3.4.3 Phylogeny based test of selection
Before the analysis using FUBAR, we assess the putative recombination sites within TLR sequences, using GARD. A total of 10 recombination sites were detected across 7 TLR sequences, mostly consisting in 1 recombination site, detected in 5 TLR sequences (data not shown ).
Among the 1739 polymorph sites observed across TLR loci (considering both P. nobilis and P. rudis ), FUBAR identified 215 sites (12%) putatively under selection, with a posterior probability of 0.9. Most of these (74%) were negatively selected sites, and as stated above, the vast majority of the positively selected sites (48 out of 56 sites) was found at the extra-cellular domain of the proteins. In most TLR sequences, the number of NSS were higher than PSS, except for the extra-cellular domain of TLR-1, TLR-13/3, TLR-2/13 and toll-like (Figure 2).
Complementary to FUBAR analysis, a DAPC performed using the putatively selected SNPs evidenced that the group of Pinna rudis and hybrids (i.e., “True Resistant” phenotype) is obviously separated from the two others, with a probability of assignment of individuals to this cluster of 100%. On the contrary, the groups of “Resistant” and “Sensitive” phenotypes overlapped, though few individuals are oppositely distributed across the second axis (Figure 4).
The relative contributions of the putatively selected SNPs to the separation of the “Resistant” and “Sensitive” clusters were weak, never exceeding 7.4% of the overall variation (data not shown ). The most contributing PSS and NSS sites to the clustering belonged to the two TLR-6 (i.e., sequences 48600 and 39119) and one toll-like protein (i.e., sequence 84580). The observed variations at PSS sites led to amino acids changes in the sequence of TLR-6: c.1613T>G which translated in p.I538R at the end of the last LRR, and c.3075A>G which translated in p.I1025M at the intracellular domain of the protein. Variations at NSS sites did not involve any amino-acids replacement. Arginine and methionine amino acids were twice more frequent in “Resistant” compared to “Sensitive” phenotypes. Noteworthy all Pinna rudis TLR-6 carried these amino-acids as well (Suppl. Table 6).