3.4 | Genetic load
At the interspecific level (S. catenatus and S. tergeminussamples combined), we identified 28,493 sites containing derived
nonsynonymous substitutions from 3,800 BUSCOs. When we analyzed the
degree of evolutionary conservation of these sites using PROVEAN, we
characterized 91% (26,035) of these sites as benign mutations, 8%
(2,189) as deleterious mutations, and only 1% (269) as loss of function
mutations. Collectively these represent Species level mutations in our
analyses.
Based on these variants, on average, the genomes of inbred S.
catenatus individuals had lower absolute numbers and lower relative
proportion of all classes of mutations compared to the genomes of
outbred S. tergeminus individuals (P < 0.001 in
all cases; Figures 6a-b). There were ~2-fold differences
in absolute numbers of benign
(S. tergeminus : mean =
7054, SE = 94; S. catenatus : mean = 4999, SE = 47) and loss of
function (S. tergeminus : mean = 65, SE = 2; S. catenatus :
mean = 24, SE < 1) mutations compared to a smaller, but still
significant, difference in deleterious mutations (S. tergeminus :
mean = 290, SE = 6; S. catenatus : mean = 253, SE = 3).
Differences in the relative proportion of deleterious variants are
highest for loss of funtion alleles (S. tergeminus : mean = 0.147,
SE = 0.003; S. catenatus : mean = 0.047, SE = 0.001), intermediate
for benign mutations (S. tergeminus : mean = 0.154, SE = 0.001;S. catenatus : mean = 0.102, SE = 0.001), and lowest for
deleterious mutations (S. tergeminus : mean = 0.071, SE = 0.001;S. catenatus : mean = 0.061, SE = 0.001) (Figure 6b).
Under the assumption that most deleterious mutations that arise in a
population are recessive (Agrawal & Whitlock, 2011) the genotypic
configuration of mutations can have a substantial impact on their
realized negative impacts on individual fitness (Mathur & DeWoody,
2021). In this respect, S. catenatus has a significantly greater
proportion of homozygotes relative to S. tergeminus ,
demonstrating greater levels of realized genetic load on a
per-individual basis for two of three classes of mutations (Figure 6c).
The proportion of deleterious homozygous genotypes was >
2-fold higher in S. catenatus compared to S. tergeminus(S. tergeminus : mean = 0.017, SE = 0.001; S. catenatus :
mean = 0.037, SE = 0.001), while benign homozygous genotypes were also
found at a significantly greater frequency in S. catenatus(S. tergeminus : mean = 0.060, SE = 0.001; S. catenatus :
mean = 0.074, SE = 0.001) (Figure 6c). The exception is loss of function
homozygous genotypes, which are significantly less often found inS. catenatus (S. tergeminus : mean = 0.043, SE =
0.003; S. catenatus : mean = 0.025, SE = 0.001; Figure 6c). Our
interpretation is that this may represent extreme purging of highly
deleterious mutations in small S. catenatus populations, as has
been found in extremely small and repeatedly bottlenecked populations of
Alpine ibex (Grossen et al., 2020).
Finally, although S. tergeminus had overall more deleterious
mutations than S. catenatus , the fitness related impacts of
mutations may be reduced because a greater proportion of all classes of
deleterious mutations are found in heterozygous genotypes in S.
tergeminus individuals (Figure 6d). This difference is highly
significant for all three mutation classes (P < 0.001).
This result supports the interpretation that the greater number of
derived deleterious mutations in S. tergeminus may have reduced
phenotypic expression because they are more often found in heterozygotes
and less often found in homozygotes.
Mean PROVEAN scores of alleles found in both homozygote and heterozygote
genotypes are also significantly lower (more deleterious) in S.
catenatus compared to S. tergeminus (Figure 6e). These results
support the interpretation that high levels of drift present in smallS. catenatus populations have allowed the persistence of
mutations with greater potential negative impacts regardless of genotype
class.
In contrast, analysis that use Population level mutations that capture
population level processes show the opposite pattern in the form of
higher levels of deleterious mutations with greater impact in S.
catenatus (Fig. 7). For example, when data is pooled across all
individuals within each species, the proportion of all variants made up
by all classes of Population specific deleterious mutations is
significantly higher in S. catenatus compared to S.
tergeminus (Fig. 7a) as is the frequency of homozygous genotypes (Fig.
7b). Patterns of variation in heterozygotes are less clear: there is the
expected increases in heterozygote frequency of beneficial mutations but
no significant different in heterozygote frequency between species for
other two more negative classes of mutations possibly due to the high
variation present among individuals (Fig. 7 c). Nonetheless, the overall
pattern is that, as a class, individual deleterious mutations that
escape the “drift sieve” at the species level are more abundant within
individual populations and potentially have more impact on individual
fitness.
Comparisons based on Population level mutations across individualS. catenatus populations also show substantial and often
significant differences in genome-wide proportion of deleterious
mutations, and homozygote and heterozygote frequency across all mutation
classes (Figure 8). If we use the proportion of each class of
deleterious mutations as a measure of load (Figure 8a-c) then there is
> 2-fold variation across populations for each class of
deleterious mutation (benign: 0.384 [KPWA] – 0.585 [MOSQ];
deleterious: 0.238 [SSSP] – 0.493 [MOSQ]; loss of function:
(0.228 [KPWA] – 0.410 [MOSQ]). These analyses identify specific
populations that have high levels of load and are potentially subject to
significant negative genetic impacts (MOSQ, CEBO, and SPVA) but also
populations with the lowest levels of load (KPWA).
Finally, to assess possible drivers of the high level of variation in
load across populations, we estimated correlations between estimates of
contemporary effective size (Sovic et al., 2019) and mean proportion of
deleterious mutations and levels of inbreeding (estimated as mean FROH)
and mean frequency of homozygote deleterious genotypes. There were
negative and marginally significant correlations between effective size
and each class of mutations, supporting the predicted impact of drift
acting through differences in population size on overall levels of
genetic load (benign: Spearman rank correlation (R ) = -0.714;P = 0.06; deleterious: R = -0.667; P = 0.08; loss
of function: R = -0.381; P = 0.3). There were also the
expected positive correlations between F ROH and
frequency of homozygotes, but none approached significance (results not
shown).