| Abstract: | Proxy measures of genome-wide heterozygosity based on approximately 10 microsatellites have been used to uncover heterozygosity fitness correlations (HFCs) for a wealth of important fitness traits in natural populations. However, effect sizes are typically very small and the underlying mechanisms remain contentious, as a handful of markers usually provides little power to detect inbreeding. We therefore used restriction site associated DNA (RAD) sequencing to accurately estimate genome-wide heterozygosity, an approach transferrable to any organism. As a proof of concept, we first RAD sequenced oldfield mice (Peromyscus polionotus) from a known pedigree, finding strong concordance between the inbreeding coefficient and heterozygosity measured at 13,198 single-nucleotide polymorphisms (SNPs). When applied to a natural population of harbor seals (Phoca vitulina), a weak HFC for parasite infection based on 27 microsatellites strengthened considerably with 14,585 SNPs, the deviance explained by heterozygosity increasing almost fivefold to a remarkable 49%. These findings arguably provide the strongest evidence to date of an HFC being due to inbreeding depression in a natural population lacking a pedigree. They also suggest that under some circumstances heterozygosity may explain far more variation in fitness than previously envisaged.It has long been known that inbreeding can reduce fitness, mainly through the unmasking of recessive or partially recessive deleterious alleles (1). Such effects are well documented in the laboratory (2, 3) but until recently have remained largely unstudied in natural populations (4, 5). This is because an individual’s inbreeding coefficient (f) can be calculated directly only by using a deep pedigree, and these pedigrees are seldom available outside the laboratory (6). However, because inbreeding increases an individual’s homozygosity, the heterozygosity of a panel of neutral genetic markers can in theory be used as a surrogate for f.Initial searches for heterozygosity fitness correlations (HFCs) (7, 8) used allozymes, but the results obtained are difficult to interpret because the proteins themselves may be under selection. With the discovery of microsatellites, an abundant class of putatively neutral genetic marker, it became possible to look for HFCs without concerns about selection on the markers themselves. A burgeoning literature now shows that HFCs based on small numbers of microsatellite loci are found in many bird and mammal species for a remarkable range of fitness traits, including neonatal survival (9), parasite susceptibility (10) and lifetime reproductive success (11), and even behavioral qualities such as territory-holding ability (12), song complexity (13) and attractiveness (14). This weight of evidence suggests that HFCs are an important and widespread phenomenon in the natural world. It is therefore important to understand their basis.Theory predicts that HFCs arise as a result of inbreeding depression, which will reduce the fitness of individuals in proportion to their inbreeding coefficient f (7). Therefore, variance in inbreeding coefficients within a population is necessary to generate HFCs. However, simulation and empirical studies indicate that the variance in f in natural populations is usually low, and estimates of f based on the small numbers of markers typically deployed are often very poor (15). As a result, HFCs will usually be weak or nonsignificant even when inbreeding actually explains a large proportion of fitness. Szulkin et al. (16) summarized this by saying that HFCs allow one to see only the “tip of the iceberg” and provided two examples in which heterozygosity explains 3% and 6% of trait variation even though inbreeding is expected to account for 24% and 30% of the variance, respectively. The most favorable situations to observe HFCs are in populations where inbred individuals are not rare due to factors such as small population size, extreme reproductive skew, and natal philopatry (15), but even in these cases using only a few markers severely curtails the power to observe the actual impact of inbreeding.To explain HFCs, authors also frequently invoke “local effects” where one or a small number of the microsatellites used as markers are by chance linked to a gene experiencing heterozygote advantage (8). Local effects are widely discussed in the literature, but their importance is unclear given that balanced polymorphisms are thought to be rare (17) and strong linkage between random pairs of loci is infrequent (16). It has also been argued that the contribution of local effects to HFCs may be overstated due to many studies having used an inappropriate statistical framework (16).One means of unambiguously testing whether inbreeding alone can explain HFCs is to deploy a larger number of genetic markers (18). If an HFC is due to inbreeding depression, the use of more markers will reduce the error variance in the estimation of genome-wide heterozygosity and thereby strengthen the correlation. In contrast, if the HFC is highly dependent on one or a few marker loci being by chance linked to a fitness locus with strong effects, as in the local effect model, adding more markers located throughout the genome will reduce the strength of the HFC as the contribution of any one marker becomes progressively diluted. Until recently, however, this approach was not available to most studies because of the prohibitive costs of developing and screening large numbers of additional microsatellites.Restriction site associated DNA (RAD) sequencing (19) has recently emerged as a rapid and economic means of genotyping thousands of single nucleotide polymorphisms (SNPs) in virtually any organism. This approach concentrates high-throughput sequencing effort around restriction enzyme cut sites that are distributed across the genome, thereby generating sufficiently deep local sequence coverage to reliably call SNPs as being either heterozygous or homozygous. Although SNPs are individually less informative than microsatellites due to their lower allelic diversity, this should be more than compensated for by the large numbers of markers screened, in principle allowing genome-wide heterozygosity to be estimated with far greater precision than previously possible.Here, we evaluated the ability of RAD sequencing to accurately estimate genome-wide heterozygosity using an experimental population of oldfield mice (Peromyscus polionotus subgriseus) with known pedigree-based inbreeding coefficients (20). We then applied RAD sequencing to a natural population of harbor seals (Phoca vitulina) to determine whether inbreeding explains previously reported HFCs for survivorship and parasite infection (21). We hypothesized that these HFCs should strengthen with the deployment of many thousands of SNPs if they are due to inbreeding. |
