Summary
Variation in immune gene sequences is known to influence resistance to infectious diseases and parasites, and hence survival and mate choice, across animal taxa. Toll-like receptors (TLRs) comprise one essential gene family in the vertebrate innate immune system and recognize evolutionarily conserved structures from all major microorganism classes. However, the causes and consequences of TLR variation in passerine birds remain largely unexplored. We examined 7 TLR genes in song sparrows (Melospiza melodia), a species that is studied across North America. We then examined sequences from 4 unduplicated TLRs (TLR1LB, TLR3, TLR4, and TLR15) from birds in 2 parts of the species’ range (N = 27, N = 6), tested for evidence of selection, and conducted pilot analyses of the role of TLR heterozygosity in survival. We identified 45 SNPs: 19 caused changes in amino acid sequences and 2 of these were likely deleterious. We found no evidence of codon-level episodic positive selection but detected purifying selection at codons in all TLRs. Contrary to expectations we found no strong correlation between heterozygosity at TLRs and inbreeding coefficient f (estimate ± standard error [SE] = −0.68 ± 0.37, R2adj = 0.08, F1,25 = 3.38, P = 0.08). In addition, pilot analyses revealed no relationship between TLR heterozygosity and survival (β ± SE: 0.09 ± 2.00, P = 0.96), possibly due to small sample size. Further analyses of genetic diversity in TLRs are likely to advance understanding of the effects of innate immune gene diversity on the fitness and persistence of wild populations.
Methodology
The song sparrow population is resident year-round on Mandarte Island, BC, Canada, (~6 ha; 48.6335°N, 123.2871°W), has comprised 4–71 annual breeding pairs from 1975 to present, is socially monogamous but genetically polygynandrous (~28% of chicks have extrapair sires), and is moderately inbred with mean f ~0.06 (Keller and Arcese 1998; Smith et al. 2006; Sardell et al. 2010; Reid et al. 2014). The population has been monitored in detail since 1975 (except 1980) to record the identity of all breeding pairs and unmated males, color-band all nestlings at 3–8 days of age, and (beginning in 1993) collect ~50 μL blood from the brachial vein of banded birds (Smith et al. 2006). These activities have allowed us to genotype all birds hatched on the island since 1993 at 160 microsatellite loci to create a precise genetic pedigree for the population from which lifetime reproductive success and f can be estimated with high precision (Sardell et al. 2010; Nietlisbach et al. 2015, 2017). Immigrants to the island are assigned f of 0 as all immigrants are assumed to be unrelated to existing residents, given that Mandarte is part of a large island meta-population (Wilson and Arcese 2008; Germain et al. 2016). Given an effective re-sighting probability of >0.99 (Wilson et al. 2007), annual survival is also estimated with high precision based on annual censuses in late April. For this study, we selected individual song sparrows for analysis to represent a broad range of genetic lineages, as well as 6 immigrants to understand better their genetic contributions to the population and as an outgroup for analysis. Immigrants to the island were assumed to be 1 year of age at arrival.
In 2016, we also collected blood samples from song sparrows breeding at 2 sites in ON, Canada: an eastern ON site (Newboro, 44.633°N, 76.330°W) and a south-western ON site (London, 43.008°N, 81.291°W). These ON sites were not physically isolated from surrounding species-suitable habitat and are within seasonal migration distances of one another. Blood samples were stored in blots on filter paper. DNA was extracted from 3 ON samples from each site using an ammonium acetate protocol (Sarquis-Adamson and MacDougall-Shackleton 2016), and from BC samples following Sardell et al. (2010).