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.