruminants, they were shown to be reliable and efficient in wild
 ungulates with a high concordance (0.8) between ELISA and the Modified
 Agglutination Test, a reference test for <i>Toxoplasma</i> in
 roe deer (Gotteland <i>et al.</i>, 2014). According to the
 manufacturer’s instructions, blood samples with antibody recognition level
 (ARL) &gt; 30% and &gt;40% were considered positive for
 <i>T. gondii</i>  and <i>C. abortus</i>
 respectively (see Sevila et al., 2014 for further details). In total, we
 obtained the <i>Toxoplasma</i> and
 <i>Chlamydia</i> serological status of 277 (with 74 repeated
 measures) and 196 (36 repeated measures) roe deer respectively (caught
 between 2008 and 2016).