Selection analyses
To test if the demographic changes driven by hunting resulted in a
reduction in the intensity of natural selection on
FIVpco , we examined selective pressure in both time
periods in each region using the RELAX hypothesis testing framework
(Wertheim et
al. 2015). The method builds upon random effects branch-site models
(BS-REL)
(Kosakovsky Pondet al. 2011) that estimates the
ω ratio (the ratio of
non-synonymous to synonymous mutations or dN/dS) along each branch from
a discrete distribution of three ω ratio classes allowing selection
pressure to vary across the phylogeny
(Wertheim et
al. 2015). A ω
ratio of one corresponds to neutral selection with values >
1 being evidence for diversifying (positive) selection along a branch,
and < 1 evidence for purifying (negative) selection along a
branch. Briefly, RELAX tests for relaxation of selection pressure by
dividing branches into three subsets; test branches (T), reference
branches (R) and unclassified branches (U) (Wertheim et al. 2015)
with ωT (resp. ωR ) being
the estimated dN/dS ratio on test (resp. reference) branches. The
discrete distribution of ω is calculated using BS-REL for each branch
class, and then branches belonging to each subset are compared. The
reference estimates of ω are raised to the power of k (an
intensity parameter) so that \(\omega_{T}=\omega_{R}^{k}\) in order
to simplify model comparison. The null RELAX model is when the ω
distribution and thus selective pressure is the same in R and T (whenk = 1). The null model is compared to an alternate model (using a
likelihood ratio test) that allows k to vary so that whenk >1 selection pressure on the test branches was
intensified or k < 1 indicating that selection pressure
has been relaxed
(Wertheim et
al. 2015). In the relaxed scenario, k < 1 branches in
R are under stronger purifying and diversifying selection compared to T
branches (e.g., ω shifts from 0.1 to 0.001 or from 10 to 2). See
Wertheim et al. (2015) for model details. T and R were selected
from leaf branches (all other branches were Unassigned, U); individuals
sampled from 2005-2011 (to the end of the lag period) were assigned to
the R set and those sampled from 2012-2014 were assigned to T set. All
branches not directly connecting to the tips were classified as ‘U’ as
the majority had low phylogenetic support (posterior probability
< 0.6). To further interrogate the sequence data to identify
individual sites under selection, we performed the MEME (mixed-effects
model of evolution) pipeline (Murrell et al. 2012). We performed
both MEME and RELAX models using the Datamonkey web application
(Weaver et
al. 2018).