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).