Biological Links between Personality and Plasticity: Testing Some Alternative Hypotheses.

AbstractWhen organisms respond behaviorally to a stimulus, they exhibit plasticity, but some individuals respond to the same stimulus consistently differently than others, thereby also exhibiting personality differences. Parent house sparrows express individual differences in how often they feed offspring and how that feeding rate changes with nestling age. Mean feeding rate and its slope with respect to nestling age were positively correlated at median nestling ages but not at hatching, indicating that individuality is primarily in plasticity. Individual differences could arise because of (1) interactions between environmental variables, (2) differences in underlying state or "quality," or (3) differences in the ability to update cues of changing nestling demand. Individual slopes were modestly repeatable across breeding attempts, hinting at the likely action of additional environmental variables, but only brood size was important. I also found few correlates suggesting quality differences. I used short-term brood size manipulations at two nestling ages to test divergent predictions between the three hypotheses. The pattern of correlations between response to the manipulation and individual slope did not fit any single hypothesis. Patterns of sparrow parental care reveal that personality and plasticity are not cleanly separable, and their biology is likely intertwined. New thinking may be needed about the factors parents use in decisions about care and the relevant fitness consequences.

The impact of parental and developmental stress on DNA methylation in the avian hypothalamic-pituitary-adrenal axis.

The hypothalamic-pituitary-adrenal (HPA) axis coordinates an organism's response to environmental stress. The responsiveness and sensitivity of an offspring's stress response may be shaped not only by stressors encountered in their early post-natal environment but also by stressors in their parent's environment. Yet, few studies have considered how stressors encountered in both of these early life environments may function together to impact the developing HPA axis. Here, we manipulated stressors in the parental and post-natal environments in a population of house sparrows (Passer domesticus) to assess their impact on changes in DNA methylation (and corresponding gene expression) in a suite of genes within the HPA axis. We found that nestlings that experienced early life stress across both life-history periods had higher DNA methylation in a critical HPA axis gene, the glucocorticoid receptor (NR3C1). In addition, we found that the life-history stage when stress was encountered impacted some genes (HSD11B1, NR3C1 and NR3C2) differently. We also found evidence for the mitigation of parental stress by post-natal stress (in HSD11B1 and NR3C2). Finally, by assessing DNA methylation in both the brain and blood, we were able to evaluate cross-tissue patterns. While some differentially methylated regions were tissue-specific, we found cross-tissue changes in NR3C2 and NR3C1, suggesting that blood is a suitable tissue for assessing DNA methylation as a biomarker of early life stress. Our results provide a crucial first step in understanding the mechanisms by which early life stress in different life-history periods contributes to changes in the epigenome of the HPA axis.

The incubation environment does not explain significant variation in heart rate plasticity among avian embryos.

The conditions an organism experiences during development can modify how they plastically respond to short-term changes in their environment later in life. This can be adaptive because the optimal average trait value and the optimal plastic change in trait value in response to the environment may differ across different environments. For example, early developmental temperatures can adaptively modify how reptiles, fish and invertebrates metabolically respond to temperature. However, whether individuals within populations respond differently (a prerequisite to adaptive evolution), and whether this occurs in birds, which are only ectothermic for part of their life cycle, is not known. We experimentally tested these possibilities by artificially incubating the embryos of Pekin ducks (Anas platyrhynchos domesticus) at constant or variable temperatures. We measured their consequent heart rate reaction norms to short-term changes in egg temperature and tracked their growth. Contrary to expectations, the early thermal environment did not modify heart rate reaction norms, but regardless, these reaction norms differed among individuals. Embryos with higher average heart rates were smaller upon hatching, but heart rate reaction norms did not predict subsequent growth. Our data also suggests that the thermal environment may affect both the variance in heart rate reaction norms and their covariance with growth. Thus, individual avian embryos can vary in their plasticity to temperature, and in contrast to fully ectothermic taxa, the early thermal environment does not explain this variance. Because among-individual variation is one precondition to adaptive evolution, the factors that do contribute to such variability may be important.

Among-population variation in telomere regulatory proteins and their potential role as hidden drivers of intraspecific variation in life history.

Biologists aim to explain patterns of growth, reproduction and ageing that characterize life histories, yet we are just beginning to understand the proximate mechanisms that generate this diversity. Existing research in this area has focused on telomeres but has generally overlooked the telomere's most direct mediator, the shelterin protein complex. Shelterin proteins physically interact with the telomere to shape its shortening and repair. They also regulate metabolism and immune function, suggesting a potential role in life history variation in the wild. However, research on shelterin proteins is uncommon outside of biomolecular work. Intraspecific analyses can play an important role in resolving these unknowns because they reveal subtle variation in life history within and among populations. Here, we assessed ecogeographic variation in shelterin protein abundance across eight populations of tree swallow (Tachycineta bicolor) with previously documented variation in environmental and life history traits. Using the blood gene expression of four shelterin proteins in 12-day-old nestlings, we tested the hypothesis that shelterin protein gene expression varies latitudinally and in relation to both telomere length and life history. Shelterin protein gene expression differed among populations and tracked non-linear variation in latitude: nestlings from mid-latitudes expressed nearly double the shelterin mRNA on average than those at more northern and southern sites. However, telomere length was not significantly related to latitude. We next assessed whether telomere length and shelterin protein gene expression correlate with 12-day-old body mass and wing length, two proxies of nestling growth linked to future fecundity and survival. We found that body mass and wing length correlated more strongly (and significantly) with shelterin protein gene expression than with telomere length. These results highlight telomere regulatory shelterin proteins as potential mediators of life history variation among populations. Together with existing research linking shelterin proteins and life history variation within populations, these ecogeographic patterns underscore the need for continued integration of ecology, evolution and telomere biology, which together will advance understanding of the drivers of life history variation in nature.

Early-life telomeres are influenced by environments acting at multiple temporal and spatial scales.

An individual's telomere length early in life may reflect or contribute to key life-history processes sensitive to environmental variation. Yet, the relative importance of genetic and environmental factors in shaping early-life telomere length is not well understood as it requires samples collected from multiple generations with known developmental histories. We used a confirmed pedigree and conducted an animal model analysis of telomere lengths obtained from nestling house sparrows (Passer domesticus) sampled over a span of 22 years. We found significant additive genetic variation for early-life telomere length, but it comprised a small proportion (9%) of the total biological variation. Three sources of environmental variation were important: among cohorts, among-breeding attempts within years, and among nestmates. The magnitude of variation among breeding attempts and among nestmates also differed by cohort, suggesting that interactive effects of environmental factors across time or spatial scales were important, yet we were unable to identify the specific causes of these interactions. The mean amount of precipitation during the breeding season positively predicted telomere length, but neither weather during a given breeding attempt nor date in the breeding season contributed to an offspring's telomere length. At the level of individual nestlings, offspring sex, size and mass at 10 days of age also did not predict telomere length. Environmental effects appear especially important in shaping early-life telomere length in some species, and more focus on how environmental factors that interact across scales may help to explain some of the variation observed among studies.

Epigenetic modification of the hypothalamic-pituitary-adrenal (HPA) axis during development in the house sparrow (Passer domesticus).

Epigenetic modifications such as DNA methylation are important mechanisms for mediating developmental plasticity, where ontogenetic processes and their phenotypic outcomes are shaped by early environments. In particular, changes in DNA methylation of genes within the hypothalamic-pituitary-adrenal (HPA) axis can impact offspring growth and development. This relationship has been well documented in mammals but is less understood in other taxa. Here, we use target-enriched enzymatic methyl sequencing (TEEM-seq) to assess how DNA methylation in a suite of 25 genes changes over development, how these modifications relate to the early environment, and how they predict differential growth trajectories in the house sparrow (Passer domesticus). We found that DNA methylation changes dynamically over the postnatal developmental period: genes with initially low DNA methylation tended to decline in methylation over development, whereas genes with initially high DNA methylation tended to increase in methylation. However, sex-specific differentially methylated regions (DMRs) were maintained across the developmental period. We also found significant differences in post-hatching DNA methylation in relation to hatch date, with higher levels of DNA methylation in nestlings hatched earlier in the season. Although these differences were largely absent by the end of development, a number of DMRs in HPA-related genes (CRH, MC2R, NR3C1, NR3C2, POMC)-and to a lesser degree HPG-related genes (GNRHR2)-predicted nestling growth trajectories over development. These findings provide insight into the mechanisms by which the early environment shapes DNA methylation in the HPA axis, and how these changes subsequently influence growth and potentially mediate developmental plasticity.

Stressors interact across generations to influence offspring telomeres and survival.

Parental stress often has long-term consequences for offspring. However, the mechanisms underlying these effects and how they are shaped by conditions offspring subsequently experience are poorly understood. Telomeres, which often shorten in response to stress and predict longevity, may contribute to, and/or reflect these cross-generational effects. Traditionally, parental stress is expected to have negative effects on offspring telomeres, but experimental studies in captive animals suggest that these effects may depend on the subsequent conditions that offspring experience. Yet, the degree to which parental stress influences and interacts with stress experienced by offspring to affect offspring telomeres and survival in free-living organisms is unknown. To assess this, we experimentally manipulated the stress exposure of free-living parent and offspring house sparrows (Passer domesticus). We found a weak, initial, negative effect of parental stress on offspring telomeres, but this effect was no longer evident at the end of post-natal development. Instead, the effects of parental stress depended on the natural sources of stress that offspring experienced during post-natal development whereby some outcomes were improved under more stressful rearing conditions. Thus, the effects of parental stress on offspring telomeres and survival are context-dependent and may involve compensatory mechanisms of potential benefit under some circumstances.

Longer telomeres during early life predict higher lifetime reproductive success in females but not males.

The mechanisms that contribute to variation in lifetime reproductive success are not well understood. One possibility is that telomeres, conserved DNA sequences at chromosome ends that often shorten with age and stress exposures, may reflect differences in vital processes or influence fitness. Telomere length often predicts longevity, but longevity is only one component of fitness and little is known about how lifetime reproductive success is related to telomere dynamics in wild populations. We examined the relationships between telomere length beginning in early life, telomere loss into adulthood and lifetime reproductive success in free-living house sparrows (Passer domesticus). We found that females, but not males, with longer telomeres during early life had higher lifetime reproductive success, owing to associations with longevity and not reproduction per year or attempt. Telomeres decreased with age in both sexes, but telomere loss was not associated with lifetime reproductive success. In this species, telomeres may reflect differences in quality or condition rather than the pace of life, but only in females. Sexually discordant selection on telomeres is expected to influence the stability and maintenance of within population variation in telomere dynamics and suggests that any role telomeres play in mediating life-history trade-offs may be sex specific.

Collision between biological process and statistical analysis revealed by mean centring.

Animal ecologists often collect hierarchically structured data and analyse these with linear mixed-effects models. Specific complications arise when the effect sizes of covariates vary on multiple levels (e.g. within vs. among subjects). Mean centring of covariates within subjects offers a useful approach in such situations, but is not without problems. A statistical model represents a hypothesis about the underlying biological process. Mean centring within clusters assumes that the lower level responses (e.g. within subjects) depend on the deviation from the subject mean (relative) rather than on the absolute scale of the covariate. This may or may not be biologically realistic. We show that mismatch between the nature of the generating (i.e. biological) process and the form of the statistical analysis produce major conceptual and operational challenges for empiricists. We explored the consequences of mismatches by simulating data with three response-generating processes differing in the source of correlation between a covariate and the response. These data were then analysed by three different analysis equations. We asked how robustly different analysis equations estimate key parameters of interest and under which circumstances biases arise. Mismatches between generating and analytical equations created several intractable problems for estimating key parameters. The most widely misestimated parameter was the among-subject variance in response. We found that no single analysis equation was robust in estimating all parameters generated by all equations. Importantly, even when response-generating and analysis equations matched mathematically, bias in some parameters arose when sampling across the range of the covariate was limited. Our results have general implications for how we collect and analyse data. They also remind us more generally that conclusions from statistical analysis of data are conditional on a hypothesis, sometimes implicit, for the process(es) that generated the attributes we measure. We discuss strategies for real data analysis in face of uncertainty about the underlying biological process.

Multiple aspects of plasticity in clutch size vary among populations of a globally distributed songbird.

Plasticity in life-history characteristics can influence many ecological and evolutionary phenomena, including how invading organisms cope with novel conditions in new locations or how environmental change affects organisms in native locations. Variation in reaction norm attributes is a critical element to understanding plasticity in life history, yet we know relatively little about the ways in which reaction norms vary within and among populations. We amassed data on clutch size from marked females in eight populations of house sparrows (Passer domesticus) from North America and Europe. We exploited repeated measures of clutch size to assess both the extent of within-individual phenotypic plasticity and among-individual variation and to test alternative hypotheses about the underlying causes of reaction norm shape, particularly the decline in clutch size with date. Across all populations, females of this multibrooded species altered their clutch size with respect to date, attempt order, and the interaction of date and order, producing a reaction norm in multidimensional environmental space. The reaction norm fits that predicted by a model in which optimal clutch size is driven by a decline with date hatched in the ability of offspring to recruit. Our results do not fit those predicted for other proposed causes of a seasonal decline in clutch size. We also found significant differences between populations in response to date and the date by attempt order interaction. We tested the prediction that the relationship with date should be increasingly negative as breeding season becomes shorter but found steeper declines in clutch size with date in populations with longer seasons, contrary to the prediction. Populations also differed in the level of among-individual variation in reaction norm intercept, but we found no evidence of among-individual variation in reaction norm slope. We show that complex reaction norms in life-history characters exhibit within- and among-population variance. The nature of this variance is only partially consistent with current life-history theory and stimulates expansions of such theory to accommodate complexities in adaptive life history.

Climate change and its effects on body size and shape: the role of endocrine mechanisms.

In many organisms, rapidly changing environmental conditions are inducing dramatic shifts in diverse phenotypic traits with consequences for fitness and population viability. However, the mechanisms that underlie these responses remain poorly understood. Endocrine signalling systems often influence suites of traits and are sensitive to changes in environmental conditions; they are thus ideal candidates for uncovering both plastic and evolved consequences of climate change. Here, we use body size and shape, a set of integrated traits predicted to shift in response to rising temperatures with effects on fitness, and insulin-like growth factor-1 as a case study to explore these ideas. We review what is known about changes in body size and shape in response to rising temperatures and then illustrate why endocrine signalling systems are likely to be critical in mediating these effects. Lastly, we discuss research approaches that will advance understanding of the processes that underlie rapid responses to climate change and the role endocrine systems will have. Knowledge of the mechanisms involved in phenotypic responses to climate change will be essential for predicting both the ecological and the long-term evolutionary consequences of a warming climate. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Variation in Embryonic Metabolic Reaction Norms and the Role of the Environment.

AbstractEarly developmental environments can shape how organisms respond to later environments, but despite the potential for this phenomenon to alter the evolution of phenotypes and their underlying mechanisms in variable environments, details of this process are not understood. For example, both temperature and parental age can alter offspring metabolic plasticity and growth within species, yet the extent of such effects is unknown. We measured the reaction norms of embryonic heart rate in response to egg temperature and the change in egg mass over the incubation period in wild house sparrows. Using Bayesian linear mixed models, we estimated covariation in the intercepts and slopes of these reaction norms among clutches and eggs. We found that heart rate intercepts, not slopes, varied among clutches and that neither intercepts nor slopes varied among eggs within clutches. In contrast, egg mass intercepts and slopes varied among clutches and eggs. Ambient temperature did not explain variance in reaction norms. Instead, individuals from older mothers were more metabolically sensitive to egg temperature and lost less mass over the incubation period than individuals from younger mothers. Nevertheless, heart rate reaction norms and egg mass reaction norms did not covary. Our results suggest that early environments influenced by parents may contribute to variation in embryonic reaction norms. The structure of variation in embryonic reaction norms that exists both among clutches and among eggs demonstrates a complexity in plastic phenotypes that should be explored in future work. Furthermore, the potential for the embryonic environment to shape the reaction norms of other traits has implications for the evolution of plasticity more broadly.

The evolutionary ecology of variation in labile traits: selection on its among- and within-individual components.

Closer integration between behavioral ecology and quantitative genetics has resulted in a recent increase in studies partitioning sources of variation in labile traits. Repeatable between-individual differences are commonly documented, and their existence is generally explained using adaptive arguments, implying that selection has shaped variation at the among- and within-individual level. However, predicting the expected pattern of non-adaptive phenotypic variation around an optimal phenotypic value is difficult, hampering our ability to provide quantitative assessments of the adaptive nature of observed patterns of phenotypic variation within a population. We argue that estimating the strength of selection on trait variation among and within individuals provides a way to test adaptive theory concerned with phenotypic variation. To achieve this aim, we describe a nonlinear selection analysis that enables the study of the selective pressures on trait means and their among- and within-individual variation. By describing an integrative approach for studying the strength of selection on phenotypic variation at different levels, we hope to stimulate empirical studies investigating the ecological factors that can shape the repeatability, heritability, and coefficients of variation of labile and other repeatedly expressed traits.

Heterozygosity predicts clutch and egg size but not plasticity in a house sparrow population with no evidence of inbreeding.

We investigated the link between heterozygosity and the reaction norm attributes of reproductive performance in female house sparrows (Passer domesticus). We collected data on clutch size, egg size, hatching success and nestling survival in 2816 nesting attempts made by 791 marked individuals over a 16-year period. Pedigree analysis revealed no evidence of inbreeding. Neither parent-offspring regression nor an animal model revealed significant heritability in clutch or egg size. We selected 42 females that laid at least seven clutches at our study site and used a survey of 21 autosomal microsatellite loci to estimate heterozygosity for each female. We controlled for phenotypic plasticity and found that both clutch and egg size showed significant positive correlations with heterozygosity. We found no evidence that heterozygosity influenced the slope of individual reaction norms. Further analysis suggested that clutch size was affected by heterozygosity across the genome, but egg size had more complex relationships, with evidence favouring the influence of multiple loci. Given the apparent lack of inbreeding and large population size, our results suggest associative overdominance as the likely mechanism for the impact of heterozygosity, but also created a puzzle about the process producing associations between neutral markers and the genes affecting clutch size or egg size. One possible explanation is a long-term residual effect of the historical bottleneck that occurred when house sparrows were introduced into North America. The existence of heterozygosity-fitness correlations in a population with considerable phenotypic plasticity and little inbreeding implies that the effects of heterozygosity may be more significant than previously thought.

A reaction norm framework for the evolution of learning: how cumulative experience shapes phenotypic plasticity.

Learning is a familiar process to most people, but it currently lacks a fully developed theoretical position within evolutionary biology. Learning (memory and forgetting) involves adjustments in behaviour in response to cumulative sequences of prior experiences or exposures to environmental cues. We therefore suggest that all forms of learning (and some similar biological phenomena in development, aging, acquired immunity and acclimation) can usefully be viewed as special cases of phenotypic plasticity, and formally modelled by expanding the concept of reaction norms to include additional environmental dimensions quantifying sequences of cumulative experience (learning) and the time delays between events (forgetting). Memory therefore represents just one of a number of different internal neurological, physiological, hormonal and anatomical 'states' that mediate the carry-over effects of cumulative environmental experiences on phenotypes across different time periods. The mathematical and graphical conceptualisation of learning as plasticity within a reaction norm framework can easily accommodate a range of different ecological scenarios, closely linking statistical estimates with biological processes. Learning and non-learning plasticity interact whenever cumulative prior experience causes a modification in the reaction norm (a) elevation [mean phenotype], (b) slope [responsiveness], (c) environmental estimate error [informational memory] and/or (d) phenotypic precision [skill acquisition]. Innovation and learning new contingencies in novel (laboratory) environments can also be accommodated within this approach. A common reaction norm approach should thus encourage productive cross-fertilisation of ideas between traditional studies of learning and phenotypic plasticity. As an example, we model the evolution of plasticity with and without learning under different levels of environmental estimation error to show how learning works as a specific adaptation promoting phenotypic plasticity in temporally autocorrelated environments. Our reaction norm framework for learning and analogous biological processes provides a conceptual and mathematical structure aimed at usefully stimulating future theoretical and empirical investigations into the evolution of plasticity across a wider range of ecological contexts, while providing new interdisciplinary connections regarding learning mechanisms.

Preserving avian blood and DNA sampled in the wild: A survey of personal experiences.

Collecting and storing biological material from wild animals in a way that does not deteriorate DNA quality for subsequent analyses is instrumental for research in ecology and evolution. Our aims were to gather reports on the effectiveness of methods commonly used by researchers for the field collection and long-term storage of blood samples and DNA extracts from wild birds. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses but were not related to season of blood sampling, access to equipment, transporting samples, temperature, and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.

Individual variation in parental care reaction norms: integration of personality and plasticity.

Personality (consistent differences between individuals in behavior) and plasticity (changes within individuals in behavior) are often viewed as separate and opposing phenomena. We tested this idea by analyzing parental care reaction norms in a bird that exhibits biparental care. Personality in provisioning behavior existed (r(ic) = 0.11) and persisted despite being reduced after accounting for individual differences in environment. Plasticity was also evident and differed between the sexes. Male visit rate was associated with changes in brood size and time of day, but female visit rate was associated with changes in nestling age and date. In both sexes changes in visit rate were positively correlated with changes in their partner's visit rate. Both sexes also exhibited multidimensional reaction norms; interaction terms revealed that within-individual visit rates increased more steeply with brood size when nestlings were older, and the effect of the partner's visit rate was sensitive to variation in date, precipitation, and the focal bird's age. Individuals also varied in how they responded (reaction norm slope) to changes in nestling age and partner visits. Moreover, parental personality was interdependent with individual plasticity in several ways. Individuals of both sexes with a high visit rate also responded more positively to changes in nestling age, and males also showed this pattern with changes in partner visit rate. Explicit use of the behavioral reaction norm integrated personality and plasticity, revealed that these are not opposing concepts, and stimulated new hypotheses about sexual conflict over care and provisioning as a life-history trait.

Most published selection gradients are underestimated: Why this is and how to fix it.

Ecologists and evolutionary biologists routinely estimate selection gradients. Most researchers seek to quantify selection on individual phenotypes, regardless of whether fixed or repeatedly expressed traits are studied. Selection gradients estimated to address such questions are attenuated unless analyses account for measurement error and biological sources of within-individual variation. Estimates of standardized selection gradients published in Evolution between 2010 and 2019 were primarily based on traits measured once (59% of 325 estimates). We show that those are attenuated: bias increases with decreasing repeatability but differently for linear versus nonlinear gradients. Others derived individual-mean trait values prior to analyses (41%), typically using few repeats per individual, which does not remove bias. We evaluated three solutions, all requiring repeated measures: (i) correcting gradients derived from classic models using estimates of trait correlations and repeatabilities, (ii) multivariate mixed-effects models, previously used for estimating linear gradients (seven estimates, 2%), which we expand to nonlinear analyses, and (iii) errors-in-variables models that account for within-individual variance, and are rarely used in selection studies. All approaches produced accurate estimates regardless of repeatability and type of gradient, however, errors-in-variables models produced more precise estimates and may thus be preferable.

Pathways to social evolution and their evolutionary feedbacks.

In the context of social evolution, the ecological drivers of selection are the phenotypes of other individuals. The social environment can thus evolve, potentially changing the adaptive value for different social strategies. Different branches of evolutionary biology have traditionally focused on different aspects of these feedbacks. Here, we synthesize behavioral ecology theory concerning evolutionarily stable strategies when fitness is frequency dependent with quantitative genetic models providing statistical descriptions of evolutionary responses to social selection. Using path analyses, we review how social interactions influence the strength of selection and how social responsiveness, social impact, and non-random social assortment affect responses to social selection. We then detail how the frequency-dependent nature of social interactions fits into this framework and how it imposes selection on traits mediating social responsiveness, social impact, and social assortment, further affecting evolutionary dynamics. Throughout, we discuss the parameters in quantitative genetics models of social evolution from a behavioral ecology perspective and identify their statistical counterparts in empirical studies. This integration of behavioral ecology and quantitative genetic perspectives should lead to greater clarity in the generation of hypotheses and more focused empirical research regarding evolutionary pathways and feedbacks inherent in specific social interactions.

Heterozygosity and extra-pair paternity: biased tests result from the use of shared markers.

Recent studies of extra-pair paternity have found support for the idea that heterozygous males have an advantage in siring offspring. Most studies use DNA microsatellite loci to determine paternity and then use the same loci to estimate individual heterozygosity. However, because the likelihood of detecting extra-pair offspring depends on the combinations of parental alleles, it is possible that biases arise from particular allele combinations. This might produce false support for the influence of heterozygosity on mating behaviour. We used a simulation model to assess how large this bias might be. We found two sources of bias. First, we found a bias in the null hypothesis of a simple statistical test commonly used to test several predictions of the heterozygosity hypothesis. The use of randomization tests could eliminate this bias. Second, we found that using the same loci for both paternity and heterozygosity can cause an increase in results supporting the heterozygosity hypothesis when no effect of heterozygosity actually exists. This bias is reduced through the use of more markers with higher levels of polymorphism and heterozygosity, but can be eliminated entirely by using a separate set of markers to determine paternity and assess heterozygosity. The two sources of bias reduce evidence favouring the heterozygosity hypothesis, but do not negate all of the studies that support it. We suggest that further studies of heterozygosity and extra-pair paternity are important and likely to be informative, but our recommendations should be incorporated by researchers to improve the reliability of their conclusions.