Songbird germline-restricted chromosome as a potential arena of genetic conflicts.

Genetic conflicts can arise between components of the genome with different inheritance strategies. The germline-restricted chromosome (GRC) of songbirds shows unusual mitotic and meiotic transmission compared with the rest of the genome. It is excluded from somatic cells and maintained only in the germline. It is usually present in one copy in the male germline and eliminated during spermatogenesis, while in the female germline, it usually occurs in two copies and behaves as a regular chromosome. Here, we review what is known about the GRC's evolutionary history, genetic content, and expression and discuss how it may be involved in different types of genetic conflicts. Finally, we interrogate the potential role of the GRC in songbird germline development, highlighting several unsolved mysteries.

Micro Germline-Restricted Chromosome in Blue Tits: Evidence for Meiotic Functions.

The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit (Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.

Genomic signatures of the evolution of a diurnal lifestyle in Strigiformes.

Understanding the targets of selection associated with changes in behavioral traits represents an important challenge of current evolutionary research. Owls (Strigiformes) are a diverse group of birds, most of which are considered nocturnal raptors. However, a few owl species independently adopted a diurnal lifestyle in their recent evolutionary history. We searched for signals of accelerated rates of evolution associated with a diurnal lifestyle using a genome-wide comparative approach. We estimated substitution rates in coding and noncoding conserved regions of the genome of seven owl species, including three diurnal species. Substitution rates of the noncoding elements were more accelerated than those of protein-coding genes. We identified new, owl-specific conserved noncoding elements as candidates of parallel evolution during the emergence of diurnality in owls. Our results shed light on the molecular basis of adaptation to a new niche and highlight the importance of regulatory elements for evolutionary changes in behavior. These elements were often involved in the neuronal development of the brain.

Local selection signals in the genome of blue tits emphasize regulatory and neuronal evolution.

Understanding the genomic landscape of adaptation is central to understanding microevolution in wild populations. Genomic targets of selection and the underlying genomic mechanisms of adaptation can be elucidated by genome-wide scans for past selective sweeps or by scans for direct fitness associations. We sequenced and assembled 150 haplotypes of 75 blue tits (Cyanistes caeruleus) of a single Central European population by a linked-read technology. We used these genome data in combination with coalescent simulations (i) to estimate an historical effective population size of ~250,000, which recently declined to ~10,000, and (ii) to identify genome-wide distributed selective sweeps of beneficial variants probably originating from standing genetic variation (soft sweeps). The genes linked to these soft sweeps, but also those linked to hard sweeps based on new beneficial mutants, showed a significant enrichment for functions associated with gene expression and transcription regulation. This emphasizes the importance of regulatory evolution in the population's adaptive history. Soft sweeps were further enriched for genes related to axon and synapse development, indicating the significance of neuronal connectivity changes in the brain potentially linked to behavioural adaptations. A previous scan of heterozygosity-fitness correlations revealed a consistent negative effect on arrival date at the breeding site for a single microsatellite in the MDGA2 gene. Here, we used the haplotype structure around this microsatellite to explain the effect as a local and direct outbreeding effect of a gene involved in synapse development.

Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Genomic Evidence for Sensorial Adaptations to a Nocturnal Predatory Lifestyle in Owls.

Owls (Strigiformes) evolved specific adaptations to their nocturnal predatory lifestyle, such as asymmetrical ears, a facial disk, and a feather structure allowing silent flight. Owls also share some traits with diurnal raptors and other nocturnal birds, such as cryptic plumage patterns, reversed sexual size dimorphism, and acute vision and hearing. The genetic basis of some of these adaptations to a nocturnal predatory lifestyle has been studied by candidate gene approaches but rarely with genome-wide scans. Here, we used a genome-wide comparative analysis to test for selection in the early history of the owls. We estimated the substitution rates in the coding regions of 20 bird genomes, including 11 owls of which five were newly sequenced. Then, we tested for functional overrepresentation across the genes that showed signals of selection. In the ancestral branch of the owls, we found traces of positive selection in the evolution of genes functionally related to visual perception, especially to phototransduction, and to chromosome packaging. Several genes that have been previously linked to acoustic perception, circadian rhythm, and feather structure also showed signals of an accelerated evolution in the origin of the owls. We discuss the functions of the genes under positive selection and their putative association with the adaptation to the nocturnal predatory lifestyle of the owls.

Negative effects of individual heterozygosity on reproductive success in a wild bird population.

The evolutionary consequences of individual genetic diversity are frequently studied by assessing heterozygosity-fitness correlations (HFCs). The prevalence of positive and negative HFCs and the predominance of general versus local effects in wild populations are far from understood, partly because comprehensive studies testing for both inbreeding and outbreeding depression are lacking. We studied a genetically diverse population of blue tits in southern Germany using a genome-wide set of 87 microsatellites to investigate the relationship between proxies of reproductive success and measures of multilocus and single-locus individual heterozygosity (MLH and SLH). We used complimentary measures of MLH and partitioned markers into functional categories according to their position in the blue tit genome. HFCs based on MLH were consistently negative for functional loci, whereas correlations were rather inconsistent for loci found in nonfunctional areas of the genome. Clutch size was the only reproductive variable showing a general effect. We found evidence for local effects for three measures of reproductive success: arrival date at the breeding site, the probability of breeding at the study site and male reproductive success. For these, we observed consistent, and relatively strong, negative effects at one functional locus. Remarkably, this marker had a similar effect in another blue tit population from Austria (~400 km to the east). We suggest that a genetic local effect on timing of arrival might be responsible for most negative HFCs detected, with carry-over effects on other reproductive traits. This effect could reflect individual differences in the distance between overwintering areas and breeding sites.

Range-wide genetic structure in the thorn-tailed rayadito suggests limited gene flow towards peripheral populations.

Understanding the population genetic consequences of habitat heterogeneity requires assessing whether patterns of gene flow correspond to landscape configuration. Studies of the genetic structure of populations are still scarce for Neotropical forest birds. We assessed range-wide genetic structure and contemporary gene flow in the thorn-tailed rayadito (Aphrastura spinicauda), a passerine bird inhabiting the temperate forests of South America. We used 12 microsatellite loci to genotype 582 individuals from eight localities across a large latitudinal range (30°S-56°S). Using population structure metrics, multivariate analyses, clustering algorithms, and Bayesian methods, we found evidence for moderately low regional genetic structure and reduced gene flow towards the range margins. Genetic differentiation increased with geographic distance, particularly in the southern part of the species' distribution where forests are continuously distributed. Populations in the north seem to experience limited gene flow likely due to forest discontinuity, and may comprise a demographically independent unit. The southernmost population, on the other hand, is genetically depauperate and different from all other populations. Different analytical approaches support the presence of three to five genetic clusters. We hypothesize that the genetic structure of the species follows a hierarchical clustered pattern.

Genes acting in synapses and neuron projections are early targets of selection during urban colonization.

When a species colonizes an urban habitat, differences in the environment can create novel selection pressures. Successful colonization will further lead to demographic perturbations and genetic drift, which can interfere with selection. Here, we test for consistent urban selection signals in multiple populations of the burrowing owl (Athene cunicularia), a species that colonized South American cities just a few decades ago. We sequenced 213 owls from three urban-rural population pairs and performed a genome-wide comparison of urban against rural birds. We further studied genome-wide associations with flight initiation distance, a measure of harm avoidance in which urban and rural birds are known to differ. Based on four samples taken over nine years from one of the urban populations, we investigated temporal allele frequency changes. The genomic data were also used to identify urban-specific signatures of selective sweeps. Single genomic sites did not reach genome-wide significance for any association. However, a gene-set analysis on the strongest signals from these four selection scans suggests a significant enrichment of genes with known functions related to synapses and neuron projections. We identified 98 genes predominantly expressed in the brain, of which many may play a role in the modulation of brain connectivity and consequently in cognitive function and motivational behaviour during urbanization. Furthermore, polymorphisms in the promoter region of the synaptic SERT gene - one of the two candidates known to correlate with urban colonization in birds - associated with the habitat in which individuals lived (urban vs. rural).

Evolution of genomic variation in the burrowing owl in response to recent colonization of urban areas.

When a species successfully colonizes an urban habitat it can be expected that its population rapidly adapts to the new environment but also experiences demographic perturbations. It is, therefore, essential to gain an understanding of the population structure and the demographic history of the urban and neighbouring rural populations before studying adaptation at the genome level. Here, we investigate populations of the burrowing owl (Athene cunicularia), a species that colonized South American cities just a few decades ago. We assembled a high-quality genome of the burrowing owl and re-sequenced 137 owls from three urban-rural population pairs at 17-fold median sequencing coverage per individual. Our data indicate that each city was independently colonized by a limited number of founders and that restricted gene flow occurred between neighbouring urban and rural populations, but not between urban populations of different cities. Using long-range linkage disequilibrium statistics in an approximate Bayesian computation approach, we estimated consistently lower population sizes in the recent past for the urban populations in comparison to the rural ones. The current urban populations all show reduced standing variation in rare single nucleotide polymorphisms (SNPs), but with different subsets of rare SNPs in different cities. This lowers the potential for local adaptation based on rare variants and makes it harder to detect consistent signals of selection in the genome.

Selection on a behaviour-related gene during the first stages of the biological invasion pathway.

Human-induced biological invasions are common worldwide and often have negative impacts on wildlife and human societies. Several studies have shown evidence for selection on invaders after introduction to the new range. However, selective processes already acting prior to introduction have been largely neglected. Here, we tested whether such early selection acts on known behaviour-related gene variants in the yellow-crowned bishop (Euplectes afer), a pet-traded African songbird. We tested for nonrandom allele frequency changes after trapping, acclimation and survival in captivity. We also compared the native source population with two independent invasive populations. Allele frequencies of two SNPs in the dopamine receptor D4 (DRD4) gene-known to be linked to behavioural activity in response to novelty in this species-significantly changed over all early invasion stages. They also differed between the African native population and the two invading European populations. The two-locus genotype associated with reduced activity declined consistently, but strongest at the trapping stage. Overall genetic diversity did not substantially decrease, and there is little evidence for new alleles in the introduced populations, indicating that selection at the DRD4 gene predominantly worked on the standing genetic variation already present in the native population. Our study demonstrates selection on a behaviour-related gene during the first stages of a biological invasion. Thus, pre-establishment stages of a biological invasion do not only determine the number of propagules that are introduced (their quantity), but also their phenotypic and genetic characteristics (their quality).

Ultradian Rhythmicity in Sleep-Wakefulness Is Related to Color in Nestling Barn Owls.

The possession of a rhythm is usually described as an important adaptation to regular changing environmental conditions such as the light-dark cycle. However, recent studies have suggested plasticity in the expression of a rhythm depending on life history and environmental factors. Barn owl ( Tyto alba) nestlings show variations in behavior and physiology in relation to the size of black feather spots, a trait associated with many behavioral and physiological phenotypes including the circadian expression of corticosterone and the regulation of body mass. This raises the possibility that individual spottiness could be associated with rhythmicity in sleep-wakefulness. Owlets showed ultradian rhythms in sleep-wakefulness, with a period length of 4.5 to 4.9 h. The period length of wakefulness and non-REM sleep was shorter in heavily compared to lightly spotted female nestlings, whereas in males, the opposite result was found. Furthermore, male and female nestlings displaying small black spots showed strong rhythmicity levels in wakefulness and REM sleep. This might be an advantage in a stable environment with predictable periodic changes in light, temperature, or social interactions. Heavily spotted nestlings displayed weak rhythms in wakefulness and REM sleep, which might enable them to be more flexible in reactions to unexpected events such as predation or might be a mechanism to save energy. These findings are consistent with previous findings showing that large-spotted nestlings switch more frequently between wakefulness and sleep, resulting in higher levels of vigilance compared to small-spotted conspecifics. Thus, nestlings with larger black feather spots might differently handle the trade-off between wakefulness and sleep, attention, and social interactions compared to nestlings with smaller black spots.

Genetic Correlates of Individual Differences in Sleep Behavior of Free-Living Great Tits (Parus major).

Within populations, free-living birds display considerable variation in observable sleep behaviors, reflecting dynamic interactions between individuals and their environment. Genes are expected to contribute to repeatable between-individual differences in sleep behaviors, which may be associated with individual fitness. We identified and genotyped polymorphisms in nine candidate genes for sleep, and measured five repeatable sleep behaviors in free-living great tits (Parus major), partly replicating a previous study in blue tits (Cyanistes caeruleus). Microsatellites in the CLOCK and NPAS2 clock genes exhibited an association with sleep duration relative to night length, and morning latency to exit the nest box, respectively. Furthermore, microsatellites in the NPSR1 and PCSK2 genes associated with relative sleep duration and proportion of time spent awake at night, respectively. Given the detection rate of associations in the same models run with random markers instead of candidate genes, we expected two associations to arise by chance. The detection of four associations between candidate genes and sleep, however, suggests that clock genes, a clock-related gene, or a gene involved in the melanocortin system, could play key roles in maintaining phenotypic variation in sleep behavior in avian populations. Knowledge of the genetic architecture underlying sleep behavior in the wild is important because it will enable ecologists to assess the evolution of sleep in response to selection.

Characterization of the genome and transcriptome of the blue tit Cyanistes caeruleus: polymorphisms, sex-biased expression and selection signals.

Decoding genomic sequences and determining their variation within populations has potential to reveal adaptive processes and unravel the genetic basis of ecologically relevant trait variation within a species. The blue tit Cyanistes caeruleus--a long-time ecological model species--has been used to investigate fitness consequences of variation in mating and reproductive behaviour. However, very little is known about the underlying genetic changes due to natural and sexual selection in the genome of this songbird. As a step to bridge this gap, we assembled the first draft genome of a single blue tit, mapped the transcriptome of five females and five males to this reference, identified genomewide variants and performed sex-differential expression analysis in the gonads, brain and other tissues. In the gonads, we found a high number of sex-biased genes, and of those, a similar proportion were sex-limited (genes only expressed in one sex) in males and females. However, in the brain, the proportion of female-limited genes within the female-biased gene category (82%) was substantially higher than the proportion of male-limited genes within the male-biased category (6%). This suggests a predominant on-off switching mechanism for the female-limited genes. In addition, most male-biased genes were located on the Z-chromosome, indicating incomplete dosage compensation for the male-biased genes. We called more than 500,000 SNPs from the RNA-seq data. Heterozygote detection in the single reference individual was highly congruent between DNA-seq and RNA-seq calling. Using information from these polymorphisms, we identified potential selection signals in the genome. We list candidate genes which can be used for further sequencing and detailed selection studies, including genes potentially related to meiotic drive evolution. A public genome browser of the blue tit with the described information is available at http://public-genomes-ngs.molgen.mpg.de.

The relationship between DRD4 polymorphisms and phenotypic correlations of behaviors in the collared flycatcher.

There is increasing evidence that the genetic architecture of exploration behavior includes the dopamine receptor D4 gene (DRD4). Such a link implies that the within-individual consistency in the same behavior has a genetic basis. Behavioral consistency is also prevalent in the form of between-individual correlation of functionally different behaviors; thus, the relationship between DRD4 polymorphism and exploration may also be manifested for other behaviors. Here, in a Hungarian population of the collared flycatcher, Ficedula albicollis, we investigate how males with distinct DRD4 genotypes differ in the consistent elements of their behavioral displays during the courtship period. In completely natural conditions, we assayed novelty avoidance, aggression and risk-taking, traits that were previously shown repeatable over time and correlate with each other, suggesting that they could have a common mechanistic basis. We identified two single-nucleotide polymorphisms (SNP554 and SNP764) in the exon 3 of the DRD4 gene by sequencing a subsample, then we screened 202 individuals of both sexes for these SNPs. Focusing on the genotypic variation in courting males, we found that "AC" heterozygote individuals at the SNP764 take lower risk than the most common "AA" homozygotes (the "CC" homozygotes were not represented in our subsample of males). We also found a considerable effect size for the relationship between SNP554 polymorphism and novelty avoidance. Therefore, in addition to exploration, DRD4 polymorphisms may also be associated with the regulation of behaviors that may incur fear or stress. Moreover, polymorphisms at the two SNPs were not independent indicating a potential role for genetic constraints or another functional link, which may partially explain behavioral correlations.

Haplotype structure, adaptive history and associations with exploratory behaviour of the DRD4 gene region in four great tit (Parus major) populations.

The assessment of genetic architecture and selection history in genes for behavioural traits is fundamental to our understanding of how these traits evolve. The dopamine receptor D4 (DRD4) gene is a prime candidate for explaining genetic variation in novelty seeking behaviour, a commonly assayed personality trait in animals. Previously, we showed that a single nucleotide polymorphism in exon 3 of this gene is associated with exploratory behaviour in at least one of four Western European great tit (Parus major) populations. These heterogeneous association results were explained by potential variable linkage disequilibrium (LD) patterns between this marker and the causal variant or by other genetic or environmental differences among the populations. Different adaptive histories are further hypothesized to have contributed to these population differences. Here, we genotyped 98 polymorphisms of the complete DRD4 gene including the flanking regions for 595 individuals of the four populations. We show that the LD structure, specifically around the original exon 3 SNP is conserved across the four populations and does not explain the heterogeneous association results. Study-wide significant associations with exploratory behaviour were detected in more than one haplotype block around exon 2, 3 and 4 in two of the four tested populations with different allele effect models. This indicates genetic heterogeneity in the association between multiple DRD4 polymorphisms and exploratory behaviour across populations. The association signals were in or close to regions with signatures of positive selection. We therefore hypothesize that variation in exploratory and other dopamine-related behaviour evolves locally by occasional adaptive shifts in the frequency of underlying genetic variants.

Two rare variants explain association with acute myocardial infarction in an extended genomic region including the apolipoprotein(A) gene.

Relatively low numbers of kringle 4 type 2 repeats in apolipoprotein(a) and specific haplotypes of the SLC22A3-LPAL2-LPA region on chromosome 6 are associated with an increased risk of coronary disease. We examined the possibility that rs3798220 and rs10455872, short variations located in LPA [the apolipoprotein(a) gene], and related to the number of kringle 4 type 2 repeats, may serve as markers for the association between haplotypes and acute myocardial infarction. Genotypes were determined with TaqMan assays in a sample of 2136 cases and 1211 controls. The minor alleles of rs3798220 and rs10455872 were associated with increased risks (rs3798220-C: adjusted OR 2.14, 95% CI, 1.37-3.33, P = 0.00080; rs10455872-G: adjusted OR 1.74, 95% CI 1.36-2.24, P < 0.00001). After adjustments were made for potential confounders, none of nine polymorphisms included in a haplotype analysis were singly related to disease. Two risk haplotypes were identified; one (CCTTGTGTG; OR 1.25, 95% CI 1.08-1.45, P = 0.0022) was correlated with rs3798220-C and the other (CCCTGGATC; OR 1.65, 95% CI 1.14-2.38, P = 0.0074) with rs10455872-G. Thus, the findings allowed for a more precise definition of risk-associated markers: specific nucleotides in LPA instead of standard haplotypes defined by noneffective variants from the extensive SLC22A3-LPAL2-LPA region.

Individual variation in sleep-wake rhythms in free-living birds.

Ultradian rhythms, such as sleep-wake periodicities, during the night might represent basic rest-activity cycles of organisms that are fundamental to the temporal organization and synchronization of behavior throughout the day. However, in contrast to circadian rhythms, little is known about the underlying oscillators and molecular mechanisms of higher-frequency rhythms. A fundamental step for the understanding of the mechanisms of these latter periodicities is the analysis of variation in sleep-wake cycles in free-living animals, which can help in estimating the relative importance of genetic and environmental influence on the rhythmicity. We analyzed variation in the level of rhythmicity and period length (τ) of behaviorally defined sleep-wake cycles in a natural population of blue tits Cyanistes caeruleus. Our results indicate that the expression of periodicity in sleep-wake patterns, but not τ, has a strong individual-specific basis. The within-individual repeatability estimate of the expression of periodicity was .45 (95% confidence interval: .35-.55) when data from males and females were combined. In addition, periodicity was influenced by specific environmental factors, such as night temperature, seasonal date, and age of the individual. Most strikingly, low nighttime temperature negatively affected periodicity of sleep-wake patterns, potentially via a hypothermic response of the birds. Our results further suggest that τ is influenced by photoperiod. Blue tits showed longer sleep-wake rhythms when the nights were longer. These observations suggest a genetic basis for the incidence of rhythmic sleep-wake behavior in addition to environmental modifications of their specific expression.

Heterozygosity-fitness correlations in zebra finches: microsatellite markers can be better than their reputation.

Numerous studies have reported associations between heterozygosity in microsatellite markers and fitness-related traits (heterozygosity-fitness correlations, HFCs). However, it has often been questioned whether HFCs reflect general inbreeding depression, because a small panel of microsatellite markers does not reflect very well an individual's inbreeding coefficient (F) as calculated from a pedigree. Here, we challenge this prevailing view. Because of chance events during Mendelian segregation, an individual's realized proportion of the genome that is identical by descent (IBD) may substantially deviate from the pedigree-based expectation (i.e. F). This Mendelian noise may result in a weak correlation between F and multi-locus heterozygosity, but this does not imply that multi-locus heterozygosity is a bad estimator of realized IBD. We examined correlations between 11 fitness-related traits measured in up to 1192 captive zebra finches and three measures of inbreeding: (i) heterozygosity across 11 microsatellite markers, (ii) heterozygosity across 1359 single-nucleotide polymorphism (SNP) markers and (iii) F, based on a 5th-generation pedigree. All 11 phenotypic traits showed positive relationships with measures of heterozygosity, especially traits that are most closely related to fitness. Remarkably, the small panel of microsatellite markers produced equally strong HFCs as the large panel of SNP markers. Both marker-based approaches produced stronger correlations with phenotypes than the pedigree-based F, and this did not seem to result from the shortness of our pedigree. We argue that a small panel of microsatellites with high allelic richness may better reflect an individual's realized IBD than previously appreciated, especially in species like the zebra finch, where much of the genome is inherited in large blocks that rarely experience cross-over during meiosis.

Correlations between heterozygosity and reproductive success in the blue tit (Cyanistes caeruleus): an analysis of inbreeding and single locus effects.

To understand the mechanisms behind heterozygosity-fitness correlations (HFC), it is necessary to employ large numbers of markers with known function and independently estimate the variation in inbreeding in the population. Here we genotyped 794 blue tits with 79 microsatellites that were distributed across 25 chromosomes and that were classified either as "functional" (N= 58) or "neutral" (N= 21). We found a positive effect of individual heterozygosity at multiple loci on clutch size, on the number of eggs sired by males, and on the number of recruits produced by males and females. We documented the occurrence of some consanguineous matings and found evidence for a particular type of population structure that can contribute to the occurrence of inbreeding. As the set of "neutral" loci provided more power to detect HFC and identity disequilibrium, we argue that "neutral" markers are better predictors of the effects of inbreeding. The number of significant effects at single loci did not exceed the expected number of false positives and no strong effects were associated with heterozygosity at "functional" markers. Thus, the HFC found here cannot be attributed to strong effects of the loci under study.