Introduction
Genetic variation is key to both the fitness of individuals and the
persistence of populations (Reed & Frankham, 2003). Loss of genetic
variation can result in inbreeding depression, loss of heterozygote
advantage, and a reduction in adaptive potential and be especially
detrimental in small or bottlenecked populations (Lande, 1995).
Therefore, understanding the factors and mechanisms that shape genetic
variation within such populations is important from both an evolutionary
and conservation perspective (Frankham, 1996).
Various interacting
evolutionary forces act to shape genetic variation within populations,
either through ‘neutral’ processes such as genetic drift, or ‘adaptive’
processes such as selection (Wright, 1931, Lande, 1976). Determining the
relative importance of these forces in shaping genetic diversity is key
to understanding the adaptive potential of populations (Lacy, 1987;
Sutton, Nakagawa, Robertson, & Jamieson, 2011). In small populations,
genetic drift is usually predominant, resulting in a decrease in genetic
variation across the genome (Robinson et al., 2016). Nevertheless,
selection can also act on functional genes, either counteracting or
reinforcing the effect of drift. Directional or purifying selection can
push alleles to fixation, resulting in a reduction in genetic variation
and reinforcing drift (Mukherjee, Sarkar-Roy, Wagener, & Majumder,
2009). In contrast, balancing selection (caused by a suite of potential
mechanisms) may maintain genetic variation and counteract the effect of
drift (Hedrick, 1998).
Pathogens can have considerable negative impact on the survival and
reproductive success of individuals (Daszak, Cunningham, & Hyatt,
2000), and are strong drivers of evolutionary change in natural
populations (Haldane, 1992). Consequently, immunogenetic loci - i.e.
those involved in the detection and combating of pathogens – are
excellent candidates in which to investigate the evolutionary forces
underlying the maintenance of genetic variation (Sommer, 2005; Croze,
Živković, Stephan, & Hutter, 2016). Indeed, pathogen-mediated selection
is thought to be a key driver of balancing selection (Spurgin &
Richardson, 2010). Three non-mutually exclusive mechanisms driving
pathogen-mediated selection have been proposed: heterozygote advantage
(Doherty & Zinkernagel, 1975), rare allele advantage (Slade &
McCallum, 1992), and fluctuating selection (Hill et al., 1991). These
three mechanisms – along with other forces such as sexual selection –
can act independently, in concert, or in trade-off with one other
(Apanius, Penn, Slev, Ruff, & Potts, 1997; Spurgin & Richardson, 2010;
Ejsmond, Radwan, & Wilson, 2014).
Immunogenetic research on wild populations has focused mainly on
receptor genes of the acquired immune system: in particular on the
exceptionally polymorphic major histocompatibility complex (MHC)
(reviewed in Piertney & Oliver, 2005). However, high levels of
diversity (Hedrick, 1994), gene duplication (Bollmer, Dunn, Whittingham,
& Wimpee, 2010), conversion, recombination (Miller & Lambert, 2004),
and epistasis (van Oosterhout, 2009) makes it hard to tease apart the
evolutionary forces driving MHC variation (Spurgin & Richardson, 2010).
In contrast, the genes involved in the innate immune response, while
still often polymorphic, exhibit relatively lower complexity.
Furthermore, the innate immune system is the host’s first line of
response to pathogens enabling a broad defence against an assortment of
organisms (Aderem & Ulevitch, 2000). Consequently, innate immune genes
can be more tractable candidates with which to study the evolutionary
forces shaping immunogenetic variation in wild populations
(Acevedo-Whitehouse & Cunningham, 2006).
Toll-Like Receptor (TLR) genes encode receptor molecules which bind to
pathogen-associated molecular patterns - evolutionary conserved
structures that are integral to the pathogen (Medzhitov, 2001). Once
bound, the TLR molecule triggers a cascade of processes associated with
the innate and adaptive immune responses (Akira, Uematsu, & Takeuchi,
2006). Vertebrate TLRs can be divided into six families, depending on
the pathogen-associated molecular patterns they detect (Roach et al.,
2005). For example, TLR3 binds to viral dsRNA (Barton, 2007),
while TLR5 binds to bacterial flagellin (Brownlie & Allan,
2011). While the majority of the TLR structure is structurally conserved
(Roach et al., 2005), there is variation in the leucine-rich repeat
domain of TLR genes, resulting in functional variation at the binding
site. Such TLR polymorphisms have been associated with resistance
(Antonides, Mathur, Sundaram, Ricklefs, & DeWoody, 2019), or
susceptibility to specific pathogens (Kloch et al., 2018), or associated
with increased survival (Grueber, Wallis, & Jamieson, 2013; Bateson et
al., 2016). TLRs can evolve rapidly as a result of pathogen-mediated
selection (Downing, Lloyd, O’Farrelly, & Bradley, 2010) and evidence of
balancing selection at TLR genes has been reported for various taxa
(e.g. Areal, Abrantes, & Esteves, 2011; Velová, Gutowska-Ding, Burt, &
Vinkler, 2018). Nevertheless, most of these studies only inferred past
selection from sequence variation and could not determine if selection
was still acting, or determine the specific mechanisms involved.
Moreover, in various bottlenecked populations, genetic drift may
override selection as the dominant evolutionary force shaping TLR
variation (Grueber et al., 2013; Gonzalez‐Quevedo, Spurgin, Illera, &
Richardson, 2015).
Here, we investigate the contemporary evolution of TLR variation in a
natural population of Seychelles warblers (Acrocephalus
sechellensis ). The last remaining population of this species on Cousin
island underwent a bottleneck in the 1900s resulting in decreased
genome-wide genetic variation (Spurgin et al., 2014). Extensive
longitudinal monitoring and a lack of dispersal (Komdeur, Piersma,
Kraaijeveld, Kraaijeveld-Smit, & Richardson, 2004) means that virtually
all individual warblers on Cousin island are sampled, marked and tracked
throughout their entire lives (Komdeur, 1992; Hammers et al., 2015).
This allows for accurate measures of survival and reproductive success
(Hammers et al., 2019). As part of a conservation programme, individuals
have been translocated from Cousin to establish populations on four
additional islands (Komdeur, 1994; Richardson, Bristol, & Shah, 2006;
Wright, Shah, & Richardson, 2014), allowing spatial TLR variation to be
investigated. A previous study found that five of seven TLR loci
examined in the Seychelles warbler were polymorphic and detected a
signature of past positive selection at two loci, one of these beingTLR3 - a viral sensing TLR (Gilroy, van Oosterhout, Komdeur, &
Richardson, 2017). A SNP at this TLR3 loci was singled out for
investigation because it is non-synonymous, found within the
functionally important leucine-rich repeat domain region, and had a
relatively high minor allele frequency (32%). However, if and how
balancing selection maintains variation at this locus has yet to be
investigated.
We first assess how the frequency of this TLR3 SNP has changed
over 25-years in the Seychelles warbler on Cousin Island. We then test
the role of selection in shaping TLR3 variation in this
population; specifically, if survival and reproductive success are
associated with individual TLR3 genotypes. Lastly, we compare
patterns of TLR3 evolution over time in, and between, the Cousin
population and the newly established (translocated) populations. These
analyses allow us to better understand which evolutionary forces shape
immunogenetic variation in small populations of conservation concern.