Western European lineage
Forty-three SNPs (22 intronic, 21 exonic) were found in Western European
populations, of which 5 are non-synonymous and 16 synonymous (Fig. 4 and
Table S6). To assess whether variation in the Tshr gene can be
explained by local seasons, SNP frequencies were correlated to different
environmental proxies. The non-synonymous SNPs were not associated with
the tested environmental proxies (Table S6). In the Western European
lineage, 3/43 SNPs (exonic, synonymous) significantly correlated with
pairwise geographical distance (Fig. 4A, C, Table S6), indicating that
there is a lack of regional equilibrium(Hutchison & Templeton, 1999),
and that an alternative approach may be used to detect selection,
classifying SNPs that show clinal variation(Endler, 1977). SNP frequency
weakly correlated to pairwise latitudinal, longitudinal and altitudinal
difference for the majority of the observed SNPs (latitude: 2 exonic
SNPs, longitude: 11 intronic and 5 exonic SNPs, altitude: 3 intronic and
3 exonic SNP) (Fig. 4D-L, Table S6). These findings show that
geographical distance, latitude and altitude by themselves are bad
predictors for genetic variation in the TSHR gene, despite the
fact that annual photoperiod-food abundance patterns depend on all these
parameters. Therefore, for each sample location, the pCPP at which grass
growth is initiated in spring (at 5-10°C ambient temperature; Cooper,
1964; Peacock, 1975; Peacock, 1976) was deduced from local annual
photoperiod-ambient temperature ellipsoids. pCPP at 6.6°C achieved
highest number of significant SNPs. Therefore, the temperature threshold
for grass growth initiation in spring was set at 6.6°C, and was used to
deduce corresponding pCPP, which were calculated to vary between 10.19
and 15.40 hours of light /24 hours (Fig. 1 and Table S1). In Western
European samples, 5 intronic and 7 exonic SNPs strongly correlated to
pairwise difference in pCPP (Fig. 4M, Table S6). FSTvalues for these specific SNPs were high (ranging from
FST = 0.032 to 0.310, mean: 0.166). All these
significant mutations were, however, synonymous SNPs. Strongest
associations with pCPP were found for intronic SNP-158
(G>C), -128 (T>C), and exonic SNP126
(A>G) (Fig. 4M-O, Table S6). It is expected that between
Orkney Island and between mainland, some of the variation reflects
isolation and genetic drift. Therefore, the same analysis was performed
excluding the Orkney Island populations and revealed similar results.
Pairwise multilocus FST-values were high for populations
that differ highly in pCPP, while FST-values were low
for populations with similar pCPP (Fig. 3).