Discussion
We
detected spatial and temporal changes in variation at the viral sensingTLR3 locus in the Seychelles warbler. On Cousin, we found a
decline in the minor allele frequency of the nonsynonymous TLR3SNP (TLR3 C allele) in the adult population over
a period of 25 years; from 40% in 1993, to 29% in 2018 (see Fig 1).
Importantly, we found differential survival associated with TLR3genotypes; individuals of either sex with theTLR3 CC genotype had a 37% increased mortality
risk compared to those with a TLR3 AC orTLR3 AA genotype. Furthermore, males - but not
females - with TLR3 CC orTLR3 AC genotypes had considerably lower overall
lifetime reproductive success (LRS) than those withTLR3 AA genotype (see Fig 3a). When separating
out the survival effects of TLR3 genotype on LRS by controlling
for survival to adulthood and for longevity, males - but not females -
with the TLR3 AC genotype had reduced
reproduction than those with the TLR3 AAgenotype (see Fig 3b). Finally, the TLR3 genotypes of
nestlings/fledglings deviated from HWE, but this deficiency of
heterozygotes was no longer significant when assessing those individuals
which survived to adulthood. We also found significant differences in
the TLR3 minor allele frequency among the different island
populations (see Fig 4). All island populations showed the same pattern
of a decrease in the minor allele frequency.
The temporal pattern in our data - with theTLR3 C allele declining in the population on
Cousin over a 25-year period - could be driven by a number of
evolutionary forces. However, the lack of migration in or out of Cousin
(Komdeur et al., 2004), means it cannot be caused by gene flow.
Importantly, our results show that individuals of either sex that were
homozygous for TLR3 C had lower survival and
that TLR3 AC males had a lower rate of
reproduction. These differences in survival (and to a lesser degree
reproductive rate) resulted, at least in males, in a considerable
reduction in LRS; males with one or two copies of theTLR3 C allele had ca half the reproductive
success of those with none (TLR3 AC: 0.63,TLR3 CC: 0.70, compared toTLR3 AA: 1.4 average independent offspring over
their lifetime). These results indicate that selection is occurring and
may explain the observed change in the TLR3 Callele frequency over time. Both TLR3 AC andTLR3 CC individuals had relatively large
selection coefficients of 0.34 and 0.46 respectively. However, it should
be noted that the added complication of overlapping generations in a
relatively long-lived species could act to dilute the observed selective
benefit of TLR3 AA genotypes in the short term.
While purifying selection in TLRs is the predominant selective mechanism
in this multigene family (Alcaide & Edwards, 2011), signatures of
positive (or balancing) selection have been detected at the codon level
in various wild vertebrate species (Areal et al., 2011; Khan et al.,
2019; Liu, Zhang, Zhao, & Zhang, 2019). Indeed, previous work in the
Seychelles warbler detected evidence of past positive selection at thisTLR3 locus (Gilroy et al., 2017). The present study now shows
that this TLR3 locus is under strong positive selection (through
both survival and reproductive success differences) in the contemporary
Cousin population.
Even if selection is acting upon the TLR3 locus in the Seychelles
warbler genetic drift will also occur. Other studies have shown that
genetic drift can override the effect of selection in driving immune
gene variation (Miller & Lambert, 2004; Sutton et al., 2011; Quemere et
al., 2015), including TLR variation (Grueber et al., 2013;
Gonzalez‐Quevedo et al., 2015). However, in the Seychelles warbler the
temporal change in allele frequencies at the TLR3 locus, aligned
as it is with the differential fitness of theTLR3 C allele, suggest that selection is
currently the prevailing force acting upon this locus in this
population. Furthermore, a previous study showed that neither neutral
microsatellite diversity, nor functional MHC allelic richness, changed
over a 18-year time period in the Cousin population, while the mean MHC
diversity per individual increased over that time (Wright et al., 2014).
This lack of a change at these other loci may suggest that the effect of
genetic drift is limited in this already genetically depauperate
(Richardson & Westerdahl, 2003; Hansson & Richardson, 2005) population
over the timeframe observed here.
While various studies have linked TLR variation with pathogen infection
(Tschirren et al., 2013; Quemere et al., 2015), few have found direct
links between TLR variation and fitness in wild populations. In the
pale-headed brushfinch (Atlapetes pallidiceps ), decreased
survival was associated with high overall TLR diversity (Hartmann,
Schaefer, & Segelbacher, 2014), whilst in song sparrows
(Melospiza melodia ) there was no relationship between survival
and TLR heterozygosity (Nelson-Flower, Germain, MacDougall-Shackleton,
Taylor, & Arcese, 2018), although in both cases the effect of specific
alleles was not tested. In the Stewart Island robin (Petroica
australis rakiura ), early life mortality was reduced in individuals
with the TLR4 BE genotype, compared to otherTLR4 genotypes, despite it being a synonymous substitution
(Grueber et al., 2013). Finally, in Attwater’s prairie-chicken
(Tympanuchus cupido attwateri ) the presence of a specificTLR1B allele was associated with reduced survival (Bateson et
al., 2016). Like the latter two studies, we found the presence of a
specific allele to confer differential survival; theTLR3 A allele conferred a selective advantage
via increased survival, predominantly in early life. Given the
importance of TLR3 as an innate immune receptor (Barton, 2007),
and that the SNP investigated causes a functional difference in the
binding region, it is likely that the survival differences seen here are
due to differential pathogen recognition.
In this study, we also found some evidence of TLR3 genotypes
conferring differential reproductive success in male, but not female
warblers. To our knowledge, this is the first-time variation at a TLR
gene has been associated with reproductive success in a wild population.
In vertebrates, longevity is generally strongly positively correlated
with lifetime reproductive success (Clutton-Brock, 1988), indeed we
found longevity to be the greatest predictor of reproductive success in
the Seychelles warbler. However, even after controlling for fitness
effects associated with offspring genotype, ability to breed, and
longevity we found an effect of male TLR3 genotype. Combined with
differential survival, this resulted in TLR3 AAmales having considerably greater overall LRS than other genotypes. This
observed difference in the reproductive output of males, but not
females, could be driven by male-male competition – with males in
better condition (through differential immune response due to theTLR3 variation) better able to outcompete others and gain more
social or extra-group offspring. For example, specific alleles at both
immune and non-immune loci have been associated with increased
competitive ability and increased reproductive success in male
vertebrates (Johnston et al., 2013; Sepil, Lachish, & Sheldon, 2013).
If female choice is
occurring based on the TLR3 variant in the Seychelles warbler
this could explain how only male, and not female, individuals had
differential reproduction associated with different TLR3genotype. Previous studies, on both the Seychelles warbler (Richardson,
Komdeur , Burke, & von Schantz, 2005; Wright et al 2016) and other
vertebrate taxa, have focused on MHC-based female mate choice (reviewed
in Milinski, 2006; Kamiya, O’Dwyer, Westerdahl, Senior, & Nakagawa,
2014). As we found a TLR3 heterozygote deficiency in offspring it
is possible that assortative mating could be taking place, whereby
individuals’ mate with individuals similar to themselves more frequently
than expected by chance (Sin et al., 2015). Likewise, as TLR3heterozygous individuals do not have higher fitness than TLR3homozygous individuals, mate choice is unlikely to be based onTLR3 heterozygosity. Further investigation should focus on ‘good
genes’ or assortative mating as potential candidate mechanisms in
driving the differential reproduction observed in this study.
A third possibility that could explain the pattern of reproductive
success linked to TLR variation is that the heterozygote deficit in
offspring is due to selection on those offspring. For example, males
with TLR3 AA genotypes are unable to produceTLR3 CC offspring (whoever they breed with), so
those males will never suffer from reduced reproductive success caused
by the higher mortality of TLR3 CC offspring,
and thus will have higher LRS. Nonetheless, if this were the sole
determinant of the differential reproductive success found in this
study, one would expect an equivalent outcome for females. However,
there was no effect of TLR3 genotype on female overall LRS or
rate of reproduction, despite females not differing from males in terms
of survival linked to the TLR3 variation. To differentiate
between the three non-mutually exclusive mechanisms outlined above,
future studies could determine if differences in competitive ability
such as body condition and immune responses, and/or differential
patterns of mating success are occurring based on this TLR3variation.
That there is contemporary positive selection acting upon theTLR3 locus in the Seychelles warbler provides insight into the
evolutionary mechanisms acting upon this important immune locus. The
decline in the TLR3 C allele demonstrated in the
current study only represents a snap-shot view of positive selection
acting upon this locus. That a selective beneficial polymorphism does
exist at this locus despite the considerable bottleneck this species has
undergone (Richardson & Westerdahl, 2003; Hansson & Richardson, 2005),
may indicate that balancing selection is acting on this locus over the
long-term. Given the role this locus plays in the innate immune
response, this is likely to be pathogen-mediated. Of the three main
mechanisms by which balancing selection is thought to maintain immune
variation (reviewed in Spurgin & Richardson, 2010), our study shows
that this is not caused by heterozygote advantage (Doherty &
Zinkernagel, 1975); TLR3 AC individuals did not
gain higher LRS or have increased survival than the homozygote
genotypes. The variation observed could potentially be driven by rare
allele advantage (Slade & McCallum, 1992), or fluctuating selection
(Hill et al., 1991), or both. However, differentiating the relative
importance of these two mechanisms in driving genetic variation, and
separating them from other evolutionary mechanisms is complicated
(Spurgin & Richardson, 2010). To do so we would first need to identify
the selective agent (pathogen) responsible and compare the presence and
change in this with the change in TLR3 variation. Secondly, we
would need to extend the present 25-year time either by including past,
or future population samples of Seychelles warbler to capture any
potential change points. Forward extrapolation from the current temporal
pattern suggests that it will take a further ca 40 years before theTLR3 C allele reaches less than 5% frequency in
the adult population. Likewise, backwards extrapolation suggests that
both TLR3 alleles were at roughly equal frequency in the
mid-1970s. It has been possible to use museum samples from 26 warblers
to examine pre-bottleneck diversity of microsatellite markers, MHC class
I alleles (Spurgin et al., 2014), and avian β-defensin genes (Gilroy,
van Oosterhout, Komdeur, & Richardson, 2016). In the future, we hope to
gain more DNA and sequence these samples to determine what TLR3variation existed prior to the bottleneck.
In the present study, we
identified a decrease in the TLR3 C allele
frequency over time across all five island populations (Fig 4) though
they did differ in rate of change. These temporal patterns ofTLR3 C loss suggest that whatever selective
agent is acting on Cousin is present on the other islands. Given their
very close proximity, and similarity to Cousin - compared to the more
isolated islands of Denis and Frégate - the weaker effect on Aride and
Cousine is surprising as one may expect close and environmentally
similar islands to contain similar pathogens. For example, Cousine (the
closest island to Cousin) is the only island to have retained (after
translocation) the single strain of the Haemoproteus
nucleocondensus pathogen that is present in the original Cousin
population (Fairfield et al., 2016). A similar pattern of
spatio-temporal change in TLR1LA diversity between translocated
populations of the New Zealand South Island saddleback,Philesturnus carunculatus , was put down to the distribution of
malaria parasites (Knafler, Grueber, Sutton, & Jamieson, 2017).
However, the distribution of the haemoproteus pathogen found in the
Seychelles warbler (not on Aride, Denis or Frégate) means that this
cannot be the selective agent here. Work is now needed to identify the
pathogen responsible, and determine why the distribution, or impact of
this pathogen, differs among the islands.
The avian TLR3 is orthologous to mammalian TLR3 and
recognises viral dsRNA (including avian pox and influenza viruses)
(Hutchens et al., 2008; Brownlie & Allan, 2011; Chen, Cheng, & Wang,
2013). Therefore, it is likely that the selective agent is a virus.
Despite this, we have found no obvious evidence of any viral illness in
the Seychelles warbler in over thirty years of study. Furthermore, while
viruses such avian pox are common in many parts of the world (van Riper
III & Forrester, 2007) there are no reports of this, or any other
virus, circulating in the passerines in the Seychelles (Hutchings,
2009). Influenza A has been reported in Procellariformes (petrels and
shearwaters) in the Seychelles (Lebarbenchon et al., 2015), but whether
this could be passed to the warblers is unknown. It is possible that we
just do not see visible signs of a pathogen that is circulating in the
warblers because of mild virulence or evolved host tolerance (Råberg,
2014, Hammers et al., 2016). Furthermore, individuals may only show
visible symptoms during the acute phase of infection when they are also
least active, consequently they may be unlikely to be observed before
recovery or death (LaPointe, Hofmeister, Atkinson, Porter, & Dusek,
2009). In the absence of any obvious symptoms, conducting virome
screening may enable us to determine if a virus is driving theTLR3 selection. While currently virome analysis is difficult for
a range of reasons, including the absence of universal primers,
difficulty in nucleic acid extraction and lack of comprehensive viral
databases (reviewed in Garmaeva et al., 2019), this could be an
important avenue of future research. Alternatively, knowing the
structural changes in the TLR3 molecule resulting from the amino
acid difference caused by the SNP, could help elucidate functional
importance and allow inference of the pathogen driving selection at this
SNP (Velová et al., 2018).
Even if there are no virulent pathogens currently in the populations,
maintaining immunogenetic variation could have important consequences
for the future success of this species. If selection continues, the SNP
investigated here will may to fixation, and potentially important
immunogenetic variation will be lost in the system. This is particularly
important given the reduced diversity already present at this, and other
innate immune genes, in the Seychelles warbler (Gilroy et al., 2016,
2017). The innate immune response is often the organism’s first line of
defence against pathogens and plays an important role in the evolution
to novel disease outbreaks (Bonneaud, Balenger, Zhang, Edwards, & Hill,
2012). Thus, knowing the underlying variation present, and understanding
the mechanisms driving evolutionary change at these key functional sites
could be important for future species conservation. This is important in
small populations and/or those of conservation concern which often have
reduced genetic variation. Managing genetic variation in such
populations could be important for their adaptive potential, while
monitoring pathogen presence may be important to identify and control
disease outbreaks - both of which may be crucial for the populations
long term survival.