Phenotype-associated inbreeding biases estimates of inbreeding depression in a wild bird population.

Inbreeding depression is usually quantified by regressing individual phenotypic values on inbreeding coefficients, implicitly assuming there is no correlation between an individual's phenotype and the kinship coefficient to its mate. If such an association between parental phenotype and parental kinship exists, and if the trait of interest is heritable, estimates of inbreeding depression can be biased. Here we first derive the expected bias as a function of the covariance between mean parental breeding value and parental kinship. Subsequently, we use simulated data to confirm the existence of this bias, and show that it can be accounted for in a quantitative genetic animal model. Finally, we use long-term individual-based data for white-throated dippers (Cinclus cinclus), a bird species in which inbreeding is relatively common, to obtain an empirical estimate of this bias. We show that during part of the study period, parents of inbred birds had shorter wings than those of outbred birds, and as wing length is heritable, inbred individuals were smaller, independent of any inbreeding effects. This resulted in the overestimation of inbreeding effects. Similarly, during a period when parents of inbred birds had longer wings, we found that inbreeding effects were underestimated. We discuss how such associations may have arisen in this system, and why they are likely to occur in others, too. Overall, we demonstrate how less biased estimates of inbreeding depression can be obtained within a quantitative genetic framework, and suggest that inbreeding and additive genetic effects should be accounted for simultaneously whenever possible.

Genetic variance components and heritability of multiallelic heterozygosity under inbreeding.

The maintenance of genetic diversity in fitness-related traits remains a central topic in evolutionary biology, for example, in the context of sexual selection for genetic benefits. Among the solutions that have been proposed is directional sexual selection for heterozygosity. The importance of such selection is highly debated. However, a critical evaluation requires knowledge of the heritability of heterozygosity, a quantity that is rarely estimated in this context, and often assumed to be zero. This is at least partly the result of the lack of a general framework that allows for its quantitative prediction in small and inbred populations, which are the focus of most empirical studies. Moreover, while current predictors are applicable only to biallelic loci, fitness-relevant loci are often multiallelic, as are the neutral markers typically used to estimate genome-wide heterozygosity. To this end, we first review previous, but little-known, work showing that under most circumstances, heterozygosity at biallelic loci and in the absence of inbreeding is heritable. We then derive the heritability of heterozygosity and the underlying variances for multiple alleles and any inbreeding level. We also show that heterozygosity at multiallelic loci can be highly heritable when allele frequencies are unequal, and that this heritability is reduced by inbreeding. Our quantitative genetic framework can provide new insights into the evolutionary dynamics of heterozygosity in inbred and outbred populations.

Bayesian paternity analysis and mating patterns in a parasitic nematode, Trichostrongylus tenuis.

Mating behaviour is a fundamental aspect of the evolutionary ecology of sexually reproducing species, but one that has been under-researched in parasitic nematodes. We analysed mating behaviour in the parasitic nematode Trichostrongylus tenuis by performing a paternity analysis in a population from a single red grouse host. Paternity of the 150 larval offspring of 25 mothers (sampled from one of the two host caeca) was assigned among 294 candidate fathers (sampled from both caeca). Each candidate father's probability of paternity of each offspring was estimated from 10-locus microsatellite genotypes. Seventy-six (51%) offspring were assigned a father with a probability of >0.8, and the estimated number of unsampled males was 136 (95% credible interval (CI) 77-219). The probability of a male from one caecum fathering an offspring in the other caecum was estimated as 0.024 (95% CI 0.003-0.077), indicating that the junction of the caeca is a strong barrier to dispersal. Levels of promiscuity (defined as the probability of two of an adult's offspring sharing only one parent) were high for both sexes. Variance in male reproductive success was moderately high, possibly because of a combination of random mating and high variance in post-copulatory reproductive success. These results provide the first data on individual mating behaviour among parasitic nematodes.

Testing evolutionary models of senescence in a natural population: age and inbreeding effects on fitness components in song sparrows.

Mutation accumulation (MA) and antagonistic pleiotropy (AP) have each been hypothesized to explain the evolution of 'senescence' or deteriorating fitness in old age. These hypotheses make contrasting predictions concerning age dependence in inbreeding depression in traits that show senescence. Inbreeding depression is predicted to increase with age under MA but not under AP, suggesting one empirical means by which the two can be distinguished. We use pedigree and life-history data from free-living song sparrows (Melospiza melodia) to test for additive and interactive effects of age and individual inbreeding coefficient (f) on fitness components, and thereby assess the evidence for MA. Annual reproductive success (ARS) and survival (and therefore reproductive value) declined in old age in both sexes, indicating senescence in this short-lived bird. ARS declined with f in both sexes and survival declined with f in males, indicating inbreeding depression in fitness. We observed a significant agexf interaction for male ARS (reflecting increased inbreeding depression as males aged), but not for female ARS or survival in either sex. These analyses therefore provide mixed support for MA. We discuss the strengths and limitations of such analyses and therefore the value of natural pedigreed populations in testing evolutionary models of senescence.

The effects of contemporary processes in maintaining the genetic structure of western song sparrows (Melospiza melodia).

Historic events and contemporary processes work in concert to create and maintain geographically partitioned variation and are instrumental in the generation of biodiversity. We sought to gain a better understanding of how contemporary processes such as movement and isolation influence the genetic structure of widely distributed vagile species such as birds. Song sparrows (Melospiza melodia) in western North America provide a natural system for examining the genetics of populations that have different patterns of geographic isolation and migratory behavior. We examined the population genetics of 576 song sparrows from 23 populations using seven microsatellite loci to assess genetic differentiation among populations and to estimate the effects of drift and immigration (gene flow) on each population. Sedentary, isolated populations were characterized by low levels of immigration and high levels of genetic drift, whereas those populations less isolated displayed signals of high gene flow and little differentiation from other populations. Contemporary dispersal rates from migratory populations, estimated by assignment test, were higher and occurred over larger distances than dispersal from sedentary populations but were also probably too low to counter the effects of drift in most populations. We suggest that geographic isolation and limited gene flow facilitated by migratory behavior are responsible for maintaining observed levels of differentiation among Pacific coastal song sparrow populations.

Batch effects in a multiyear sequencing study: False biological trends due to changes in read lengths.

High-throughput sequencing is a powerful tool, but suffers biases and errors that must be accounted for to prevent false biological conclusions. Such errors include batch effects; technical errors only present in subsets of data due to procedural changes within a study. If overlooked and multiple batches of data are combined, spurious biological signals can arise, particularly if batches of data are correlated with biological variables. Batch effects can be minimized through randomization of sample groups across batches. However, in long-term or multiyear studies where data are added incrementally, full randomization is impossible, and batch effects may be a common feature. Here, we present a case study where false signals of selection were detected due to a batch effect in a multiyear study of Alpine ibex (Capra ibex). The batch effect arose because sequencing read length changed over the course of the project and populations were added incrementally to the study, resulting in nonrandom distributions of populations across read lengths. The differences in read length caused small misalignments in a subset of the data, leading to false variant alleles and thus false SNPs. Pronounced allele frequency differences between populations arose at these SNPs because of the correlation between read length and population. This created highly statistically significant, but biologically spurious, signals of selection and false associations between allele frequencies and the environment. We highlight the risk of batch effects and discuss strategies to reduce the impacts of batch effects in multiyear high-throughput sequencing studies.

Immigration and the ephemerality of a natural population bottleneck: evidence from molecular markers.

Population bottlenecks are often invoked to explain low levels of genetic variation in natural populations, yet few studies have documented the direct genetic consequences of known bottlenecks in the wild. Empirical studies of natural population bottlenecks are therefore needed, because key assumptions of theoretical and laboratory studies of bottlenecks may not hold in the wild. Here we present microsatellite data from a severe bottleneck (95% mortality) in an insular population of song sparrows (Melospiza melodia). The major findings of our study are as follows: (i) The bottleneck reduced heterozygosity and allelic diversity nearly to neutral expectations, despite non-random survival of birds with respect to inbreeding and wing length. (ii) All measures of genetic diversity regained pre-bottleneck levels within two to three years of the crash. This rapid recovery was due to low levels of immigration. (iii) The rapid recovery occurred despite a coincident, strong increase in average inbreeding. These results show that immigration at levels that are hard to measure in most field studies can lead to qualitatively very different genetic outcomes from those expected from mutations only. We suggest that future theoretical and empirical work on bottlenecks and metapopulations should address the impact of immigration.

Development of SNP markers identifying European wildcats, domestic cats, and their admixed progeny.

Introgression can be an important evolutionary force but it can also lead to species extinction and as such is a crucial issue for species conservation. However, introgression is difficult to detect, morphologically as well as genetically. Hybridization with domestic cats (Felis silvestris catus) is a major concern for the conservation of European wildcats (Felis s. silvestris). The available morphologic and genetic markers for the two Felis subspecies are not sufficient to reliably detect hybrids beyond first generation. Here we present a single nucleotide polymorphism (SNP) based approach that allows the identification of introgressed individuals. Using high-throughput sequencing of reduced representation libraries we developed a diagnostic marker set containing 48 SNPs (Fst > 0.8) which allows the identification of wildcats, domestic cats, their hybrids and backcrosses. This allows assessing introgression rate in natural wildcat populations and is key for a better understanding of hybridization processes.

Microsatellite primers for the four Galápagos mockingbird species (Mimus parvulus, Mimus macdonaldi, Mimus melanotis and Mimus trifasciatus).

Nineteen di- and tetranucleotide and one trinucleotide microsatellite DNA markers were isolated from the Galápagos mockingbird (Mimus parvulus) and tested for cross-species amplification in the other three mockingbird species in the Galápagos. In addition, primers for two microsatellite loci previously developed for Mimus polyglottos were redesigned to obtain shorter amplification fragments. The number of alleles per locus and species ranged from 1 to 8, and expected heterozygosity varied from 0.0 to 0.809. These microsatellite markers will be useful to study levels of inbreeding in different island populations.

Interactive effects of environmental stress and inbreeding on reproductive traits in a wild bird population.

1. Conservation biologists are concerned about the interactive effects of environmental stress and inbreeding because such interactions could affect the dynamics and extinction risk of small and isolated populations, but few studies have tested for these interactions in nature. 2. We used data from the long-term population study of song sparrows Melospiza melodia on Mandarte Island to examine the joint effects of inbreeding and environmental stress on four fitness traits that are known to be affected by the inbreeding level of adult birds: hatching success, laying date, male mating success and fledgling survival. 3. We found that inbreeding depression interacted with environmental stress to reduce hatching success in the nests of inbred females during periods of rain. 4. For laying date, we found equivocal support for an interaction between parental inbreeding and environmental stress. In this case, however, inbred females experienced less inbreeding depression in more stressful, cooler years. 5. For two other traits, we found no evidence that the strength of inbreeding depression varied with environmental stress. First, mated males fathered fewer nests per season if inbred or if the ratio of males to females in the population was high, but inbreeding depression did not depend on sex ratio. Second, fledglings survived poorly during rainy periods and if their father was inbred, but the effects of paternal inbreeding and rain did not interact. 6. Thus, even for a single species, interactions between the inbreeding level and environmental stress may not occur in all traits affected by inbreeding depression, and interactions that do occur will not always act synergistically to further decrease fitness.

Comparative landscape genetics and the adaptive radiation of Darwin's finches: the role of peripheral isolation.

We use genetic divergence at 16 microsatellite loci to investigate how geographical features of the Galápagos landscape structure island populations of Darwin's finches. We compare the three most genetically divergent groups of Darwin's finches comprising morphologically and ecologically similar allopatric populations: the cactus finches (Geospiza scandens and Geospiza conirostris), the sharp-beaked ground finches (Geospiza difficilis) and the warbler finches (Certhidea olivacea and Certhidea fusca). Evidence of reduced genetic diversity due to drift was limited to warbler finches on small, peripheral islands. Evidence of low levels of recent interisland migration was widespread throughout all three groups. The hypothesis of distance-limited dispersal received the strongest support in cactus and sharp-beaked ground finches as evidenced by patterns of isolation by distance, while warbler finches showed a weaker relationship. Support for the hypothesis that gene flow constrains morphological divergence was only found in one of eight comparisons within these groups. Among warbler finches, genetic divergence was relatively high while phenotypic divergence was low, implicating stabilizing selection rather than constraint due to gene flow. We conclude that the adaptive radiation of Darwin's finches has occurred in the presence of ongoing but low levels of gene flow caused by distance-dependent interisland dispersal. Gene flow does not constrain phenotypic divergence, but may augment genetic variation and facilitate evolution due to natural selection. Both microsatellites and mtDNA agree in that subsets of peripheral populations of two older groups are genetically more similar to other species that underwent dramatic morphological change. The apparent decoupling of morphological and molecular evolution may be accounted for by a modification of Lack's two-stage model of speciation: relative ecological stasis in allopatry followed by secondary contact, ecological interactions and asymmetric phenotypic divergence.

No evidence for inbreeding avoidance in a natural population of song sparrows (Melospiza melodia).

We studied mate choice and inbreeding avoidance a natural population of song sparrows (Melospiza melodia) on Mandarte Island, Canada. Inbreeding occurred regularly: 59% all matings were between known relatives. We tested for inbreeding avoidance by comparing the observed levels of inbreeding to those expected if mate choice had been random with respect to relatedness. Independent of our assumptions about the availability of mates in the random mating model, we found that the expected and observed distributions of inbreeding coefficients were similar, as was the expected and observed frequency of close (f >/= 0.125) inbreeding. Furthermore, there was no difference in relatedness observed pairs and those that would have resulted had birds mated instead with their nearest neighbors. The only evidence to suggest any inbreeding avoidance was a reduced rate of parent-offspring matings as compared to one random mating model but not the other. Hence, despite substantial inbreeding depression in this population, we found little evidence for inbreeding avoidance through mate choice. We present a simple model to suggest that variation in inbreeding avoidance behaviors in birds may arise from differences in survival rates: in species with low survival rates, the costs of forfeiting matings to avoid inbreeding may exceed the costs of inbreeding.

Selection against inbred song sparrows during a natural population bottleneck.

The genetic and demographic consequences of population subdivision have received considerable attention from conservation biologists. In particular, losses of genetic variability and reduced viability and fecundity due to inbreeding (inbreeding depression) are of concern. Studies of domestic, laboratory and zoo populations have shown inbreeding depression in a variety of traits related to fitness. Consequently, inbreeding depression is widely accepted as a fact. Recently, however, the relative impact of inbreeding on the viability of natural populations has been questioned. Work on the cheetah (Acinonyx jubatus), for example, has emphasized the overwhelming importance of environmental factors on mortality in the wild. Here we report that song sparrows (Melospiza melodia) that survived a severe population bottleneck were a non-random subset of the pre-crash population with respect to inbreeding, and that natural selection favoured outbred individuals. Thus, inbreeding depression was expressed in the face of an environmental challenge. Such challenges are also likely to be faced by inbred populations of endangered species. We suggest that environmental and genetic effects on survival may interact and, as a consequence, that their effects on individuals and populations should not be considered independently.

Heritability of morphological traits in Darwin's finches: misidentified paternity and maternal effects.

We studied the influence of extra-pair paternity on heritability estimates of morphological traits in a population of the Medium Ground Finch (Geospiza fortis) on Isla Daphne Major, Galápagos. Data from eight microsatellite loci were used to determine parentage. Six morphological traits measured on each finch were represented by two separate principal components analyses, one for the three bill measurements and one for the body size measurements. Heritabilities were calculated using weighted regressions of offspring on their parents and also offspring on their grandparents. We found that 20% of all offspring were extra-pair young but all offspring matched their mothers. Heritabilities derived from midparent-offspring regressions were all high and significantly different from zero. Removing all extra-pair young from the data set increased father-offspring regressions by an average of 21%, but mother-offspring resemblance still exceeded father-offspring resemblance by up to 42%. These results and grandparent-offspring regressions provide evidence for maternal effects, comparable in magnitude to those reported in other studies of wild birds.

Neutral locus heterozygosity, inbreeding, and survival in Darwin's ground finches (Geospiza fortis and G. scandens).

Comprehensive long-term studies of isolated populations provide valuable comparative data that may be used to evaluate different methods for quantifying the relationship between genetic diversity and fitness. Here, we report on data collected from large and well-characterized cohorts of the two numerically dominant species of Darwin's finches on Isla Daphne Major, Galápagos, Ecuador - Geospiza fortis and G. scandens. Multilocus microsatellite (SSR) genetic diversity estimates (heterozygosity and d2) and pedigree-based estimates of the inbreeding coefficient (f) were compared to each other and to two fitness components: lifespan and recruitment. In the larger sample of G. fortis, heterozygosity (H) was correlated with both fitness components, but no relationship was detected in the smaller sample of G. scandens. Analyses of the inbreeding coefficient detected highly significant relationships between f and recruitment, but no relationship between f and overall lifespan. The d2 statistic showed no relationship to either fitness component. When the two SSR-based estimators were compared to f, d2 was correlated with f in G. fortis in the predicted direction, while in G. scandens the relationship was positive. Multilocus heterozygosity was correlated with f in G. fortis but not in the G. scandens sample. A pedigree simulation demonstrated that the variation in true autozygosity can be large among individuals with the same level of inbreeding. This observation may supplement the interpretation of patterns relevant to the local (locus-specific) and general (genome-wide) effects hypotheses, which have been proposed to explain the mechanism responsible for associations between genetic diversity and fitness.